Affymetrix®GeneChip® Operating Software With AutoLoader, Version 1.4 P/N 701439 Rev. 5 For research use only. Not for use in diagnostic procedures. Trademarks ® , GeneChip®, HuSNP®, GenFlex®, Flying Objective™, Affymetrix®, CustomExpress®, CustomSeq™, NetAffx™, Tools To Take You As Far As Your Vision®, and The Way Ahead™ are trademarks of Affymetrix, Inc. GeneArray® is a registered trademark of Agilent Technologies. All other trademarks are the property of their respective owners. Limited License Notice Limited License. Subject to the Affymetrix terms and conditions that govern your use of Affymetrix products, Affymetrix grants you a non-exclusive, non-transferable, non-sublicensable license to use this Affymetrix product only in accordance with the manual and written instructions provided by Affymetrix. You understand and agree that except as expressly set forth in the Affymetrix terms and conditions, that no right or license to any patent or other intellectual property owned or licensable by Affymetrix is conveyed or implied by this Affymetrix product. In particular, no right or license is conveyed or implied to use this Affymetrix product in combination with a product not provided, licensed or specifically recommended by Affymetrix for such use. Patents Scanner products may be covered by one or more of the following patents: U.S. Patent Nos. 5,578,832; 5,631,734; 5,834,758; 5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,171,793; 6,185,030; 6,201,639; 6,207,960; 6,218,803; 6,225,625; 6,252,236; 6,335,824; 6,403,320; 6,407,858; 6,472,671; 6,490,533, and other U.S. or foreign patents. Software products may be covered by one or more of the following patents: U.S. Patent No's. 5,733,729; 5,795,716; 5,974,164; 6,066,454; 6,090,555, 6,185,561 6,188,783, 6,223,127; 6,228,593; 6,229,911; 6,242,180; 6,308,170; 6,361,937; 6,420,108; 6,484,183; 6,505,125; 6510,391; 6,532,462; 6,546,340; 6,687,692; and other U.S. or foreign patents. Fluidics stations Products may be covered by U.S. Patent No. 6,114,122; and 6,391,623; 6,422,249; and other U.S. or foreign patents. AutoLoader products may be covered by one or more of the following patents: U.S. Patent Nos. 6,511,277; 6,604,902, and other U.S. or foreign patents. Copyright © 2003-2005 Affymetrix, Inc. All Rights Reserved. iii Table of Contents CHAPTER 1 CHAPTER 2 CHAPTER 3 Welcome to the GeneChip® Operating System 3 INTRODUCING GENECHIP® OPERATING SOFTWARE 3 CONVENTIONS USED IN THIS GUIDE 3 RESOURCES 5 Installing GCOS 9 INSTALLING GCOS FOR NEW USERS 9 Affymetrix GeneChip® Library Install INSTALLING LIBRARY FILES ON A NEW GCOS INSTALL 19 19 INSTALLING LIBRARY FILES ON AN UPGRADED GCOS INSTALL CHAPTER 4 GeneChip® System Overview 25 37 SYSTEM OVERVIEW 37 GENECHIP® ASSAY 38 DATABASES 47 iv CHAPTER 5 CHAPTER 6 CHAPTER 7 Affymetrix® GeneChip® Operating Software User’s Guide Getting Started 53 STARTING GCOS 53 CHECKING THE GCOS DATA STORAGE SETTING 55 THE GCOS TRANSFER AND ANALYSIS SERVICES 56 GCOS USER INTERFACE 61 RENAMING A PROJECT, SAMPLE, OR EXPERIMENT 67 GCOS DATA 68 FILTERING DATA 71 CONFIGURING THE FLUIDICS STATION & SCANNER 73 Setting Up an Experiment 79 REGISTERING A SAMPLE & DEFINING AN EXPERIMENT 80 VIEWING OR EDITING AN EXPERIMENT 85 Sample History & Workflow Monitor 91 SAMPLE HISTORY 91 WORKFLOW MONITOR 95 Contents CHAPTER 8 CHAPTER 9 Controlling the Instruments v 105 THE FLUIDICS STATION 105 SCANNING A PROBE ARRAY–GENECHIP® SCANNER 3000 121 TRACKING CELL ANALYSES AND DATA TRANSFERS 133 Working with Images 141 INTRODUCTION 141 THE IMAGE WINDOW WITH CEL AND DAT FILES 145 VIEWING AND ALIGNING THE GRID AND SUBGRIDS 147 THE CELL SUMMARY REPORT 178 COMPUTING CELL INTENSITIES 195 ADJUSTING THE IMAGE SETTINGS 196 INTENSITY DISPLAY 204 CALCULATING AVERAGE INTENSITY FOR USER SPECIFIED PROBE CELLS 205 VIEWING PROBE CELL DATA 206 VIEWING PROBE SET INFORMATION 207 BOOKMARKS 212 OUTLIERS 215 THE IMAGE VIEWER WITH JPG FILES 217 vi CHAPTER 10 CHAPTER 11 Affymetrix® GeneChip® Operating Software User’s Guide Gene Expression Analysis 225 OVERVIEW OF EXPRESSION ANALYSIS 226 EXPRESSION ANALYSIS SETTINGS 231 SINGLE-ARRAY EXPRESSION ANALYSIS 232 COMPARISON EXPRESSION ANALYSIS 235 EXPRESSION ANALYSIS TABULAR DATA 242 EXPRESSION ANALYSIS MEASURED IMAGES 256 EXPRESSION ANALYSIS GRAPHS 259 EXPRESSION REPORT 283 Batch Analysis 297 OPENING THE BATCH ANALYSIS WINDOW 298 SELECTING DATA FOR BATCH ANALYSIS 298 RENAMING RESULTS FROM BATCH ANALYSIS 306 REMOVING DATA FROM THE BATCH ANALYSIS WINDOW 308 RUNNING A BATCH ANALYSIS 309 EXPORTING A BATCH FILE 309 IMPORTING A BATCH FILE 310 Contents CHAPTER 12 CHAPTER 13 CHAPTER 14 CHAPTER 15 Publishing Data vii 315 SELECTING THE PUBLISH DATABASE 316 SPECIFYING THE TASK 316 PUBLISHING OPTIONS 320 PUBLISHING, CANCELING, OR RESTARTING A TASK 321 Defaults 327 VIEWING DEFAULT SETTINGS 327 FLUIDICS SETTINGS 333 SCANNER SETTINGS 334 ANALYSIS SETTINGS 335 Printing 341 PRINTING IMAGE OR CELL INTENSITY DATA 341 PRINTING PROBE ANALYSIS DATA 342 PRINTING AN EXPRESSION REPORT 343 GCOS Manager 347 GETTING STARTED 347 PROCESS TAB 356 viii CHAPTER 16 Appendix A Appendix B Affymetrix® GeneChip® Operating Software User’s Guide PUBLISH TAB 381 IMPORT TAB 396 USERSET TAB 415 TEMPLATE TAB 425 ROLES TAB 441 GCOS Administrator 455 GETTING STARTED 455 REGISTER OR UNREGISTER A GCOS SERVER 459 CHANGING A DATABASE PASSWORD 461 AUTOMATIC PROCESS DATABASE BACK UP 462 MANAGING WORKSTATION DATABASES 464 Client Testing 471 GCOS MANAGER 1.4 TESTING 471 GCOS 1.4 TESTING 493 DATA MINING TOOL 3.1 TESTING 518 GCOS MANAGER ADDITIONAL TESTING 523 Installing Data Mining Tool 3.1 DATA MINING TOOL 3.1 531 531 Contents Appendix C GCOS Instrument Installation ix 541 THE AFFYMETRIX® SCANNER 3000 WITH FLUIDICS STATION INSTALLATION 541 Appendix D Appendix E FLUIDICS STATION INSTALLATION (ONLY) 548 GENECHIP® SCANNER 3000 INSTALLATION (ONLY) 553 Using the GeneChip® Scanner 3000 561 INTRODUCTION 562 SAFE OPERATION 564 MAINTENANCE 568 CONNECTIONS AND INDICATOR LIGHTS 569 USING THE BARCODE READER 573 SCANNER 3000 SPECIFICATIONS 576 TROUBLESHOOTING 577 CE MARK DECLARATION OF CONFORMITY 583 REGULATORY 584 Configuring the GCOS Services 587 REQUIREMENTS FOR USING THE GCOS SERVICES 587 CONFIGURING THE GCOS SERVICE SETTINGS 589 SELECTING THE GCOS SERVICES OPTIONS 597 x Appendix F Appendix G Appendix H Affymetrix® GeneChip® Operating Software User’s Guide SETTING THE START/STOP SERVICE POLICY 601 SETTING THE ACT AS PART OF THE OPERATING SYSTEM POLICY 612 Expression Algorithm, Expression Analysis Metrics & Settings627 EXPRESSION ANALYSIS ALGORITHM 627 EXPRESSION ANALYSIS METRICS 636 EXPRESSION ANALYSIS SETTINGS 644 CELL SUMMARY REPORT ALGORITHM 679 GCOS Data Types 685 PROBE INFORMATION (LIBRARY) DATA 685 FLUIDICS PROTOCOL DATA 685 EXPERIMENT DATA 686 Base Codes and Amino Acid Abbreviations 691 IUPAC BASE CODES 691 AMINO ACID ABBREVIATIONS 691 xi Contents Appendix I Appendix J Appendix K Appendix L Toolbars, Hot Keys, & Windowpanes 695 TOOLBARS 695 HOT KEYS 702 WORKING WITH WINDOWPANES & COLUMNS 705 RESIZING OR HIDING COLUMNS IN THE EAW 706 Database Management & Data Source Name Descriptions709 PERFORMANCE TUNING FOR SQL SERVER DATABASES 709 CLIENT TUNING 711 Troubleshooting 715 TROUBLESHOOTING QUESTIONS 715 GCOS 1.4 TROUBLESHOOTING 716 GCOS MANAGER 1.4 TROUBLESHOOTING 721 LIBRARY FILES TROUBLESHOOTING 727 Using the GeneChip® AutoLoader 731 SETTING UP THE AUTOLOADER 732 INSTALLING AND CONFIGURING THE E-MAIL SYSTEM 739 xii Appendix M Affymetrix® GeneChip® Operating Software User’s Guide OPERATING THE SCANNER-AUTOLOADER 746 QUICK REFERENCE WALKTHROUGH 747 THE AUTOLOADER RUN 751 CLEANING AND MAINTENANCE 787 TROUBLESHOOTING 787 GENECHIP® SCANNER 3000 - AUTOLOADER SPECIFICATIONS 794 CE MARK DECLARATION OF CONFORMITY 795 REGULATORY 796 Data Transfer Tool Guide 801 USING THE DATA TRANSFER TOOL 801 ERROR MESSAGES AND TROUBLESHOOTING GUIDE 854 GCOS DATA COMPATIBILITY TABLE 865 Index 871 Chapter 1 Welcome to the GeneChip® Operating System Chapter 1 3 Welcome to the GeneChip® Operating System Welcome to the Affymetrix® GeneChip® Operating Software 1.4 User’s Guide. This manual explains how to use the following applications: • GeneChip® Operating Software (GCOS) 1.4 (formerly Affymetrix Microarray Suite) • GCOS Manager (formerly Affymetrix LIMS Manager) • GCOS Administrator Introducing GeneChip® Operating Software GCOS 1.4 manages Affymetrix GeneChip array data. GCOS 1.4 manages data on the local work station using a Microsoft® Data Engine database on the workstation. GCOS also has the ability to connect with Affymetrix GeneChip Array Operating Software Server, a separate application for large scale, network accessible databases. GCOS, GCOS Manager, and GCOS Administrator are a trio of applications that: • Automate control of the GeneChip instruments. • Capture and analyze the array image. • Provide workflow tracking of experiment data (image, cell intensities, probe analysis data). • Manage experiment data. • Automate basic expression analysis and publishing. • Provide end user administration tools for workstation databases. Conventions Used in This Guide This manual provides a detailed outline for all tasks associated with Affymetrix® GCOS, GCOS Manager, and GCOS Administrator. Various conventions are used throughout the manual to help illustrate the procedures described. Explanations of these conventions are provided below. 4 Affymetrix® GeneChip® Operating Software User’s Guide Steps Instructions for procedures are written in a step format. Immediately following the step number is the action to be performed. On the line below the step there may be the following symbol: ⇒. This symbol defines the system response or consequence that happens as a result of user action; what you see and what has happened. Following the response additional information pertaining to the step may be found and is presented in paragraph format. For example: 1. Click Yes to continue. The Delete task proceeds. In the lower right pane the status is displayed. To view more information pertaining to the delete task, right-click Delete and select View Task Log from the shortcut menu that appears. Font Styles Bold fonts indicate names of commands, buttons, options or titles within a dialog box. When asked to enter specific information, such input appears in italics within the procedure being outlined. For example: Click the Find button or select Edit → Find from the menu bar. The Find dialog box appears. 2. Enter AFFX-BioB-5_at in the Find what box, then click Find Next to view the first search result. 3. Continue to click Find Next to view each successive search result. 1. Screen Captures The steps outlining procedures are frequently supplemented with screen captures to further illustrate the instructions given. The screen captures depicted in this manual may not exactly match the windows displayed on your screen. chapter 1 | Welcome to the GeneChip® Operating System 5 Additional Comments Information presented in Tips provide helpful advice or shortcuts for completing a task. The Note format presents important information pertaining to the text or procedure being outlined. Caution notes advise you that the consequence(s) of an action may be irreversible and/or result in lost data. Warnings alert you to situations where physical harm to person or damage to hardware is possible. Resources ONLINE DOCUMENTATION The CD with GCOS includes an electronic version of this user’s guide. The online documentation is in Adobe Acrobat® format (a *.pdf file) and is readable with the Adobe® Acrobat Reader® software, available at no charge from Adobe at http://www.adobe.com. The online help contains a version of this user’s guide as a Microsoft® HTML help file. 6 Affymetrix® GeneChip® Operating Software User’s Guide TECHNICAL SUPPORT Affymetrix provides technical support to all licensed users via phone or E-mail. To contact Affymetrix® Technical Support: Affymetrix, Inc. 3380 Central Expressway Santa Clara, CA 95051 USA E-mail: [email protected] Tel: 1-888-362-2447 (1-888-DNA-CHIP) Fax: 1-408-731-5441 Affymetrix UK Ltd., Voyager, Mercury Park, Wycombe Lane, Wooburn Green, High Wycombe HP10 0HH United Kingdom UK and Others Tel: +44 (0) 1628 552550 France Tel: 0800919505 Germany Tel: 01803001334 E-mail: [email protected] Tel: +44 (0) 1628 552550 Fax: +44 (0) 1628 552585 Affymetrix Japan, K. K. Mita NN Bldg 16 Floor, 4-1-23 Shiba, Minato-ku, Tokyo 108-0014 Japan Tel: +81 (03) 5730-8222 Fax: +81 (03) 5730-8201 www.affymetrix.com Chapter 2 Installing GCOS Chapter 2 9 Installing GCOS This chapter provides detailed instructions for installing the GeneChip® Operating Software (GCOS) for new users. For information on installing the library files, see Chapter 3, Affymetrix GeneChip® Library Install, on page 19. For information on installing the instruments, see Appendix C, GCOS Instrument Installation, on page 541. Installing GCOS for New Users You must be logged in as an administrator to install GCOS. The screen captures depicted in this manual may not exactly match the windows displayed on your screen. At least 1024 MB of available disk space is recommended for the installation. Insert the GCOS CD-ROM. 2. If the autorun feature does not start the program: 1. Click Start → Run. B. Type <cd drive letter>:\setup.exe. C. Click OK. The Affymetrix Software Setup window appears (Figure 2.1). A. 10 Affymetrix® GeneChip® Operating Software User’s Guide Figure 2.1 Install window Click Install GCOS. 4. In the Welcome window (Figure 2.2), click Next. The License Agreement windows appear (Figure 2.3). 3. Figure 2.2 Welcome to GCOS setup chapter 2 | Installing GCOS Figure 2.3 License Agreement Window 5. Review the contents and click Yes to accept the terms of the licensing agreement. The Customer Information window appears (Figure 2.4). 11 12 Affymetrix® GeneChip® Operating Software User’s Guide Figure 2.4 Customer Information window 6. Enter your Name, Company, and Serial Number. The serial number is located on the Affymetrix® Software Product Registration card. If you do not have a serial number, contact Affymetrix Technical Support. 7. Click Next. The GCOS Server Name window appears (Figure 2.5). chapter 2 | Installing GCOS Figure 2.5 GCOS Server Name window Enter the name of the remote GCOS server that you want to connect to. If you do not have one, leave the server name blank. 9. Click Next. The Choose Destination Locations window opens (Figure 2.6). 8. 13 14 Affymetrix® GeneChip® Operating Software User’s Guide Figure 2.6 Choose Destinations Locations window 10. Select the destination where GCOS will be installed. C:\GeneChip is the default location. If you are upgrading from Microarray Suite, we recommend installing in the same directory. 11. Enter and confirm the password for the MSDE system administrator (sa). The password should be at least six characters long and must contain at least two digits. 12. Click Next. The installation begins. When the install is completed, the GCOS Software Setup Completed window appears (Figure 2.7). chapter 2 | Installing GCOS 15 Figure 2.7 GeneChip Operating Software Setup Completed window 13. Choose Yes, I want to restart my computer now, and click Finish. The system reboots. If you are not prompted to reboot your computer, the installation is complete. If you are upgrading from a version of Microarray Suite, it is necessary to run the library migrate program to migrate the library files into the local database. If GCOS will be used with a GCOS server, it is important to ensure the library migration has been completed on the GCOS server. 16 Affymetrix® GeneChip® Operating Software User’s Guide Chapter 3 Affymetrix GeneChip® Library Install Chapter 3 19 Affymetrix GeneChip® Library Install This chapter provides detailed instructions for installing Affymetrix® GeneChip® probe array library files using the library install program. The probe information or library data include the probe array design characteristics, probe utilization and content, and scanning and analysis parameters. Library data are unique to each probe array type. The library install program includes a full install and an update program. The full install should be used when installing the Affymetrix GeneChip Operating Software (GCOS) on a new system. If upgrading from a previous version of Affymetrix Microarray Suite, use the library update program. This chapter contains the following sections: • Installing Library Files on a New GCOS Install (see below) • Installing Library Files on an Upgraded GCOS Install (see page 25) Installing Library Files on a New GCOS Install This section of the manual guides you through the full library install program. The full install is required on a GCOS system that was not upgraded from a previous version of Affymetrix® Microarray Suite. 1. Insert the library files CD-ROM for the appropriate probe array type. 20 Affymetrix® GeneChip® Operating Software User’s Guide The Affymetrix Library Files Setup window appears (Figure 3.1). Figure 3.1 Library Files Setup window 2. Click Full Library Install. chapter 3 | Affymetrix GeneChip® Library Install 21 The InstallShield Wizard for Affymetrix Library Files starts (Figure 3.2). Figure 3.2 InstallShield Wizard Welcome window GCOS does not allow you to modify the location of the library file directory. By default, all probe array type information is installed in the library subdirectory of the GCOS installation. Do not move or delete the library directory or the files in the directory. This will render the GCOS installation invalid. 3. Click Next. 22 Affymetrix® GeneChip® Operating Software User’s Guide The Select Probe Array Types window appears (Figure 3.3). Figure 3.3 Select Probe Array Types window Click Select All to select all array types for installation; or Place a check mark next to the array type(s) that you want to install. For example, in Figure 3.3, the HG-U133A and HG-U133B probe arrays will be installed on the system. 5. Click Next. The Start Installing Files window appears (Figure 3.4). This window shows the install options selected, including the: - probe array types that will be installed on the system - location of the library directory where the probe array library data will be saved - location of the protocol directory where the fluidic station script files will be saved. 4. chapter 3 | Affymetrix GeneChip® Library Install Figure 3.4 Start Installing Files window 6. Click Next. The install process begins. 23 24 Affymetrix® GeneChip® Operating Software User’s Guide After the installation is complete, the software displays the Finish window (Figure 3.5). Figure 3.5 Finish window 7. Choose Yes, I want to view the Audit Report file now, and click Finish. The installation completes. If an error occurs, the software displays an error dialog box (Figure 3.6). Figure 3.6 Error dialog box chapter 3 | Affymetrix GeneChip® Library Install 8. 25 Click Yes to view the SQL.log and Audit XXX.log that contain information about the error that occurred. Forward this file to Affymetrix® technical support to obtain a solution for the problem. Installing Library Files on an Upgraded GCOS Install The library install program includes a full install and an update program. Use the update program when you upgrade from a version of Affymetrix® Microarray Suite to GCOS. The update program migrates probe array type information from the files in the library directory that was used with Microarray Suite. 1. Insert the library files CD_ROM for the appropriate probe array type. The Affymetrix Library Files Setup window appears (Figure 3.7). Figure 3.7 Library Files Setup window 2. Click Update Library Files. 26 Affymetrix® GeneChip® Operating Software User’s Guide The InstallShield Wizard for Affymetrix Library Files starts (Figure 3.8). Figure 3.8 InstallShield Wizard Welcome window 3. Click Next. chapter 3 | Affymetrix GeneChip® Library Install 27 The Select Source Library Directory window appears (Figure 3.9). Figure 3.9 Select Source Library Directory window 4. To select the location of the source library directory from which to migrate the information into the GCOS database, click Browse. Click Next. This library directory is the directory that was used with Affymetrix Microarray suite. It contains .CIF and .CDF files that contain information about the probe array types used in the system. 28 Affymetrix® GeneChip® Operating Software User’s Guide The Select Source Protocol Directory window appears (Figure 3.10). Figure 3.10 Select Source Protocol Directory window Click Browse and select the location of the fluidics protocols directory from which to migrate the information into the GCOS database. 6. Click Next to display the probe array types available for migration. 5. chapter 3 | Affymetrix GeneChip® Library Install 29 The Select Probe Array Types window appears (Figure 3.11). To migrate a probe array type from a previous version of Microarray Suite software, the library directory must contain the .CIF and .CDF files. The update program will not install the gene descriptions, sequences, or annotations. These should exist in the GCOS database from the previous Microarray Suite install. To migrate fluidics scripts from the protocols directory, the .BIN file must exist along with the .MAC file. If the .BIN file is missing, the software will not update the database with the associated script. However, if a .BIN file exists in the directory without the associated .MAC file (for example, the “Home” script that can be executed without selecting an experiment) the update program will copy the file to the new protocols directory. Figure 3.11 Select Probe Array Types window 7. Select the probe array types that you want to migrate from the library directory. Click Select All to select all of the array types or 30 Affymetrix® GeneChip® Operating Software User’s Guide place a check mark next to the probe array type(s) that you want to install. For example, in Figure 3.11, the RG-U34A probe array will be installed on the system. 8. Click Next. The Select Protocols window appears and displays the fluidics protocols that will be migrated to the GCOS installation (Figure 3.12). Figure 3.12 Select Protocols window Select the fluidics scripts that you want to migrate from the protocols directory. To select all of the scripts, click Select All. To select scripts individually, place a check mark next to the script. For example, in Figure 3.12, the scripts EukGE-WS1v4, EukGEWS2v4, FlexGE-WS1v4, FlexGE-WS2v4 will be installed on the system. 10. Click Next. 9. chapter 3 | Affymetrix GeneChip® Library Install 31 The Start Importing Library Files window appears (Figure 3.13). It shows the: - probe array types and fluidics scripts that will be installed - location of the library directory for the probe array type data - location of the protocol directory for the fluidics station scripts Figure 3.13 Start Importing Library Files window 11. Click Next to start the installation. 32 Affymetrix® GeneChip® Operating Software User’s Guide After the installation is complete, the software displays the Finish window (Figure 3.14). Figure 3.14 Finish window 12. Choose Yes, I want to view the Audit Report file now and click Finish. The installation completes. If an error occurs, the software displays an error dialog box (Figure 3.15). Figure 3.15 Error dialog box chapter 3 | Affymetrix GeneChip® Library Install 13. Click 33 Yes to view the sql.log and updateXXX.log files that contain information about the error that occurred. Forward this file to Affymetrix® technical support to obtain a solution for the problem. 34 Affymetrix® GeneChip® Operating Software User’s Guide Chapter 4 GeneChip® System Overview Chapter 4 37 GeneChip® System Overview This chapter provides background information on the Affymetrix® GeneChip® array and instrument platform. It provides a brief overview of the software applications that acquire, analyze, and manage the data. This chapter contains the following sections: • System Overview (see below) • GeneChip® Assay (see page 38) • Databases (see page 47) System Overview The Affymetrix® GeneChip® platform uses high-density GeneChip oligonucleotide probe arrays to efficiently acquire and analyze genetic information. This integrated and complete system includes the: • GeneChip® Hybridization Oven 640 • GeneChip® Fluidics Station • GeneChip® Scanner 3000 scanner • Computer workstation • GeneChip® Operating Software (GCOS) 1.4 (formerly Microarray Suite) • GCOS Manager (formerly LIMS Manager) • GCOS Administrator • GeneChip® Operating Software Server (GCOS Server) (formerly LIMS) • Affymetrix® Data Mining Tool (DMT) • GeneChip® Sequence Analysis Software (GSEQ) • GeneChip® Genotyping Analysis Software (GTYPE) 38 Affymetrix® GeneChip® Operating Software User’s Guide GeneChip® Assay The GeneChip® Operating Software (GCOS) automates instrument control and provides a central infrastructure to acquire, track, and analyze experiment data. Table 4.1 shows the major steps and software functions in a GeneChip® assay. Table 4.1 GeneChip® Assay Assay Step Output Software Function Experiment information GCOS adds the sample and experiment information to the process database. GCOS tracks the sample and the associated experiment data (image, cell intensities, and probe analysis data) in the workflow monitor. Prepare the target. Register the sample and set up an experiment. Hybridize, wash, and stain the probe array. Scan the probe array. GCOS automates control of the fluidics station and scanner. Image data GCOS automates control of the scanner, acquires and saves the scan data. Cell intensity data GCOS automatically computes and saves the cell intensities from the image data. GCOS tracks the image and cell intensity data in the process database. chapter 4 | GeneChip® System Overview 39 Table 4.1 GeneChip® Assay Assay Step Output Software Function Analyze the cell intensities. Probe analysis The GCOS Statistical Expression algorithm analyzes the cell intensities of expression arrays, and saves the probe analysis or chip data. The GSEQ algorithms analyze the cell intensities of resequencing GeneChip® arrays, and saves the probe analysis or chip data. The GTYPE algorithms analyze the cell intensities of other types of GeneChip® genotyping and Universal Tag arrays, and saves the probe analysis or chip data. Generate a report. Report (.rpt) GCOS generates and saves the Expression report (.rpt). GSEQ and GTYPE generate and save a report (.rpt) for array types other than expression. Publish the sample and experiment data. GCOS copies expression experiment data (cell intensity and probe analysis data) to the publish database where the data are accessible to the Affymetrix® Data Mining Tool (DMT) software and other third party analysis tools. GCOS provides a workflow-based tracking system for the data. The workflow monitor (Figure 4.1) tracks sample or experiment status through a series of steps or queue that includes: • hybridization • scan • grid alignment • cell intensity analysis (for image data files that require manual grid alignment) 40 Affymetrix® GeneChip® Operating Software User’s Guide • probe array analysis for expression assay • publish expression probe array analysis data You can easily confirm the status of an experiment by checking the workflow monitor. For example, the Hybridization tab shows experiments to be processed in the fluidics station. Figure 4.1 Workflow monitor, Hybridization tab The workflow is further described in the following sections: • Preparing the Target Sample, on page 41 • Registering a Sample and Setting Up an Experiment, on page 41 • Hybridizing, Washing, and Staining a Probe Array, on page 43 • Scanning a Probe Array, on page 43 • Computing Cell Intensities, on page 44 • Expression Analysis, on page 45 • Publishing Experiment Data, on page 46 chapter 4 | GeneChip® System Overview 41 PREPARING THE TARGET SAMPLE Refer to the appropriate GeneChip® probe array package insert, the Expression Analysis Technical Manual, or to other Affymetrix® technical documentation relevant to your particular project. REGISTERING A SAMPLE AND SETTING UP AN EXPERIMENT You must identify or register a sample (target) and define an experiment in GCOS before processing a probe array in the fluidics station or scanning an array. You register the sample by entering the sample type (required) and sample information (attributes), and associating the sample with a project (which may include several different samples) (Figure 4.2). The registration process adds the sample information to the process database. Now GCOS tracks the sample and the associated experiment data (image, cell intensities, probe analysis data) in the workflow monitor. 42 Affymetrix® GeneChip® Operating Software User’s Guide Figure 4.2 Sample and experiment information To define an experiment, you specify the type of probe array to which the target sample will be hybridized as well as other information relevant to the experiment (Figure 4.2). You can also associate a barcode with an experiment. If an Affymetrix® barcode is entered (manually or using a keyboard wedge reader), GCOS automatically reads and displays the probe array type, lot number, and expiration date. GCOS uses the experiment information to select scanner specifications for the probe array and identify the user-modifiable algorithm parameters for data analysis. You can also use a template (defined in GCOS Manager) to register a sample and define an experiment. For more information on creating a template, see Template Tab, on page 425. chapter 4 | GeneChip® System Overview 43 HYBRIDIZING, WASHING, AND STAINING A PROBE ARRAY During hybridization, the probe array is incubated with a hybridization cocktail containing the labeled target and control oligonucleotides in a buffer optimized for each type of probe array (refer to the appropriate GeneChip® probe array package insert). The hybridization reaction occurs in the GeneChip® fluidics station, except for assays requiring longer hybridization periods in which case the hybridization reaction occurs in the GeneChip® Hybridization Oven 640. During hybridization in the fluidics station, the probe array is repeatedly filled and drained with the hybridization cocktail. The strongest hybridization occurs between the array probes and sequences in the target that are most nearly complementary. After hybridization, the probe array undergoes a series of stringent washes (specifically optimized for each type of probe array) in the fluidics station. If the target was labeled with a fluorescent tag, the array may be scanned at this point using the GeneChip® Scanner 3000. In applications that use a biotin-labeled target, the probe array must be stained with a streptavidin-conjugated fluorescent stain and requires antibody amplification prior to scanning. GCOS controls the fluidics station using pre-programmed fluidics protocols for hybridizing, washing, and staining the probe arrays. The computer workstation running GCOS can simultaneously control up to eight fluidics stations. A fluidics station contains four modules and each module can independently process a probe array using different fluidics protocols. Some of the fluidics protocols may be customized. Utilities for station maintenance are also included. SCANNING A PROBE ARRAY After the GeneChip® probe array has undergone hybridization, washing, and staining, it is ready to be scanned. GCOS automates control of the scanner and uses the experiment information to manage the scanner settings for each type of probe array. The software enables you to start a scan and view the intensity 44 Affymetrix® GeneChip® Operating Software User’s Guide data as it is collected during scanning. After the scan is completed, GCOS displays a picture of the image data in the Image window (Figure 4.3). GCOS tracks the image data and the scan parameters in the process database. For more information on image data, see Chapter 9, Working with Images, on page 141. The Affymetrix® GeneChip® Scanner 3000 requires about 4 to 7 minutes to scan an array depending on the size of the scan region, the number of scans, the autofocusing adjustment, and other parameters. The Affymetrix® Scanner 3000 cannot scan fluorescein labeled arrays. COMPUTING CELL INTENSITIES After the scan is completed, GCOS displays a picture of the scan image in the image window. The software represents the fluorescence intensity values from each pixel on the array in a grayscale or pseudocolor mode and superimposes a grid on the image to delineate the probe cells (features). GCOS automatically: • analyzes the image data and computes a single intensity value for each probe cell on an array • saves the cell intensity data (Figure 4.3) GCOS tracks the cell intensity data and the parameters used in the cell intensity analysis in the process database. GCOS can be configured to archive the scan image locally or on a network location. For more information, see Setting Archiving Options for DAT files, on page 599. chapter 4 | GeneChip® System Overview 45 Figure 4.3 Image window, scan image (left) and computed cell intensity data (right) EXPRESSION ANALYSIS The Statistical Expression algorithm processes the cell intensity data. A single-array analysis examines the cell intensity data from one experiment (GeneChip® probe array). For each transcript represented on the array, the algorithm computes the detection call (present, absent, or marginal (unable to call the transcript present or absent), or no call) and other call metrics. A comparison analysis compares the cell intensities of the same probe set on two different probe arrays (an experiment and baseline of the same probe array type) to determine the relative change in the expression level of a transcript. For each transcript represented on the array, the algorithm computes the change call (increase, marginal increase, no change, marginal decrease, decrease) or no call, and other call metrics. 46 Affymetrix® GeneChip® Operating Software User’s Guide GCOS saves the probe analysis data and automatically displays it in the Expression Analysis window (Figure 4.4). GCOS tracks the probe analysis data and the analysis parameters in the process database. Figure 4.4 Expression Analysis window, probe analysis data GCOS analyzes expression cell intensity data only. GTYPE analyzes cell intensity data from Mapping and Universal Tag probe arrays. GSEQ analyzes cell intensity data from Resequencing probe arrays. PUBLISHING EXPERIMENT DATA GCOS copies or publishes expression experiment data (cell intensity and probe analysis data) in AADM format to the publish database (see Figure 4.5 on page 49). The publish database is accessible to the Affymetrix® Data Mining Tool (DMT) or several other third party chapter 4 | GeneChip® System Overview 47 analysis tools. The DMT is a separate application that provides powerful query and analysis tools. Databases You can use GCOS installed as a stand-alone workstation or with a networked GCOS Server. If used as a stand-alone workstation, GCOS installs Microsoft® Data Engine (MSDE) databases on the workstation. Table 4.2 shows the GCOS databases and their functions. Alternatively, the Affymetrix® GeneChip® Operating Software Server (GCOS Server) installs Microsoft® SQL Server or Oracle® databases on a network accessible server. GCOS also provides the end user interface to GCOS Server. GCOS Server enables users to register samples and set up experiments, and access and analyze data from any client workstation on the network. Table 4.2 GCOS databases Database Name Contents Process Data tracking information about samples and experiments, and information about the image, intensity, and probe analysis data for a particular sample and experiment. Publish Expression data in the Affymetrix® Analysis Data Model (AADM) database schema that can be accessed by the Affymetrix® Data Mining Tool (DMT) and other software. Gene Information Accession numbers, probe set descriptions, target sequence, and user annotations accessible to DMT. DATABASE MANAGEMENT & ADMINISTRATION GCOS Manager is a separate application that helps you manage your expression data. This software tool provides a convenient way to: • Manage the process and publish databases. 48 Affymetrix® GeneChip® Operating Software User’s Guide • Delete process or publish data. • Create templates for entering sample and experiment attributes. • Create a userset (a predefined set of Statistical Expression algorithm parameter settings) for expression analysis. • Archive image, cell intensity, and probe analysis data to a network drive. • Restore image, cell intensity, and probe analysis data to a network drive. • Export an experiment and the associated data files to a userspecified location on the workstation. • Define user privileges (roles) for the GCOS server. • Apply additional file security to the GCOS server. For more information, see Chapter 15, GCOS Manager, on page 347. GCOS Administrator enables you to: • Monitor free and used workstation disk space. • Change the location of a publish database. • Change the password for the process database or a publish database. For more information, see Chapter 16, GCOS Administrator, on page 455. Together GCOS, GCOS Manager, and GCOS Administrator provide full functionality for the GeneChip® instrument and array platform as well as basic expression analysis (Figure 4.5). chapter 4 | GeneChip® System Overview Figure 4.5 GCOS, GCOS Manager, GCOS Administrator, and Data Mining Tool software 49 50 Affymetrix® GeneChip® Operating Software User’s Guide Chapter 5 Getting Started Chapter 5 53 Getting Started This chapter explains how to: • Start the GeneChip® Operating Software (GCOS) (see below) • Check the GCOS data storage setting (see page 54) • Specify filters that determine the data displayed by the system (see page 71) • Configure the fluidics station and scanner (see page 73) The chapter also introduces: • The optional GCOS Services for automated data management (see page 56) • The GCOS interface (see page 61) • GCOS data types (see page 68) Starting GCOS 1. Turn on the power for the computer workstation. If the workstation is connected to the instruments, also turn on the power for the GeneChip® Fluidics Station 400 and the Affymetrix® GeneChip® Scanner 3000. Laser in use during scanning. The scanner laser should be turned on and warmed up at least 10 minutes before use. For more information, refer to Appendix D, Using the GeneChip® Scanner 3000, on page 559. 2. After the computer startup is complete, press Ctrl+Alt+Delete to open the logon dialog box. Enter your user name, password, and domain. 54 Affymetrix® GeneChip® Operating Software User’s Guide Click the Microsoft® Windows® Start menu button , then select Programs → Affymetrix → GeneChip Operating Software. When GCOS starts, the program displays the shortcut bar, data tree, and status log (Figure 5.1). The GCOS User interface is described in the following sections: • Shortcut Bar (see below) 3. • Data Tree (see page 64) • Status Log (see page 67) • Scan Status Window (see page 760) After you have completed the above steps, you can configure the GCOS software to: • Run with a network accessible GeneChip Operating Software (GCOS) server (see below). • Use the optional GCOS Services for automated data management (see page 56). chapter 5 | Getting Started 55 Menu bar Toolbar Data Tree displays system data in the process database that resides on the local workstation or GCOS server. The data may be filtered. Main display area Shortcut bar has three sections: GeneChip Software Instrument Control Settings Scan Status log Status log Figure 5.1 GCOS user interface (local database) Checking the GCOS Data Storage Setting The GCOS software may be configured to run with a network accessible GeneChip® Operating Software (GCOS) Server or with an MSDE database on the workstation. To check the database setting to make sure GCOS is properly configured for your system: 1. In the Settings shortcut bar, click the Defaults button Select Tools → Defaults from the menu bar. The Defaults dialog box appears. 2. Click the Database tab (Figure 5.2). ; or 56 Affymetrix® GeneChip® Operating Software User’s Guide Figure 5.2 Defaults dialog box, Database tab The GCOS Server data storage option is available only if all windows are closed and no instrument is active. 3. To store experiment data on the GCOS server, choose the Affymetrix GCOS option. If the GCOS Server option is not selected, the data are stored in the local MSDE database. 4. Click OK. The GCOS Transfer and Analysis Services GCOS provides services that help you automate your data management: • GCOS Analysis Service performs cell intensity analyses. If you are using GCOS with a networked GCOS Server, you can chapter 5 | Getting Started 57 configure the software to run the analysis on the GCOS Server or on the local instrument control client. When analyzing data on the client (in local mode or in GCOS Server mode), the software uses the service that runs on the local workstation. When analyzing data on the GCOS Server (in server mode), the software uses the analysis service on the remote GCOS Server. • GCOS Transfer Service lets you automate the archiving of DAT files. If you are performing a scan on a system configured to work with a GCOS Server, and if GCOS is set up to analyze the DAT to CEL data on the GCOS Server, you cannot use the transfer service to archive data. This is the default mode. You have several options for configuring the services, depending upon the type of setup you have: • Configuring the Analysis and Transfer Services on the Client (see page 57) • Configuring the Analysis Service on the GCOS Server (see page 60) CONFIGURING THE ANALYSIS AND TRANSFER SERVICES ON THE CLIENT The analysis/transfer service settings can be configured by opening the service settings dialog from the Archive Settings tab or the Analysis Settings tab on the GCOS Defaults dialog box (Figure 5.3). 58 Affymetrix® GeneChip® Operating Software User’s Guide Click here or here to open the GCOS Service Settings dialog box Figure 5.3 Defaults dialog box, Archive Settings and Analysis Settings tabs, and GCOS Service Settings dialog box The transfer service and analysis service use the same settings. Changes made to one while using the service settings dialog will automatically apply to the other service. chapter 5 | Getting Started 59 There are two possible workstation configurations: • Local analysis of DAT to CEL data on a workstation configured to work without a GCOS Server • Local analysis of DAT to CEL data on a workstation configured to work with a GCOS Server Local analysis of DAT to CEL data on a workstation configured to work without a GCOS Server The workstation configuration can be determined by looking at the Database tab of the GCOS Defaults dialog. If the server name is empty, the workstation was configured to work without a GCOS server. For more information, see Checking the GCOS Data Storage Setting, on page 55. The services can run under the Windows SYSTEM account if archiving is OFF (for information on setting the archive options see Setting Archiving Options for DAT files, on page 599). If archiving is ON, the service must be configured to run under the user credential who has access to the Universal Naming Convention (UNC) path where data is to be archived. Any changes to the analysis service user settings are applied automatically to the transfer service settings. For more information, see Configuring the GCOS Service Settings on a Stand Alone System (No GCOS server), on page 591. Local analysis of DAT to CEL data on a workstation configured to work with a GCOS Server The analysis /transfer service must be reconfigured to run as a user who has admin rights on the local machine and is in the GCOS Administrator's role on the GCOS Server. The list is populated automatically with a list of users who have the above mentioned rights. 60 Affymetrix® GeneChip® Operating Software User’s Guide For more information, see Configuring the GCOS Service Settings on a Client Configured to Work with GCOS Server, on page 593. CONFIGURING THE ANALYSIS SERVICE ON THE GCOS SERVER The analysis service settings can be configured by opening the service settings dialog from the control panel of Microsoft Windows (Figure 5.4). Open the Control Panel and click the GeneChip Analysis and Transfer icon to open the GCOS Service Settings dialog box for the GCOS Server Figure 5.4 Control Panel and GCOS Service Settings dialog box The analysis service must be reconfigured to run as a user who has admin rights on the local machine and is in the GCOS Administrator's role on the GCOS Server. The list is populated automatically with a list of users who have the above mentioned rights. For more information, see Configuring the GCOS Service Settings on a GCOS Server, on page 595. chapter 5 | Getting Started 61 For more information on the following topics, refer to Appendix E, Configuring the GCOS Services, on page 587: • The requirements for using the Analysis and Transfer services (see below) • How to configure the GCOS Service settings (see page 589) GCOS User Interface The GCOS User interface is described in the following sections: • Shortcut Bar (see below) • Data Tree (see page 64) • Status Log (see page 67) • Scan Status Window (see page 760) SHORTCUT BAR The shortcut bar provides quick alternatives to menu bar commands. The shortcut bar has three sections: • GeneChip Software • Instrument Control • Settings • To display a particular section of the shortcut bar, click the shortcut bar section name (Figure 5.5 through Figure 5.6). GeneChip® Software Shortcut Bar Figure 5.1 shows the GeneChip Software shortcut bar at startup. Additional buttons may be displayed in the shortcut bar, depending on the data types that are open (Figure 5.5). For example, if a scan image or cell intensity data are open, the shortcut bar includes the Image Views button . Click this button to view the cell intensity image in the main display area. 62 Affymetrix® GeneChip® Operating Software User’s Guide p Shortcut Bar Functions Click a button to change the display of open data in the main display area. Set up an experiment or display experiment attributes Display open image or cell intensities Open the Batch Analysis window Open the Publish window Open the sample history view Open the workflow monitor Display open probe analysis data in the Expression Analysis window Display an open report (.rpt) Figure 5.5 GeneChip® Software shortcut bar chapter 5 | Getting Started 63 Instrument Control Shortcut Bar Instrument Control Shortcut Bar Functions Click a button to perform the following functions. Open Station Selection & Fluidics Station dialog box Open the Scanner dialog box Figure 5.6 Instrument control shortcut bar The instrument control shortcut bar does not display instrument buttons if the workstation is not connected to the instruments, or if the default configuration settings specify no fluidics station or scanner installed (select Tools → Defaults from the menu bar to view the default settings). 64 Affymetrix® GeneChip® Operating Software User’s Guide Settings Shortcut Bar Settings Shortcut Bar Functions Click a button to perform the following functions. Open the Defaults dialog box Open the Filters dialog box Open the Expression Analysis Settings dialog box Open the Expression Report dialog box Figure 5.7 Settings shortcut bar DATA TREE The data tree displays the data in the system (process database), including: • experiments • image data • cell intensities • analysis results chapter 5 | Getting Started 65 There are two different views of the data tree (Figure 5.8): • Classic View organizes the experiment by data type. • Project/Sample view organizes the experiment data by sample, and samples are organized by project. Classic View Project/Sample View Project Sample Experiment Experiment data includes image, cell intensity, and probe analysis data Figure 5.8 Data tree, Classic View (left), Project/Sample View (right) 66 Affymetrix® GeneChip® Operating Software User’s Guide Data Tree Display Options • To select the data tree view, right-click the data tree and choose Classic View or Project/Sample View from the shortcut menu that appears. • To collapse or expand the list of data types in the tree, click the minus [-] or plus [+] sign next to each data type icon. • To hide (or display) the data tree, click the Data Tree button Data Tree Shortcut Menu • To view a shortcut menu of commands (specific to the data type), right-click a data name in the tree (Figure 5.9). Figure 5.9 Data tree shortcut menu for cell intensities From the data tree you can: • Open an experiment, an image, cell intensity data, or probe analysis data. • Update the data source tree to show new information in the system. • Display information about the data (attributes). . chapter 5 | Getting Started 67 • Rename an experiment, sample, or project. • Analyze an image (generate the cell intensity data). • Analyze the cell intensity data from an expression probe array (generates the probe analysis data). • Generate an expression report. STATUS LOG The status log displays system status messages (Figure 5.1). To clear the messages, right-click the status log and select Clear Messages in the shortcut menu that appears. 2. To mute the error message sound, right-click the status log and remove the check mark from the Play Error Sound option. 3. Click the Status Log button in the main toolbar to hide (or display) the status log. 1. Renaming a Project, Sample, or Experiment In the data tree project/sample view, you can rename an project, sample, or experiment. In the data tree classic view, you can rename an experiment. 1. Right-click the data tree, and select Project/Sample View from the shortcut menu that appears (Figure 5.10). 68 Affymetrix® GeneChip® Operating Software User’s Guide Figure 5.10 Data tree, project/sample view 2. Expand the data tree, and right-click the item (project, experiment, or sample) that you want to rename. 3. Select Rename Project (Rename Experiment or Rename Sample) in the shortcut menu that appears. A Rename dialog box appears (Figure 5.11). Figure 5.11 Rename Project dialog box 4. Enter a new name and click OK. GCOS Data There are three types of GCOS data: • Probe information or library files are unique for each probe array type and contain information about the probe array design chapter 5 | Getting Started 69 characteristics, scanning parameters, and default analysis parameters. • Fluidics protocol files define the hybridization, wash, or stain protocols run by the GeneChip® Fluidics Station. • Experiment data include the sample and experiment created by the user when an experiment is defined, as well as the other data types GCOS generates during an analysis: image data, cell intensity data, and probe analysis data. For more information about the data types, see Appendix G, GCOS Data Types, on page 685. OPENING DATA AND VIEWING INFORMATION You can view information (attributes) about experiments, images, cell intensity data, and probe analysis data. In the data tree, double-click the data name; or Right-click the data name in the tree and select Open from the shortcut menu that appears. This displays the data in the main display area. Many data types may be open at the same time, but you can view only one at a time in the main display area (except for a cascade or tile view of images or cell intensity data). 2. Click a GeneChip Software shortcut bar button (Figure 5.5) to toggle the view between the different types of open data. 1. To view information about the data, right-click the data name in the data tree and select Information in the shortcut menu that appears. An Information dialog box appears (Figure 5.12 and Figure 5.13). 4. To copy experiment data information to the system clipboard, click Copy. 3. 70 Affymetrix® GeneChip® Operating Software User’s Guide Figure 5.12 Experiment information (left) and image information (right) chapter 5 | Getting Started 71 Figure 5.13 Cell intensity data information (left) and probe analysis data information (right) Filtering Data You may apply filters to the experiment data. The filters determine the data that GCOS displays in the data tree, the sample history view, workflow monitor, and the instrument control dialog boxes. Filters are applied on a per user basis (identified by the logon name). The filters you specify do not affect the filters specified by other users. 1. Select Tools → Filters from the menu bar. The Filters dialog box appears (Figure 5.14). 72 Affymetrix® GeneChip® Operating Software User’s Guide Figure 5.14 Filters dialog box Make selections from the drop-down lists to specify the filters. 3. If you want to view the experimental data for a specific time period, choose the Date From or To options and click the dropdown arrows to select calendar dates. 4. Click OK when finished to close the Filters dialog box and apply the filters. The status bar indicates that filters are applied (Figure 5.15). 2. chapter 5 | Getting Started 73 Figure 5.15 Status bar in the lower right indicates filters applied Configuring the Fluidics Station & Scanner Before you use the fluidics station, check the fluidics station configuration and prime the fluidics station with appropriate buffer. For information on how to prime the fluidics station, and set up and run a fluidics protocol, see Chapter 8, Controlling the Instruments, on page 105. 74 Affymetrix® GeneChip® Operating Software User’s Guide The configuration process is described in the following sections: • Configuring the Fluidics Station (see below) • Configuring the Scanner (see page 75) CONFIGURING THE FLUIDICS STATION Before running a fluidics protocol, check to make sure the fluidics station(s) is properly configured. 1. Select Tools → Defaults from the menu bar. The Defaults dialog box appears (Figure 5.16). Figure 5.16 Defaults dialog box, Fluidics tab Click the Fluidics tab. 3. Confirm the number of fluidics stations installed is correct or enter a new value. 4. If desired, check mark Notification Message when protocol is complete to display a notification message when a fluidics protocol is completed. 2. chapter 5 | Getting Started 5. 75 Click OK to close the Defaults dialog box. CONFIGURING THE SCANNER 1. Select Tools → Defaults from the menu bar. The Defaults dialog box appears (Figure 5.17). Figure 5.17 Defaults dialog box, Scanner tab Click the Scanner tab. 3. If the workstation is connected to the scanner, choose the Scanner Installed option. 4. To automatically turn the scanner laser on at startup, choose the Turn on Laser at startup option. 2. If the AutoLoader is present and you would like to disable the autorun feature and add cartridges one by one, choose the Enable Manual Mode option. 6. If the AutoLoader is present and you would like to disable it and use the GCOS software without the AutoLoader, choose Disable AutoLoader option. 5. 76 Affymetrix® GeneChip® Operating Software User’s Guide Chapter 6 Setting Up an Experiment Chapter 6 79 Setting Up an Experiment This chapter explains how to set up an experiment. You must define an experiment in GCOS before processing a probe array in the fluidics station or scanning a probe array. GCOS relies on the experiment information to direct the scanner and apply the correct cell and data analysis algorithms. Register sample & define experiment Process probe array in fluidics station Scan probe array & save image data Compute cell intensity data from image data & save cell intensity data Analyze expression cell intensity data & save expression probe analysis data Publish expression data Generate an expression report Figure 6.1 Assay & analysis flow chart This chapter contains the following sections: • Registering a Sample & Defining an Experiment (see page 80) • Viewing or Editing an Experiment (see page 85) 80 Affymetrix® GeneChip® Operating Software User’s Guide Registering a Sample & Defining an Experiment You identify, or register, a sample by entering sample information (attributes) and associating the sample with a project (which may include several different samples) (Figure 6.3). To define an experiment, specify the type of probe array to which the target (sample) will be hybridized as well as other information relevant to the experiment. You may define one or more experiments per sample (for example, the different probe arrays of a multiple probe array set hybridized to the same sample). You can associate a barcode with an experiment. If you entered an Affymetrix® barcode (manually or using a keyboard wedge reader) GCOS automatically reads and displays the probe array type, lot number, and the expiration date. GCOS uses the experiment information to select scanner specifications for the probe array and identify the user-modifiable algorithm parameters for data analysis. You can also use a template (defined in GCOS Manager) to register a sample and define an experiment. For more information on creating a template, see Template Tab, on page 425. GCOS automatically adds the sample and experiment information to the process database. This enters the experiment in the workflow and enables you to track its status in the workflow monitor. After you define an experiment, the Hybridization tab of the workflow monitor displays the experiment and associated sample (Figure 6.2). The Hybridization tab displays experiments that have not yet been processed in the fluidics station. For more information about the workflow monitor, see Workflow Monitor, on page 95. The software uses sample and experiment information to maintain relationships among the experiment data (image, cell intensity data, probe analysis data), manage the workflow queue, and identify the user-modifiable algorithm parameters for data analysis. GeneChip® Operating Software Server supports expression, Resequencing, Mapping, and Universal data. chapter 6 | Setting Up an Experiment 81 Sample Umb 243 is registered and associated with the ESC project. Workflow monitor Workflow monitor tabs. Hybridization tab shows the ESC1 experiment is defined, but not yet processed in the fluidics station. Figure 6.2 Workflow monitor, hybridization tab 1. In the GeneChip Software shortcut bar, click Experiment Info , or select File → New Experiment from the menu bar. The Experiment Information tab appears (Figure 6.3). 82 Affymetrix® GeneChip® Operating Software User’s Guide Enter information here to register the sample Enter information here to define the experiment Figure 6.3 Experiment Information tab In the Experiment Information tab, items in bold are required entries. Enter a new sample name (up to 64 alphanumeric characters) or select an existing sample name from the drop-down list. This is required by GCOS. If you enter a new sample name, the sample type and project are also required. Or, if you want to use a sample template, make a selection from the Sample Template drop-down list and enter the sample information. A template specifies the information fields available in the Experiment Information tab. You can define a template in GCOS Manager. For more information on creating templates, see Template Tab, on page 425. 3. Enter a unique experiment name (up to 64 alphanumeric characters). This is required by GCOS. Or, if you want to use an 2. chapter 6 | Setting Up an Experiment 83 experiment template, make a selection from the Experiment Template drop-down list and enter the experiment information. The experiment name also serves as the name for the subsequent data types generated during the analysis. For a description of GCOS data types see Appendix G, GCOS Data Types, on page 685. Make a selection from the Probe Array Type drop-down list (required by GCOS). 5. You may add a barcode (up to 64 characters). Enter a barcode value in the dialog box, or use a barcode reader to read the barcode directly from the probe array cartridge. If you use a barcode reader to read an Affymetrix® barcode from the probe array cartridge: - GCOS automatically fills in the probe array type, probe array lot and expiration date fields. - The Probe Array Type drop-down list is disabled. 4. GCOS has the ability to store barcodes. You can enter barcode information in either of two places: in the Experiment tab (Figure 6.3) or in the Fluidics dialog box (see Figure 8.4 on page 110). When you enter a valid barcode (maximum of 64 characters), GCOS stores the barcode with the experiment. 6. To automatically publish expression probe analysis data: Make a selection from the User Set drop-down list. A user set is defined in GCOS Manager. The userset specifies the values for the user-modifiable expression algorithm parameters. For more information about user sets, see Userset Tab, on page 415. B. Make a selection from the Publish Database drop-down list. For more information about publishing data see Chapter 12, Publishing Data, on page 315. A. 84 Affymetrix® GeneChip® Operating Software User’s Guide You must specify both a user set and a publish database, otherwise the probe analysis data will not be published. If you specify only a user set, the analysis proceeds using the analysis settings in the Expression Analysis Settings dialog box, but the data are not published. To view the Expression Analysis Settings dialog box, click Expression Settings in the Settings shortcut bar. C. To include the cell intensity data in the publish database, choose the Publish Intensities option. Publishing cell intensity data uses large amounts of computer memory. For example, publishing the probe analysis data for a high density probe array requires approximately 2 MB of disk space compared to 30 MB for both the probe analysis and cell intensity data. 7. Click the Save button . This saves the experiment and displays it in the data tree. If you specified a user set and publish database during experiment setup, the software prompts you for the publish database password before the experiment is saved. After the probe array is processed in the fluidics station and scanned, click the Instrument Info tab to view information about the fluidics protocol and scanning parameters captured by GCOS (Figure 6.4). The instrument information page is blank until the probe array has been processed in the fluidics station. chapter 6 | Setting Up an Experiment 85 Figure 6.4 Instrument Info tab Viewing or Editing an Experiment 1. To display an experiment, double-click the experiment name in the data tree; or Right-click the experiment name and select Open from the shortcut menu that appears. 86 2. Affymetrix® GeneChip® Operating Software User’s Guide To edit an entry, highlight the entry and enter the new information; or Right-click to open a shortcut menu of edit commands (Figure 6.5). Figure 6.5 Experiment information and shortcut menu of edit commands 3. To clear all fields, select Edit → Clear from the menu bar (equivalent to closing the experiment without saving). When connected to a GCOS server database, only users authorized by the System Administrator may delete data from the system using the GCOS Manager software. When operating using the local MSDE database, there is no data security. 4. When finished, click the Close button in the upper right corner of the window or select File → Close from the menu bar. chapter 6 | Setting Up an Experiment Before the experiment is closed, GCOS prompts you to save changes. 87 88 Affymetrix® GeneChip® Operating Software User’s Guide Chapter 7 Sample History & Workflow Monitor Chapter 7 91 Sample History & Workflow Monitor This chapter explains how to use the sample history feature and workflow monitor to track samples and experiment data. It contains the following sections: • Sample History (see below) • Workflow Monitor (see page 95) Sample History The sample history feature provides two different views of the samples in the system: • Data view shows the experiment data derived from a particular sample. • Process view shows all stages (sample registration, experiment setup, hybridization, scan, grid alignment, cell intensity analysis, probe array analysis, and publishing) that are completed or pending for a sample. The following sections describe the sample history feature: • Displaying Sample History (see below) • Displaying Sample History Process View (see page 93) DISPLAYING SAMPLE HISTORY 1. In the GeneChip Software shortcut bar, click Sample History . The sample history data view appears (Figure 7.1). The data view is the default. If the data view is not displayed, select View → Data from the menu bar. The sample tree displays all assay samples. The expression folder contains all registered expression analysis samples. If filters have been applied, not all registered samples may be displayed. For more information about filters, see Filtering Data, on page 71. 92 Affymetrix® GeneChip® Operating Software User’s Guide Sample history, data view Click an item to display property values Data tree Sample tree Click a sample in the tree to display experiment data derived from the sample Property list Figure 7.1 Sample history, data view 2. To display a tree of the experiment data derived from a sample, click the sample name in the sample tree. 3. To display a list of data properties and their values (bottom right pane), click an item in the sample history tree. chapter 7 | Sample History & Workflow Monitor 93 DISPLAYING SAMPLE HISTORY PROCESS VIEW The process view shows all completed or pending stages for a particular sample. 1. In the GeneChip Software shortcut bar, click Sample History The sample history view appears. . 2. To display the process view (Figure 7.2), select View → Processes from the menu bar. The expression folder in the sample tree contains registered expression analysis samples. If filters have been applied, not all registered samples may be displayed. For more information about filters, see Filtering Data, on page 71. 94 Affymetrix® GeneChip® Operating Software User’s Guide Data tree Sample tree Sample history, process view Click a sample in the tree to display processes (completed or pending) for the sample Click an item to display property values Property list Figure 7.2 Sample history, process view To display the sample history tree, click a sample name in the sample tree. Gray bullets indicate completed processes and green bullets indicate those that are pending. 4. Click the plus sign [+] to the left of a process name to expand the information in the sample history tree (Figure 7.2). Use the scroll bars to the right or below the process tree to view the expanded information. 3. chapter 7 | Sample History & Workflow Monitor 95 The tree displays the inputs and outputs for each process. For example, cell intensity data is the input to the probe array analysis process that generates probe analysis data. 5. To display a list of data properties (bottom right pane), click one of the following in the sample history tree (Figure 7.2): - sample name - experiment name - a process input or output Workflow Monitor When you define an experiment, it automatically enters the workflow and may be tracked through the sequential tabs in the workflow monitor (see Table 7.1). Table 7.1 Workflow monitor tabs Workflow Monitor Tab Displays... Hybridization Experiments that have not been processed in the fluidics station. Scan Experiments that have been processed in the fluidics station, but have not been scanned. Grid Alignment Image data that require manual grid alignment. Cell Intensity Analysis Image data that have been manually aligned, but have not been analyzed by the cell analysis algorithm (no cell intensity data exists) Probe Array Analysis Cell intensity data that have not been analyzed by the expression analysis algorithm. 96 Affymetrix® GeneChip® Operating Software User’s Guide Table 7.1 Workflow monitor tabs Workflow Monitor Tab Displays... Publish Expression probe array analysis data that have not been published. Note: The GeneChip® Genotyping Analysis Software (GTYPE) handles mapping and Universal Tag probe array data for publishing queue purposes. The GeneChip® Sequence Analysis Software (GSEQ) handles resequencing probe array data for publishing queue purposes. The following sections describe the workflow monitor: • Opening the Workflow Monitor (see below) • Workflow Monitor Tabs (see page 98) OPENING THE WORKFLOW MONITOR 1. In the GeneChip Software shortcut bar, click Workflow Monitor . The workflow monitor appears (Figure 7.3). If filters have been applied, not all registered samples or experimental data may be displayed. For more information about filters, see Filtering Data, on page 71. chapter 7 | Sample History & Workflow Monitor 97 Data tree Workflow monitor Shortcut bar Workflow monitor tabs Figure 7.3 Workflow monitor, details view 2. To update the display, select View → Refresh from the menu bar. This refreshes the workflow monitor so that it displays any new items added to the system since it was last opened. To view a list of experiments, click the List button . To view sample and project information for each experiment, click the Details button . 4. To view experiment or data information, right-click the item and select Open Item from the shortcut menu that appears; or Select View → Open Item from the menu bar. 3. 98 Affymetrix® GeneChip® Operating Software User’s Guide WORKFLOW MONITOR TABS Click a tab to view the items in that part of the workflow queue. 2. To update the workflow monitor, select View → Refresh from the menu bar. 1. Hybridization Tab The Hybridization tab displays experiments that have not been processed in the fluidics station. An experiment is automatically moved to the Scan tab after it has been processed in the fluidics station. If a probe array is not processed (hybridized, or washed and stained) in the fluidics station, the experiment must be manually advanced to the Scan tab. To manually advance an experiment to the Scan tab: • Right-click the experiment and click Advance to Scan in the shortcut menu that appears; or Click the experiment and select View → Advance to Scan from the menu bar. Scan Tab The Scan tab displays experiments that have been processed in the fluidics station, but have not been scanned. After the scan and grid alignment processes are completed, the cell intensity analysis is automatically run and the experiment is moved to the Probe Array Analysis tab. If the grid was not successfully aligned by the Alignment algorithm, the Grid Alignment tab displays the experiment. Grid Alignment Tab The Grid Alignment tab displays image data that require manual grid alignment. To manually align the grid: 1. Right-click the image data in the Grid Alignment tab and click Open Item in the shortcut menu that appears. The image is displayed. chapter 7 | Sample History & Workflow Monitor 2. 99 Align the grid at each corner of the image: Click the button in the Image window task bar and choose a corner. B. Place the mouse arrow over the grid (or sub-grid) perimeter (the arrow becomes a double arrow, ). The diagonal orientation of the double arrow along the perimeter of a corner probe cell indicates horizontal and vertical adjustments can be made simultaneously using the click-anddrag method or by using the keyboard arrow keys. A. Use the click-and-drag method or the keyboard arrow keys to adjust the horizontal or vertical position of the grid so that it is aligned over the corner of the image (Figure 7.4). 3. After you align the grid at each corner of the image, click the Save button (Figure 7.4). The grid is saved. C. Figure 7.4 Manual grid alignment: align the grid at each corner of the image and click the Save button 100 Affymetrix® GeneChip® Operating Software User’s Guide Cell Intensity Analysis Tab The Cell Analysis tab displays image data after the grid has been manually aligned. GCOS does not automatically generate the cell intensity data from an image that requires manual grid alignment. As a result, no cell intensity data exist for the image data under the Cell Analysis tab. To generate the cell intensity data: • In the Cell Analysis tab, right-click the image and click Analyze Item in the shortcut menu that appears. This generates the cell intensity data and places it under the Probe Array Analysis tab. To generate the cell intensity data and probe array analysis data: In the Cell Analysis tab, right-click the image and click Open Item in the shortcut menu that appears. This displays the image data. 2. Select Run Analysis from the menu bar. This generates the cell intensity and probe analysis data. The Publish tab displays the probe analysis data. 1. Probe Array Analysis Tab The Probe Array Analysis tab displays cell intensity data that have not been analyzed by the expression analysis algorithm (no probe array analysis data exists). To analyze cell intensity data: • In the Probe Array Analysis tab, right-click the cell intensity data and click Analyze Item in the shortcut menu that appears; or Select View → Analyze Item from the menu bar. After the cell intensity data have been analyzed and the probe analysis data have been generated, the experiment is moved to the Publish tab. chapter 7 | Sample History & Workflow Monitor 101 Publish Tab The Publish tab displays expression probe analysis data that have not been published. The probe analysis data is removed from the Publish tab and the workflow monitor after it is published. 102 Affymetrix® GeneChip® Operating Software User’s Guide Chapter 8 Controlling the Instruments Chapter 8 105 Controlling the Instruments This chapter describes how to use the GeneChip® Operating Software (GCOS) to control the GeneChip® Fluidics Station and the Affymetrix® GeneChip® Scanner 3000 (Figure 8.1). Register sample & define experiment Process probe array in fluidics station Scan probe array & save image data Compute cell intensity data from image data & save cell intensity data Analyze expression cell intensity data & save expression probe analysis data Publish expression data Generate an expression report Figure 8.1 Assay & analysis flow chart This chapter contains the following sections: • The Fluidics Station (see below) • Scanning a Probe Array–GeneChip® Scanner 3000 (see page 121) • Tracking Cell Analyses and Data Transfers (see page 133) The Fluidics Station The fluidics station hybridizes, washes, and stains the probe arrays. One workstation can control up to eight fluidics stations. Each fluidics station contains four modules and each module can independently 106 Affymetrix® GeneChip® Operating Software User’s Guide process a probe array using a different fluidics protocol. Refer to the GeneChip® Fluidics Station User’s Guide for a description of the instrument, its components, and set up. Before you use the fluidics station, check the fluidics station configuration and prime the fluidics station with appropriate buffer. For more information, see Configuring the Fluidics Station & Scanner, on page 73. The steps needed to use the fluidics station are described in the following sections: • Priming the Fluidics Station (see below) • Setting Up a Fluidics Protocol (see page 109) • Running the Fluidics Protocol (see page 110) • Running a Maintenance Protocol in All Modules (see page 111) • Resuming a Fluidics Protocol (see page 111) • Bypassing Steps in a Fluidics Protocol (see page 112) • Editing a Fluidics Protocol (see page 113) • Editing the Fluidics ID Associated with a Fluidics Station (see page 115) PRIMING THE FLUIDICS STATION Priming fills the fluidics station lines with wash buffers and deionized water. The GeneChip® Fluidics Station must be primed before it can be used to run assay protocols. Prime the fluidics station when: • the fluidics station is first turned on • a wash solution is changed • the fluidics station is to be used again after a shutdown has been performed chapter 8 | Controlling the Instruments 107 • a module LCD window informs you that the module is not primed To prime the fluidics station: Click the Fluidics button in the Instrument Control shortcut bar or the main toolbar; or Select Run → Fluidics from the menu bar. If more than one fluidics station is installed on the workstation, the Station Selection dialog box appears (Figure 8.2). If only one fluidics station is installed on the workstation, the Fluidics Station dialog box appears (Figure 8.3). 2. If more than one fluidics station is installed on the workstation, select the number designation of the current fluidics station from the Station Number drop-down list. 1. Figure 8.2 Station Selection dialog box 3. Click OK to close the Station Selection dialog box. The Fluidics Station dialog box for the currently selected station appears (Figure 8.3). 108 Affymetrix® GeneChip® Operating Software User’s Guide Figure 8.3 Fluidics Station dialog box, Prime protocol selected 4. Select Prime from the Protocol drop-down list and No Probe Array from the Experiment drop-down list for each module to be used. Fill the intake buffer reservoirs A and B with the appropriate priming buffer. (Refer to the appropriate GeneChip® probe array package insert.) 6. Empty the waste bottle and fill the water reservoir with deionized water. 5. Load an empty, standard 1.5 mL microcentrifuge tube in the sample holder of each module to be primed. 8. Click Run for each module to be primed and follow the prompts in the fluidics station dialog box (also shown in the module LCD window); or To prime all modules, choose the All Modules option. Click Run to start the priming on all modules and follow the prompts in the Fluidics Station dialog box. The Fluidics Station dialog box and the module LCD window display the status of the procedure. The fluidics station is ready to 7. chapter 8 | Controlling the Instruments 109 use when priming is completed and Priming done, Ready appears in the module LCD window. SETTING UP A FLUIDICS PROTOCOL To set up the fluidics protocol: 1. In the Instrument Control shortcut bar, click Fluidics ; or Select Run → Fluidics from the menu bar. If more than one fluidics station is installed on the workstation, the Station Selection dialog box appears (Figure 8.2). If only one fluidics station is installed on the workstation, the Fluidics Station dialog box appears (Figure 8.4). If more than one fluidics station is installed on the workstation, select the number designation of the current fluidics station from the Station Number drop-down list. 3. In the Fluidics Station dialog box (Figure 8.4): 2. Select the current fluidics station module. B. Choose an experiment and protocol from the drop-down lists. (Refer to the appropriate GeneChip® probe array package insert.) C. If a barcode was entered when the experiment was created, enter the barcode using the keyboard or scan the barcode with an external barcode reader, and press the Tab key. This selects the experiment associated with the barcode from the database. 4. If the selected experiment has no barcode, you can associate a barcode with the experiment by entering the barcode using the keyboard or an external barcode reader. A. 110 Affymetrix® GeneChip® Operating Software User’s Guide If filters have been applied, the Experiment drop-down list may not include all experiments. Click Filter to open the Filters dialog box and view or specify filters. Choose the Include Hybridized Experiments option to include hybridized experiments in the drop-down list. Figure 8.4 Fluidics Station dialog box Click View to display information about the selected experiment. 6. Click Run to start the protocol on the selected module. 7. Repeat as necessary for other modules in the fluidics station(s). 5. RUNNING THE FLUIDICS PROTOCOL To run the fluidics protocol: 1. Load the probe array and sample vial holder containing the appropriate solution in each active module. chapter 8 | Controlling the Instruments 111 Sensors in the fluidics station detect when the probe array and sample vial holder have been loaded. The process will proceed automatically from this point, although some protocols may require removal and substitution of the sample vial and solution. The Fluidics Station dialog box and each module LCD window display the status of the procedure. 2. After the protocol is finished, remove the probe array and inspect the probe array window for air bubbles. If air bubbles are present, reinsert the probe array into the fluidics station to automatically drain and refill the probe array with the last wash buffer used. (Refer to the appropriate GeneChip® probe array package insert.) If no bubbles are present, the probe array is ready to be scanned. RUNNING A MAINTENANCE PROTOCOL IN ALL MODULES The software can start a maintenance protocol in all modules of the fluidics station using a single button click. A maintenance protocol (for example, CLEAN, DRAIN, HOME, PRIME, RECOVER, SHUTDOWN, or BLEACH) does not require an experiment. To run the maintenance protocol in all modules of the station: Select a maintenance protocol from the Protocol drop-down list. 2. Select the All Modules option. 3. Click Run. 1. RESUMING A FLUIDICS PROTOCOL GCOS tracks the progress of a fluidics protocol run. If the protocol stops before completion, it can be resumed at the point where it was interrupted. 112 Affymetrix® GeneChip® Operating Software User’s Guide The resume feature is only available for fluidics protocols that display multiple steps in the Step drop-down list of the Fluidics Station dialog box. If you exit GCOS while a fluidics protocol is running, the resume feature will be unavailable upon startup of GCOS. To resume a fluidics protocol: When ready to resume the protocol, select the appropriate protocol from the Protocol drop-down list in the Fluidics Station dialog box. 2. Select the All Modules option. 3. Click Run. The selected protocol is started in modules one through four of the fluidics station. 1. BYPASSING STEPS IN A FLUIDICS PROTOCOL Some multi-step fluidics protocols can be started at any step, so that part of a protocol can be bypassed. To bypass steps: 1. In the Instrument Control shortcut bar, click Fluidics Select Run → Fluidics from the menu bar. The Fluidics Station dialog box appears (Figure 8.5). ; or chapter 8 | Controlling the Instruments 113 Figure 8.5 Fluidics Station dialog box, bypassing protocol steps 1 and 2 2. Select the desired experiment, module, and protocol from the drop-down lists in the Fluidics Station dialog box. The bypass function is only available for fluidics protocols that display multiple steps in the Step drop-down list of the Fluidics Station dialog box. 3. Select the desired beginning step from the Step drop-down list (Figure 8.5). 4. Click Run to start the fluidics protocol at the selected step. EDITING A FLUIDICS PROTOCOL You can edit some hybridization and wash (Hybwash) protocols. 114 Affymetrix® GeneChip® Operating Software User’s Guide Modifications to a Hybwash protocol must be completed before it is run. Protocol changes made during a run do not affect the run in progress. To edit the protocol: 1. Select Tools → Edit Protocol from the menu bar. The Fluidics Protocol dialog box appears (Figure 8.6). Figure 8.6 Fluidics Protocol dialog box 2. Choose the fluidics protocol you want to edit from the Protocol Name drop-down list. Only the protocols in this list may be edited. All others are defined for specific applications and cannot be customized. 3. Highlight the parameter value you want to change and enter the new value (Parameters values must be within the ranges in Table 8.1). Enter a Hybridization Time of zero if only a wash is chapter 8 | Controlling the Instruments 115 desired. To omit Wash A or B, enter zero for the Number of Wash A or Wash B cycles. Table 8.1 Valid ranges for hybridization or stain protocol parameters Parameter Valid Range Hybridization or stain time 0 - 86,399 seconds Temperature 15 - 50° C Number of Wash cycles 0 - 99 Mixes per Wash cycle 1 - 99 To save the parameters under the same protocol name (overwrites the old protocol), click Save. 5. To save the parameters under a new protocol name, enter a new name in the Protocol Name field, then click Save. This adds the new protocol name to the drop-down list. 6. Click Defaults to return the parameter settings to the default values. 7. Click Delete to delete the currently selected protocol from the system. 4. EDITING THE FLUIDICS ID ASSOCIATED WITH A FLUIDICS STATION GCOS has the ability to determine the fluidics station identity (ID) and module number used to wash and stain a probe array. The local or GCOS server database keeps track of the fluidics station ID and the module number that processes an experiment (probe array). To enable this feature, you must configure the registry using the regedit application so that GCOS can read the fluidics station ID and module number information from the registry. To edit the fluidics ID: 1. Click the Microsoft® Windows® Start menu button select Run. and 116 Affymetrix® GeneChip® Operating Software User’s Guide The Run box appears (Figure 8.7). Figure 8.7 Run box 2. Enter regedit at the command prompt and click OK. The Registry Editor appears (Figure 8.8). Figure 8.8 Registry Editor Open the HKEY_LOCAL_MACHINE registry hive. 4. Select the Software → Affymetrix → GeneChip → Fluidics node under the HKEY_LOCAL_MACHINE registry hive (Figure 8.9). 3. chapter 8 | Controlling the Instruments 117 Figure 8.9 Registry Editor 5. In the right window, right-click and select New → DWORD Value from the shortcut menu to create a new name/data pair. Repeat for each fluidics station whose ID is to be captured (Figure 8.10). GCOS allows a maximum of eight fluidics stations to be controlled by a single workstation. 118 Affymetrix® GeneChip® Operating Software User’s Guide Figure 8.10 Registry Editor. create new name/data pair 6. Select the first newly created Name/Data pair, right-click the name, and select Rename from the shortcut menu. Rename the entry to “ID1”. The example in Figure 8.11 shows four Name/Data pairs that have been renamed ID1, ID2, ID3, and ID4. chapter 8 | Controlling the Instruments 119 Figure 8.11 Registry Editor, four newly created Name/Data pairs renamed ID1, ID2, ID3, and ID4 120 7. Affymetrix® GeneChip® Operating Software User’s Guide To edit the value of an entry, double-click the entry. The Edit DWORD Value box appears (Figure 8.12). Figure 8.12 Edit DWORD Value box Select the Decimal option and enter a new value for the station ID. 9. Repeat step 7 and 8 to edit the value of each entry. In this example, stations one through four have the ID 1001, 1002, 1003, and 1004 respectively (Figure 8.13). When a fluidics protocol is run on any of these stations, the fluidics station ID and the module number will be tracked in the process database. 8. chapter 8 | Controlling the Instruments 121 Figure 8.13 Registry Editor Scanning a Probe Array–GeneChip® Scanner 3000 This section shows you how to scan a GeneChip® probe array using the Affymetrix® GeneChip® Scanner 3000. Before you scan a probe array, make sure to read and understand: • Appendix D, Using the GeneChip® Scanner 3000 (see page 561) • Appendix L, Using the GeneChip® AutoLoader, on page 731 The steps needed to use the scanner are described in the following sections: • Using Tough-Spots® to Prevent Leaks (see below) • Scanning a Probe Array (see page 124) • Stopping a Scan (see page 132) • Shutting Down the Scanner (see page 133) 122 Affymetrix® GeneChip® Operating Software User’s Guide USING TOUGH-SPOTS® TO PREVENT LEAKS Tough-Spots® are chemically inert polyvinyl labels that adhere to all plastics. Affymetrix recommends using 3/8-inch circle diameter Tough-Spots to prevent leakage from the cartridge septa. Before loading the probe array cartridge, follow this procedure to prevent the leaking of fluids from the cartridge during scanning. Even if you have already applied Tough-Spots to the cartridge prior to hybridization or after washing, you must remove the old Tough-Spots and apply new ones before you load them into the AutoLoader. Affymetrix recommends the use of Tough-Spots® obtained from Affymetrix P/N 64-0158 or from USA Scientific, Inc. P.O. Box 3565 Ocala, FL 34478 (800)LABTIPS P/N 9185-0000 To reduce the risk of leakage, do not use excessively large pipette tips to pierce the septa. To use Tough-Spots: 1. On the back of the probe array cartridge, clean excess fluid from around septa (Figure 8.14). chapter 8 | Controlling the Instruments 123 Figure 8.14 The GeneChip® probe array cartridge 2. Carefully apply one Tough-Spot over each of the two septa. Press to ensure that the spots remain flat. If a Tough-Spot does not apply smoothly; that is, if you observe bumps, bubbles, tears or curled edges, do not attempt to smooth them out. Remove the spot and apply a new one (Figure 8.15). TM Tough-Spots Figure 8.15 Applying Tough-Spots® to cartridge septa 124 Affymetrix® GeneChip® Operating Software User’s Guide SCANNING A PROBE ARRAY To scan a probe array: 1. Press the on/off (I/O) switch on the front panel. The scanner’s on board computer will boot up. The bootup process takes a couple of minutes. During this time both the yellow and green lights will be on. 2. Click the Start Scanner button in the Instrument Control shortcut bar or the main toolbar: or Select Run → Start Scanner from the menu bar. The Scanner dialog box appears (Figure 8.16). Note that the dialog also displays barcode information if you entered it when you created the experiment or set up the fluidics protocol. Figure 8.16 Scanner dialog box, laser off (left) and laser on (right) 3. To include scanned experiments in the Experiment Name dropdown list, choose the Include Scanned Experiments option. chapter 8 | Controlling the Instruments 125 By default, the Experiment Name drop-down list displays the experiments in the current directory that have not been scanned (no image data exists for the experiments). If filters have been applied, the drop-down list may not include all experiments. Click Filter to open the Filters dialog box and view or specify filters. 4. Select the experiment name of the probe array to be scanned from the Experiment Name drop-down list: or Set the focus to the barcode field. If a barcode was associated with the experiment to be scanned, scan or manually enter the barcode from the probe array. This automatically retrieves the experiment from the database. The Probe Array Type field automatically displays the probe array type that was entered during experiment setup. If a barcode was entered when the experiment was created or hybridized, the barcode field displays the barcode. GCOS sets the Number of Scans per probe array type (generally one for the Scanner 3000). You may change the number of scans in the Scanner dialog box (Figure 8.16). Multiple scans are statistically averaged to create the image data. Increasing the number of scans increases the scan time as well as the amount of fluorophore bleaching and may result in lower fluorescence intensities. 5. If it is necessary to rescan a probe array, select the experiment name from the Experiment Name drop-down list. Also select Include Scanned Experiments. If you find it necessary to rescan a chip, GCOS appends an “_#” to a new DAT file for the rescan operation 126 6. Affymetrix® GeneChip® Operating Software User’s Guide Click Start in the Scanner dialog box. When the software displays the Laser is On button in the scanner dialog box, the Start button is activated and you can start a scan. The GeneChip Scanner dialog box appears (Figure 8.17). Figure 8.17 Scanner dialog box The scanner door opens and the chip transport mechanism raises to accept a probe array. 7. Load the probe array (Figure 8.18) into the scanner chip transport mechanism. Insert the probe array into the chip transport mechanism such that the front of the probe array (label side) faces to the rear of the scanner (Figure 8.19). The scanner will enter Park mode if it is unattended for 15 minutes and will enter standby, or sleep, mode if it is unattended for 60 minutes (45 minutes after entering Park mode). The green light will turn off and the yellow light will turn on. To reactivate the laser, click Turn Laser On in the scanner dialog box (Figure 8.16) and wait 10 minutes. chapter 8 | Controlling the Instruments 127 Figure 8.18 Affymetrix® GeneChip® probe arrays: note the location of the flange. The scanner will accept the probe array in only one orientation. Do not force the probe array cartridge. If the cartridge does not drop easily into the chip transport mechanism, eject the cartridge and try again. If this does not remedy the situation, see Troubleshooting, on page 577 or call Affymetrix technical support. If a probe array becomes lodged in the scanner, you can manually remove it. See Manually Removing a Lodged Probe Array Cartridge, on page 579. You cannot modify the scanner settings. GCOS automatically selects the appropriate settings based on the probe array type specified during experiment setup. 128 Affymetrix® GeneChip® Operating Software User’s Guide Figure 8.19 Loading the probe array into the chip transport mechanism. Note that the front of the probe array faces to the rear of the scanner. 8. Click OK in the GeneChip Scanner dialog box to start scanning the probe array. During the scan, the green light will flash, and the yellow light will be off. After the scan starts, the software will start with the autofocus routine. Data collection starts after successful completion of autofocus. During the pre-scan state, when autofocus is complete, chapter 8 | Controlling the Instruments 129 but before data collection has started, the software will count downwards. If the scan in progress feature is enabled (select View → Scan in Progress from the menu bar), the Image window automatically opens in the main display area when a scan starts. It displays the fluorescence intensity of the probe array at approximately 50 lines at a time as the scan progresses. To enable (or disable) this option, select View from the menu bar and place (or remove) a check mark next to Scan in Progress. When multiple emission filters are used during the scan process, the software will display the scan with a letter followed by the percentage of scan completed. The letter identifies the scan associated with the emission filter. After the scan is completed, GCOS: - Saves the image data (displayed in the main display area). - Aligns a grid on the image to identify the probe cells. - Saves the image data. - Generates a JPG image from the DAT data. - Ejects the probe array. - Automatically closes the open image window. - Submits the DAT file for archiving, if the archiving option has been enabled in the Archive tab of the GCOS defaults dialog. For more information, see Setting Archiving Options for DAT files, on page 599. If you are using GCOS on a workstation connected to a networked GCOS Server, you can perform the CEL intensity analysis on the workstation or on the GCOS Server. When performing the analysis on the workstation, the DAT data is submitted to the analysis service for computing CEL intensity analysis. The DAT data and CEL data is transferred to the server after completion of the CEL intensity analysis using the transfer service. 130 Affymetrix® GeneChip® Operating Software User’s Guide When performing the analysis on the GCOS Server, the DAT and JPG data is transferred immediately to the GCOS server. After the data is copied, the software will submit the DAT data to the analysis service for computing CEL intensity analysis. For more information about configuring GCOS to work with a networked GCOS Server, see The GCOS Transfer and Analysis Services, on page 56. If automation was used with this experiment, the transfer and analysis services are not used for transferring the DAT data or for generating the CEL intensity data If you leave the scanner idle for an additional 15 minutes, the scanner will also enter “Park” mode. The yellow light will be off and the green light on.The chip transport mechanism will retract and the scanner door will close. You must click the Eject Chip button to open the scanner door and raise the chip transport mechanism. SCANNING FOUR-COLOR ARRAYS The four color scans are performed on GeneChip arrays that have been configured for use with four emission filters. The four emission filters are specified in the GCOS scan parameters. when performing a multifilter scan, GCOS scans the array with different emission filters, using the order specified for the array. A DAT file is created for each of the emission filter scans. To distinguish the different scans, GCOS appends a suffix of A, B, C, or D to the DAT files. Different file naming conventions are used in the case of a rescan of an array, depending upon whether the array was manually loaded (see below) or loaded using the autoloader (see page 131). chapter 8 | Controlling the Instruments 131 When running multiple scans on an array, the scanner performs autofocus only once, prior to the first scan. This is true whether the scans are performed as part of a four-color scan or as a re-scan. Aborting a Scan with a Manually Loaded Array If scanning is aborted on a manually loaded array, the emission filter scan in progress continues until it is complete, and the DAT data for the completed scans are saved. When the scan is resumed, GCOS autofocuses the scanner and then re-scans and re-creates a DAT file for each emission filter scan, overwriting the previously created DAT files. Aborting a Scan when Using the Autoloader If scanning is aborted on an array loaded using the autoloader, the emission filter scan in progress continues until it is complete, and the DAT data for the completed emission scans are saved. When the scan is resumed, GCOS autofocuses the scanner and then re-scans and recreates a DAT file for each emission filter scan. GCOS does not overwrite the previously created DAT data, but creates a new set of DAT files, adding a suffix of “_nX” to the DAT file name, where n identifies the rescan number and 'X' is the letter assigned for the emission filter. A 4-color array scanned a second time will produce DAT files with the following suffixes: _2A, _2B, _2C, _2D. When using the “Add Chips Now” function of the autoloader, if the door is opened in the middle of a scan acquisition, GCOS treats the scan as an abort request. After the emission filter scan in progress completes, scanning halts and the DAT and CEL files for the completed emission filter scans are saved. Upon resume, the GCOS autofocuses the scanner and rescans the array for each emission filter. To avoid the rescan of the array, it is recommended that the “Add Chips Later” function be used. 132 Affymetrix® GeneChip® Operating Software User’s Guide STOPPING A SCAN Click the STOP button or select Run → Stop Scanner from the menu bar. 2. At the prompt, click Yes to stop the scanner or No to cancel stopping (Figure 8.20). 1. Figure 8.20 Stop scanner prompt If you click Yes, the data from a partial scan will be lost. This is different from using earlier software or scanner versions where you could save the data from a partial scan. If you rescan a probe array that has been partially scanned, the previously scanned area of the probe array may experience fluorophore bleaching. This will result in non-uniform fluorescence intensity across the probe array. 3. After you stop a scan, the scanner will automatically eject the chip. The scanner dialog box (Figure 8.16 on page 124) has an eject chip button. This is reserved for ejecting a chip after the scanner goes into sleep mode or when you must manually eject the chip for any other reason. If the probe array cartridge becomes stuck, see Troubleshooting, on page 577 or call Affymetrix technical support. chapter 8 | Controlling the Instruments 133 SHUTTING DOWN THE SCANNER Close the GCOS software. 2. Press the I/O (on/off) button on the front panel to turn off the instrument. 1. The laser also has a sleep mode that activates after 1 hour of inactivity. Tracking Cell Analyses and Data Transfers The Analysis and Transfer Status window (Figure 8.21) allows you to monitor the progress of cell intensity analyses and the archiving and transferring of other DAT and CEL files. For information about configuring the analysis and archive options see Appendix E, Configuring the GCOS Services, on page 587. To open the Analysis and Transfer Status window: • Select Tools → Analysis and Transfer Status from the main menu. The Analysis and Transfer Status window opens (Figure 8.21). Figure 8.21 Analysis and Transfer Status window 134 Affymetrix® GeneChip® Operating Software User’s Guide The window displays the analyses and transfers as: • Tasks: the analysis and transfer performed for each DAT file. • Task items: the different steps used to complete each task (Figure 8.22). Task Task Items Figure 8.22 Task and task items The window has the following columns: Task Items being processed by the analysis and transfer services. For more information, see Task Column, on page 134. Status The status of the analysis or transfer. For more information, see Status Column, on page 135. Description Description of the task and reason for failure, if necessary. For more information, see Description Column, on page 136. The Restart Selected Task(s) button allows you to restart an interrupted or failed task or task item (see Restarting an Interrupted Task, on page 137). ANALYSIS AND TRANSFER STATUS MESSAGES Task Column The Task column displays tasks and their associated task items. chapter 8 | Controlling the Instruments 135 Task For a task, the Task column displays the following information: • <name of user who submitted task> - <start time> Task Items For a task item, the Task column displays the type of process: • Analysis: the cell intensity analysis • Transfer: transfer of DAT files to the archive location Status Column The Status column displays information about the progress of tasks and task items. Task For tasks, the status column may display the following messages: ACTIVE The task is active. WAIT The task is waiting for processing. ERROR There was an error. CANCELED This task has been cancelled by the user SUBMITING ITEMS GCOS is in the process of submitting to the task, this task is not available for processing. Task Items For task items, the status column may display the following messages: ACTIVE This item is active. WAIT This item is waiting. ERROR There was an error. COMPLETE This item is complete. CANCELED This item has been cancelled by the user. 136 Affymetrix® GeneChip® Operating Software User’s Guide FILE WAIT This item can not be processed because it is waiting for a dependent file. Description Column The Description column includes information about the analyses and transfers in progress, including the reasons for failure if necessary. Analysis Descriptions The description includes the following information for cell intensity analyses: • Analyze <number of files> files including <first file name> • Analyze <file name> When the cell intensity analysis is unsuccessful, the description also includes a text description of the failure and its causes. Transfer Descriptions The description includes the following information for data transfers: • Transfer <file name> to <path> • Archive <file name> to <path> When the transfer is unsuccessful, the description includes a text description of the failure and its cause (Figure 8.23). chapter 8 | Controlling the Instruments 137 Figure 8.23 Transfer error Message RESTARTING AN INTERRUPTED TASK An interrupted task can be restarted using the Analysis and Transfer status controls. To restart an interrupted task or task item: Click on the task or task item to select it. To select adjacent items, press and hold the Shift key while you click the first and last item in the selection. To select non-adjacent items, press and hold the Ctrl key while you click the items. 2. Click the Restart Selected Task button ; or Right-click on the selected task or task item and select Restart Selected Task(s) from the shortcut menu (Figure 8.24). 1. 138 Affymetrix® GeneChip® Operating Software User’s Guide Figure 8.24 Shortcut menu The task or task item is restarted. Chapter 9 Working with Images Chapter 9 141 Working with Images The Image Window in GCOS 1.4 allows you to: • View the Image data (.DAT) • View a JPG file of the Image data • Make adjustments to the gridding on the DAT file when necessary • View the Cell Intensity data (.CEL) This chapter contains the following sections: • Introduction (see below) • The Image Window with CEL and DAT Files (see page 145) • Viewing and Aligning the Grid and Subgrids (see page 147) • The Cell Summary Report (see page 178) • Computing Cell Intensities (see page 195) • Adjusting the Image Settings (see page 196) • Intensity Display (see page 204) • Calculating Average Intensity for User Specified Probe Cells (see page 205) • Viewing Probe Cell Data (see page 206) • Viewing Probe Set Information (see page 207) • Bookmarks (see page 212) • Outliers (see page 215) • The Image Viewer with JPG Files (see page 217) Introduction You can open the Image Window by selecting the following file types in the data tree or the Open Dialog box: • .DAT files • .JPG image of the DAT file 142 Affymetrix® GeneChip® Operating Software User’s Guide • .CEL files You can also set GCOS up so the Image Window automatically displays the resulting DAT file during a probe array scan (for more information, see Scanning a Probe Array–GeneChip® Scanner 3000, on page 121). Scroll bar Image window task bar Scroll bar Figure 9.1 Image data in the Image window The Image window displays the data name (same as the experiment name entered during experiment setup) in the upper left corner. • Use the scroll bars at the bottom and right side of the Image window to navigate the scan image. You can learn more about using the Image window in the following sections: chapter 9 | Working with Images 143 • Opening Multiple Image Windows, on page 143 • Automatically Archiving Image Data, on page 144 • The Image Window with CEL and DAT Files, on page 145 • The Image Viewer with JPG Files, on page 217 OPENING MULTIPLE IMAGE WINDOWS You can open and view more than one Image window in the main display area. After opening multiple files: 1. Select Window → Cascade from the menu bar to display an overlapping cascade of the open Image windows. Select Window → Tile to display the open Image windows side by side (Figure 9.2). Figure 9.2 Multiple image windows, cascade view (left) and tile view (right) 2. If other data types are open (for example, an experiment or probe analysis data) and displayed in the main display area, click Image 144 Affymetrix® GeneChip® Operating Software User’s Guide Views in the GeneChip Software shortcut bar to display the open Image windows. AUTOMATICALLY ARCHIVING IMAGE DATA DAT files may be stored locally or to a network location after the scan is complete. A JPG file is always created, even if the image is not archived. The JPG file is a much smaller representation of the DAT file and is meant to show large scale features in the image such as defects (bubbles or scratches) and the general quality of the hybridization. For more information about viewing JPG files, see The Image Viewer with JPG Files, on page 217. for more information about the archiving options, see Setting Archiving Options for DAT files, on page 599. chapter 9 | Working with Images 145 The Image Window with CEL and DAT Files The Image window can be used to display DAT and CEL files (Figure 9.3). Image window task bar Figure 9.3 Image data in the Image window You can learn more about using the Image window with DAT and CEL files in the following sections: • Commands in the Image Window Task Bar (see page 146) • Viewing and Aligning the Grid and Subgrids (see page 147) • Computing Cell Intensities (see page 195) • Adjusting the Image Settings (see page 196) • Intensity Display (see page 204) 146 Affymetrix® GeneChip® Operating Software User’s Guide • Calculating Average Intensity for User Specified Probe Cells (see page 205) • Viewing Probe Set Information (see page 207) • Bookmarks (see page 212) • Outliers (see page 215) COMMANDS IN THE IMAGE WINDOW TASK BAR Figure 9.4 Task bar for CEL and DAT files When viewing CEL and DAT files, the Image Window displays the following controls in the task bar (Figure 9.4): Auto Automatically scales the intensity of the current image based on the range of intensity levels in a selected region of the scan image. See Adjusting the Image Settings, on page 196. Adj. Enables the subgrid adjustment. Grid Toggles the grid on and off the scan image. (Press the G key.) See Viewing the Grid and Subgrids, on page 151. Mask Masks user selected areas of the scan image. Masked probe cells are excluded from the analysis. See Outliers, on page 215. Avg Calculates the average intensity and standard deviation for a user selected region of the scan image. See Calculating Average Intensity for User Specified Probe Cells, on page 205. In Incrementally zooms in on the scan image. Press the I key: or Use a click-and-drag operation to draw a rectangle over the area of interest, then click In to zoom directly to the selected area. Out Zooms out incrementally from the scan image (press the O key). Full Zooms out completely to the full scan image view (press the Shift+O keys). chapter 9 | Working with Images 147 Enables the user to select and zoom directly to each corner of the scan image or selected subgrid to check grid alignment. See Viewing and Aligning the Grid and Subgrids, on page 147. SELECTING THE VIEW To zoom in on an image: • Click the In button in the task bar; or Press the I key. Holding the I key down performs a continuous zoom. To zoom out on an image: • Click the Out button in the task bar; or Press the O key. Holding the O key down performs a continuous zoom. To zoom into a selected region: Use the cursor to select the area of interest in the image. A dashed box indicated the selected area. 2. Click the In button; or Press the I key. To zoom out completely: • Click the Full button; or Press the <Shift> + O keys 1. Viewing and Aligning the Grid and Subgrids The Alignment algorithm uses the checkerboard image of the control probes, located at the corners of the probe array, to superimpose a grid on the scan image. The algorithm aligns the grid so that each square in the grid delineates a probe cell. Some arrays use a single main grid (Figure 9.5). 148 Affymetrix® GeneChip® Operating Software User’s Guide Figure 9.5 Array with a single grid Other arrays use a main grid with multiple subgrids on the scan image (Figure 9.6). The position of each sub-grid can be adjusted independent of the other sub-grids. chapter 9 | Working with Images 149 Figure 9.6 Main grid with subgrids Each subgrid is identified by its row and column in the main grid. The corners of each subgrid are marked on the chip by specific alignment patterns; the anchors of the subgrid are aligned to these patterns (Figure 9.7). 150 Affymetrix® GeneChip® Operating Software User’s Guide corner Figure 9.7 Corner patterns of a subgrid The software uses the corner patterns at the 4 corners of the array to come up with the initial main grid. The main grid is then divided into smaller subgrids. The subgrid is aligned on the grid patterns in the final steps of the gridding algorithm and in manual alignment. For more information, see Aligning Subgrids, on page 162. The alignment of the grid and subgrids usually takes place automatically after scanning the chip. If the alignment algorithm fails you can perform a manual alignment of the main grid and subgrids. Learn more about working with grids and subgrids in: • Viewing the Grid and Subgrids (see below) • Aligning the Main Grid (see page 154) • Aligning Subgrids (see page 162) chapter 9 | Working with Images 151 VIEWING THE GRID AND SUBGRIDS To toggle the grid on or off, do one of the following: - Click Grid in the Image window task bar (Figure 9.1). - Press the G key. - Select View → Grid from the menu bar. Figure 9.8 Main grid with subgrids You can zoom in to view the grid or subgrids in more detail. When viewing an array that uses a single grid, the individual cells are displayed at higher magnifications (Figure 9.9). 152 Affymetrix® GeneChip® Operating Software User’s Guide Figure 9.9 Zoom view of array with single grid, showing the individual cells When viewing an array that uses subgrids, the Image window does not display individual cells as a grid—instead, at smaller scales it displays a small crosshair to mark the center of each cell of the grid (Figure 9.10). chapter 9 | Working with Images 153 Figure 9.10 Zoom view of chip with subgridding, feature centers indicated by crosshairs You can change the color used to display the grid. For more information, see Selecting Color Settings, on page 199. 154 Affymetrix® GeneChip® Operating Software User’s Guide ALIGNING THE MAIN GRID If the main grid is misaligned, you will see a notice after you scan a probe array or try to open a DAT file (Figure 9.11). Figure 9.11 Notice of main grid misalignment You have two options for fixing an alignment problem: • You can run the gridding algorithm again (see below). • You can align the grid manually (see below) Running the Gridding Algorithm In some cases you can realign the grid by running the alignment algorithm again. To run the alignment algorithm again on an array that uses a single grid: • Right-click on the image and select Realign Grid from the shortcut menu (Figure 9.12). chapter 9 | Working with Images 155 Figure 9.12 Array with single grid and shortcut menu To run the alignment algorithm again on an array that uses sub-grids: • Right-click on the image and select Realign All Grids from the shortcut menu (Figure 9.13). This will align the main grid and all the subgrids. 156 Affymetrix® GeneChip® Operating Software User’s Guide Figure 9.13 Array with subgrids and shortcut menu After the alignment algorithm runs, click the Save button select File → Save from the menu bar. or chapter 9 | Working with Images 157 Aligning the Grid Manually If the grid alignment fails, an error message appears (Figure 9.14). Figure 9.14 Notice of main grid misalignment If you see the error message, you can manually adjust the main grid by using the following procedure: 1. Click the OK button in the error message. The main grid is displayed in the Image window for an array with a single grid (Figure 9.15) or an array using subgrids (Figure 9.16). 158 Affymetrix® GeneChip® Operating Software User’s Guide Figure 9.15 Misaligned main grid on an array with one grid Subgrids will not be displayed if the main grid is misaligned. chapter 9 | Working with Images 159 Figure 9.16 Misaligned main grid for probe array using subgrids You can display the grid and subgrids for any DAT file by clicking the Grid button in the Image window task bar. 2. Align the grid at each corner of the image: A. Click the a corner. button in the Image window task bar and choose 160 Affymetrix® GeneChip® Operating Software User’s Guide You may need to zoom out on the image to locate the grid boundary. Place the mouse arrow over the grid perimeter (the arrow becomes a double arrow, ). The diagonal orientation of the double arrow along the perimeter of a corner probe cell indicates horizontal and vertical adjustments can be made simultaneously using the click-anddrag method or by using the keyboard arrow keys. C. Use the click-and-drag method or the keyboard arrow keys to adjust the horizontal or vertical position of the grid so that it is aligned over the corner of the outermost corner checkerboard. For arrays that use a single grid, the software will display a box around each feature when editing the grid (Figure 9.17). B. chapter 9 | Working with Images 161 Figure 9.17 Aligning on a chip with a single grid When an array uses sub-grids and the main grid has failed to align itself, then the software will display it with a single box (Figure 9.18). 162 Affymetrix® GeneChip® Operating Software User’s Guide corner Figure 9.18 Aligning on an array with subgrids Repeat the above steps for the other corners of the main grid. 3. After you align the grid, click the Save button or select File → Save from the menu bar. The main grid is aligned. For arrays with a single grid, you can now proceed to generate the CEL file. For arrays with subgrids, you can now check the alignment of the subgrids. Users have to select Realign Sub-grids to automatically create the sub-grids using the main grid. D. ALIGNING SUBGRIDS Sometimes one or more subgrids may require manual alignment. You will be informed by the following notice when the subgrid alignment fails (Figure 9.19): chapter 9 | Working with Images 163 Figure 9.19 Notice of subgrid misalignment(s) If the subgrid alignment fails you can: • Run the subgrid alignment algorithm (see below). • Perform a manual alignment (see below). Running the Subgrid Alignment Algorithm You can run the subgrid alignment algorithm if some of the subgrids are misaligned or if you have manually aligned the main grid. To run the subgrid alignment algorithm: 1. Right-click the image and select Realign SubGrids from the shortcut menu. 2. After the alignment algorithm runs, click the Save button select File → Save from the menu bar. or 164 Affymetrix® GeneChip® Operating Software User’s Guide Manually Aligning the Subgrids The boundaries of a subgrid are indicated by the alignment patterns at the four corners of the subgrid. A small checkerboard may mark the corner of two or more subgrids, depending upon its position in the main grid (Figure 9.20). Figure 9.20 Subgrid borders chapter 9 | Working with Images 165 If the sub-grids are not aligned correctly, the following error message appears after scanning an array or opening a DAT file: Figure 9.21 Notice of subgrid misalignment(s) You can perform a manual alignment of the subgrids with the following procedure: 1. Click OK in the Subgrid Alignment Failure dialog box (Figure 9.21). The Manual Alignment alert opens (Figure 9.22). Figure 9.22 Manual alignment alert The subgrids are displayed in the Image window with the misaligned subgrids highlighted by a red “X” (Figure 9.23). You can display the grid and subgrids for any DAT file by clicking the Grid button in the Image window task bar. 166 Affymetrix® GeneChip® Operating Software User’s Guide Misaligned subgrids Figure 9.23 Misaligned subgrids You can change the color used to display the grid, the misaligned subgrids, and the selected subgrid. For more information, see Selecting Color Settings, on page 199. 2. Click Yes in the manual alignment dialog box (Figure 9.22) to perform a manual alignment. The Manual Subgrid Alignment dialog box opens (Figure 9.24). chapter 9 | Working with Images 167 Selected subgrid Defective subgrids Figure 9.24 the Manual Subgrid Alignment dialog box with misaligned and selected subgrids You can also open the Manual Subgrid Alignment dialog box by clicking the Adj. button in the Image window task bar. The Manual Subgrid Alignment dialog box (Figure 9.25) allows you to select a subgrid for adjustment and navigate to the corners of the subgrid. 168 Affymetrix® GeneChip® Operating Software User’s Guide Select the corner of the subgrid to be aligned Select the subgrid to be aligned Figure 9.25 Manual Subgrid Alignment dialog box The dialog box has the following controls: Subgrid List Displays the subgrid and its position in the main grid by row and column. Go To Corner buttons Allows you to go to the subgrid corner you want to adjust. Show All Subgrids Check to display all subgrids in the subgrid list (misaligned subgrids are highlighted by a red X). Subgrid Displays the selected subgrid and its position in the grid Corner Displays the selected corner. Close Closes the dialog box. You can use the + and - keys to step through the items in the subgrid list. chapter 9 | Working with Images 169 Click on the subgrid you wish to align in the list. 4. A zoomed-in view of the subgrid appears in the Image window. 3. Figure 9.26 Selected subgrid 5. Align the subgrid at each corner: A. Click the Go To Corner button for the corner you wish to align. A zoomed-in view of the corner of the subgrid appears (Figure 9.27). 170 Affymetrix® GeneChip® Operating Software User’s Guide When any one of the Go To Corner buttons is highlighted, the software places a box around each feature to help with the grid alignment. If the software is displaying the centers of features, as explained on page 152, then the centers will not be displayed for grids that have been manually aligned. Figure 9.27 The upper right subgrid corner B. Place the mouse arrow over the grid perimeter (the arrow becomes a double arrow, ). The diagonal orientation of the double arrow along the perimeter of a corner probe cell indicates horizontal and vertical adjustments can be made simultaneously using the click-anddrag method or by using the keyboard arrow keys. chapter 9 | Working with Images C. 171 Use the click-and-drag method or the keyboard arrow keys to adjust the horizontal or vertical position of the subgrid so that it is aligned over the outside corner of the small checkerboard pattern (Figure 9.28). Figure 9.28 Dragging the subgrid boundary Repeat steps a through c for the other corners. E. Continue manually aligning all misaligned subgrids from the subgrid list. 6. After you align the grid, click the Save button or select File → Save from the menu bar. The grid is aligned. You are now ready to recompute the cell intensities (see below). D. 172 Affymetrix® GeneChip® Operating Software User’s Guide TIPS FOR MANUALLY ALIGNING SUBGRIDS ON CUSTOMSEQ FOR RESEQUENCING ARRAYS Why would it be necessary to manually adjust grids on subimages? A subgrid may need to be manually gridded due to incorrect corner placement, weak signals, debris, or other factors. 2. How would one know if there is a subgrid that needs to be manually adjusted? The Cell Summary Report in GCOS 1.4 provides a listing of control probes which have unexpected intensities. These unexpected intensities could be seeing bright intensities on expected dim probes or vice versa. Examine the features on the image that correspond to the control probes with unexpected intensities. If they are not due to image debris then it is possible they may be due to misgridding. 1. 3. What do properly placed grids look like? Properly placed grids are shown in Figure 9.29. chapter 9 | Working with Images Figure 9.29 Correctly placed grids 4. What is the key item to look for when manually placing grids? The key item for placing grids vertically is the checkerboard pattern (Figure 9.30). 173 174 Affymetrix® GeneChip® Operating Software User’s Guide Figure 9.30 To position grids vertically chapter 9 | Working with Images The key item for placing the corner grids horizontally is the sequence in the adjacent rows (Figure 9.31). Figure 9.31 To position grids horizontally 175 176 Affymetrix® GeneChip® Operating Software User’s Guide Please be careful when placing the subgrids manually because only the checkerboard is available. The intensities of the extreme corner cells are the only reference for manual alignment (Figure 9.32). In fact, it is possible for the subgrid to look correct and be placed incorrectly by two features. Figure 9.32 Sub images only have the checkers for horizontal placement 5. What do improperly placed grids look like? Improperly placed grids are shown in Figure 9.33. chapter 9 | Working with Images Figure 9.33 Improperly placed grids 177 178 Affymetrix® GeneChip® Operating Software User’s Guide The Cell Summary Report The Cell Summary Report is a tool for monitoring the performance of the hybridization and grid alignment of arrays. The report allows you to detect problems in these steps and compare the performance of different chips. The Cell Summary Report uses the control features on the chip. Control features are cells with special probes; the corresponding targets are spiked into the sample cocktail. The resulting patterns of bright and dark cells are used in grid alignment and other processes. You can learn more about the Cell Summary reports in the following sections: • Generating a Cell Summary Report (see below) • Cell Summary Report Components for Non-Resequencing Chips (see page 181) • Cell Summary Report Components for Resequencing Chips (see page 186) • Cell Summary Report Settings (see page 189) • Cell Summary Report Options (see page 190) • Editing a Cell Summary Report (see page 192) • Saving a Cell Summary Report (see page 193) For more information about the algorithm used to generate the Cell Summary report, see Cell Summary Report Algorithm, on page 679. GENERATING A CELL SUMMARY REPORT You must have a .GRC file for the chip type in the AffyData/Library directory. The GRC files will be installed during the library installation. When working with a server, the GRC files need to be located in the library folder on the server. chapter 9 | Working with Images 179 To generate a cell-summary report: 1. To generate the report, in the data tree, right-click the cell intensity data for the report and select Report from the shortcut menu that appears. You can also select File → Report from the menu bar. The Report dialog box opens (Figure 9.34). Figure 9.34 Report dialog box A. From the Data of Type list, select CEL Data (*.CEL). Select the Cell Intensity data for the report from the Data Name list C. Click OK. This displays the Cell Summary Report in the main display area. Figure 9.35 show an example Cell Summary Report. B. A previously generated report will be overwritten if the new report is saved under the same name. To prevent overwriting old files, save the report under a new name. For more information, see Saving a Cell Summary Report, on page 193. 180 Affymetrix® GeneChip® Operating Software User’s Guide You can also look at the failed cells in the Image window (see Outliers, on page 215) Report Header Report Summary Metrics List of failed cells by subgrid row and column Figure 9.35 Expression Cell Summary report chapter 9 | Working with Images 181 CELL SUMMARY REPORT COMPONENTS FOR NON-RESEQUENCING CHIPS The Cell Summary Reports for all types of chips except Resequencing (Expression, Mapping, and others) (Figure 9.35) provides information on the following features: • Non-synthesized features • OligoB1 features • OligoB2 features Refer to the relevant manual for the GeneChip array for more information about these features. The report contains the following sections: • Report Header (see below) • Summary (see page 182) • Metrics (see page 183) • Failed Cell Locations (see page 184) Report Header Figure 9.36 Expression Cell Summary report: header The report header (Figure 9.36) lists basic information about the Cell Summary report: Report Type Used to distinguish types of reports (Cell Summary Report, Algorithm Report, etc.). Date Time and date report was generated. 182 Affymetrix® GeneChip® Operating Software User’s Guide Filename Input .CEL data file name that was used to generate the report. GRC Filename Name of the .GRC file used to generate the report. Probe Array Type Array type used for the .CEL file. Summary Figure 9.37 Expression Cell Summary report: Summary The Summary (Figure 9.37) displays information: For the following types of features: Non-synthesized features Bright control probes Dim control probes Total control features The summary lists the following values: Failures The number of features in the category that failed. Total The total number of features in the category. Failure Rate The percentage of features in the category that failed. chapter 9 | Working with Images 183 Metrics Figure 9.38 Expression Cell Summary report: metrics The metrics section (Figure 9.38) displays additional data about the B1 and B2 oligos. 184 Affymetrix® GeneChip® Operating Software User’s Guide Failed Cell Locations Figure 9.39 Expression Cell summary report, failed cell locations If the cell summary report was performed on a chip that used subgridding, the report (Figure 9.39) displays the locations of the failed features by row and column of the subgrid (Figure 9.40): Row Subgrid row. Column Subgrid column. Total Failures Total failures in the subgrid. chapter 9 | Working with Images 185 To view these probes on a DAT file, the probes can be marked as Masked. For more information about this option, see Cell Summary Report Settings, on page 189. Column Row 1 2 3 4 1 2 3 4 5 6 7 8 9 10 11 12 13 Figure 9.40 Rows and columns in the subgrid 5 6 7 8 9 10 11 12 13 186 Affymetrix® GeneChip® Operating Software User’s Guide CELL SUMMARY REPORT COMPONENTS FOR RESEQUENCING CHIPS Resequencing chips do not have oligoB1 features. Instead, it provides information on: • Non-synthesized features. • OligoB2 checker features (oligoB2 features arranged in a checkerboard pattern). • OligoB2 Extended features (oligoB2 features arranged in a Resequencing pattern). Refer to the relevant manual for the GeneChip array for more information about these features. The report (Figure 9.41) contains the following sections: • Report Header (see below) • Summary (see page 188) • Metrics (see page 189) chapter 9 | Working with Images Report Header Report Summary Metrics Figure 9.41 Cell Summary report for resequencing chip Report Header Figure 9.42 Resequencing Cell Summary report: header 187 188 Affymetrix® GeneChip® Operating Software User’s Guide The report header (Figure 9.42) lists basic information about the Cell Summary report: Report Type Used to distinguish types of reports (Cell Summary Report, Algorithm Report, etc.). Date Time and date report was generated. Filename Input .CEL data file name that was used to generate the report. GRC Filename Name of the .GRC file used to generate the report. Probe Array Type Array type used for the .CEL file. Summary Figure 9.43 Resequencing Cell Summary report: Summary The Summary (Figure 9.43) displays information: For the following types of features: Non-synthesized features OligoB2 Checker OligoB2 Extended control probes Total control features The summary lists the following values: Failures The number of features in the category that failed. Total The total number of features in the category. Failure Rate The percentage of features in the category that failed. chapter 9 | Working with Images 189 Metrics Figure 9.44 Resequencing Cell Summary report: metrics The metrics section (Figure 9.44) displays additional information about the non-synthesized and oligoB2 features. USING THE CELL SUMMARY REPORT The Cell Summary report provides clues to problems in the fluidics, grid alignment, and other issues with the cell file. It can be used to establish a baseline for performance, which can then be used for quality control for subsequent experiments. CELL SUMMARY REPORT SETTINGS To change the masking of failed features: 1. From the Menu bar, select Options -> Cell Summary Report. The Cell Summary report dialog box opens (Figure 9.45). 190 Affymetrix® GeneChip® Operating Software User’s Guide Figure 9.45 Cell Summary report dialog box Click the Mask control Features checkbox. 3. Click OK. The failed features will be displayed as masked features in the Image window. 2. CELL SUMMARY REPORT OPTIONS You can perform the following operations on the Cell Summary report: • Changing Font and Tab Stops (see below) • Searching the Report (see page 192) • Editing a Cell Summary Report (see page 192) • Performing a Search and Replace on the Report (see page 192) • Saving a Cell Summary Report (see page 193) Changing Font and Tab Stops The Cell Summary report looks best when the default font and tab settings of 10 point Arial font and 10 point tab stops are used. To change the font: 1. Select View → Set Font from the menu bar. The Font dialog box opens (Figure 9.46). chapter 9 | Working with Images 191 Figure 9.46 Font dialog box Select the font, style, and size from the dialog box. 3. Click OK. To change the tab stops and adjust the horizontal spacing of items on a line: 2. 1. Select View → Set Tab Stops from the menu bar. The Set Tab Stops dialog box opens (Figure 9.47). Figure 9.47 Set Tab Stops dialog box Enter a tab value in the Tab Stop box. 3. Click OK. 2. 192 Affymetrix® GeneChip® Operating Software User’s Guide Searching the Report The Find feature searches the report. 1. Click the Find button or select Edit → Find from the menu bar. The Find dialog box appears (Figure 9.48). Figure 9.48 Find dialog box Enter the text for the search (up to 256 alphanumeric characters including spaces) in the Find what box, then click Find Next to view the first search result. 3. Click Find Next or the Find Next button to view each successive search result. 2. Editing a Cell Summary Report Edit commands for the cell summary report are available in the: • Edit menu bar • Report window toolbar • Shortcut menu (right-click the report) Performing a Search and Replace on the Report The replace feature performs a text search and replaces the search item with specified text. 1. Select Edit → Replace from the menu bar. The Replace dialog box appears (Figure 9.49). chapter 9 | Working with Images 193 Figure 9.49 Replace dialog box 2. Enter the text for the search in the Find what box and enter the replacement text in the Replace with box. 3. Click Find Next to begin the search, then click Replace to replace the search item with the specified text. Repeat to find and replace successive instances of the search item; or Click Replace All to find and replace all occurrences of the search item in the report. Saving a Cell Summary Report When GCOS generates a report for alignment probe analysis data, it overwrites a previously generated report. To prevent a previous report from being overwritten, save it under a new name. 1. To open the report that you want to save under a new name, click the Open button . The Open dialog box appears (Figure 9.50). 194 Affymetrix® GeneChip® Operating Software User’s Guide Figure 9.50 Open dialog box Double-click the report name. The report is displayed. 3. Select File → Save As from the menu bar. The Save As dialog box appears (Figure 9.51). 2. Figure 9.51 Save As dialog box 4. Click the report to be saved, then enter a new name for the report in the File name box. 5. Click Save. chapter 9 | Working with Images 195 Computing Cell Intensities GCOS automatically generates the cell intensity data from the image data (Figure 9.53). The Cell Analysis algorithm analyzes the image data, computes a single intensity value for each probe cell on the array, and saves the cell intensity data. Register sample & define experiment Process probe array in fluidics station Scan probe array & save image data Compute cell intensity data from image data & save cell intensity data Analyze expression cell intensity data & save expression probe analysis data Publish expression data Generate an expression report Figure 9.52 Assay & analysis flow chart 196 Affymetrix® GeneChip® Operating Software User’s Guide Figure 9.53 Image window, scan image data (left) and computed cell intensity data (right) REANALYZING IMAGE DATA GCOS automatically generates the cell intensity data from the image data. In some cases you may want to generate new cell intensity data (for example, if the grid has been realigned). To reanalyze image data: • Right-click the open image and select Recalculate Cell Intensity from the shortcut menu that appears. Adjusting the Image Settings You can adjust image settings (intensity range, color, coordinates, and highlights) for image or cell intensity data. The image settings may be adjusted for the current data only or globally for all subsequently opened image or cell intensity data. chapter 9 | Working with Images 197 MAKING ADJUSTMENTS TO THE CURRENT IMAGE OR CELL INTENSITY DATA ONLY To adjust the settings for the current image or intensity data only, do one of the following: • Click the Image settings button . • Press the S key. • Select Edit → Image Settings from the menu bar. This displays the Image Settings dialog box for the active image or cell intensity data (Figure 9.54). All settings (except the color setting) in the Image Settings dialog box apply only to the current image or cell intensity data and do not affect other image or intensity data. The settings (except for the color setting) are not saved after you close the image or intensity data. 198 Affymetrix® GeneChip® Operating Software User’s Guide Click the S key Select Tools → Defaults from the menu bar Settings for the active image or cell intensity data only. Default settings for subsequently opened image or cell intensity data Figure 9.54 Image Settings dialog box for the active image or cell intensity data (left); Defaults dialog box, Image Settings tab (right) MAKING GLOBAL ADJUSTMENTS TO SUBSEQUENTLY OPENED IMAGE OR CELL INTENSITY DATA To adjust settings globally: • Select Tool → Defaults from the menu bar. This displays the Image Settings tab of the Defaults dialog box (Figure 9.54). The settings specified in the Image Settings tab of the Defaults dialog box do not affect open image or cell intensity data, but are applied to all subsequently opened image or cell intensity data. These default image settings are saved when the image is closed. chapter 9 | Working with Images 199 SETTING THE INTENSITY RANGE GCOS records a dynamic intensity range from 0 to 65,000 and provides 256 colors from black through white for image display. The intensity range for image or cell intensity data is divided into 256 bins and each bin is assigned a color or gray scale. You may enter a new lower or upper limit for the intensity range associated with image or cell intensity data and apply this subset of the dynamic range to the image. Lowering the upper limit increases the image brightness and raising the lower limit decreases the brightness. This process is called image scaling and can be used to adjust the display of image or cell intensity data for optimum viewing. Alternatively, GCOS can automatically scale the image using the minimum and maximum pixel intensities of the image. To autoscale the current image, choose the Autoscale option in the Image Settings dialog box (Figure 9.54) 2. Click OK to automatically scale the image. When the Image Settings dialog box is reopened, it displays the minimum and maximum pixel intensities used to scale the image. 3. To return the intensity settings (autoscale option and intensity range) to the defaults in the Defaults dialog box, click Defaults in the Image Settings dialog box. 1. SELECTING COLOR SETTINGS To display the image or cell intensity data in: • Gray scale - choose the Gray color option (Figure 9.54). • Pseudo color - choose the Pseudo Color option. This applies rainbow colors to the intensity scale. To display a gray scale or pseudo color bar at the top of the Image window: • Choose the Color Bar option. 200 Affymetrix® GeneChip® Operating Software User’s Guide SELECTING DISPLAY COORDINATES • Click the image to display image coordinates. A red cross hairs marks the location. The status bar (lower left in the main window) displays the intensity data and x,y coordinates of a probe cell or pixel indicated by the cross hairs. If you choose the Cell option in the Image Settings dialog box (Figure 9.54), the status bar and a pop-up tool tip display the x,y coordinates of the cell, the cell intensity, and the pixel intensity (Figure 9.55). If you choose the Pixel option, the pop-up tool tip and the status bar display the x,y coordinates of the pixel and the pixel intensity (Figure 9.55). Only the Cell option is available for cell intensity data. chapter 9 | Working with Images Cell X = 186, Y = 41, Cell Intensity = 22561.8, Pixel Intensity = Figure 9.55 Image data, probe cell coordinates (top) and pixel coordinates (bottom) 201 202 Affymetrix® GeneChip® Operating Software User’s Guide Pixel X = 1727, Y = 573, Intensity = 22409 Figure 9.55 Image data, probe cell coordinates (top) and pixel coordinates (bottom) SPECIFYING HIGHLIGHT COLORS You may specify new highlighting colors for the grid, masked cells, outlier cells, or highlighted probe array cells (features). 1. Click Highlights in the Image Settings dialog box (Figure 9.54 on page 198). The Highlight Colors dialog box appears (Figure 9.56). chapter 9 | Working with Images 203 Figure 9.56 Highlight Colors dialog box 2. To change the color of a particular item (for example, the grid overlay): Click the associated color box in the Highlight Colors dialog box. The Color palette is displayed (Figure 9.57). A. 204 Affymetrix® GeneChip® Operating Software User’s Guide Custom color Luminosity scale Figure 9.57 Color palette To select a predefined color, click one of the basic colors. C. To define a custom color, click Define Custom Colors, then use the click-and-drag method to move the cross hairs in the custom color field. Adjust the color brightness using the luminosity scale to the right. When finished, click Add to Custom Colors to apply the color. The new color is applied to the selected item. D. Click OK to close the Color palette. B. Intensity Display You can view the probe cell intensity in the image data as a measured or difference image. Open the image data. 2. Select View → Image from the menu bar, then choose one of the two viewing options: Measured or Average. 1. chapter 9 | Working with Images 205 MEASURED IMAGE VIEW The measured image view (Figure 9.58) displays the raw data from a scanned image. Figure 9.58 Measured image view AVERAGE IMAGE VIEW The average image view (Figure 9.59) displays the probe cell intensity values calculated by the Cell Analysis algorithm. Figure 9.59 Average image view Calculating Average Intensity for User Specified Probe Cells GCOS can calculate the average intensity for a user-specified group of probe cells in the image data. 1. In the image, use the click-and-drag method to select the probe cells of interest. The selected probe cells are outlined in the image. 2. Click AVG in the Image window task bar (Figure 9.1 on page 142). 206 Affymetrix® GeneChip® Operating Software User’s Guide The status bar displays the average intensity and the standard deviation. 3. Position the mouse arrow inside the selected area of the image. A pop-up tool tip displays the average intensity and the standard deviation for the selected group of probe cells (Figure 9.60). Average Intensity = 17328, Std Dev = Figure 9.60 Average intensity and standard deviation for user-specified probe cells in the image data (.dat) Viewing Probe Cell Data You can click a probe cell (feature) in the image or cell intensity data to obtain more information about the feature and the associated gene. To display the probe cell data for image or cell intensity data, do one of the following: • Double-click the probe cell of interest. • Click the probe cell of interest; right-click the image, and select View Probe Cell Data in the shortcut menu that appears. • Click a probe cell of interest and select View → Probe Cell Data from the menu bar. The Probe Cell Data dialog box appears (Figure 9.61). Click other probe cells in the image to update the displayed data. chapter 9 | Working with Images 207 Figure 9.61 Probe cell data, GeneChip® Hu6800 probe array Cell Coordinates The probe cell x and y coordinates in cell units. Intensity The cell intensity calculated by the cell analysis algorithm. Probe Set The identifier of the probe set containing the probe cell. Direction The direction (sense or antisense) of the target (sample). Target Base The expected nucleotide at the position that corresponds to the substitution position of the probe. Probe Base The actual nucleotide located at the substitution position of the probe. Position The numbered position of the perfect match and mismatch probe pair within the probe set (for example, 1-20 for a probe set consisting of 20 probe pairs). Tile The identification number for the probe set. Viewing Probe Set Information You can learn about viewing probe set information in: • Viewing Probe Array Tiling Information (see below) • Viewing Probe Cell Data for a Highlighted Probe Set(s) (see page 209) • Highlighting Tiles on the Probe Array (see page 211) 208 Affymetrix® GeneChip® Operating Software User’s Guide VIEWING PROBE ARRAY TILING INFORMATION You can view probe array tiling information for the current image or cell intensity data in the Image window. 1. Right-click the image or cell intensity data and click View Probe Tiling in the shortcut menu that appears: or Select View → Probe Tiling from the menu bar. The View Probe Tiling dialog box appears (Figure 9.62). Figure 9.62 View Probe Tiling dialog box The Features tab of the View Probe Tiling dialog box lists all probe sets tiled on the probe array for the current image or cell intensity data. 2. In the Features tab, press and hold the Ctrl key while you click the features of interest (Figure 9.62). Click Apply when the selection is completed. This highlights the probe sets in the image or cell intensity data that correspond to the selected features so that hybridization data may be quickly located (Figure 9.63). chapter 9 | Working with Images 209 Figure 9.63 Highlighted probe cells in the image data Turn off the grid (press the G key) so that the highlighted probe cells can be easily seen. The highlighting color for the selected probe cells may be changed. See Specifying Highlight Colors, on page 202. 3. Click OK to close the View Probe Tiling dialog box. VIEWING PROBE CELL DATA FOR A HIGHLIGHTED PROBE SET(S) 1. In the image or cell intensity data, click a probe cell of interest. This places the red cross hairs over the feature. 210 2. Affymetrix® GeneChip® Operating Software User’s Guide Right-click and select View Probe Cell Data from the shortcut menu that appears; or Select View → Probe Cell Data from the menu bar. This displays the probe cell data (Figure 9.64). If you click another probe cell, GCOS automatically updates the probe Cell Data dialog box. Figure 9.64 Probe cell data for a selected feature chapter 9 | Working with Images 211 To place the red cross hairs over the next feature of the highlighted probe set, click the image (to return focus to the Image window) and do one of the following: - Press the N key. - Right-click the image and select View Next Highlight in the shortcut menu that appears. - Select View → Next Highlight from the menu bar. If it is open, the Probe Cell Data dialog box is automatically updated with the new probe cell data. 4. To return the cross hairs to a previous feature, do one of the following: - Press the P key. - Right-click the image and select View Previous Highlight in the shortcut menu that appears. - Select View → Previous Highlight from the menu bar. 3. 5. To remove all highlights, do one of the following: - Press the E key. - Right-click the image and select Clear Highlights from the shortcut menu that appears. - Select View → Clear Highlights from the menu bar. Highlights are automatically cleared when you close the image or cell intensity data. HIGHLIGHTING TILES ON THE PROBE ARRAY You can highlight: • all tiles or a particular tile on the probe array (by tile number) • all positions or a particular position of all tiles or a selected tile 212 Affymetrix® GeneChip® Operating Software User’s Guide Right-click the image or cell intensity data and click View Probe Tiling in the shortcut menu that appears: or Select View → Probe Tiling from the menu bar. The View Probe Tiling dialog box appears (Figure 9.65). 2. Click the Positions tab, select a Position and Tile, then click Apply. This highlights the selection in the image. Figure 9.65 shows two examples: all positions of tile number 3000 highlighted and only position 15 of tile 3000 highlighted. 1. Figure 9.65 Cell intensity data, all positions of tile 3000 highlighted (left), position 15 of tile 3000 highlighted (right) Bookmarks A bookmark denotes a specific area of the image or cell intensity data at a particular magnification level. Bookmarks make it easy to quickly view the hybridization data at a specific location in different images. chapter 9 | Working with Images 213 When you select a bookmark, the Image window automatically displays the image location at the magnification specified by the bookmark. You can learn more about using bookmarks in: • Creating a Bookmark (see below) • Viewing Bookmarks (see page 214) CREATING A BOOKMARK Use the task bar zoom commands and scroll bars in the Image window to locate the image area of interest at the desired magnification level. 2. Right-click the image and select View Bookmarks → Add Bookmark from the shortcut menu that appears; or Select View → Bookmarks → Add Bookmark from the menu bar. The Add Bookmark dialog box appears (Figure 9.66). 1. Figure 9.66 Add Bookmark dialog box Enter a name in the Bookmark Name box or use the default name Bookmark 1, 2,3.... 4. Click OK to create the bookmark. 3. 214 Affymetrix® GeneChip® Operating Software User’s Guide VIEWING BOOKMARKS To view bookmarks, do either of the following: • Right-click the image and select View Bookmarks → Edit Bookmarks from the shortcut menu that appears; or Select View → Bookmarks → Edit Bookmarks from the menu bar. The Edit Bookmarks dialog box displays a list of all the bookmarks (Figure 9.67). • Select View → Bookmarks from the menu bar. (A maximum of 20 bookmark names are displayed here.) Figure 9.67 Edit Bookmarks dialog box Commands in the Edit Bookmarks Dialog Box Highlight the bookmark name in the Edit Bookmarks dialog box, then click the command of interest. Delete Permanently removes the selected bookmark. Rename Allows the selected bookmark to be renamed. Go To Displays the selected bookmark in the image. Close Closes the Edit Bookmarks dialog box. chapter 9 | Working with Images 215 Outliers Outliers are probe cells that are obscured or non-uniform in intensity (for example, probe cells with bright or dark streaks). The Outlier Detection algorithm automatically determines whether a probe cell is an outlier. You can exclude or mask outliers from a probe array in an expression analysis. The Statistical Expression algorithm (generates the probe analysis data from the cell intensity data) ignores the probe cell data from masked outliers. Learn more about working with outliers in: • Viewing Outliers (see below) • Masking Outliers (see page 216) • Unmasking Outliers (see page 217) When an array uses sub-grids, outliers will not be displayed in the image. VIEWING OUTLIERS 1. Open the image or cell intensity data of interest. 2. Right-click in the Image window and select View → Cells Masked from the shortcut menu; or Select View → Cells → Outliers from the menu bar. This highlights outlier probe cells with diagonal lines (Figure 9.69). 216 Affymetrix® GeneChip® Operating Software User’s Guide Figure 9.68 Highlighted cells The color that highlights outliers may be changed. See Specifying Highlight Colors, on page 202. MASKING OUTLIERS Open the image or cell intensity data of interest. 2. Select Edit → Mask All Outliers from the menu bar. All outliers identified by the Outlier Detection algorithm are masked. 3. To mask user-specified probe cells: 1. Use the click-and-drag method to select a probe cell or area of the image to be masked. B. Select Edit → Mask Cells from the menu bar. A. Turn off the grid (press the G key) to easily view the masked probe cells highlighted with diagonal lines. 4. To view masked probe cells, right-click in the Image window and select View Cells → Masked from the shortcut menu that appears. chapter 9 | Working with Images 217 UNMASKING OUTLIERS Select Edit → Unmask All Cells from the menu bar. This removes all masking. 2. To unmask only a selected probe cell or area: 1. Use the click-and-drag method to select a probe cell or area of interest. B. Select Edit → Unmask Cells from the menu bar. A. If you manually adjust the grid or change the probe array type in the experiment, the mask or outlier data will be lost when new cell intensity data are generated. The Image Viewer with JPG Files DAT files may be stored locally or to a network location. A JPG format file is created to allow the user to look at the image for basic QC purposes. The JPG file takes up much less memory than the DAT file and is meant to show large scale features in the image such as defects (bubbles or scratches) and the general quality of the hybridization. You can display these JPG files in the Image window to evaluate hybridization quality. The Image window functions are described in the following sections: • Opening a JPG Image (see below) • The JPG View Task Bar (see page 219) • Selecting the View (see page 220) • Adjusting Brightness and Contrast (see page 221) 218 Affymetrix® GeneChip® Operating Software User’s Guide OPENING A JPG IMAGE To open a JPG image: • Right-click on the DAT file and select Open JPG from the shortcut menu that appears (Figure 9.69); or Figure 9.69 Selecting the JPEG file in the data tree • Select File → Open from the main menu. The Open dialog box opens. Select JPG Data (*.JPG) from the Data of Type list. B. Select the JPG file you wish to open from the File Name list. C. Click OK. The JPG image is displayed in the JPG Viewer (Figure 9.70). A. chapter 9 | Working with Images 219 JPG Image window task bar Figure 9.70 JPG image in the Image window THE JPG VIEW TASK BAR When viewing JPG files, the Image window displays the following controls in the task bar (Figure 9.70): In Zooms in incrementally on the image. Out Zooms out incrementally on the image. Full Zooms out to the full scan image. Figure 9.71 JPG View task bar 220 Affymetrix® GeneChip® Operating Software User’s Guide SELECTING THE VIEW You can click the In and Out buttons to magnify the image, or the Full button to return to the full scan image view. To zoom in on a particular area: 1. Select View → Zoom → Zoom to Selected Region from the main menu; or Right-click in the image and select Zoom to Selected Region from the shortcut menu (Figure 9.72). Figure 9.72 Shortcut menu in JPG view 2. Use the cursor to select the area of interest in the image. A dashed box indicated the selected area. chapter 9 | Working with Images 221 When you release the mouse button, the selected area fills the Image Viewer. To zoom in without selecting an area: • Select View → Zoom → Zoom In from the main menu; or Click the In button in the Image window task bar. To zoom out on an area: • Select View → Zoom → Zoom Out from the main menu; or Click the Out button in the Image Window task bar. To display the entire image in the window: • Select View → Zoom → Best fit from the main menu; or Click the Full button in the Image window task bar. To shift the area displayed in the viewer: Select View → Zoom → Pan Image from the main menu; or Right-click in the image and select Pan Image from the shortcut menu. 2. Click on the image and drag to display the area of interest. 1. You can also use the horizontal and vertical sliders to move the image. ADJUSTING BRIGHTNESS AND CONTRAST You can adjust the brightness and contrast to make features easier to see. To adjust the brightness and contrast: 1. Select Edit → Adjust Image Brightness and Contrast... from the main menu; or Right-click in the image and select Adjust Image Brightness and Contrast... from the shortcut menu. The Brightness and Contrast dialog box opens (Figure 9.73). 222 Affymetrix® GeneChip® Operating Software User’s Guide Figure 9.73 Brightness and Contrast dialog box Adjust the sliders for Brightness and Contrast; or Enter values in the Brightness and Contrast boxes. You can enter values between ±1000 in the boxes 3. Click OK. 2. Chapter 10 Gene Expression Analysis Chapter 10 225 Gene Expression Analysis The Expression Analysis algorithm analyzes cell intensity data from GeneChip® expression arrays (Figure 10.1). Register sample & define experiment Process probe array in fluidics station Scan probe array & save image data Compute cell intensity data from image data & save cell intensity data Analyze expression cell intensity data & save expression probe analysis data Publish expression data Generate an expression report Figure 10.1 Assay & analysis flow chart This chapter explains how to: • Perform single-array or comparison array expression analysis. • View the expression probe analysis data in graphical or tabular format in the Expression Analysis window (EAW). • Generate the following types of reports: - Expression reports - Grid Reports This chapter contains the following sections: • Overview of Expression Analysis (see below) 226 Affymetrix® GeneChip® Operating Software User’s Guide • Expression Analysis Settings (see page 231) • Single-Array Expression Analysis (see page 232) • Comparison Expression Analysis (see page 235) • Expression Analysis Tabular Data (see page 242) • Expression Analysis Measured Images (see page 256) • Expression Analysis Graphs (see page 259) • Expression Report (see page 283) Overview of Expression Analysis There are two different types of expression analysis available in GCOS: • Single-Array Expression Analysis (see below) • Comparison Expression Analysis of an Experiment & Baseline (see page 229) SINGLE-ARRAY EXPRESSION ANALYSIS A single-array expression analysis examines the cell intensity data from one experiment (GeneChip® probe array). For each transcript represented on the probe array, the expression algorithm computes the: • Detection call (present, absent, or marginal (unable to call the transcript present or absent), or no call) • Detection p-value • Signal (background-subtracted and adjusted for noise) • Stat Pairs • Stat Pairs Used GCOS saves the probe analysis or chip data. chapter 10 | Gene Expression Analysis 227 To run a single-array analysis (Figure 10.2): Confirm the defaults or specify new values in the Expression Analysis Settings dialog box 2. In the data tree, select the cell intensity data for the analysis. 1. 3. Analyze the cell intensity data and view the probe analysis data in the Expression Analysis window (EAW). 228 Affymetrix® GeneChip® Operating Software User’s Guide 1. Expression Analysis Settings In the Settings shortcut bar, click the shortcut button to open the Expression Analysis Settings dialog box. Confirm or specify new analysis settings. Do not choose Use Baseline Comparison File option. 2. Select & Analyze Cell Intensity Data In the data tree, right-click the cell intensity data and select Analyze from the shortcut menu. 3. View Probe Analysis Data Expression Analysis window (EAW) displays single-array probe analysis data. Figure 10.2 Single-array expression analysis chapter 10 | Gene Expression Analysis 229 COMPARISON EXPRESSION ANALYSIS OF AN EXPERIMENT & BASELINE A comparison analysis compares the cell intensities of the same probe set on two different probe arrays (an experiment and baseline of the same probe array type) to determine the relative change in the expression level of a transcript. For each transcript represented on the probe array, the expression algorithm computes the: • Change call (increase, marginal increase, no change, marginal decrease, decrease) or no call • Change Detection p-value • Signal Log Ratio (background-subtracted and adjusted for noise) • Signal Log Ratio Low • Signal Log Ratio high • Stat Common Pairs GCOS saves the probe analysis data. To run a comparison analysis (Figure 10.3): Select the baseline probe analysis data. 2. Confirm the default expression analysis settings or specify new values. 3. Select the experiment cell intensity data from the data tree. 4. Analyze the experiment cell intensity data and view the comparison probe analysis data in the EAW. 1. 230 Affymetrix® GeneChip® Operating Software User’s Guide Comparison Array Expression Analysis Compares an Experiment & Baseline 1. Select the baseline data: In the Settings shortcut bar, click the shortcut button to open the Expression Analysis Settings dialog box. Choose the Use Baseline Comparison Data option. Click Browse to select the baseline probe analysis data. 2. Expression Analysis Settings In the Expression Analysis Settings dialog box. Confirm or specify new analysis settings. 3. Select & Analyze Cell Intensity Data In the data tree, right-click the cell intensity data and select Analyze from the shortcut menu. 4. View Probe Analysis Data Expression Analysis window (EAW) displays comparison analysis output. Figure 10.3 Comparison expression analysis of an experiment and baseline chapter 10 | Gene Expression Analysis 231 Expression Analysis Settings The Statistical Expression algorithm relies on the expression analysis settings to derive biologically meaningful results from the hybridization intensity data. The Expression Analysis Settings dialog box (Figure 10.4) displays: • The type of expression probe array for the analysis • A scaling or normalization factor (for more information, see Appendix F, Expression Algorithm, Expression Analysis Metrics & Settings, on page 627) • The probe mask definition (for more information, see Appendix F, Expression Algorithm, Expression Analysis Metrics & Settings, on page 627) • The baseline data selected for a comparison analysis • The user-modifiable parameters of the expression analysis algorithm (for more information, see Appendix F, Expression Algorithm, Expression Analysis Metrics & Settings, on page 627) Figure 10.4 Expression Analysis Settings dialog box, Baseline tab 232 Affymetrix® GeneChip® Operating Software User’s Guide Before running a single-array or comparison analysis, confirm the defaults or specify new values for the expression analysis settings. To open the Expression Analysis Settings dialog box (Figure 10.4), click Expression in the Settings shortcut bar. 2. Click a tab to view each type of setting. For more information about the settings and how to change them, see Expression Analysis Settings, on page 644. The expression analysis settings are saved on a per user basis. Changes made by one user (identified by the logon name) do not affect the settings of other users. 1. To compare the probe analysis data of several experiments, scale the experiments to the same target intensity using the All Probe Sets or Selected Probe Sets scaling option. See Scaling Tab, on page 645. Single-Array Expression Analysis A single-array expression analysis analyzes the cell intensity data of a GeneChip® expression probe array. For each transcript represented on the probe array, the algorithm computes a Detection call, Detection pvalue, and Signal. An expression analysis run from the data tree, menu bar, toolbar, or workflow monitor uses the expression analysis settings in the Expression Analysis Settings dialog box. The userset specified during experiment setup is not applied. For more information on usersets, see Userset Tab, on page 415. chapter 10 | Gene Expression Analysis 1. 233 Open the Expression Analysis Settings dialog box (Figure 10.4), and: Select the probe array type for the baseline from the Probe Array Type drop-down list. B. Click the Baseline tab and confirm that the Use Baseline Comparison File option is not chosen. A. C. 2. Confirm or change the other expression analysis settings as desired. To analyze cell intensity data, do one of the following: - In the data tree, right-click the cell intensity data name (or image data name), then click Analyze in the shortcut menu that appears. - Select File → Analyze from the menu bar and choose the cell intensity data that you want to analyze from the Analyze dialog box that appears. - If the cell intensity data or image data is open, click the Analyze ; or Select Run → Analysis from the menu bar. The Save Results As dialog box appears (if chosen as a default option, see Analysis Settings, on page 335) and displays the probe analysis data default name (same as the experiment name specified during experiment setup) (Figure 10.5). Figure 10.5 Save Results As dialog box 234 Affymetrix® GeneChip® Operating Software User’s Guide 3. If an analysis was previously run and you want to save the current results without overwriting the previous results, enter a new name in the Save Results As dialog box. 4. Click OK. This closes the Save As dialog box and runs the single-array analysis. The Expression Analysis Settings dialog box appears next if chosen as a default option. For more information on this default setting, see Analysis Settings, on page 335. When the analysis is finished, the Expression Analysis window (EAW) displays the probe analysis data (Figure 10.6). The status log (Figure 10.6) displays: - the name of the probe analysis data - the location of the probe analysis data - a message indicating when the analysis is completed If the status log is not displayed, click the Status Log button or select View → Status Bar from the menu bar. If the EAW is already open, the results are added to the open window and it may be necessary to use the scrollbars at the bottom and right side of the EAW to see the newly added results. chapter 10 | Gene Expression Analysis 235 Data tree EAW Shortcut bar Status log Figure 10.6 Expression Analysis Window (EAW), pivot tab, single-array analysis data Comparison Expression Analysis A comparison expression analysis compares the cell intensity data of an experiment to a baseline GeneChip® expression probe array (of the same probe array type). The comparison analysis identifies the relative change in the expression level of each transcript represented on the probe array. For each transcript represented on the array, the Statistical Expression algorithm computes a Change call, Change p-value, Signal Log Ratio, Signal Log Ratio low, and Signal Log Ratio high. 236 Affymetrix® GeneChip® Operating Software User’s Guide A single-array analysis of the baseline must be run prior to running a comparison analysis of the experiment and baseline. Selecting the Baseline 1. Open the Expression Analysis Settings dialog box (Figure 10.7) (click Expression in the Settings shortcut bar). 2. Select the probe array type for the baseline from the Probe Array Type drop-down list. Figure 10.7 Expression Analysis Settings dialog box 3. Click the Baseline tab and choose the Use Baseline Comparison File option. 4. Click Browse. The Open dialog box appears (Figure 10.8). chapter 10 | Gene Expression Analysis 237 Figure 10.8 Open dialog box 5. Select the baseline data and click OK. Running the Comparison Analysis 1. To run the comparison analysis of an experiment and baseline, do one of the following: - In the data tree, right-click the experiment cell intensity data or image, and click Analyze in the shortcut menu that appears. - Select File → Analysis from the menu bar and choose the experiment cell intensity data from the Analyze dialog box that appears. - If the cell intensity data or image is open, click the Analyze button ; or Select Run → Analysis from the menu bar. The Save Results As dialog box appears (if chosen as a default option, see Analysis Settings, on page 335) and displays the default name for the probe analysis data (same as the experiment name specified during experiment setup) (Figure 10.9). 238 Affymetrix® GeneChip® Operating Software User’s Guide Figure 10.9 Save Results As dialog box 2. Enter a new name in the Save Results As dialog box if an analysis was previously run and you want to save the current results without overwriting the previous results. Click OK. The Expression Analysis Settings dialog box appears next if chosen as a default option (Figure 10.10). For more information on this default setting, see Analysis Settings, on page 335. Figure 10.10 Expression Analysis Settings dialog box, baseline tab chapter 10 | Gene Expression Analysis 3. 239 If the Expression Analysis Settings dialog box appears, review and confirm the settings for the probe array type and baseline data: In the Baseline tab, select the probe array type for the baseline from the Probe Array Type drop-down list (Figure 10.10). B. Choose the Use Baseline Comparison Data option, then click Browse The Open dialog box appears (Figure 10.11). A. Figure 10.11 Open dialog box C. Double-click the baseline data; or Click the baseline data, then click Open or OK. The Expression Analysis Settings dialog box displays the selected baseline data (.chp) (Figure 10.12). 240 Affymetrix® GeneChip® Operating Software User’s Guide Figure 10.12 Expression Analysis Settings, baseline tab displays the baseline data for the comparison analysis 4. In the Expression Analysis Settings dialog box (Figure 10.12), click the normalization tab and specify a normalization option for the experimental data. You need only specify a normalization option for the experiment, not the baseline. You may use the scaling option instead of the normalization option. If you use the scaling option, scale both the experiment and the baseline. For more details on normalization and scaling, see Appendix F, Expression Algorithm, Expression Analysis Metrics & Settings, on page 627. 5. Confirm or specify other probe array analysis parameters as desired. For more details on the parameters, see Appendix F, Expression Algorithm, Expression Analysis Metrics & Settings, on page 627. 6. Click OK. The Expression Analysis Settings dialog box closes and the comparison analysis runs. chapter 10 | Gene Expression Analysis 241 When the analysis is finished, the Expression Analysis window (EAW) displays the probe analysis data (Figure 10.13). The status log (Figure 10.13) displays: - the name of the probe analysis data - the location of the probe analysis data - a message indicating when the analysis is completed If the status log is not displayed, click the Status Log button or select View → Status Bar from the menu bar. If the EAW is already open, the results are added to the open window and it may be necessary to use the scrollbars at the bottom and right side of the EAW to see the newly added results. 242 Affymetrix® GeneChip® Operating Software User’s Guide Figure 10.13 Expression Analysis Window (EAW), comparison probe analysis data, pivot tab Expression Analysis Tabular Data The Expression Analysis window (EAW) has three tables that display the tabular and graphic analysis output for gene expression assays: • Analysis information table (see page 243) • Metrics table (see page 245) • Pivot table (see page 249) The EAW opens after you complete an expression analysis or when you open expression probe analysis data. chapter 10 | Gene Expression Analysis 243 You can open more than one set of probe analysis data in the EAW. Use the EAW scroll bars to view all of the results. A second toolbar and additional menu bar commands are available in the EAW (see Appendix I, Toolbars, Hot Keys, & Windowpanes, on page 695). The tabular data display is described further in: • Analysis Information Table (see below) • Metrics Table (see page 245) • Pivot Table (see page 249) • Finding Probe Set Information (see page 253) • Sorting Probe Set Information (see page 254) • Reordering or Removing Probe Set Information (see page 255) • Hiding Probe Set Information (see page 256) ANALYSIS INFORMATION TABLE The analysis information table (Figure 10.14) displays experiment and sample information entered during experiment setup, values for usermodifiable expression analysis algorithm settings, and other information. Each column in the analysis information table represents one analysis (probe array). 244 Affymetrix® GeneChip® Operating Software User’s Guide Figure 10.14 Expression Analysis window, analysis information table Exporting Analysis Information You can export the data in the analysis information table to a tab delimited text file (.txt) or Microsoft® Excel file (.xls). To export the data: 1. If it is not already open, open the EAW (in the data tree, doubleclick the probe analysis data that you want to export). Click the Analysis Information tab. 3. To export a user-selected portion of the data table, highlight the cells of interest. 2. chapter 10 | Gene Expression Analysis 4. 245 Click the Save button ; or Select File → Save As from the menu bar. The Save As dialog box appears (Figure 10.15). Figure 10.15 Save As dialog box 5. Choose a Save Option: - Save All exports all of the analysis information table data to the text file. - Save Selected exports only the user-selected portion of the analysis information table to the text file. Enter a name for the text file and confirm or select another directory for the text file. 7. Click Save to export the experiment information table or selected experiment data. 6. METRICS TABLE The metrics table (Figure 10.16) displays data for each probe set in the selected probe analysis data (Table 10.1). For definitions of the Statistical expression metrics see Expression Analysis Metrics, on page 636. 246 Affymetrix® GeneChip® Operating Software User’s Guide Table 10.1 Statistical expression algorithm metrics Single-Array Analysis Comparison Analysis Stat Pairs Stat Common pairs Stat Pairs Used Signal Log Ratio Signal Signal Log Ratio low Detection Signal Log Ratio high Detection p-value Change Change p-value Figure 10.16 Expression Analysis window, metrics tab, single-array probe analysis data for experiments N002 and N004 Exporting Metrics Data You can export metrics data and the associated analysis information as a text file (.txt) or Microsoft® Excel file (.xls). chapter 10 | Gene Expression Analysis 247 Setting Metrics Export Preferences Click the Analysis Options button . The Analysis Options dialog box appears (Figure 10.17). 2. Click the metrics tab to view export preferences. 1. Figure 10.17 Analysis Options, Metrics tab Choose the Save Analysis Info option to include the analysis information with the metrics data for export. 4. Choose the Save All Metric Results option to view the save preferences: - The Save all analyses to one file option exports the analysis information and metrics data for all analyses in the EAW to one text file (.txt). - The Save each analysis to a separate file option exports the analysis information and metrics data for each analysis in the EAW to a separate text file. 3. 248 Affymetrix® GeneChip® Operating Software User’s Guide The save preferences set in the metrics tab do not affect the save function in the Pivot or Analysis Information tab in the EAW. 5. Click OK to close the Analysis Options dialog box. Exporting Metrics Data If it is not already open, open the EAW (in the data tree, doubleclick the probe analysis data of interest), then click the Metrics tab. 2. To export: 1. - All of the metrics table, click the Save button ; or Select File → Save As from the menu bar. - A selected portion of the metrics table, highlight the rows of interest, then click the Save button ; or Select File → Save As from the menu bar. Do not choose the Save All Metric Results option (Figure 10.17) if you want to save selected results. The Save As dialog box appears (Figure 10.18). chapter 10 | Gene Expression Analysis 249 Figure 10.18 Save As dialog box Enter a name for the text file and confirm or select another directory for the text file. 4. Click Save to export the metrics data according to the preferences set in the Analysis Options (Metrics tab) (Figure 10.17). 3. PIVOT TABLE To view the pivot table: • Open probe analysis data (double-click the data in the data tree). • Click the Pivot tab in the EAW. Each row in the pivot table (Figure 10.19) begins with a probe set name and each set of columns displays the results from one probe analysis. The pivot table displays probe analysis data and descriptions for each transcript represented on the probe array. If several sets of probe analysis data from the same probe array type are open in the EAW, each row of the pivot table displays the data for one probe set from the different experiments. The pivot table is organized so that probe analysis data from different experiments may be easily compared. 250 Affymetrix® GeneChip® Operating Software User’s Guide Figure 10.19 Expression Analysis window (EAW), pivot table, single-array probe analysis data for experiments N002 and N006 Pivot Table Display Options Single-array or comparison analysis output You can select the types of single-array or comparison analysis output (columns) displayed in the pivot table. 1. Click the Analysis Options button .; or Select Analysis → Options from the menu bar. The Analysis Options dialog box appears (Figure 10.20). 2. Click the Pivot tab. The pivot table includes column headers for the items selected in the Analysis Options dialog box. You can also view these items by selecting View → Pivot Data from the menu bar. chapter 10 | Gene Expression Analysis 251 Select the output for display in the Pivot table. Figure 10.20 Analysis Options dialog box, Pivot tab The pivot table can also display probe analysis data for experiments analyzed using the Empirical expression analysis algorithm (in Microarray Suite versions lower than 5.0). 3. Click an unchecked item to add it to the pivot table or click a checked item to remove it from the table. 4. To reorder an experiment result column in the pivot table, use the drag-and-drop method to move the column header in the table. Probe Set Descriptions To view probe set descriptions, do either of the following: • Scroll to the Descriptions (far right column) in the pivot table. • Double-click the row of interest in the pivot table. 252 Affymetrix® GeneChip® Operating Software User’s Guide The Description dialog box is displayed (Figure 10.21). Figure 10.21 EAW, probe set Description dialog box Obtaining Further Information You can search for further information about a probe set at an Internet site. To obtain further information about a probe set in the metrics or pivot table: Right-click the probe set (row) of interest. 2. Select External Database → NetAffx™ (or Entrez) from the shortcut menu that appears. This starts the default Internet browser (for example, Microsoft® Internet Explorer or Netscape® Communicator®) and automatically opens the selected Internet site. 1. Exporting the Pivot Table You can export the pivot table as a text file (.txt) or Excel file (.xls). If it is not already open, open the EAW (in the data tree, doubleclick the probe analysis data of interest). 2. Click the Pivot tab. 1. chapter 10 | Gene Expression Analysis 253 To export only a user-selected portion of the data table, highlight the cells of interest. 4. Click the Save button or select File → Save As from the menu bar. The Save As dialog box appears (Figure 10.22). 3. Figure 10.22 Save As dialog box 5. Choose a save option: - Save All places all of the pivot table in the text file. - Save Selected places only the selected pivot data in the text file. Enter a name for the text file and confirm or select another directory for the text file. 7. Click Save to export the pivot table or selected pivot data. 6. FINDING PROBE SET INFORMATION You can perform text searches in the EAW. 1. To start a text search, do one of the following: - Click the Find button . 254 Affymetrix® GeneChip® Operating Software User’s Guide - Select Edit → Find from the menu bar. - Right-click the metrics or pivot table and select Find in the shortcut menu that appears. The Find dialog box appears (Figure 10.23). Figure 10.23 Find dialog box Enter the text string for the search (up to 256 alphanumeric characters). 3. Choose the Match or Direction search option, then click Find Next. Click Find Next again to continue the search. 2. The Find command finds all strings that match the text string for the search. For example, using the Find command to search for the text string biob would find AFFX-BioB-5 as well as other occurrences of BioB. SORTING PROBE SET INFORMATION You can sort the metrics or pivot table by any category of data displayed in the table. Single Column Sort In the EAW, click the Metrics or Pivot tab. 2. Right-click the column header of interest and click Sort Ascending or Sort Descending in the shortcut menu that appears. 1. chapter 10 | Gene Expression Analysis 255 Multiple Column Sort In the EAW, click the Metrics or Pivot tab. 2. Click the Sort button ; or Select Edit → Sort from the menu bar. The Sort dialog box appears (Figure 10.24). 1. Figure 10.24 Sort dialog box Select the desired sort category from the first (top) drop-down list and choose an ascending or descending sort order. Up to three additional sort categories may be specified. 4. Click OK when finished specifying the sort. The sort category and order are displayed at the bottom of the EAW. 3. REORDERING OR REMOVING PROBE SET INFORMATION Reordering Columns in the Metrics or Pivot Table In the Metrics or Pivot tab, click the column header of interest. 2. Use the drag-and-drop method to move the column to a new position in the table. 1. 256 Affymetrix® GeneChip® Operating Software User’s Guide Reordering Rows in the Metrics or Pivot Table Use the Edit commands in the menu bar to remove or reorder tabular data in the metrics or pivot table: Edit → Move Selected to Top Moves a selected row(s) to the top of the metric or pivot table. Edit → Remove Analyses Edit → Select All When the pivot table contains only one analysis, removes the analysis from the table (equivalent to closing the probe analysis data). When the pivot table includes more than one analysis, this displays an Open dialog box that enables you to select the analyses you want to remove from the table. Highlights all rows in the metric or pivot table. HIDING PROBE SET INFORMATION You can hide tabular data in the metrics or pivot table. Use the View commands in the menu bar to hide (unhide) selected rows or all of the tabular data. Alternatively, right-click the metrics or pivot table to display a shortcut menu of hide commands: View → Hide Selected View → Hide Unselected View → Unhide All Hides the currently selected row(s) in the metrics or pivot table. The status bar at the bottom of the EAW displays the remaining number of rows in the table. Hides all unselected rows in the metrics or pivot table. The status bar at the bottom of the EAW displays the remaining number of rows in the data table. Displays all previously hidden rows at the bottom of the data table. Expression Analysis Measured Images A measured image displays the probe set hybridization intensity data (from the image data). chapter 10 | Gene Expression Analysis 257 Measured images are displayed in the following sections: • Displaying Measured Images (see below) • Clearing Measured Images (see page 258) DISPLAYING MEASURED IMAGES To display measured images: 1. Click the desired probe set name(s) in the metrics or pivot table. To select adjacent probe set names, press and hold the Shift key and click the first and last probe set name in the selection. To select non-adjacent probe set names, press and hold the Ctrl key while you click the desired names. 2. Click the Image button ; or Select Graphs → Measured Images from the menu bar. The graph pane (Intensity tab) opens and displays the measured image and the intensity signal range for the selected probe set (Figure 10.25). When an additional image is created, the graph pane automatically scrolls to the new image. Use the scroll bar to view all of the measured images created during a session. For information about resizing the graph pane see Working with Windowpanes & Columns, on page 705. 258 Affymetrix® GeneChip® Operating Software User’s Guide Probe set measured images in the graph pane Figure 10.25 EAW, measured images in the graph pane (top) CLEARING MEASURED IMAGES You can remove all or selected images from the graph pane. To remove all measured images from the graph pane, right-click the graph pane and select Clear All Graphs from the shortcut menu that appears; or Select Graph → Clear All Graphs from the menu bar. 2. To remove selected images from the graph pane: 1. Double-click the measured image. B. Right-click the image and select Clear Selected Graphs from the shortcut menu that appears; or Select Graph → Clear Selected Graphs from the menu bar. A. chapter 10 | Gene Expression Analysis 259 Expression Analysis Graphs GCOS plots three types of expression analysis graphs (Table 10.2). The graphs are displayed in the graph pane that appears at the top of the EAW (Figure 10.26). Table 10.2 Expression analysis graphs Expression Graph Type Plots... Scatter A user-specified metric for all probe sets from two experiments (of the same probe array type) using a traditional scatter plot. For more information, see Scatter Graph, on page 260. Series A user-specified metric for each probe set on the array in a bar or line graph format. For more information, see Series Graph, on page 274. Intensity The intensities of the probe pairs in a probe set using a bar graph format. For more information, see Intensity Bar Graph, on page 279. The graph pane has three tabs: • Scatter Graph (see page 260) • Series Graph (see page 274) • Intensity (see page 279) Click the graph pane tabs to toggle the display among the different graph types. For information about resizing the graph pane see Working with Windowpanes & Columns, on page 705. The intensity graph and images functions are unavailable until a probe set(s) is highlighted in the metrics or pivot table. 260 Affymetrix® GeneChip® Operating Software User’s Guide Scatter graph in the graph pane Pivot table Figure 10.26 Expression Analysis window (EAW), Scatter graph tab of the graph pane (top) and Pivot tab (bottom) SCATTER GRAPH The scatter graph plots user-selected expression data for all probe sets from one or more pairs of analyses using a traditional scatter plot (Figure 10.26). Each point in the scatter graph represents a probe set common to both experiments and is defined by the intersection of the analysis result value on the x and y axes for the common probe set. The chapter 10 | Gene Expression Analysis 261 scatter graph displays fold change lines (four pairs) that help identify probe sets in the comparison that change expression levels. Plotting a Scatter Graph 1. Click the Scatter graph button ; or Select Graph → Scatter Correlation Graph from the menu bar. The Scatter Graph dialog box displays the pivot table columns available for the scatter graph (Figure 10.27). For the scatter plot, select pairs of analyses that are derived from the same probe array type. The order of the analyses is critical. For example, the analyses from a multiple probe array set must be ordered identically along the x and y-axes since the scatter graph compares the first set of analysis outputs on the x-axis with the first set of analysis outputs on the y-axis (and so forth). If the analysis outputs are not in identical order, many probe sets will not be compared and plotted (only the probe sets in common such as the controls). 262 Affymetrix® GeneChip® Operating Software User’s Guide This box shows the pivot table columns available for the scatter graph Figure 10.27 Scatter graph dialog box 2. To select a column for the x-axis, do either of the following: - Use the drag-and-drop method to move the column name from the Available Columns box to the Select X Axis Columns box (Figure 10.27). - Highlight the column name in the Available Columns box, then click the down arrow (on the left side) to place the column in the Select X Axis Columns box. 3. To select a column for the y-axis, do either of the following: - Use the drag-and-drop method to move the column name from the Available Columns box to the Select Y Axis Columns box (Figure 10.27). - Highlight the column name in the Available Columns box and click the down arrow (on the right side) to place the column in the Select Y Axis Columns box. chapter 10 | Gene Expression Analysis 4. 263 To change the order of columns, do either of the following: - Use the drag-and-drop method to move the column to the desired location in the list. - Highlight the column name, then use the up and down arrows located to the inside of the Select Axis Columns boxes. To select linear scaling, remove the check mark from the Log Scale check box. Logarithmic scaling is the default. 6. Click OK to generate the scatter graph. The graph pane (Scatter Graph tab) displays the scatter graph (Figure 10.26). 5. Working with the Scatter Graph Right-click the scatter graph to display a shortcut menu of commands that include: Points Displays the absolute call combinations and the color assigned to each. The scatter graph displays the check marked items. Click an item to remove or add a check mark. The scatter graph is immediately updated. Copy Graph Copies the Scatter graph to the system clipboard. Close Graph Pane Closes the graph pane. Graphs created during the session are not saved. Full Out Zoom Restores the graph to the original magnification view. Redraw Graph Redraws the scatter graph so that it plots only the points that correspond to rows displayed in the pivot table. Points that correspond to hidden rows are not plotted. Select Highlighted Points Selects rows in the Pivot and Metric tables that correspond to points selected (roped) in the Scatter graph. Options Displays the Analysis Options dialog box. 264 Affymetrix® GeneChip® Operating Software User’s Guide Magnifying the Graph Press and hold the Shift key while you use the click-and-drag method to draw a rectangle over the graph area of interest (Figure 10.28). 2. Release the mouse key. This magnifies the area selected by the rectangle (Figure 10.29). 1. 3. To zoom out and restore the graph, right-click the graph and select Full Out Zoom from the shortcut menu that appears. Figure 10.28 Scatter graph, rectangle selects an area to magnify chapter 10 | Gene Expression Analysis 265 Figure 10.29 Magnified area in the scatter graph Displaying Probe Set and Gene Information 1. Click a point in the scatter graph. This displays the probe set name, parameter value, detection call, and a brief description of the probe set (Figure 10.30). The corresponding row in the pivot table is highlighted. 266 Affymetrix® GeneChip® Operating Software User’s Guide Database drop-down list Figure 10.30 Scatter graph, click a point to display corresponding probe set and gene information 2. To obtain further gene information, select a database from the drop-down list and click Info. The Internet browser window opens and displays information for the selected gene (Figure 10.31). chapter 10 | Gene Expression Analysis 267 Figure 10.31 Internet browser window Selecting Points in the Scatter Graph 1. Click the Lasso button ; or Select Graph → Lasso Points from the menu bar. The mouse pointer changes to a cross hairs (+) when it is positioned over the scatter graph 2. To rope points of interest, position the cross hairs near the group of points, then do either of the following: - Use the click-and-drag method to draw a circle around the points, then release the mouse button near the starting point. 268 Affymetrix® GeneChip® Operating Software User’s Guide - Click the mouse, move it to draw a line segment, then click the mouse again to start drawing a new line segment. Repeat until you return the cross hairs to the starting point and the line segments enclose the points of interest. This completes the lasso operation (Figure 10.32). The scatter graph displays the selected points in orange. The metrics and pivot tables highlight the corresponding probe sets. Press the Esc key to cancel the lasso operation. chapter 10 | Gene Expression Analysis 269 Figure 10.32 Roping points in the scatter graph highlights the corresponding probe set rows in the metrics and pivot tables After you select points in the scatter graph, hide the unselected rows in the pivot table, then use the Redraw Graph command to display only the selected points in the scatter graph. 270 Affymetrix® GeneChip® Operating Software User’s Guide Hiding Points in the Scatter Graph You can hide points in the scatter graph according to detection or Change call (if the scatter graph points are color coded using the Color by Detection/Absolute Call or Color by Change/Difference Call option, see Point Options, on page 271. 1. Right-click the scatter graph and select Points from the shortcut menu that appears. This displays a key for the scatter graph point colors (Figure 10.33). Figure 10.33 Scatter graph shortcut menu, point color combinations for detection calls 2. Click a point color combination to remove (or add) a check mark. This hides (unhides) the selected points in the scatter graph. Scatter Graph Options You can modify some display features of the scatter graph. The Analysis Options dialog box displays the user-modifiable features (Figure 10.34). 1. To open the Analysis Options dialog box, do one of the following: - Click the Options button . - Right-click the scatter graph and select Options from the shortcut menu that appears. chapter 10 | Gene Expression Analysis 271 - Select Analysis → Options from the menu bar. The Analysis Options dialog box appears (Figure 10.34). 2. Click the Scatter Graph tab. Figure 10.34 Analysis Options, Scatter Graph tab Point Options Point Size Determines the size of the graph points. Enter a larger point size number for easier viewing, but use a smaller point size for high resolution graphs. 272 Affymetrix® GeneChip® Operating Software User’s Guide Color by Detection/ Displays the graph points using the colors assigned to the singleAbsolute Call array call combination of the x and y-axis analyses. There are nine possible call combinations: Absent in Y Marginal in Y Present in Y A-A M-A A-P Marginal in X M-A M-M M-P P-M P-P Absent in X Present in X P-A Color coding the combinations can help quickly determine whether a highly expressed gene in one experiment was present or absent in another experiment. If the call is No Call in either analysis, the point is displayed using the No Call Point Color (the default is black). Color by Change/ Difference Call Displays the data points using the colors assigned to the comparison analysis call of the y-axis analysis. There are five possible calls: Decrease (D), Marginal Decrease (MD), No Change (NC), Marginal Increase (MI), and Increase (I). Use Point Color Displays points using the color assigned to the Point Color. Fold Change Lines The default fold change lines are defined in pairs: y = 2x and y = 1/2x, y = 3x and y = 1/3x, y = 10x and y = 1/10 x, y = 30x and y = 1/30x. The fold change lines (four pairs) are drawn using the values entered in the edit boxes. Only integer values may be entered. Removing a check mark turns off the display of that pair of fold change lines. Colors You can change the colors assigned to the: • single-array analysis call categories chapter 10 | Gene Expression Analysis 273 • comparison analysis call categories • scatter graph points • graph background • selected (roped) scatter graph points • fold change lines 1. In the Analysis Options dialog box (Figure 10.34), click the color square associated with the item you want to change. This displays the color palette (Figure 10.35). Custom color Luminosity scale Figure 10.35 Color palette To select a predefined color, click one of the basic colors. 3. To define a custom color, click Define Custom Colors, then use the click-and-drag method to move the cross hairs in the custom color field. Adjust the color brightness using the luminosity scale to the right. When finished, click Add to Custom Colors to apply the color. 4. Click OK to close the color palette. The color choices are saved on a per user basis. 2. 274 Affymetrix® GeneChip® Operating Software User’s Guide Defaults Click Defaults to return all analysis option settings to the factory set defaults. SERIES GRAPH The series graph plots a line or bar graph of user-selected expression data for all analyses or for all probe sets of analyses open in the EAW (Figure 10.36 and Figure 10.37). The line graph format is the default. Hidden probe sets are not included in the series graph. Figure 10.36 Series line graph, signal chapter 10 | Gene Expression Analysis Figure 10.37 Series bar graph, signal Plotting a Series Graph 1. Click the Series Graph button ; or Select Graph → Series Graph from the menu bar. The Series Graph dialog box appears (Figure 10.38). 275 276 Affymetrix® GeneChip® Operating Software User’s Guide Figure 10.38 Series Graph dialog box 2. Select the pivot table columns for the series graph. This generates the series line graph for the probe sets displayed in the data tables (Figure 10.36). The line graph format is the default. For information on how to change the bar graph format, see Series Graph Options, on page 277. Working with the Series Graph Place the mouse arrow over a probe set name, data point, or bar to view a pop-up tool tip that displays the analysis or probe set name and parameter value. 2. Right-click the series graph to display a shortcut menu of commands that include: 1. Copy Graph Copies the graph to the system clipboard. Close Graph Pane Closes the graph pane (graphs created during the session are not saved). Options Displays the Analysis Options dialog box. chapter 10 | Gene Expression Analysis 277 Series Graph Options Some display features of the series graph may be modified. The Analysis Options dialog box displays the user-modifiable features (Figure 10.39). 1. To open the Analysis Options dialog box, do one of the following: - Click the Options button . - Right-click the scatter graph and select Options from the shortcut menu that appears. - Select Analysis → Options from the menu bar. The Analysis Options dialog box appears (Figure 10.39) 2. Click the Series Graph tab and choose the Bar Graph or Line Graph option. Figure 10.39 Analysis Options, Series Graph tab 278 Affymetrix® GeneChip® Operating Software User’s Guide Series Bar Graph Options Probe Set Width (%) Determines the graph bar width. X-Axis Parameters Visible The number of probe sets displayed on the x-axis within the viewable portion of the graph. Series Line Graph Options X-Axis Select Probe Sets or Columns (from the X-Axis drop-down list) for display on the x-axis. Point Size Determines the size of the graph points. X-Axis Parameters Visible Specifies the number of probe sets or columns displayed on the xaxis within the viewable portion of the graph. Colors The color of the points or background in the series line graph may be changed. 1. In the Analysis Options dialog box (Figure 10.39), click the Point Color or Background color square. This displays the color palette (Figure 10.40). chapter 10 | Gene Expression Analysis Custom color 279 Luminosity scale Figure 10.40 Color palette To select a predefined color, click one of the basic colors. 3. To define a custom color, click Define Custom Colors, then use the click-and-drag method to move the cross hairs in the custom color field. Adjust the color brightness using the luminosity scale to the right. When finished, click Add to Custom Colors to apply the color 4. Click OK to close the color palette. The color choices are saved on a per user basis. 2. INTENSITY BAR GRAPH The intensity bar graph displays the intensities of the probe pairs of a probe set in a bar graph format (Figure 10.41). The intensity bar graph is a useful way to: • Evaluate the relative performance of the probe pairs in a probe set. • Determine whether an image or probe mask has been applied to a probe pair. 280 Affymetrix® GeneChip® Operating Software User’s Guide Figure 10.41 Intensity bar graph of probe set X54925_at (from experiment N004) Plotting the Intensity Bar Graph Highlight a row or cell in the metrics or pivot table. 2. To plot the intensity bar graph(s) for the highlighted probe set(s), do either of the following: 1. - Click the Intensity Bar Graph button . - Select Graph → Intensity Bar Graph from the menu bar. 3. If necessary, use the scroll bar to view all of the intensity bar graphs created during a session. Intensity Bar Graph Options You can modify some display features of the intensity bar graph. The Analysis Options dialog box displays the user-modifiable features (Figure 10.42). 1. To open the Analysis Options dialog box, do one of the following: - click the Options button - right-click the intensity bar graph, then click Options in the shortcut menu that appears - select Analysis → Options from the menu bar The Analysis Options dialog box appears (Figure 10.42) 2. Click the Intensity Graph tab. chapter 10 | Gene Expression Analysis 281 Figure 10.42 Analysis Options, Intensity Graph tab Show Values Choose the Show Values option to display numerical intensity values on the intensity bar graph. Scale Relative to Pair Scales each probe pair set independently. The scale for each PM and MM pair is 0 → max (PM, MM). Relative to Probe Set Scales each probe pair relative to all probe pairs of a particular probe set. The scale is determined by all pairs of the probe set and ranges from 0 → max (all PM/MM probe pairs in the set). Absolute Scales all probe pairs according to a user specified intensity range (for example, 10 to 1000 in Figure 10.42). 282 Affymetrix® GeneChip® Operating Software User’s Guide Colors The color code specifies the colors for: Match Perfect match probe intensities Mismatch Mismatch probe intensities Image Mask Probe pairs included in an image mask Probe Mask Probe pairs included in a probe mask Not in Average Probe pairs excluded from the Average Difference calculation (Empirical expression algorithm only) To change the colors specified by the color code: 1. Click the color square of the item you want to change (for example, Match in Figure 10.42). This displays the color palette (Figure 10.43). Custom color Luminosity scale Figure 10.43 Color palette To select a predefined color, click one of the basic colors. 3. To define a custom color, click Define Custom Colors, then use the click-and-drag method to move the cross hairs in the custom color field. Adjust the color brightness using the luminosity scale 2. chapter 10 | Gene Expression Analysis 283 to the right. When finished, click Add to Custom Colors to apply the color 4. Click OK to close the color palette. The color choices are saved on a per user basis. Defaults Click Defaults to restore the factory set defaults for all intensity bar graph options. Clearing Intensity Bar Graphs To remove all intensity bar graphs from the graph pane, rightclick the graph pane and select Clear All Graphs from the shortcut menu that appears; or Select Graph → Clear All Graphs from the menu bar. 2. To remove user-selected intensity bar graphs from the graph pane: 1. Double-click the graph(s) you want to remove. B. Right-click the selected graph and select Clear Selected Graphs from the shortcut menu that appears; or Choose Graph → Clear Selected Graphs from the menu bar. A. Expression Report The expression report summarizes information about the expression analysis settings, algorithm settings, and probe set hybridization intensity data. You can learn more about expression reports in the following sections: • Generating an Expression Report (see below) • Expression Report Components (see page 287) • Expression Report Settings (see page 289) • Expression Report View Options (see page 291) • Editing an Expression Report (see page 291) 284 Affymetrix® GeneChip® Operating Software User’s Guide • Saving an Expression Report (see page 292) GENERATING AN EXPRESSION REPORT 1. Confirm or change the user-modifiable report settings. See the next section Expression Report Settings. 2. To generate the report, do either of the following: - In the data tree, right-click the probe analysis data for the report and select Report from the shortcut menu that appears. - Select File → Report from the menu bar, then in the Report dialog box (Figure 10.44) that appears, double-click the probe analysis data for the report. Figure 10.44 Report dialog box This displays the expression report in the main display area. Figure 10.45 and Figure 10.46 show an example expression report. 3. To best view the expression report: A. Use Arial font (10 point) (select View → Set Font from the menu bar). chapter 10 | Gene Expression Analysis B. 285 Set the tab stops set at 10 (select View → Set Tab Stops from the menu bar). A previously generated report will be overwritten. To save a previous report, rename it and save it under the new name. See the Saving an Expression Report, on page 292. Figure 10.45 Expression report, comparison analysis, Statistical Expression algorithm (page 1) 286 Affymetrix® GeneChip® Operating Software User’s Guide Figure 10.46 Expression report, comparison analysis, Statistical Expression algorithm (page 2) chapter 10 | Gene Expression Analysis 287 EXPRESSION REPORT COMPONENTS Statistical Expression Algorithm Probe Pair Threshold Alpha1 (α1) The minimum number of probe pairs a probe set must have in order for the probe set data to be included in the calculation of the report statistics The significance level for the Detection p-value in an analysis. Alpha1 is a user-modifiable parameter that is set in the Parameters tab of the Expression Analysis Settings. See Expression Analysis Settings, on page 644. If the probe set Detection p-value < alpha1, the call is present. Alpha2 (α2) The second significance level for the Detection p-value in an analysis. Alpha2 is a user-modifiable parameter set in the Parameters tab of the Expression Analysis Settings. See Expression Analysis Settings, on page 644. If the probe set Detection p-value ≥ alpha2, the call is absent. If alpha1 ≤ Detection p-value < alpha2, the call is marginal. Tau (τ) Tau is a user-modifiable parameter that is set in the Parameters tab of the Expression Analysis Settings See Expression Analysis Settings, on page 644. Ideally, tau should be set to a value that is a little larger than the median of the discrimination scores of the probe sets whose targets are absent to avoid false detected calls. Noise (Raw Q) The degree of pixel-to-pixel variation among the probe cells used to calculate the background. See Statistical Expression Algorithm, on page 636. Scale Factor (SF) The scale factor specified in the Scaling tab of the Expression Analysis Settings dialog box or computed by the algorithm. See Expression Analysis Settings, on page 644. TGT Value The user-specified target signal for scaling of the experiment probe array. The target signal is set in the Scaling tab of the Expression Analysis Settings dialog box. See Expression Analysis Settings, on page 644. Norm Factor (NF) The normalization factor specified in the Normalization tab of the Expression Analysis Settings dialog box or computed by the algorithm. See Expression Analysis Settings, on page 644. 288 Affymetrix® GeneChip® Operating Software User’s Guide Gamma1H (γ1H) Gamma2H (γ2H) Gamma1L (γ1L) Gamma2L (γ2L) The small significance level for Change calls at high intensities. Gamma1H is a user-modifiable parameter that is set in the Parameters tab of the Expression Analysis Settings. See Expression Analysis Settings, on page 644. The large significance level for Change calls at high intensities. Gamma2H is a user-modifiable parameter that is set in the Parameters tab of the Expression Analysis Settings. See Expression Analysis Settings, on page 644. The small significance level for Change calls at low intensities. Gamma1L is a user-modifiable parameter that is set in the Parameters tab of the Expression Analysis Settings. See Expression Analysis Settings, on page 644. The large significance level for Change calls at low intensities. Gamma2L is a user-modifiable parameter that is set in the Parameters tab of the Expression Analysis Settings. See Expression Analysis Settings, on page 644. Perturbation A user-modifiable expression algorithm parameter that is set in the parameters tab of the Expression Analysis Settings. See Expression Analysis Settings, on page 644. Perturbation influences the p-value computed for a probe set in a comparison analysis. Baseline Noise (Raw Q) The degree of pixel-to-pixel variation among the probe cells used to calculate the background in the baseline probe array. See Statistical Expression Algorithm, on page 636. Baseline Scale Factor (SF) The scale factor specified for the baseline probe array in the Scaling tab of the Expression Analysis Settings dialog box or computed by the algorithm. See Expression Analysis Settings, on page 644. Background Minimum, maximum, average, and standard deviation of the background intensity calculated for the probe array. Noise The minimum, maximum, average, and standard deviation of the noise calculated for the probe array. Corner+ The average cell intensity for the sense probe cells used in the grid alignment process. Corner- The average cell intensity of the antisense probe cells used in the grid alignment process. Central+ The average cell intensity for the nine probe cells that comprise the cross at the center of a sense probe array. Central- The average cell intensity for the nine probe cells that comprise the cross at the center of an antisense probe array. chapter 10 | Gene Expression Analysis 289 Total Probe Sets The number of probe sets on the array that exceed the probe pair threshold and are not called No Call. Average Signal The average signal for all probe sets that exceed the probe pair threshold and are not called No Call. Controls The expression report includes the signal and call data for the probe sets that correspond to the housekeeping or spike control transcripts. Separate signal and call data are reported for the probe pairs specific to the 5’, middle (M), and 3’ regions of the control transcripts. Sig(all) The average signal for all control probe sets. Sig(3′/5′) For a probe set, Sig(3′)/Sig(5′). EXPRESSION REPORT SETTINGS The expression report has user-modifiable settings. 1. To view the user-modifiable settings, click Settings in the shortcut bar, then click Expression Report . The Expression Report dialog box appears (Figure 10.47). Figure 10.47 Expression Report dialog box 290 Affymetrix® GeneChip® Operating Software User’s Guide Probe Pair Threshold The minimum number of probe pairs a probe set must have in order for the probe set data to be included in the calculation of the report statistics. Antisense Probe Sets Choose this option for probe arrays that have antisense control probes. (Refer to the probe array product insert.) Sense Probe Sets Choose this option for probe arrays that have sense control probes. (Refer to the probe array package insert.) Housekeeping Controls Housekeeping controls are housekeeping gene transcripts that are usually constitutively expressed in the sample. The transcripts serve as endogenous controls and are useful for monitoring the quality of the target. The report includes the signal (Sig) for probe sets that are designed to be specific to the 5′, middle, or 3′ portion of the transcript. These data are informative about the reverse transcription and IVT steps in sample preparation. Differences greater than three fold between the Sig 3′ and Sig 5′ (Sig 3′/5′) for a housekeeping control indicate the target may be degraded and should be prepared again. To delete a housekeeping control from the expression report: Double-click the probe set name. 2. Right-click probe set and select Delete from the shortcut menu that appears. 1. Spike Controls Spike controls are transcripts that are added to or spiked in the sample. These are exogenous transcripts that are useful for monitoring assay procedures such as hybridization, washing, and staining. To delete a spike control from the expression report: 1. Double-click the probe set name. chapter 10 | Gene Expression Analysis 2. 291 Right-click probe set and select Delete from the shortcut menu that appears. Other Controls You can choose any probe set on the array as a control. Enter the probe set name in the Other Controls box. The expression report includes the signal and detection call for user-specified controls. EXPRESSION REPORT VIEW OPTIONS Find Feature The Find feature searches the report. 1. Click the Find button or select Edit → Find from the menu bar. The Find dialog box appears (Figure 10.48). Figure 10.48 Find dialog box Enter the text for the search (up to 256 alphanumeric characters including spaces) in the Find what box, then click Find Next to view the first search result. 3. Click Find Next or the Find Next button to view each successive search result. 2. EDITING AN EXPRESSION REPORT Edit commands for the expression report are available in the: • Edit menu bar 292 Affymetrix® GeneChip® Operating Software User’s Guide • Report window toolbar • Shortcut menu (right-click the report) Replace Feature The replace feature performs a text search and replaces the search item with specified text. 1. Select Edit → Replace from the menu bar. The Replace dialog box appears (Figure 10.49). Figure 10.49 Replace dialog box Enter the text for the search in the Find what box and enter the replacement text in the Replace with box. 3. Click Find Next to begin the search, then click Replace to replace the search item with the specified text. Repeat to find and replace successive instances of the search item; or Click Replace All to find and replace all occurrences of the search item in the report. 2. SAVING AN EXPRESSION REPORT When GCOS generates a report for expression probe analysis data, it overwrites a previously generated report. To prevent a previous report from being overwritten, save it under a new name. 1. To open the report that you want to save under a new name, click the Open button . chapter 10 | Gene Expression Analysis 293 The Open dialog box appears (Figure 10.50). Figure 10.50 Open dialog box Double-click the report name. The report is displayed. 3. Select File → Save As from the menu bar. The Save As dialog box appears (Figure 10.51). 2. Figure 10.51 Save As dialog box 4. Click the report to be saved, then enter a new name for the report in the File name box. 5. Click Save. 294 Affymetrix® GeneChip® Operating Software User’s Guide Chapter 11 Batch Analysis Chapter 11 297 Batch Analysis Batch analysis is a convenient way to analyze the cell intensity data from many different experiments and to generate probe analysis data with unattended operation. Register sample & define experiment Process probe array in fluidics station Scan probe array & save image data Compute cell intensity data from image data & save cell intensity data Analyze expression cell intensity data & save expression probe analysis data Publish expression data Generate an expression report Figure 11.1 Assay & analysis flow chart This chapter contains the following sections: • Opening the Batch Analysis Window (see below) • Selecting Data for Batch Analysis (see page 298) • Renaming Results from Batch Analysis (see page 306) • Removing Data from the Batch Analysis Window (see page 308) • Running a Batch Analysis (see page 309) • Exporting a Batch File (see page 309) • Importing a Batch File (see page 310) 298 Affymetrix® GeneChip® Operating Software User’s Guide Opening the Batch Analysis Window To open the Batch Analysis window: • In the GeneChip Software shortcut bar, click Batch Analysis • Select Run → Batch Analysis from the menu bar. The Batch Analysis window opens (Figure 11.2). Figure 11.2 Batch Analysis window Selecting Data for Batch Analysis You may select cell intensity data or probe analysis data for batch analysis. If you select cell intensity data, GCOS automatically places the parent cell intensity data and baseline probe analysis data (if applicable) in the Batch Analysis window. Batch analysis will overwrite previous probe analysis data. . chapter 11 | Batch Analysis 299 Using the Drag-and-Drop Method 1. In the data tree, select the cell intensity data or probe analysis data that you want to analyze. To select adjacent items, press and hold the Shift key while you click the first and last item in the selection. To select non-adjacent items, press and hold the Ctrl key while you click the items. 2. Drag the selected data from the data tree to the Batch Analysis window (Figure 11.3). Data tree Shortcut bar Figure 11.3 Batch Analysis window Batch analysis window 300 Affymetrix® GeneChip® Operating Software User’s Guide Using the Toolbar or Menu Commands 1. Click the Add button or select Edit → Add Item from the menu bar. The Add Cell/Chip Data dialog box appears (Figure 11.4). Click Details to display experiment and sample information for the cell intensity or probe analysis data. Figure 11.4 Add Cell/Chip Data dialog box 2. To filter the data in the Add Cell/Chip Data dialog box: Click Filter. The advanced filter appears (Figure 11.5). B. Make selections from the drop-down lists. C. Click Apply. A. chapter 11 | Batch Analysis 301 Figure 11.5 Add Cell/Chip Data dialog box, advanced filter 3. Select the data for the batch analysis, then click OK. To select adjacent items, press and hold the Shift key while you click the first and last item in the selection. To select non-adjacent items, press and hold the Ctrl key while you click the items. The selected cell intensity data are added to the Batch Analysis window (Figure 11.6). Batch analysis will overwrite previous probe analysis data. The Batch Analysis window displays previously analyzed cell intensity data in red to indicate that the existing probe analysis data will be overwritten. To reanalyze experiments without overwriting existing probe analysis data, specify a different name 302 Affymetrix® GeneChip® Operating Software User’s Guide for the .chp from the batch analysis. See Renaming Results from Batch Analysis, on page 306. Figure 11.6 Batch Analysis window, cell intensity data (.cel) selected for batch analysis You can also use the Batch Analysis window for: • Selecting a Userset for Expression Analysis (see below) • Selecting a Baseline for a Comparison Expression Analysis (see page 303) SELECTING A USERSET FOR EXPRESSION ANALYSIS A userset specifies the expression analysis settings (scaling, normalization, probe masks, baseline experiment, values for usermodifiable Statistical Expression algorithm parameters). For more information about usersets, see Userset Tab, on page 415. In a batch analysis, the settings for the default userset are specified in the Expression Analysis Settings dialog. The default userset is not the userset specified during experiment setup. 1. To select other than the default userset, double-click the Userset column. This displays a drop-down list of available usersets (Figure 11.7). chapter 11 | Batch Analysis 303 If a userset with a baseline experiment is selected and the field under the baseline column is empty, the baseline experiment from the userset is used during the analysis. Figure 11.7 Batch Analysis window, drop-down list of usersets 2. Select a userset from the drop-down list; or Highlight the cell intensity data of interest, select Edit → Select Userset from the menu bar, then make a selection from the dropdown list. SELECTING A BASELINE FOR A COMPARISON EXPRESSION ANALYSIS 1. In the Batch Analysis window, double-click in the baseline column of the cell intensity data for the comparison expression analysis (Figure 11.8); or Select the .cel of interest and choose Edit → Select Baseline from the menu bar. The Select Baseline Data dialog box appears (Figure 11.9). 304 Affymetrix® GeneChip® Operating Software User’s Guide Figure 11.8 Batch Analysis window Figure 11.9 Select Baseline dialog box, List view (left) and Details view (right) Click Details to display experiment and sample information for the data. 3. To filter the data in the Select Baseline dialog box: 2. Click Filter. This displays the advanced filter (Figure 11.10). B. Make selections from the drop-down lists. A. C. Click Apply. chapter 11 | Batch Analysis Figure 11.10 Select Baseline dialog box, advanced filter 4. Double-click the baseline data. The Batch Analysis window displays the baseline data (Figure 11.11). Figure 11.11 Batch Analysis window, baseline selected 305 306 5. Affymetrix® GeneChip® Operating Software User’s Guide To clear a baseline from the batch analysis window, right-click the baseline data and select Clear Baseline from the shortcut menu that appears; or Click the baseline experiment and select Edit → Clear Baseline from the menu bar. Renaming Results from Batch Analysis To reanalyze experiments without overwriting existing analysis output, rename the output .chp data from the batch analysis using one of the following methods: • Renaming Results Automatically (see below) • Renaming Results Individually (see page 307) RENAMING RESULTS AUTOMATICALLY If you select .cel data for batch analysis, GCOS can automatically rename all of the output .chp data by adding a user-specified prefix and/or suffix to the data name. If you select .chp data for batch analysis, the prefix or suffix are not applied to the output name. 1. Click the Options button or select View → Options from the menu bar. The Batch Analysis Options dialog box appears (Figure 11.12). chapter 11 | Batch Analysis 307 Figure 11.12 Output Naming Options dialog box 2. To add a prefix to the .chp name, choose the Prefix option and enter a prefix for the name in the associated box. To add a suffix to the .chp name, choose the Suffix option and enter a suffix for the name in the associated box. 4. Click OK. When cell intensity data are placed in the Batch Analysis window, the user-specified prefix and/or suffix is automatically added to the output analysis name. 3. RENAMING RESULTS INDIVIDUALLY You can individually rename an output results from the batch analysis. Editing the Output Name To edit the default name, do either of the following: • Double-click the name in the Batch Analysis window and enter a new name. • Select the cell intensity data of interest in the Batch Analysis window, select Edit → Rename Output Data from the menu bar, and enter a new name. 308 Affymetrix® GeneChip® Operating Software User’s Guide Adding a Prefix or Suffix to the Output Name 1. In the Batch Analysis window, use one of the following methods to select one or more outputs. - To select all data, right-click a name and click Select All in the shortcut menu that appears; or Select View → Select All from the menu bar. - To select adjacent data, press and hold the Shift key while you click the first and last name in the selection. - To select non-adjacent data, press and hold the Ctrl key while you click the names. 2. Right-click the selected outputs and select Set Prefix/Suffix from the shortcut menu that appears; or Click the selection and select Edit → Set Prefix/Suffix from the menu bar. The Set Prefix/Suffix dialog box appears (Figure 11.13). Figure 11.13 Set Prefix/Suffix dialog box 3. Enter a prefix and/or suffix, then click OK. The prefix and/or suffix are added to the selected output names. Removing Data from the Batch Analysis Window To remove cell intensity data from the Batch Analysis window, do either of the following: • Select the data name and click the Remove button . chapter 11 | Batch Analysis 309 • Select the data name, then select Edit → Remove Item from the menu bar. Running a Batch Analysis To run the batch analysis, click the Analyze button ; or Select Edit → Start Analysis from the menu bar. 2. To stop a batch analysis in progress, click the Stop button Select Edit → Stop Analysis from the menu bar. The software completes the current analysis and cancels the remaining data in the batch analysis queue. 1. ; or RESUMING A BATCH ANALYSIS If the batch analysis is interrupted before all analyses are completed, GCOS can resume the run and analyze the remaining .cel data in the batch. To resume a batch analysis: Open the Batch Analysis window. The Batch Analysis window displays the remaining unanalyzed data. 2. Click the Analyze button ; or Select Edit → Start Analysis from the menu bar. 1. Exporting a Batch File The Batch Analysis window contents can be exported to a text file (tab delimited .txt). The batch file provides a record of the analysis and can be imported for a subsequent batch analysis. 1. Select Edit → Export Batch from the menu bar. The Export Batch dialog box appears (Figure 11.14). 310 Affymetrix® GeneChip® Operating Software User’s Guide Figure 11.14 Export Batch dialog box 2. Enter a name for the batch file, then click Save. The batch file (.txt) is created (Figure 11.15). Figure 11.15 Batch file (.txt) Importing a Batch File GCOS can import a batch file (tab delimited .txt) that follows the format shown in Figure 11.15. To import a batch file: 1. Open the Batch Analysis window. 2. Select Edit → Import Batch from the menu bar. The Import Batch dialog box appears (Figure 11.16). chapter 11 | Batch Analysis Figure 11.16 Import Batch dialog box 3. Double-click the batch file (.txt) you want to import. The batch file is imported to the Batch Analysis window. 311 312 Affymetrix® GeneChip® Operating Software User’s Guide Chapter 12 Publishing Data Chapter 12 315 Publishing Data GCOS can copy expression sample and experiment data, including cell intensity data and probe analysis data from the process database to a publish database. This is called publishing. Published expression data can be queried by the Affymetrix® Data Mining Tool (DMT) software or several other third party analysis tools. Register sample & define experiment Process probe array in fluidics station Scan probe array & save image data Compute cell intensity data from image data & save cell intensity data Analyze expression cell intensity data & save expression probe analysis data Publish expression data Generate an expression report Figure 12.1 Assay & analysis flow chart This chapter includes the following sections: • Selecting the Publish Database (see below) • Specifying the Task (see page 316) • Publishing Options (see page 320) • Publishing, Canceling, or Restarting a Task (see page 321) 316 Affymetrix® GeneChip® Operating Software User’s Guide Selecting the Publish Database 1. In the GeneChip Software shortcut bar, click Publish ; or Select Run → Publish from the menu bar. This displays the publish window (Figure 12.2). The top windowpane displays all publish databases. The bottom windowpane displays a status log of tasks. Figure 12.2 Publish window 2. Click the publish database destination for the data. A yellow icon indicates the currently selected database. A gray icon indicates an unselected database. Each database requires a login password before publishing can proceed. Specifying the Task A task consists of all data selected for publishing. Each experiment or type of data to be published is called a task item. Table 12.1 shows the data that are published for each type of task item. chapter 12 | Publishing Data 317 Table 12.1 Data published for different types of task items Task Item Data Published Experiment All cell intensity data associated with the experiment. All probe analysis data associated with the experiment. Image data All cell intensity data associated with the experiment. All probe analysis data associated with the experiment. Cell intensity data The associated experiment. The cell intensity data. All probe analysis data associated with the cell intensity data. Probe analysis data The associated experiment. The probe analysis data. All cell intensity data associated with the probe analysis data. To select the tasks, use either of the following methods: Using the Drag-and-Drop Method Drag the task item that you want to publish from the data tree onto a database icon in the top pane of the publish window. 2. Repeat the drag-and-drop operation for the remaining task items to be published (Figure 12.5). 1. Using the Add Toolbar Button 1. Click the Add button or select Publish → Add Item from the menu bar. The Open dialog box appears (Figure 12.3). 318 Affymetrix® GeneChip® Operating Software User’s Guide Figure 12.3 Open dialog box, list view (left) and details view (right) 2. Click Details to display experiment and sample information for each data type. 3. To filter the list of data types in the Open dialog box, click Filter The advanced filters are displayed (Figure 12.4). chapter 12 | Publishing Data 319 Figure 12.4 Open dialog box, advanced filter Make selections from the drop-down lists and click Apply to filter the list. 5. Make a selection from the Data Type drop-down list and select the data to be published. To select adjacent data, press and hold the Shift key while you click the first and last name in the selection. To select non-adjacent data, press and hold the Ctrl key while you click the names. 6. Click OK to add the data to the selected publish database (Figure 12.5). 7. To remove a task item from the publish window, select the task item in the publish window and click the Remove button ;or Choose Publish → Remove Item from the menu bar. 4. 320 Affymetrix® GeneChip® Operating Software User’s Guide Figure 12.5 Publish window Publishing Options You may publish intensity data or overwrite a previously published task item. You must be authorized by the GCOS administrator to overwrite data. The status bar at the bottom of the publish window displays the publishing options (Figure 12.5). You can select from the following options: • Publishing Intensity Data (see below) • Overwriting Data (see page 321) PUBLISHING INTENSITY DATA Cell intensity data (.cel) are not automatically published. Publishing .cel data uses large amounts of computer memory. For example, publishing the probe analysis data for a high density probe array requires approximately 2 MB of disk space compared to 30 MB for both the probe analysis and cell intensity data. chapter 12 | Publishing Data 321 The image data are not published. To include cell intensity data in the publish database, select Publish → Options → Publish Intensities from the menu bar. The Publish Intensities menu item is check marked. 2. To not include cell intensity data in the publish database, select Publish → Options → Publish Intensities from the menu bar to remove the check mark. 1. OVERWRITING DATA A previously published task item can be overwritten. Users must be authorized by the GCOS administrator to overwrite data. • Select Publish → Options → Overwrite Data from the menu bar. Publishing, Canceling, or Restarting a Task You can use the Publish window for: • Publishing the Task (see below) • Canceling a Task (see page 322) • Restarting a Publishing Task (see page 322) It is recommended that users not publish and restore to the same publish database simultaneously. Sometimes the restore will be rejected and sometimes the publishing will be rejected. If the restore of a publish database fails, an error message is displayed. PUBLISHING THE TASK 1. After you have specified the task, click the Publish button ; or 322 Affymetrix® GeneChip® Operating Software User’s Guide Select Publish → Publish from the menu bar. The Publish Login dialog box appears if this is the first time during the session that an item has been published to the database (Figure 12.6). Figure 12.6 Publish Database Login 2. Enter the password for the database. The task is displayed in the task log and will be published during the time frame specified by the GCOS administrator. CANCELING A TASK You can cancel tasks waiting to be published (the status in the task log is WAIT). Select the task in the task log. 2. Click the Cancel button ; or Select Publish → Monitor → Cancel Publish from the menu bar. The task is now canceled (the status is CANCELED). 1. RESTARTING A PUBLISHING TASK You can reinstate or restart a canceled task and return it to the publishing queue. 1. Select the canceled task in the task log. 2. Click the Restart button ; or Select Publish → Monitor → Restart Publish from the menu bar. chapter 12 | Publishing Data The task is now restarted (the status is WAIT) and will be published during the time frame specified by the GCOS administrator. 323 324 Affymetrix® GeneChip® Operating Software User’s Guide Chapter 13 Defaults Chapter 13 327 Defaults This chapter explains how to change the factory set defaults for: • image settings • data locations • fluidics station configuration • analysis prompts • experiment data storage This chapter contains the following sections: • Viewing Default Settings (see below) • Fluidics Settings (see page 333) • Scanner Settings (see page 334) • Analysis Settings (see page 335) The following Defaults settings are described in Chapter 5, Getting Started, on page 53: • Cell Intensity Analysis Options (see page 598) • Cell Intensity Analysis Options (see page 598) • Setting Archiving Options for DAT files (see page 599) Viewing Default Settings • Select Tools → Defaults from the menu bar. The Defaults dialog box appears (Figure 13.1). 328 Affymetrix® GeneChip® Operating Software User’s Guide Figure 13.1 Defaults dialog box, Image Settings tab IMAGE SETTINGS The image settings (Figure 13.1) affect the display of image and cell intensity data in the Image window. For more information on this window, see Introduction, on page 141. The image settings specified in the Defaults dialog box (except for the color option) are applied to subsequently opened image or cell intensity data and do not affect active, open data. Intensity Range GCOS software records a dynamic intensity range from 0 to 65,000 and provides 256 colors from black through white for image display. The intensity range of an image is divided into 256 bins and each bin is assigned a color or gray scale. You may enter a new lower or upper limit for the intensity range associated with an image and apply this subset of the dynamic range chapter 13 | Defaults 329 to the image. Lowering the upper limit increases the image brightness and raising the lower limit decreases the brightness. Using this image scaling process, you can adjust the display of a data or cell intensity file for optimum viewing. Alternatively, GCOS can automatically scale the image using the minimum and maximum pixel intensities of the image. To automatically scale an image, choose the Autoscale option in the Defaults dialog box (Figure 13.1) and click OK. 2. In the Image Settings dialog box for the current image or cell intensity data, you may click Defaults to return the intensity parameters (Autoscale option and Intensity range) to the settings in the Image Settings tab of the Defaults dialog box. For more information about the Image Settings dialog box, see Making Adjustments to the Current Image or Cell Intensity Data Only, on page 197. 1. Color You can view the pixel intensity values in gray scale or pseudo color (generated by applying rainbow colors to the intensity scale). Choose the Color Bar option (Figure 13.1) to display a gray scale or pseudo color bar at the top of the Image window. Coordinates When you click the image, a red cross hairs marks the location. The status bar at the lower left of the main window displays the intensity data and x-y coordinates of a probe cell or pixel indicated by the cross hairs. If you choose the Cell option in the Defaults dialog box (Figure 13.1), a pop-up tool tip and the status bar display the x-y coordinates of the cell, the cell intensity, and the pixel intensity (Figure 13.2). If you choose the Pixel option, the pop-up tool tip and the status bar display the x-y coordinates of the pixel and the pixel intensity (Figure 13.2). 330 Affymetrix® GeneChip® Operating Software User’s Guide Cell X = 186, Y = 41, Cell Intensity = 22561.8, Pixel Intensity = Figure 13.2 Image data, probe cell coordinates (top) and pixel coordinates (bottom) chapter 13 | Defaults 331 Pixel X = 1727, Y = 573, Intensity = 22409 Figure 13.2 Image data, probe cell coordinates (top) and pixel coordinates (bottom) Highlight Colors You may specify new highlighting colors for the grid, masked cells, outlier cells, or highlighted probe array cells (features). 1. Click Highlights in the Defaults dialog box (Figure 13.1). The Highlight Colors dialog box appears (Figure 13.3). 332 Affymetrix® GeneChip® Operating Software User’s Guide Figure 13.3 Highlight Colors dialog box 2. To change the color of a particular item (for example, the grid overlay): Click the associated color box in the Highlight Colors dialog box. The Color palette is displayed (Figure 13.4). A. Custom color Figure 13.4 Color palette Luminosity scale chapter 13 | Defaults 333 To select a predefined color, click one of the basic colors. C. To define a custom color, click Define Custom Colors, then use the click-and-drag method to move the cross hairs in the custom color field. Adjust the color brightness using the luminosity scale to the right. When finished, click Add to Custom Colors to apply the color. The new color is applied to the selected item. B. D. Click OK to close the Color palette. Grid Overlay Choose the Grid Overlay option (Figure 13.1) to automatically display the grid on a image or cell intensity data. Remove the check mark to toggle the grid overlay off. Alternatively, click Grid in the Image window task bar (or press the G key) to toggle the grid on or off. Fluidics Settings In the Fluidics tab (Figure 13.5) you can: • Specify the number of fluidics stations installed on the system (up to eight fluidics stations may be simultaneously controlled by one work station). • Specify each fluidics station’s number and ID. 334 Affymetrix® GeneChip® Operating Software User’s Guide Figure 13.5 Defaults, Fluidics tab Notification Message when protocol is complete Choose this option to display a notification of completion at the end of a protocol Scanner Settings In the Fluidics tab (Figure 13.6) you can: • specify whether a scanner is installed on the system • if a scanner is installed, choose the option to turn on the laser at startup If the AutoLoader is installed, you can: • choose to use it in manual mode • choose to disable it. chapter 13 | Defaults 335 Figure 13.6 Defaults, Scanner tab Scanner Installed Choose this option after the scanner is installed. Turn on Laser at startup Confirm this option is chosen (default) so that the laser automatically turns on when GCOS is started. Enable Manual Mode Choose this option if you are using a scanner equipped with the AutoLoader and would like to disable the AutoLoader’s autorun feature. This will allow you to add and scan cartridges one at a time. Disable AutoLoader Choose this option if you are using a scanner equipped with the AutoLoader and would like to disable the AutoLoader completely. This will allow you to use the software without communicating with the AutoLoader. Analysis Settings The Analysis Settings tab (Figure 13.7) displays two options that are available when a batch analysis is run. The other settings of the Analysis Settings tab are described in Cell Intensity Analysis Options, on page 598. 336 Affymetrix® GeneChip® Operating Software User’s Guide Figure 13.7 Defaults, Analysis Settings tab PROMPT FOR OUTPUT DATA NAME 1. Choose the Prompt for output data name option (Figure 13.7) to display a Save Results As dialog box (Figure 13.8) at the start of an analysis. The default name for the probe analysis data is the same as the experiment name. The Save Results As dialog box displays the default name. Figure 13.8 Save Results As dialog box chapter 13 | Defaults 2. 337 Click OK to keep the default data name; or Enter a new name and click OK. DISPLAY SETTINGS WHEN ANALYZING DATA Choose the Display settings when analyzing data option (Figure 13.7) to display the Expression Analysis Settings dialog box (Figure 13.9) at the start of an analysis. Figure 13.9 Expression Analysis Settings dialog box 338 Affymetrix® GeneChip® Operating Software User’s Guide Chapter 14 Printing Chapter 14 341 Printing This chapter explains how to print: • image data • cell intensity data • probe analysis data • reports It contains the following sections: • Printing Image or Cell Intensity Data (see below) • Printing Probe Analysis Data (see page 342) • Printing an Expression Report (see page 343) Printing Image or Cell Intensity Data 1. When the Image window is open, click the Print button select File → Print from the menu bar. The Print dialog box appears (Figure 14.1). No print range options are available. Figure 14.1 Print dialog box in the Image window or 342 2. Affymetrix® GeneChip® Operating Software User’s Guide Click OK to print the image or cell intensity data. Printing Probe Analysis Data You may print tabular data and graphs from the Expression Analysis window (EAW). 1. When the EAW is open, click the Print button → Print from the menu bar. The Print dialog box appears (Figure 14.2). or select File Figure 14.2 Print dialog box, EAW The All and Selection options apply only to the Experiment Information, Metrics, and Pivot tables. If the Graphics option is chosen, the displayed graphs are automatically printed. 2. Choose Results and: - All to print the entire table in a tab (Experiment Information, Metrics, or Pivot). - Selection to print only highlighted table rows. chapter 14 | Printing 343 Printing an Expression Report You may print all or selected pages from an expression report. 1. When a report is open in the main display area, click the Print button or select File → Print from the menu bar. The Print dialog box appears (Figure 14.3). Figure 14.3 Print dialog box, report (.rpt) 2. Choose: - All to print all pages of a report. - Pages and enter a page range to print selected pages of a report. 344 Affymetrix® GeneChip® Operating Software User’s Guide Chapter 15 GCOS Manager Chapter 15 347 GCOS Manager The GCOS Manager application provides software tools to: • Manage the GeneChip® data in the process and publish databases. • Define and manage templates for sample registration and experiment setup. • Create usersets for analysis. • Create and manage publish databases. • Import data to the process database. • Export experiment and analysis data. • Archive and restore analysis data. • Create and manage roles that define user access privileges (available only to users connected to a GCOS server). This chapter explains the GCOS Manager functions and how to use them. It contains the following sections: • Getting Started (see below) • Process Tab (see page 356) • Publish Tab (see page 381) • Import Tab (see page 396) • Userset Tab (see page 415) • Template Tab (see page 425) • Roles Tab (see page 441) Getting Started To start the GCOS Manager: • Click the Microsoft® Windows® Start menu button , then select Programs → Affymetrix → GCOS Manager. The GCOS Manager starts and displays the user interface (Figure 15.1). 348 Affymetrix® GeneChip® Operating Software User’s Guide The software functions are organized in the following tabs: - Process (see page 356) - Publish (see page 381) - Import (see page 396) - Userset (see page 415) - Template (see page 425) - Roles (see page 441) The Roles tab is available when the GCOS Manager is connected to a GCOS server. Table 15.1 lists the functions found in each tab. To select the active server: • Make a selection from the server drop-down list in the toolbar (Figure 15.1 and Figure 15.2). chapter 15 | GCOS Manager Menu bar Server drop-down list Toolbar Active server Tabs Active server Process database Figure 15.1 GCOS Manager user interface, Microsoft® MSDE workstation database Roles tab is only available to users connected to a GCOS server Figure 15.2 GCOS Manager user interface, GCOS server database 349 350 Affymetrix® GeneChip® Operating Software User’s Guide Table 15.1 GCOS Manager tab functions for Affymetrix® GeneChip® data GCOS Manager Tab Functions in This Tab Process Archive or restore experiment data (.dat, .cel, .chp). Delete process data. Export experiment data. Assume ownership of data (available only to users connected to a GCOS server). Print an experiment. Register or unregister the local workstation or GCOS server. Publish Delete published data or a probe array type from a publish database. Create a publish database. Import Import experiment data to the process database Userset tab Create or modify a userset (a set of predefined parameter settings for the Statistical Expression algorithm). Delete a probe array type from a userset. Delete a userset. Template Create a template (a predefined form used to register samples and define experiments in GCOS 1.4). Deactivate or activate a template for use in GCOS 1.4. Rename a template. Roles (available only to users connected to a GCOS server) Create a role (a defined set of user privileges). Delete a role. You can learn more about GCOS in the following sections: • GCOS Manager Icons (see below) • Terminology (see page 352) GCOS MANAGER ICONS Table 15.2 shows the icons used in GCOS Manager. chapter 15 | GCOS Manager Table 15.2 GCOS Manager Icons Sample Template Properties information Project Global Gray bullet indicates processes that have been completed. Experiment information Server Green bullet indicates processes waiting to commence. Scanned probe array image data (.dat) Process database (yellow) Computed cell intensities (.cel) Publish database (gray) Export status Probe analysis data (.chp) Import to database Import status Probe array type Domain Delete status Chip User group Archive status Vessel User Create publish database status Userset Role Filter Red check mark on an experiment, .dat, .cel, or .chp icon(s) indicates that the data already exist in the publish database. 351 352 Affymetrix® GeneChip® Operating Software User’s Guide TERMINOLOGY Table 15.3 lists terms that are used frequently throughout this chapter. For your convenience, the terms are listed under the tabs where they are most frequently found. Table 15.3 Terminology common to all tabs Data & Object Types Across All Tabs Definition Automate A monitored task whose status, either on or off, is viewed in the Local Task pane. Automate On indicates GCOS will automatically track an experiment through data analysis after the probe array has been scanned. Sample A set of experiments. Experiment Specifies the type of probe array to which the target (sample) will be hybridized as well as other information relevant to the experiment. The experiment name provides the name for the data types that are subsequently generated in the analysis. Note: The Import tab shows experiment, cell intensity, or probe analysis data that can be imported or copied to the process database. In GCOS, the process database includes the experiment and sample data. Image data (.dat) The .dat file contains the image data of the scanned probe array data acquired in GCOS. The image data along with the image attributes stored in the database define the raw image data. Cell intensity data (.cel) The cell file (.cel) of computed intensities that GCOS automatically generates from the image data file (.dat). GCOS can publish the .cel to a publish database where it is accessible to the Affymetrix® Data Mining Tool (DMT). chapter 15 | GCOS Manager 353 Table 15.3 Terminology common to all tabs Data & Object Types Across All Tabs Definition Probe analysis data (.chp) The probe analysis output or chip file that is generated by the GeneChip data algorithms. GCOS can publish expression .chp to a publish database where it is accessible to the Affymetrix® DMT. Table 15.4 Terminology in the Process tab Process Tab Terms Definition Active server More than one GCOS server may be registered. The active server refers to the GCOS server where the displayed process database resides. Archive Move files from the process database to an external location. Unarchive Restore archived files to the process database. Export Copy data from the process database and send to an external location. Note: For an export from GCOS to Microarray Suite (MAS) 5.x, data created in GCOS are used to generate data files that are added to the default file location in MAS. Process database Database residing on the workstation or GCOS server that contains experiment and sample data. There is only one process database per workstation or GCOS server. Project A set of experiments with a common bond that are grouped together by their affiliation with a project. A project is created during sample registration in GCOS. 354 Affymetrix® GeneChip® Operating Software User’s Guide Table 15.5 Terminology in the Publish tab Publish Tab Terms Definition Affymetrix® Analysis Data Model (AADM) The AADM is the relational database schema that Affymetrix uses to store experiment data (.dat, .cel, .chp) in the publish database so that results may accessed by the Affymetrix® Data Mining Tool and other third party analysis tools. The AADM includes additional tables to support expression results. The AADM is publicly available to support open access to experiment information generated and managed by Affymetrix® software. Cell intensities The computed intensities that GCOS automatically generates from the image data (.dat). GCOS can copy or publish the cell intensity data to a publish database. Publish database An AADM database residing on a server that contains select published process data. More than one publish database may reside on a single server. Publish To copy or migrate experiment data to an AADM database on the workstation or GCOS server. Table 15.6 Terminology in the Import tab Import Tab Terms Definition Create Sample Creating a sample captures sample information and identifies the relationship between the experiment and experiment data (.cel, .chp) that are being imported. Import Copies experiment data (.cel, .chp) created in GCOS to the process database on the workstation or the GCOS server. Note: For an import to GCOS from Microarray Suite (MAS) 5.X, data are extracted from the experiment data files created in MAS and are added to the process data base. chapter 15 | GCOS Manager 355 Table 15.7 Terminology in the Userset tab Userset Tab Terms Definition Userset A predefined set of values for modifiable parameters in the Statistical Expression algorithm. Independent parameter Static settings defined in the userset that are not influenced by the probe array type. Dependent parameters Settings defined in the userset that are specific to a particular probe array type. Table 15.8 Terminology in the Template tab Template Tab Terms Definition Activate Restores an inactive (deactivated) attribute or template. Template A form created by the administrator that defines the attributes and values which are required to register samples and define experiments in GCOS. Deactivate attribute Renders an attribute inactive. The attribute is not permanently removed from the system. A deactivated attribute does not appear in GCOS until it is restored by the Activate command. Deactivate template Permanently deletes a template from the system if it is not currently used by an experiment or sample. Otherwise, the template is deactivated and does not appear in GCOS until it is restored by the Activate command. Table 15.9 Terminology in the Roles tab Roles Tab Terms Definition Privileges The functions or permissions that define a role. 356 Affymetrix® GeneChip® Operating Software User’s Guide Table 15.9 Terminology in the Roles tab Roles Tab Terms Definition Role Definitions Identifies the first branch under the active server where the role names are listed. Role A defined set of user access privileges. User Definitions Identifies the branch under the process database where role membership is defined. Process Tab This section of the manual explains the layout of the Process Tab window, its functions, and how to accomplish tasks here. The Process tab shows you the data in the system for all registered servers, where the data are found, and environmental property information about the data. The Process tab window is divided into three windowpanes (Figure 15.3). You can learn more about the Process Tab in the following sections: • Process Tab Windowpanes (see page 357) • Process Tab Functions (see page 358) chapter 15 | GCOS Manager 357 Active server Process database tree Selected data Task status/ Properties Figure 15.3 Process tab windowpanes PROCESS TAB WINDOWPANES The Process Tab window (Figure 15.3) has the following panes: Process database tree Shows the active server, process database, and data types (samples, projects, and types of probe arrays). The data type subfolders contain samples , projects , or probe array types . Click a sample or project to display the associated experiment data (.dat, .cel, .chp) in the data pane. Selected data Shows the experiment data of the sample or project selected in the process database tree. The first time you click an experiment in the tree of selected data, the information is read and a + sign appears next to the experiment name. Click the + sign to display the associated data. 358 Affymetrix® GeneChip® Operating Software User’s Guide Task Displays the property information or local task view, depending on the status/ item selected in process database tree or the selected data pane. Property Properties information is defined by the template that was used during sample registration in GCOS. The task status view shows the status of processes that may be occurring in the database (for example, export, import, delete, archive, or create). In the task status view, you can monitor these jobs while you work on other tasks in the system. PROCESS TAB FUNCTIONS The primary tasks in the Process tab are: • Archive data (see page 359) • Unarchive data (see page 361) • Change ownership of data (see page 362) • Delete process data (see page 364) • Deleting a probe array type (see page 366) • Export data (see page 368) You can also use the tools in the Process tab to: • Register a GCOS server (see page 369) • Unregister a GCOS server (see page 371) • View Server Events (see page 371) • View and manage Publish tasks (see page 372) • Search the Process database (see page 376) • Filter samples displayed in the data tree (see page 380) The primary data types in the Process tab are: • sample • project • experiment • image data, cell intensity data, and probe analysis data chapter 15 | GCOS Manager 359 The tasks associated with each data type are found under Process in the menu bar. Alternatively, right-click an item to view a shortcut menu (Figure 15.4). Sample shortcut menu Probe array type shortcut menu Project shortcut menu File shortcut menu Figure 15.4 Process tab shortcut menus Archiving Data Archiving data moves a user-selected data type to an external storage location. An archived data type no longer exists in the process database; however, its representative icon remains in the data tree and appears gray to indicate the data are available through the unarchive process. 1. In the Process tab, click the sample or project that contains the data you want to archive. 360 2. Affymetrix® GeneChip® Operating Software User’s Guide In the data pane, right-click the data that you want to archive and select Archive from the shortcut menu that appears (Figure 15.4); or Click the data and select Process → Archive Data Path from the menu bar. The Select Archive Data Path dialog box appears (Figure 15.5). Figure 15.5 Select Archive Data Path dialog box Select a drive from the Drive drop-down list and navigate to the directory where you want to archive the data. 4. Click OK. The data are archived in the specified directory. 3. To view more information about the archive task status, right-click in the Task Status/Properties pane and select View Task Log from the shortcut menu that appears (Figure 15.6); or Double-click to view the archive task log (Figure 15.7). chapter 15 | GCOS Manager 361 Figure 15.6 Task Status/Properties pane Figure 15.7 Archive task log Use the Find function to generate a list of files associated with a specific project. Next, SHIFT-select and archive the data in one quick step. This is only available for .dat, .cel, and .chp files in the process database. Unarchiving Data The icon for archived data appears gray. The unarchive command restores archived data to the process database. 1. In the Process tab, click the sample or project that contains the file you want to unarchive. 362 2. Affymetrix® GeneChip® Operating Software User’s Guide In the data pane, right-click the data that you want to unarchive and select Unarchive from the shortcut menu that appears (Figure 15.4 on page 359); or Click the data and select Process → Unarchive from the menu bar. Selecting the parent data includes all related child data in the unarchive process. If GCOS Manager cannot find the archived file, a Data Location dialog box appears. Enter the location of the data you want to unarchive in this dialog box. The data are unarchived and restored to the process database on the active server. The Process window does not automatically refresh upon completion of the Archive or Unarchive task. Therefore, if you unarchive data, the icon remains gray even though the task was completed successfully. To refresh the view, select View → Refresh from the menu bar. Changing Data Ownership If needed for security or management purposes, you can assume or assign ownership of data (change the user attribute of data). This feature is only available to users connected to a GCOS server. If the Take Ownership Process Data option is defined for a user in the Roles tab, the user can: • assume ownership of samples, experiments, or analyses. • assign ownership of samples, experiments, or analyses to a user who is also a member of a user group that the assignor belongs to. (For more information on role definition, see Roles Tab, on page 441). chapter 15 | GCOS Manager 363 Data ownership means you have permission to overwrite a file with new contents. For example, an owner can reanalyze cell intensity data using different parameter settings for the Statistical Expression algorithm to generate new probe analysis data. Assuming Ownership In the Process tab, right-click the sample, project, or data of interest. 2. Select Assume Ownership from the shortcut menu that appears. Your user name is displayed in the Task Status/Properties pane (Figure 15.9). 1. Assigning Ownership 1. In the Process tab, right-click the sample, project, or data of interest. 2. Select Assign Ownership from the shortcut menu that appears. The Assign Owner dialog box appears (Figure 15.8). Figure 15.8 Assign Owner dialog box 3. Enter the name of the new owner, and click OK. The user name of the new owner is displayed in the Task Status/ Properties pane (Figure 15.9). 364 Affymetrix® GeneChip® Operating Software User’s Guide Data owner Task status/ Properties Figure 15.9 Process tab, Task status/Properties pane shows the user name of the owner of the selected data Deleting Process Data You can delete samples, experiments, experiment data (.cel, .chp), or usersets. Deleting a sample deletes all associated experiments and analyses (.cel, .chp). Deleting an experiment deletes the associated analyses (.cel, .chp). The Delete option permanently removes data from the database. There is no restore function to recover deleted data. chapter 15 | GCOS Manager 1. 365 Right-click the item that you want to delete, and select Delete from the shortcut menu that appears; or Click the item and select Process → Delete from the menu bar. A warning/confirmation box appears (Figure 15.10). Figure 15.10 Delete warning/confirmation box 2. Click Yes to continue. The Task Status/Properties pane displays the delete local task status. To view more information about the delete task status, right-click in the Task Status/Properties pane and select View Task Log from the shortcut menu that appears (Figure 15.11); or Double-click to view the delete task log (Figure 15.12). Figure 15.11 Task Status/Properties pane 366 Affymetrix® GeneChip® Operating Software User’s Guide Figure 15.12 Delete task log Deleting process data is different from deleting publish data (see Chapter 12, Publishing Data, on page 315). Deleting published data removes only the data that has been published to a publish database. The data source in the process database remains available. Deleting a Probe Array Type You can remove an unused or unwanted probe array type from the process database. Deleting a probe array type deletes the associated usersets. The data created with these usersets remain, but are not accessible. 1. In the process pane data tree, expand the assay folder (for example, Expression) under the Probe Array Type folder (Figure 15.13). chapter 15 | GCOS Manager 367 Figure 15.13 Process pane 2. Right-click the probe array type that you want to delete and select Delete from the shortcut menu that appears; or Click the probe array type and select Process → Delete from the menu bar. A warning/confirmation box appears (Figure 15.14). Figure 15.14 Delete warning/confirmation box 3. Click Yes to continue. The selected probe array type is removed from the process database. 368 Affymetrix® GeneChip® Operating Software User’s Guide Exporting Data You can export (copy) sample or experiment data (.dat, .cel, .chp) to an external storage location. Sample and experiment information are exported to an Affymetrix® Microarray Suite 5.x .exp file. 1. In the Data pane, right-click the item that you want to export and select Export from the shortcut menu that appears; or Select the item and click the Data Path button or select Tools → Data Path from the menu bar. Selecting an object includes all of the data that are associated with it. For example, selecting image data (.dat) includes any related cell intensity data (.cel) and probe analysis data (.chp). The Select Export Data Path dialog box appears (Figure 15.15). Figure 15.15 Select Export Data Path dialog box 2. Choose a drive from the Drive drop-down list and a directory path from the Directories box. Click Network to map a network drive to a drive letter. 4. Click OK. 3. chapter 15 | GCOS Manager 369 The Task Status/Properties pane displays the export local task status. To view more information about the export task status, right-click in the Task Status/Properties pane and select View Task Log from the shortcut menu that appears (Figure 15.16); or Double-click to view the export task log (Figure 15.17). Figure 15.16 Task Status/Properties pane Figure 15.17 Export task log Registering a GCOS Server Registering a GCOS server makes it available to GCOS Manager. 1. Click the button or select Tools → Register GCOS Server from the menu bar. 370 Affymetrix® GeneChip® Operating Software User’s Guide The Register GCOS Server dialog box appears (Figure 15.18). Figure 15.18 Register GCOS Server dialog box 2. Select the server that you want to register and click OK. The selected server is added to the drop-down list on the toolbar and becomes the active server. If the selected server is not GCOS aware, an error message is displayed (Figure 15.19). Figure 15.19 Register GCOS Server error message chapter 15 | GCOS Manager 371 The error message indicates that there is no GCOS Server software installed, or that there is an installation error. For further information, please refer to the Affymetrix® GeneChip® Operating Software Server Installation and Administration Guide. Unregistering a GCOS Server • In the process pane, right-click the GCOS server that you want to remove and select Unregister GCOS Server from the shortcut menu that appears. Viewing Server Events The server events option displays a log of the events that occurred on the active server. • To display the Event Viewer, click the Server Events button or select Tools → Server Events from the menu bar (Figure 15.20). 372 Affymetrix® GeneChip® Operating Software User’s Guide Figure 15.20 Event Viewer shows an event log for the active server Viewing the Publish Tasks You can use the Server Task option to view the publishing queue on the active server. Click the Server Tasks button or select Tools → Server Tasks from the menu bar. The task list for the active server appears and shows all items in the publishing queue (Figure 15.21). 2. To sort the task list in ascending order, click a column header to define the primary sort order (for example, the Started column header). To sort the task list in descending order, click the same column header again. 1. chapter 15 | GCOS Manager Click to view the task list menu or right-click a task in the list. 373 Select a column header to define the primary sort order. Click the header once to sort the task list in ascending order. Click again to sort in descending order. Service status Figure 15.21 Task list displays the publishing queue on the active server Task List Items & Commands indicates a new task in the queue waiting to be published blue check mark indicates a task that is being published green check mark indicates a completed (published) task indicates an incomplete task that was not published due to an error To view the task list commands, click the icon (upper left corner of the window) (Figure 15.21) or right-click a task to display a shortcut menu. 374 Affymetrix® GeneChip® Operating Software User’s Guide Table 15.10 Task list menu commands Command Use this command to... View Tasks Display the status of each task in the publishing queue on the active server (Figure 15.22). View Task Items Display all items for a selected task (Figure 15.22). View All Items Display all items for all tasks in the Task List window. Cancel Cancel a pending publish task. Restart Reactivate the publish process for a cancelled task. Save As Save the task list to a text file (.txt). Delete Task Items Remove all task items from the Task List window. Figure 15.22 Server Task window, View Task Items command displays all items for a selected task Saving the Task List You can save the items in the Server Task window to a text file (.txt). chapter 15 | GCOS Manager 1. 375 Right-click a task in the Server Task window and select Save As from the shortcut menu that appears: or Click the icon in the upper left corner of the Task List window and select Save As from the drop-down menu. The Save As dialog box appears (Figure 15.23). Figure 15.23 Save As dialog box From the Save in drop-down list, navigate to the destination where you want to save the .txt file. 3. Name the file and click Save. The task list is saved as a text file in the defined destination. 2. Deleting Task Items To remove all of the task items from the Server Task Window: 1. Right-click a task and select Delete Task Items from the shortcut menu that appears; or Click the icon in the upper left corner of the Task List window and select Delete Task Items from the drop-down menu. A warning/confirmation window appears (Figure 15.24). 376 Affymetrix® GeneChip® Operating Software User’s Guide Figure 15.24 Task Delete warning/confirmation 2. Click Yes to delete all tasks on and before the date and time indicated. The items are deleted and Server Task window is empty. Cancelling or Restarting an Active Publish Task • To stop the publish process, right-click a pending task and select Cancel from the shortcut menu that appears. • To reactive the publish process for a task that has been cancelled or stopped due to error, right-click the task and select Restart from the shortcut menu that appears. Find Function You can use the Find feature to search the process database for samples, experiments, or experiment data (.dat, .cel, .chp), then apply the Export, Archive, Assume Ownership or Delete command to the objects returned by the search. The action affects only the selected objects and does not affect related children objects. The Find function searches only the process database. 1. Click the Find button ; or Select Tools → Find from the menu bar. The Find window appears (Figure 15.25). The Find window has three tabs: chapter 15 | GCOS Manager 377 - Select Object - Advanced Filter - Date Filter All three Find tabs work in union. For example, if you select sample (object type) in the Select Object tab, then only the boxes that pertain to finding samples will be active in the Advanced Filter tab. You may open multiple Find windows. Figure 15.25 Find window 378 Affymetrix® GeneChip® Operating Software User’s Guide Select Object tab Data Filter tab Advanced Filter tab Figure 15.25 Find window Click the Select Object, Advanced Filter, or Data Filter tab and make selections from the drop-down lists, or enter text for the search. Further refine your search by specifying additional search parameters in the other Find tabs. 3. Click Find Now. The search results are displayed at the bottom of the window (Figure 15.26). 2. chapter 15 | GCOS Manager 379 Figure 15.26 Find window, search results displayed To sort the search results, choose a column header to define the primary sort order. Click the column header to sort the results in ascending order. Click the header again to sort in descending order. 5. To apply the Export, Archive/Unarchive, Delete, or Assume Ownership command to the returned search objects: 4. A. Select the objects for the command. 380 Affymetrix® GeneChip® Operating Software User’s Guide To select adjacent objects, press and hold the Shift key, and click the first and last object in the selection. To select nonadjacent objects, press and hold the Ctrl key while you click the objects of interest. B. Right-click the selection and choose the command that you want to apply in the shortcut menu that appears (Figure 15.26); or Select the command from the Find menu in the menu bar. When you apply a command to selected search results, the command affects only the selected objects and does not affect any associated data (related children objects). Filtering Samples You can filter the samples that are displayed in the Process tab data tree (Figure 15.27). Sample filters are available in the Process or Import tab when you click a subfolder of the Samples directory. The filters remain active from session to session until the parameters are changed. In the data tree, click the Samples folder and the assay folder (for example, Expression). The data tree displays the samples and the Filter button becomes available. 2. Click the Filter button . The Sample Filter dialog box appears (Figure 15.27). 3. Select filter parameters from the drop-down boxes. The Role and User filter options are mutually exclusive. You may select one or the other, but not both. 1. 4. Click Apply. The sample filters are applied to the data tree. chapter 15 | GCOS Manager 381 Specify sample filters that determine the samples displayed Figure 15.27 Selections in the Sample Filters dialog box (top) determine the samples displayed in the process pane data tree (bottom) Publish Tab In the Publish tab you can create new publish databases and delete published data from a database. This section of the manual explains 382 Affymetrix® GeneChip® Operating Software User’s Guide the layout of the Publish tab window, its functions, and how to accomplish tasks here. • Click the Process tab. The three Process tab windowpanes are displayed (Figure 15.28). Create database Database tree This pane is blank until you initiate a create database command. Task status/ Properties Figure 15.28 Publish tab windowpanes You can learn more about the Publish tab in the following sections: • Publish Tab Windowpanes (see below) • Publish Tab Functions (see page 383) chapter 15 | GCOS Manager 383 PUBLISH TAB WINDOWPANES The Publish tab window (Figure 15.28) has the following panes: Publish database Displays a tree that shows the active server, the publish databases on tree the server, and the supported probe array types and their contents experiment data and analysis data (.cel, .chp) that have been published for that probe array type). Create database Shows the Create Database dialog box when you initiate a create database command. Task Status/ Properties pane Displays the status of processes that may be occurring in the database (for example, export, import, delete, archive, or create). In the task status view, you can monitor these jobs while you work on other tasks in the system. PUBLISH TAB FUNCTIONS The primary tasks in the Publish tab are: • Access a publish database (see page 384) • Delete a probe array type from the publish database (see page 385) • Delete data from the publish database (see page 386) • Create a publish database on the workstation or GCOS Server (see page 388) The tasks associated with a publish database, published data, or a probe array type are found under Publish in the menu bar; or Right-click an item to view a shortcut menu that appears (Figure 15.29). 384 Affymetrix® GeneChip® Operating Software User’s Guide Figure 15.29 Publish tab shortcut menus Accessing a Publish Database Each publish database is password protected. The password is defined during the creation of the database. You can change the password for a database on the GCOS server using the GCOS Server Sync utility. (For more information on the GCOS Server Sync utility, see the Affymetrix® GeneChip® Operating Software Server Installation and Administration Guide.) In the publish pane, click the database that you want to access. A Publish Database Login window appears (Figure 15.30). 2. Enter the database password and click Login. 1. Passwords are case-sensitive. The publish database tree expands and displays the Probe Array Type folder (Figure 15.30). chapter 15 | GCOS Manager 385 Figure 15.30 Publish Database Login box Deleting a Probe Array Type Probe array types that are no longer used may be removed from a publish database. When you delete a probe array type, all of the associated published data (from the parent down) are also deleted. Click the Probe Array Type folder under the publish database of interest. 2. Right-click the probe array type that you want to delete and select Delete from the shortcut menu that appears; or Click the probe array type and select Publish → Delete from the menu bar. A warning window appears (Figure 15.31). 1. Figure 15.31 Probe array delete warning/confirmation box 3. Click Yes to continue. 386 Affymetrix® GeneChip® Operating Software User’s Guide The probe array type and all associated data are removed from the publish database. Deleting Published Data Deleting publish data removes selected cell intensity or probe analysis data from the publish database. When you delete published data, all children data are also removed. Deleting publish data permanently removes the data from the publish database. If the information still resides on the process database, the data can be restored to the publish database by repeating the publish procedure in GCOS. In the server drop-down list, select the server where the publish database resides. 2. In the publish pane, select the database of interest and expand the tree. 3. Right-click the data that you want to delete and select Delete from the shortcut menu that appears; or Click the data and select Publish → Delete from the menu bar. 4. On the menu bar select Publish → Delete. A warning/confirmation box appears (Figure 15.32). 1. Figure 15.32 Analysis delete warning / confirmation window 5. Click Yes to continue. chapter 15 | GCOS Manager 387 The Delete task proceeds. The task status/properties pane displays the status of the delete task. To view more information about the delete task status, right-click in the Task Status/Properties pane and select View Task Log from the shortcut menu that appears (Figure 15.33); or Double-click to view the delete task log (Figure 15.34). Figure 15.33 Task Status/Properties pane Figure 15.34 Delete task log 388 Affymetrix® GeneChip® Operating Software User’s Guide Creating a Publish Database In the Publish tab, you can create a new publish database. Table 15.11 shows the location, size, and storage capacity options for a publish database. Along with the new database, GCOS Manager also creates the ODBC DSNs that are required for publishing data. Several key pieces of information are needed to create a publish database, including: • the server where the database will reside • a username and password with appropriate permissions for creating databases on the selected server • the database size • the proper drive and path for the database files The server name is represented by an ODBC data source name description. This ODBC DSN specifies the location of an administration database that is used to create the actual publish database. This DSN is usually created during installation of the software. The initial size of a Microsoft® SQL Server 2000 publish database on the GCOS server can range from 128 to 1024 MB. The size can easily be increased after the database is created. For information on increasing the size of a publish database on the GCOS server, see the GCOS Server Administration and Installation Guide. A MSDE publish database on the workstation has a 2 GB size limit. If the database reaches this limit, create a new publish database. MSDE cannot create a publish database on a network drive. chapter 15 | GCOS Manager 389 Table 15.11 Publish database location, size, and storage capacity options Publish Database/ Size Options Location Storage Capacity* Microsoft® MSDE/ workstation 8 MB per experiment (includes experiment information, .cel, .chp) up to 2 GB 8 MB per library file 128 MB database = 4 library files & 12 experiments Microsoft® SQL Server 2000/GCOS server 128-1024 MB initial size range 8 MB per experiment (includes experiment information, .cel, .chp) 8 MB per library file 128 MB database = 4 library files & 12 experiments 1024 MB database = 16 library files & 110 experiments Oracle®/GCOS server * Based Small (~6 GB) Large ~500 experiments ~1000 experiments on a probe array with 270x270 features, 1800 probe sets per array. Creating a Microsoft® MSDE Publish Database on the Workstation Select the server where the publish database will reside from the drop-down list on the toolbar (Figure 15.35). 2. In the publish pane, right-click the server name in the data tree and select Create MSDE Database from the shortcut menu that appears; or Click the server name and select Publish → Create MSDE Database from the menu bar. The Create Database dialog box appears (Figure 15.35). 1. 390 Affymetrix® GeneChip® Operating Software User’s Guide Select the server for the publish database Create Database dialog box Database tree Figure 15.35 Publish tab, create a MSDE publish database 3. In the Create Database dialog box: A. Enter a name for the new database. The database name is limited to 22 characters in length. Enter a password for the database. Re-enter the password to confirm it. The publish database can only be accessed with this password. 4. To select the path for the database, do either of the following: - Enter the path for the publish database - Click Browse and select a folder from the Choose Folder dialog box (Figure 15.36). B. chapter 15 | GCOS Manager 391 Figure 15.36 Choose Folder dialog box 5. Click Install. It takes several minutes to create the database and allocate the amount of drive space requested (2 -15 minutes or more, depending on the speed of the workstation and drive). Once it is created, the database is available for publishing without further configuration. Creating a Microsoft® SQL Server 2000 Publish Database on the GCOS Server 1. Select the server where the publish database will reside from the drop-down list on the toolbar (Figure 15.37). 2. In the database tree, right-click the server name and select Create SQL Server Database from the shortcut menu that appears; or Click the server name and select Publish → Create SQL Database from the menu bar. The Create Database dialog box appears (Figure 15.37). 392 3. Affymetrix® GeneChip® Operating Software User’s Guide In the Create Database dialog box: Enter a name for the new database. The database name is limited to 22 characters in length. B. Enter a password for the database. Re-enter the password to confirm it. The publish database can only be accessed with this password. A. Select the server for the publish database Database tree Figure 15.37 Publish tab, create a Microsoft® SQL Server publish database Create Database dialog box chapter 15 | GCOS Manager 4. 393 Enter the following information in the Data device, Index device, and Log device sections: - Path: Use the complete drive description, including the drive letter, colon, and backslash (for example, E:\Affymetrix DBDevices). The drive letter specifies the drive on the actual publish database server. If you are creating the publish database on a remote server, make sure the selected drive letter is available on that server. - Size (MB): The size depends on the number of experiments that will be published to the database and the amount of available space on the selected drive. A Microsoft® SQL Server database is easy to expand after creation; therefore, creating a smaller database and expanding it as necessary is recommended. 5. Click Install. It takes several minutes to create the database and allocate the amount of drive space requested (12-15 minutes or more, depending on the speed of the server and drive). Once created, the database is available for publishing without further configuration. If the new database does not appear in the data tree, verify that the database and ODBC DSN were created properly. Creating an Oracle® Publish Database on the GCOS Server An Oracle® publish database is much larger than the default Microsoft SQL Server database. Before you create an Oracle database, verify that there is adequate space available on the server. The small size Oracle publish database requires 6 GB and the large size requires 12 GB. These sizes have capacity for approximately 500 and 1,000 published experiments respectively. 1. Select the server where the publish database will reside from the drop-down list on the toolbar (Figure 15.38). 394 Affymetrix® GeneChip® Operating Software User’s Guide 2. In the database tree, right-click the server name in the data tree and select Create Oracle Database from the shortcut menu that appears; or Click the server name and select Publish → Create Oracle Database from the menu bar. The Create Database dialog box appears (Figure 15.38). 3. In the Create Database dialog box: Enter a name for the new database. The database name is limited to 22 characters in length. B. Enter a password for the database. Re-enter the password to confirm it. The publish database can only be accessed with this password. A. chapter 15 | GCOS Manager Select the server for the publish database Create Database dialog box Figure 15.38 Publish tab, create Oracle® database 4. In the Data Device section enter the following information: - Server Device Path: This is the complete path for the database files. Use the complete drive description for Microsoft® Windows® 2000 (for example, E:\Affymetrix DBdevices\). Be sure to include the trailing slash. 395 396 Affymetrix® GeneChip® Operating Software User’s Guide The data file path directly specifies the file location on the actual publish database server. If you are creating a publish database on a remote server, make sure the specified path is available on that server. The data file path directory used specifies the file location on the actual publish database server. If the publish database is being created on a remote server, make sure the path specified is available on that server. - Database Size: make a selection from the drop-down list. The Small database requires approximately 6 GB of drive space and the Large requires approximately 12 GB. 5. Click Install. It takes several minutes to create the database and allocate the amount of drive space requested (15 - 30 minutes or more, depending on the speed of the server and drive). Once it is created, the database is available for publishing without further configuration. If the newly created database does not appear in the data tree, verify that the database and the ODBC DSN were created properly. Import Tab In the Import tab you can copy experiment data (.exp, .cel, .chp created in MAS 5.x) into the GCOS or GCOS server process database. This section of the manual explains the layout of the Import tab window and how to accomplish tasks here. • Click the Import tab. The four windowpanes of the Import tab are displayed (Figure 15.39). chapter 15 | GCOS Manager 397 Active server Staging area Available data Sample tree Task status/ Properties Figure 15.39 Import tab windowpanes You can learn more about the Import tab in the following sections: • Filtering Samples (see page 397) • Import Tab Windowpanes (see page 400) • Importing Data (see page 400) FILTERING SAMPLES You can filter the samples that are displayed in the sample tree (Figure 15.39). Sample filters are available in the Import or Process 398 Affymetrix® GeneChip® Operating Software User’s Guide tab when you click a subfolder of the Samples directory. The filters remain active from session to session until the parameters are changed. 1. In the sample tree, click the Samples folder and the assay folder (for example, Expression). The sample tree displays the samples and the Filter button becomes available. Click the Filter button . The Sample Filter dialog box appears (Figure 15.27). 3. Select filter parameters from the drop-down boxes. The Role and User filter options are mutually exclusive. You may select one or the other, but not both. 4. Click Apply. The sample filters are applied to the data tree. 2. chapter 15 | GCOS Manager 399 Specify sample filters that determine the samples displayed in the sample tree. Figure 15.40 Selections in the Sample Filters dialog box (top) determine the samples displayed in the sample data tree (bottom) 400 Affymetrix® GeneChip® Operating Software User’s Guide IMPORT TAB WINDOWPANES The Import tab window (Figure 15.39) has the following panes: Sample tree Shows the active server, process database, and samples in the system. Staging area Shows the sample(s) that will be associated with experiment data for import. Data available for import Displays the experiment data located at the defined import data path. Task status/ properties status/ Properties Displays the property information or local task view, depending on the item selected in process database tree or the selected data pane. Property information is defined by the template that was used during sample registration in GCOS. The task status view shows the status of processes that may be occurring in the database (for example, export, import, delete, archive, or create). In the task status view, you can monitor these jobs while you work on other tasks in the system. IMPORTING DATA The Import function imports or copies experiment data created in MAS 5.x and moves them to a process database on the workstation or a GCOS server. You can import data to multiple GCOS servers. Importing data is a three-step process: Select the data path that specifies the location of the MAS 5.x experiment data (.exp, .dat, .cel, .chp) that you want to import (see Selecting an Import File Path, on page 401). 2. Create a new sample or select an existing sample to associate with the experiment data (the data will be also be associated with the project specified by the sample information) (see Specifying a Sample for Imported Data, on page 402). 3. Associate the experiment data with the selected sample so that GCOS Manager can track the data in the process database (see Associating Data & Samples, on page 408). 1. chapter 15 | GCOS Manager 401 Selecting an Import File Path 1. Click the data path button ; or Select Tools → Data Path from the menu bar. The Select Import Data Path dialog box appears (Figure 15.41). Figure 15.41 Select Import Data Path dialog box Make a selection from the Drives from the drop-down list and navigate to the data that you want to import. 3. Click OK. The defined data path and the experiment data at that location are displayed in the available data windowpane (upper right) in the Import tab (Figure 15.42). 2. 402 Affymetrix® GeneChip® Operating Software User’s Guide Import data Figure 15.42 Import tab, available data windowpane shows data available for import at the current import data path The default data path is the same path assigned to Affymetrix® GCOS experiment data. Specifying a Sample for Imported Data The experiment data for import are not associated with a sample or project. You must associate the data with a sample and a project so that GCOS Manager can track the data in the process database. You can associate the imported data with a new sample or an existing sample. Creating & Editing a New Sample 1. Right-click the appropriate assay folder (for example, Expression) in the process database tree and select Create Sample from the shortcut menu that appears; or Click the assay folder and select Import → Create Sample from the menu bar. chapter 15 | GCOS Manager 403 The Create Sample dialog box appears (Figure 15.43). Table 15.12 and Table 15.13 list the data entry rules for the fields in the Create Expression Sample dialog box. Table 15.12 Rules for data input Data Input Rule Character length < 64 characters Invalid characters \ / : * ? “ < > | ‘ ~ ´, { } [ ] Required data fields Required fields are indicated by blue titles and may not be left blank. Data fields A name may not duplicate an existing name. Table 15.13 Valid field lengths Sample Attribute Length (characters) Sample project 64 Sample type 64 Description 64 Comments 64 Sample name 64 2. Enter the following required information (or make selections from the drop-down lists): - Sample name - Sample project name - Sample type 3. Enter any descriptions or comments. 404 Affymetrix® GeneChip® Operating Software User’s Guide If the sample name, project name, and sample type do not match the information stored in the experiment file, the software notifies you and directs you to the import log to determine inconsistencies. 4. Click OK. The new sample appears in the import staging area (middle pane). Staging area Figure 15.43 Import tab window and Create Sample dialog box chapter 15 | GCOS Manager 405 If necessary, you can edit the information for a new sample before you start the data import. The sample name cannot be edited. You can only edit a new sample prior to data import. After data import, a sample cannot be edited. Samples created in GCOS cannot be edited. 5. To edit the sample information prior to data import: A. Right-click the sample name and select Edit from the shortcut menu that appears (Figure 15.44); or Click the sample name and select Import → Edit from the menu bar. The Create Sample dialog box appears (Figure 15.43). Figure 15.44 Sample for imported data, shortcut menu 406 Affymetrix® GeneChip® Operating Software User’s Guide B. Enter the new information in the Create Sample dialog box and click OK when finished. Selecting an Existing Sample 1. In the process database tree, expand the sample folder and click the sample of interest (Figure 15.45). 2. Drag the sample to the staging area. The sample and all of the associated data are displayed with red check marks that indicate the data already exist in the database. chapter 15 | GCOS Manager 407 Data available for import Staging Area Figure 15.45 Import tab, drag an existing sample to the Staging Area Removing a Sample From the Staging Area You can remove samples from the Staging Area. • Right-click the sample that you want to remove, and select Remove (for new samples) or Remove All (for existing samples) from the shortcut menu that appears (Figure 15.44); or Click the sample and select Import → Remove or Import → Remove All from the menu bar. 408 Affymetrix® GeneChip® Operating Software User’s Guide A new sample or an existing sample and its children files are removed from the staging area. Associating Data & Samples The last step in the import process is to associate the experiment data (.cel, .chp) with a sample. You can only associate data from probe array types installed in the process database. If you attempt to associate data from a probe array type that is not installed in the process database, an error is displayed. The names of imported data must conform to the naming conventions in GCOS (see Table 15.13 on page 403). If necessary, rename a file so that the name complies with the naming conventions. Associating .exp with a Sample In the available data pane (Figure 15.46), the EXP tab displays the experiment data (.exp) available for import at the designated data import path. When you associate an experiment (.exp) with a sample, the .dat, .cel, and .chp with the same prefix (if they exist) are also automatically associated with the sample. To associate a .exp with a sample, drag the data to the sample in the staging area (Figure 15.46). The .exp is added to the below the sample along with the .cel and .chp with the same prefix (Figure 15.47). 2. To associate another .exp and its children data with the sample, repeat step 1. 1. chapter 15 | GCOS Manager File path that specifies location of experiment data available for import Staging area Figure 15.46 Import tab, drag the .exp to associate it with a sample in the staging area Available data 409 410 Affymetrix® GeneChip® Operating Software User’s Guide Figure 15.47 Staging Area showing N004.exp and children data associated with sample N008 Associating Additional .cel or .chp with a Sample You can associate additional .cel or .chp that have different prefixes with a sample that is already associated with an .exp. This is done in a hierarchical manner by adding the .cel to the .exp that is associated with the sample of interest, and adding the .chp to the .cel. To associate a .cel with a sample: Select the CEL tab in the available data pane. 2. Drag the .cel to the .exp that is associated with the sample of interest in the staging area (Figure 15.48). Repeat this step to associate another .cel with the sample. The .cel is displayed below the .exp (Figure 15.49). To associate a .chp with a sample: 1. 1. Select the CHP tab in the available data pane. chapter 15 | GCOS Manager 2. 411 Drag the .chp to the .cel that is associated with the sample of interest in the staging area. Repeat this step to associate another .cel with the sample. The .chp is displayed below the .cel (Figure 15.49). To avoid having to associate a .cel and .chp with each .exp imported into the process database, simply import the .exp, then regenerate the.cel and .chp in GCOS using batch analysis. This minimizes potential errors from dragging a .chp to the wrong .cel. For more information, see Chapter 11, Batch Analysis, on page 297. 412 Affymetrix® GeneChip® Operating Software User’s Guide Staging area Figure 15.48 Import tab Available data chapter 15 | GCOS Manager 413 Additional .cel and .chp with different prefixes that are associated with sample N008 Figure 15.49 Staging area showing .cel and .chp data associated with the sample N008 Removing Data from the Staging Area You can remove experiment data (.exp, .cel, or .chp) from the staging area. Use either of the following methods to remove data: • Right-click the sample that you want to remove, and select Remove (for data without children) or Remove All (for data and associated children data) from the shortcut menu that appears (Figure 15.44); or Click the sample and select Import → Remove or Import → Remove All from the menu bar. The selected data or data and children data are removed from the staging area. • Drag the data from the staging area to the available data pane. This must be done in an ascending hierarchical manner by dragging the .chp to the CHP tab, the .cel to the CEL tab, and the .exp to the EXP tab. 414 Affymetrix® GeneChip® Operating Software User’s Guide Starting the Import 1. Right-click the process database, a sample, or one of the data items and select Start Import from the shortcut menu that appears; or Click one of these items and select Import → Start Import from the menu bar. The import task proceeds and the task status/properties pane displays the import status. 2. To view more information about the import task status, right-click in the Task Status/Properties pane and select View Task Log from the shortcut menu that appears (Figure 15.50); or Double-click . The import task log is displayed (Figure 15.51). Figure 15.50 Task Status/Properties pane Figure 15.51 Import task log chapter 15 | GCOS Manager 415 Userset Tab This section of the manual explains how to create, modify, or delete a userset. A userset is a set of modifiable Statistical Expression algorithm parameters with predefined values. For more information about the expression algorithm parameters, see Expression Analysis Algorithm, on page 627. A userset is defined by an administrator or other authorized user. It provides a convenient way to select analysis parameters without having to set each parameter individually before an analysis. A userset is applied in GCOS when you analyze expression cell intensity data. You can learn more about the Userset tab in the following sections: • Userset Tab Windowpanes (see below) • Creating a Userset (see page 417) • Modifying a Userset (see page 421) • Deleting a Userset (see page 424) USERSET TAB WINDOWPANES • Click the Userset tab. The three Userset windowpanes are displayed (Figure 15.39). 416 Affymetrix® GeneChip® Operating Software User’s Guide Create/Update pane Userset tree Task status/ Properties Figure 15.52 Userset tab windowpanes The Userset tab window (Figure 15.52) has the following panes: Userset tree Shows the active server, process database, user sets and associated probe array types. Create/ Shows new userset information that can be specified or existing userset Update pane information that can be modified (Figure 15.53). chapter 15 | GCOS Manager Task status/ properties 417 Displays the property information or local task view, depending on the item selected in process database tree or the selected data pane. Property information is defined by the template that was used during sample registration in GCOS. The task status view shows the status of processes that may be occurring in the database (for example, export, import, delete, archive, or create). In the task status view, you can monitor these jobs while you work on other tasks in the system. Probe array types included in an existing userset. Or probe array types to include in a new user set Probe array types in the process database Existing userset names New userset name Independent parameters Dependent parameters Figure 15.53 Create/Update pane CREATING A USERSET Creating a userset includes the following steps: 1. Enter a name for the userset. 2. Select the probe array type(s) that the userset will analyze. 418 Affymetrix® GeneChip® Operating Software User’s Guide Confirm the defaults or enter new values for the independent parameters. 4. Confirm the defaults or enter new values for the dependent parameters. To create a userset: 3. In the Create set name box, enter a unique name for the userset. 2. In the Probe array types box, select a probe array type and click the arrow button >>. Repeat this step to add other probe array types for the userset. The selected probe array type(s) is displayed in the Probe array types used box and the independent and dependent parameters are set to the default values (Figure 15.54). 3. If you want to use the independent and dependent parameter defaults, click Create Set. The userset is created and the name appears in the Existing userset name(s) box. If you do not want to accept the defaults, see the following sections on modifying the independent and dependent parameters. 1. chapter 15 | GCOS Manager Dependent parameters 419 Independent parameters Figure 15.54 Create/Update pane Modifying Independent or Dependent Parameter Values The statistical expression algorithm parameter settings are independent parameters. These are static settings that are not influenced by the probe array type. A dependent parameter is a setting that is specific to a particular probe array type. Dependent parameters include the scaling, normalization, probe mask, and baseline settings. For more information on these parameters, see Expression Analysis Settings, on page 644. 1. To modify an independent parameter: A. In the independent parameters box, click the parameter that you want to modify. 420 Affymetrix® GeneChip® Operating Software User’s Guide The parameter name and current setting are displayed in the box above (Figure 15.55). Current value for the selected independent parameter. Enter a new value here. Figure 15.55 Create/Update pane Enter a new value in the box and click Modify. The new parameter value is displayed in the box below. C. Repeat step b to modify other independent parameters. 2. To modify a dependent parameter: B. Click the tab for the parameter you want to modify (Scaling, Normalization, Probe Mask, or Baseline). B. Choose the analysis option that you want to include in the userset and enter any additional necessary information. A. 3. Click Create Set after you finish modifying the parameter values. The userset is created and the name appears in the Existing userset name(s) box (Figure 15.55). chapter 15 | GCOS Manager 421 The new parameter values are applied after you click Create Set. MODIFYING A USERSET You can modify the independent or dependent parameter values for a userset, or add or remove a probe array type(s) from a userset. 1. In the Existing userset name(s) box, click the userset that you want to modify. The Create/Update pane shows the parameter values for the selected userset (Figure 15.56). Figure 15.56 Create/Update pane 422 Affymetrix® GeneChip® Operating Software User’s Guide 2. To add a probe array type to the userset, select a probe array type in the Probe array types box, and click the arrow button >>. Repeat this step to add other probe array types for the userset. The selected probe array type(s) is displayed in the Probe array types used box (Figure 15.56). 3. To remove a probe array type from the userset, do either of the following: - Select a probe array type in the Probe array types used box, and click the arrow button <<. The selected probe array type is removed from the Probe array types used box. - In the userset tree, expand the Statistical folder and the userset that you want to modify. Right-click the probe array of interest and select Delete from the shortcut menu that appears (Figure 15.57). If you remove a probe array type from the process database in the Process tab, the probe array is also removed from any userset that includes this array type in its Probe Array Types Used list. chapter 15 | GCOS Manager 423 Figure 15.57 Userset tab window 4. To modify an independent or dependent parameter value, see the section Modifying Independent or Dependent Parameter Values, on page 419. 5. Click Update Set after you finish modifying the userset. The modifications are saved to the userset and the task status/ properties pane displays the Statistical Expression algorithm properties (Figure 15.58). 424 Affymetrix® GeneChip® Operating Software User’s Guide Figure 15.58 Task Status/Properties pane This information is metadata that exists in the process database and is used internally by GCOS and the GCOS server to distinguish different algorithms in the system. DELETING A USERSET 1. In the userset tree, right-click the userset that you want to delete and select Delete from the shortcut menu that appears; or Click the userset and select Userset → Delete from the menu bar. A warning/confirmation box appears (Figure 15.59). Figure 15.59 Delete userset warning/confirmation box 2. Click Yes to delete the userset. The userset is removed from the userset tree and the Existing userset name(s) box in the create/update pane. chapter 15 | GCOS Manager 425 Template Tab A template is a form that defines the attributes and values needed to register a sample or set up an experiment in GCOS. A sample or experiment template is created by the administrator or other authorized user. This section of the manual explains how to create and manage sample or experiment templates. You can learn more about the Template tab in the following sections: • Template Tab Windowpanes (see below) • MIAME Sample Template (see page 427) • Working with Templates (see page 428) • Working with Attributes (see page 439) 426 Affymetrix® GeneChip® Operating Software User’s Guide TEMPLATE TAB WINDOWPANES Template tree Attributes This pane is blank until you select a template in the template tree or initiate a create template command. Task status/ Properties Figure 15.60 Template tab windowpanes The Template tab window (Figure 15.60) has the following panes: Template tree Shows the active server, process database, and the experiment and sample templates that reside there. Click a sample or project to display the associated experiments and data (.dat, .cel, .chp) in the data pane. Select a template to display the attributes and values in the attribute pane. Attributes Displays the template selected in the template tree. Displays a blank template when a Create Template command is initiated. chapter 15 | GCOS Manager 427 Task status/ Displays the property information or local task view, depending on the Properties item selected in process database tree or the selected data pane. Property information is defined by the template that was used during sample registration in GCOS. The task status view shows the status of processes that may be occurring in the database (for example, export, import, delete, archive, or create). In the task status view, you can monitor these jobs while you work on other tasks in the system. MIAME SAMPLE TEMPLATE GCOS Manager provides a sample template based on the Minimum Information About a Microarray Experiment (MIAME).1 The MIAME sample template includes 23 types of sample attributes. For each attribute, you may: • define the data type • specify whether the attribute information is required for sample registration • specify the values available for a controlled value attribute To view the MIAME Sample Information template, click the MIAME Sample Information template in the Sample Templates folder (Figure 15.61). The Attributes pane displays the MIAME Sample Information template. 2. To enter template information, follow step 5 to step 12 in the section Creating a Template, on page 428. 1. 1 A. Brazma, P. Hingamp, J. Quackenbush, G. Sherlock, P. Spellman, C. Stoeckert, J. Aach, W. Ansorge, C.A. Ball, H.C. Causton, T. Gaasterland, P. Glenisson, F.C. P. Holstege, I.F. Kim, V. Markowitz, J.C. Matese, H. Parkinson, A. Robinson, U. Sarkans, S. Schulze-Kremer, J. Stewart, R Taylor, J. Vilo, and M. Vingron. Minimum information about a microarray experiment (MIAME)- toward standards for microarray data. Nature Genetics, 29:365-371. 428 Affymetrix® GeneChip® Operating Software User’s Guide Figure 15.61 MIAME Sample Information template WORKING WITH TEMPLATES Creating a Template When you create a template, you specify: • the attributes included in the template • the data type for each attribute (for example, an integer or a character string) chapter 15 | GCOS Manager 429 • whether the attribute is required • value options for a controlled data attribute 1. To create an experiment template, right-click the Experiment Templates folder and select Create from the shortcut menu that appears; or Click the folder and select Template → Create from the menu bar (Figure 15.62). To create a sample template, right-click the Sample Templates folder and select Create from the shortcut menu that appears; or Click the folder and select Template → Create from the menu bar. The Create Template box appears (Figure 15.63). Figure 15.62 Template tree, shortcut menu for experiment templates 430 Affymetrix® GeneChip® Operating Software User’s Guide Figure 15.63 Create Template box 2. Enter a name for the template. A blank template appears in the attribute pane (Figure 15.64) and the template name is added to the appropriate folder (experiment or sample). Click the folder and expand it to view the newly added template name. chapter 15 | GCOS Manager 431 Figure 15.64 Template tab, experiment template 3. In the template, double-click the first blank cell under the Name column. The mouse pointer becomes an I-beam and the other cells in the row are blocked (Figure 15.65). 432 Affymetrix® GeneChip® Operating Software User’s Guide Figure 15.65 Attributes pane 4. Enter an attribute name and press ENTER. The attribute name is inserted and numbered (Figure 15.66). During the process of defining an attribute, you can click Undo to remove the entries from the Attributes pane. chapter 15 | GCOS Manager 433 Name attribute inserted Figure 15.66 Attributes pane 5. 6. Click the cell under the Type column and click the drop-down arrow that appears. A drop-down list of data type options for the attribute appears (Figure 15.66). Integer Whole number Float Floating point number String Limited to 255 characters Date Date Time Time Controlled Limits user response to the value options listed in the template. Make a selection from the Type drop-down list. The selected data type is displayed in the cell (Figure 15.67). 434 Affymetrix® GeneChip® Operating Software User’s Guide Figure 15.67 Attributes pane 7. Click the cell under the Required column and click the drop-down arrow that appears. 8. In the drop-down list that appears, click Yes if the information is required or No if it is not (Figure 15.68). If the information is required, GCOS requires the user to enter a value during the sample registration or experiment set up process. chapter 15 | GCOS Manager 435 Figure 15.68 Attributes pane 9. If the attribute data type is controlled, double-click the cell under the Values column. The Controlled Values Edit List box appears (Figure 15.69). 436 Affymetrix® GeneChip® Operating Software User’s Guide Figure 15.69 Attribute pane and the Controlled Values Edit List box 10. To define a controlled value: Enter a value option in the box. B. Press Enter. C. Repeat steps a and b to enter additional controlled values. D. Click OK when finished. The controlled values appear in the Values drop-down list (Figure 15.70). A. chapter 15 | GCOS Manager 437 Figure 15.70 Attributes pane and the drop-down list of controlled values for the attribute 11. Click Apply. The defined attributes are now in effect. 12. To define additional attributes for the template, repeat step3 through step 11. Editing a Template You can edit an existing template. In the template tree, click the template you want to edit. The attributes pane displays the selected template. 2. To edit an attribute name, double-click the current entry and enter a new name. 1. 3. Follow step 3 to step 11 in the section Creating a Template, on page 428 to edit the attribute definition. 438 Affymetrix® GeneChip® Operating Software User’s Guide Deactivating or Activating a Template You can deactivate a template to make it unavailable in GCOS. An inactive template can be activated to restore it for use in GCOS. 1. To deactivate a template: Right-click the template of interest in the template tree and select Deactivate from the shortcut menu that appears; or Click the template and select Template → Deactivate from the menu bar. B. Click OK in the warning window that appears. The template icon appears gray in the template tree to indicate the template is inactive and is not available to GCOS. 2. To activate a template: A. A. Right-click the template of interest in the template tree and select Activate from the shortcut menu that appears; or Click the template and select Template → Activate from the menu bar. The template icon is restored to full color in the template tree to indicate the template is active and is available to GCOS. Renaming a Template 1. In the template tree, right-click the template that you want to rename and select Rename from the shortcut menu that appears; or Click the template and select Template → Rename from the menu bar. 2. Enter a name for the template and press ENTER. You cannot duplicate an existing name for the same type of template. chapter 15 | GCOS Manager 439 WORKING WITH ATTRIBUTES To view a shortcut menu of attribute commands: • Right-click an attribute number (Figure 15.71); or Click an attribute number and select Template → Attribute from the shortcut menu that appears. Attribute number Figure 15.71 Attributes pane, shortcut menu To select an attribute for a command: • Click the attribute number. To select a group of adjacent attributes: • Press and hold the Shift key while you click the number of the first and last attribute in the group. To select non-adjacent attributes: • Press and hold the Ctrl key while you click the attribute numbers. 440 Affymetrix® GeneChip® Operating Software User’s Guide Inserting an Attribute You can insert a blank row in the template. 1. Right-click the attribute number below the insertion point and select Insert from the shortcut menu that appears; or Click the attribute number and select Template → Insert from the menu bar. A blank row is inserted in the template. 2. Define the attribute and click Apply. Deactivating an Attribute A deactivated attribute is not displayed in the template for sample registration or experiment setup in GCOS. Right-click the number of the attribute that you want to deactivate and select Deactivate from the shortcut menu that appears; or Click the attribute number and select Template → Attribute → Deactivate from the menu bar. The attribute remains in the template, but appears grayed-out. 2. Click Apply. A warning message appears. 3. Click OK to deactivate the attribute. 1. In GCOS, the attribute is displayed in samples and experiments that were saved using the attribute. These cannot be modified. A deactivated attribute is not available for new samples or experiments. Activating an Attribute 1. Right-click the number of the attribute that you want to activate and select Activate from the shortcut menu that appears; or chapter 15 | GCOS Manager 441 Click the attribute number and select Template → Attribute → Activate from the menu bar. The attribute is no longer grayed-out. 2. Click Apply. The attribute becomes active and is available in the template. Cutting, Copying or Pasting an Attribute 1. To cut an attribute, right-click the attribute number and select Cut from the shortcut menu that appears; or Click the attribute number and select Template → Attribute → Cut from the menu bar. The attribute information is copied to the system clipboard. To copy an attribute, right-click the attribute number and select Copy from the shortcut menu that appears; or Click the attribute number and select Template → Attribute → Copy from the menu bar. The attribute information is copied to the system clipboard. 3. To paste an attribute, right-click the attribute number and select Paste from the shortcut menu that appears; or Click the attribute number and select Template → Attribute → Paste from the menu bar. The attribute information on the system clipboard is pasted on the template at the end of the attribute list. 4. Click Apply. 2. Roles Tab The Roles tab is only available to GCOS users connected to a GCOS server. 442 Affymetrix® GeneChip® Operating Software User’s Guide A role is a defined set of user access privileges. The administrator or other authorized user can create roles with different levels of access privilege for different users or groups of users. In this way, the roles function provides data security. This section of the manual explains how to create and manage roles. You can learn more about the Roles tab in the following sections: • Roles Tab Window (see below) • Creating a Role (see page 444) • Defining Role Membership (see page 448) • Deleting Roles (see page 451) chapter 15 | GCOS Manager 443 ROLES TAB WINDOW Roles Definitions tree Privilege/Domain pane Task status/ Properties Figure 15.72 Roles tab windowpanes The Roles tab window (Figure 15.72) has the following panes: Role Definitions Displays the active server, the process database, the roles created on tree the server, and the members of each role (Figure 15.73). Privileges pane Displays a list of the domains that reside on the active server. Shows the privileges that are associated with a role selected in the role definitions tree. Displays a template of privileges that is used to define privileges for a new role. 444 Affymetrix® GeneChip® Operating Software User’s Guide Task status/ Properties Displays the property information or local task view, depending on the item selected in process database tree or the selected data pane. Property information is defined by the template that was used during sample registration in GCOS. The task status view shows the status of processes that may be occurring in the database (for example, export, import, delete, archive, or create). In the task status view, you can monitor these jobs while you work on other tasks in the system. Active server List of role definitions Process database List of member definitions Roll member Figure 15.73 Role definitions tree CREATING A ROLE A role defines a set of user access privileges. Each GCOS user is assigned to a role. Creating a role is a three-step process: Name the role. 2. Define the role privileges (what the user can and cannot do). 3. Define role membership (add users to the role). 1. Naming the Role 1. Right-click an existing role in the role definitions tree and select Create Role from the shortcut menu that appears; or Click the role and select Role → Create Role from the menu bar. The Create Role box appears (Figure 15.74). chapter 15 | GCOS Manager 445 Figure 15.74 Create Role box 2. Enter a name for the role and click OK. The role name appears in the role definitions tree (Figure 15.75). The role name is required and cannot duplicate an existing name. Added role name Figure 15.75 Role definitions tree 3. In the role definitions tree, drag the role name to the process database. The role is added to the list of member definitions under the process database. 446 Affymetrix® GeneChip® Operating Software User’s Guide Figure 15.76 Drag the role to the process database Defining Privileges 1. In the role definitions tree, click the role that you want to define. The Privileges pane displays a template of rights and privileges (Figure 15.77). Figure 15.77 Default privilege settings In the privilege template, the function list shows the privileges that can be granted to a role member with respect to their own data. The second column called Other User Data Access defines the chapter 15 | GCOS Manager 447 rights of the role member with respect to other users’ data. Table 15.14 defines the privilege functions. 2. Click the check box next to a function to select that privilege for the role with respect to the user’s own data. 3. Click Yes/No in the right column to toggle between the two options and define the user privilege with respect to other users’ data. Other User Data Access is always Yes and cannot be toggled for the following functions: Take Ownership, Maintain Roles, Import Process Data, and Delete Publish Data. Table 15.14 Role privileges Function Allows a user to... Read Process Database Browse sample, experiment or analyses information in the process database. Update Process Database Modify sample and experiment properties information in the process database. Update Publish Database Overwrite experiment and analyses information in the publish database. Archive Process Data Archive .dat, .cel, or .chp data to an user-specified location. Delete Process Data Delete sample, experiment, analyses, userset and probe array type information from the process database. Delete Publish Data Delete experiment and analyses information from any publish database. Import Process Data Migrate Affymetrix® GCOS data files into the process database. Export Process Data Copy sample, experiment, and analysis data from the GCOS server to a defined location. Take Ownership of Process Data Assume ownership of a sample, experiment or analysis information with the ability to overwrite a file with new contents (for example, reanalyze a .cel with different expression analysis parameter settings). Maintain Roles Manage role, role membership, and set privileges. 448 Affymetrix® GeneChip® Operating Software User’s Guide Table 15.14 Role privileges Function Allows a user to... Delete Server Tasks Delete publish tasks on or before a given date. Maintain Templates Manage templates and their attributes. Maintain Usersets Manage expression analysis parameter sets. Create Publish Database Create a publish database. DEFINING ROLE MEMBERSHIP You add a user to a role in order to assign the user the privileges that are defined for the role. You can also remove a user from a role. Adding a User to a Role 1. In the role definitions tree, click the role of interest in the list of member definitions (under the process database). The privilege/domain pane displays the domain name tree (Figure 15.78). chapter 15 | GCOS Manager 449 Privilege/Domain pane Roles Definitions tree Task status/ Properties Figure 15.78 Roles tab, domain names Expand the domain names and locate the user name of interest. The tree displays the user groups that reside on the domain. 3. Drag the user name to the role of interest in the member definitions list. The user is added to the role and the role name displays the new member name (Figure 15.79). 2. Click and drag the icon to add an entire user group to a role. 450 Affymetrix® GeneChip® Operating Software User’s Guide Figure 15.79 Roles tab Deleting a User from a Role In the role definitions tree (under the process database), expand the user list for the role of interest. 2. Right-click the member that you want to delete and select Remove User from the shortcut menu that appears (Figure 15.80); or Click the user and select Roles → Remove User from the menu bar. 1. chapter 15 | GCOS Manager 451 Figure 15.80 Role definitions tree 3. The user name is removed from the role membership. DELETING ROLES LIMS User and LIMS Administrator are default roles and cannot be deleted. 1. In the role definitions tree (under the active server), right-click the role that you want to delete and select Delete Role from the shortcut menu that appears (Figure 15.81); or Click the role and select Roles → Delete Role from the menu bar. 452 Affymetrix® GeneChip® Operating Software User’s Guide Figure 15.81 Role definitions tree 2. In the warning/confirmation box that appears, click Yes to proceed. The role is deleted and is removed from the role definitions tree. Chapter 16 GCOS Administrator Chapter 16 455 GCOS Administrator The GCOS Administrator provides tools to backup a database as well as data and help you manage your workstation disk space. This chapter explains how to use the GCOS Administrator to: • Automatically backup the process database on the workstation. • Monitor available space on a workstation drive or database. • Relocate a publish database between workstation drives. • Register or unregister a GCOS server. This chapter contains the following sections: • Getting Started (see below) • Register or Unregister a GCOS Server (see page 459) • Changing a Database Password (see page 461) • Automatic Process Database Back Up (see page 462) • Managing Workstation Databases (see page 464) Getting Started 1. To start the GCOS Administrator, click the Microsoft® Windows® Start menu button , then select Programs → Affymetrix → GCOS Administrator. The GCOS Administrator starts and displays the user interface. Figure 16.1 shows the user interface at startup when the workstation is the active server and Figure 16.2 shows the user interface at startup when a GCOS server is selected. 456 Affymetrix® GeneChip® Operating Software User’s Guide Toolbar Data source selected in the server dropdown list Space Manager Monitors available space on a workstation drive or database. Available only when connected to the local GCOS MSDE database. Figure 16.1 GCOS Administrator user interface at startup, workstation server selected chapter 16 | GCOS Administrator 457 Server drop-down list Figure 16.2 GCOS Administrator user interface at startup, GCOS server selected 2. To select the active server, make a selection from the server dropdown list in the toolbar. 3. To display the Space Manager window: Select Local from the active server drop-down list. B. Click the Space Manager button . A data tree and property pane are displayed (Figure 16.3). A. 458 Affymetrix® GeneChip® Operating Software User’s Guide The Space Manager functions are only available for the workstation database and not for a GCOS server. Shortcut bar Data tree Property pane Space Manager window Figure 16.3 GCOS Administrator, Space Manager window for the workstation database You can learn more about the GCOS Administrator interface in the following sections: • GCOS Administrator Shortcut Bar & Windowpanes (see below) chapter 16 | GCOS Administrator 459 • Register or Unregister a GCOS Server (see page 459) • Register or Unregister a GCOS Server (see page 459) GCOS ADMINISTRATOR SHORTCUT BAR & WINDOWPANES The GCOS Administrator window (Figure 16.3) has the following panes: Shortcut bar Click the Space Manager button to display the Space Manager window for the workstation database. Data tree Space Manager window (for the workstation): displays the publish databases on the active server, workstation drives, and the locations of the probe information (library) files and experiment data. (The file locations are in GCOS.) Property pane Space Manager window (for the workstation): displays property information for the item selected in the data tree, the free and used space for a selected database or drive, or directory size. Register or Unregister a GCOS Server GCOS Administrator can register or unregister a GCOS server. The GCOS server can be the source of objects (a publish database or data) that you want to backup or the destination for objects that you want to restore. This enables the transfer of a publish database or data between a workstation and a GCOS server. You may have more than one registered GCOS server; however, GCOS, GCOS Manager, and GCOS Administrator can connect to only one GCOS server at a time. To learn more, see: • Registering a GCOS Server (see below) • Unregistering a GCOS Server (see page 460) 460 Affymetrix® GeneChip® Operating Software User’s Guide REGISTERING A GCOS SERVER 1. Click the Server button ; or Select Tools → Register GCOS Server from the menu bar. The Register GCOS Server box appears (Figure 16.4). Figure 16.4 Register GCOS Server box 2. Select the server that you want to register and click OK. The GCOS server name is added to the server drop-down list. UNREGISTERING A GCOS SERVER 1. Select the GCOS server that you want to unregister in the server drop-down list (Figure 16.5). chapter 16 | GCOS Administrator 461 GCOS server selected in the server drop-down list Figure 16.5 Server drop-down list 2. Press the Delete key or select Tools → Unregister GCOS Server from the menu bar. The GCOS server is unregistered and the server name is removed from the server drop-down list. Changing a Database Password You can change the password for the process database or a publish database on the workstation. 1. Click the Password button . The Administrator Password Dialog opens (Figure 16.6). 462 Affymetrix® GeneChip® Operating Software User’s Guide Figure 16.6 Administrator Password Dialog 2. 3. 4. 5. 6. Make a selection from the Database drop-down list. Enter the current password for the selected database. Enter a new password for the database. Re-enter the password to confirm it. Click OK. Automatic Process Database Back Up When the workstation is turned on, the GCOS Administrator automatically starts and runs in the background. The system tray displays the GCOS Administrator icon. For example, Figure 16.7 shows the GCOS application open and the GCOS Administrator running in the background. The GCOS Administrator monitors the number of analyses that are added to the process database on the workstation. After every 50 analyses added to the process database, the software automatically backs up the process database to the GCOS directory (\backup subfolder). chapter 16 | GCOS Administrator 463 The automated backup feature is only available for a process database on the workstation. It is not available for a process database on a GCOS server. System tray GCOS Administrator icon in the system tray indicates the application is running in the background Figure 16.7 GCOS application window is open and GCOS Administrator runs in the background 464 Affymetrix® GeneChip® Operating Software User’s Guide • To maximize the GCOS Administrator application, double-click the icon. • To access a shortcut menu for the GCOS Administrator, rightclick the GCOS Administrator icon in the system tray. Shortcut Menu Command Function Maximize Maximizes the GCOS Administrator window. Minimize Minimizes the GCOS Administrator window. Exit Exit the GCOS Administrator application. If you exit the GCOS Administrator application, the process database will not be automatically updated. Managing Workstation Databases GCOS Administrator provides tools to help you manage your process and publish databases on the workstation. • To open the space management window (Figure 16.8), click the Space Manager button . In this window, you can: • View the properties of a workstation database, drive, or GeneChip directory. • Monitor free and used space on a workstation database, drive, or directory. • Relocate a database between workstation drives. The Space Manager functions are only available for the workstation. chapter 16 | GCOS Administrator Shortcut bar Data tree Property pane Figure 16.8 GCOS Administrator, Space Manager window To learn more, see: • Viewing Property & Space Information (see below) • Relocating a Publish Database (see page 466) • Publish Database Statistics (see page 467) 465 466 Affymetrix® GeneChip® Operating Software User’s Guide VIEWING PROPERTY & SPACE INFORMATION • To view information about a workstation database, drive, or directory, click the item of interest in the data tree. The property pane displays information about the selected item (top) and a pie chart of space information. Table 16.1 lists the types of information that are available. Table 16.1 Space management information Item Property Pane Displays... Database Database files names Physical location of the database files Disk space allocated to each database file Pie chart of free and used database space Drive Databases on the drive Size of each database Create date for each database Pie chart of free and used drive space GeneChip Data Directory Number of files in the directory Directory size Pie chart of drive size and data size RELOCATING A PUBLISH DATABASE GCOS Administrator can move a workstation publish database to a user-specified drive. In the data tree, click the publish database that you want to relocate. The property pane displays the location and space allocation for the files that comprise the database (data device, log device, and index device) (Figure 16.9). For more information on these files, see Performance Tuning for SQL Server Databases, on page 709. 2. In the property pane, make a selection from the Location dropdown list for the database file that you want to move. 1. chapter 16 | GCOS Administrator 467 Click outside the drop-down list (to lose focus). The file is relocated to the selected drive. 4. Repeat Step 2 and Step 3 for each database file to be moved. 3. Shortcut bar Data tree Property pane Figure 16.9 GCOS Administrator, Space Manager window PUBLISH DATABASE STATISTICS 1. To view publish database statistics, click a publish database in the data tree. 468 Affymetrix® GeneChip® Operating Software User’s Guide The property pane displays all of the databases that comprise the publish database. For each database, it shows the: - database name - allocated size - create date - probe array type - number of published experiments - estimated number of experiments that can be published (with intensity data) - estimated number of experiments that can be published (without intensity data) 2. To find out how many experiments can be published using a particular probe array type, select the array type from the probe array drop-down list. Appendix A Client Testing Appendix A 471 Client Testing After installing the different client software applications, testing of the software must be completed to verify the functionality of the software components. Following the checklist below will complete the testing process. The screen captures depicted in this section may not exactly match the windows displayed on your screen. If you encounter problems during testing, please refer to Appendix K, Troubleshooting, on page 715. This appendix contains the following sections: • GCOS Manager 1.4 Testing (see below) • GCOS 1.4 Testing (see page 493) • Data Mining Tool 3.1 Testing (see page 518) • GCOS Manager Additional Testing (see page 523) GCOS Manager 1.4 Testing For more details about the GCOS Manager 1.4 client application, refer to Chapter 15, GCOS Manager, on page 347. 1. To perform the testing, login to the workstation as a user who has Administrator permissions. 2. Launch GCOS Manager 1.4 (Start → Programs → Affymetrix → GCOS Manager) 3. Verify that the a server is registered on the left hand side. 4. Click the Process, Publish, Import, Userset, Template and Roles tabs to verify that they are accessible. 472 Affymetrix® GeneChip® Operating Software User’s Guide The Roles tab is only available when GCOS Manager is connected to a GCOS server. PROCESS TAB 5. Click the Process tab. The Process tab shows the administrator the data is in the system. In this tab you can archive, unarchive, delete, export, assume ownership, and print data. 6. Expand the Samples folder. 7. Verify that the Expression folder appears. 8. Expand the Expression folder. If this is an upgrade, existing samples should appear. PUBLISH TAB 9. Click the Publish tab In this tab, users with access can create new publish databases or delete published data in any publish database on the system. 10. In the Publish tab, highlight the name of the active server. 11. Go to the menu bar and select Publish → Create SQL Server Database (if using SQL Server). If using Oracle®, go to step 16. You can create a publish database on the workstation by following the steps for creating a database on a SQL server. appendix A | Client Testing 473 Figure A.1 Create SQL Server Publish Database window 12. Enter the following information: Database - Database Name - The name of the publish database to be created (in this example, MyPub). - Database Password - Enter password for access to the publish database. - Confirm Database Password - Re-enter password for confirmation. The publish database is password protected. To access a publish database, users are required to enter the appropriate password. 474 Affymetrix® GeneChip® Operating Software User’s Guide Data Device - Path – Full path location for the Data Device to reside on the GCOS server (for example, F:\GeneChipDBDevices). - Size – Size of the Data Device (128MB). Index Device - Path – Full path location for the Index Device to reside on the GCOS server (i.e. F:\GeneChipDBDevices). - Size – Size of the Index Device (128MB). Log Device - Path – Full path location for the Log Device to reside on the GCOS server (for example, F:\GeneChipDBDevices). - Size – Size of the Log Device (128MB). The Microsoft® SQL Server database that you create must be between 128MB to 1024 MB. If you want to expand the size after the database is created, use SQL Enterprise Manager to expand the database. 13. Click Install after all information is entered. This may take a few minutes depending on the size of the database created. Creating a Publish database only needs to be tested once from a client. It does not need to be tested on additional clients, as publish databases are quite large. The path location for the devices must already exist on the server, otherwise creating the database will fail. 14. Click Close when the database is created. 15. Go to Step 19. 16. To create an Oracle® publish database, select Publish → Create Oracle Database from the menu bar. appendix A | Client Testing 475 The Create Oracle Publish Database window appears (Figure A.2). You can create an Oracle publish database when the GCOS Manager is connected to a GCOS server running Oracle. Figure A.2 Create Oracle® Publish Database window 17. Enter the following information: Database: - Database name – Name of the Publish database (in this example, MyPub) - Database password – Enter password for access to publish database - Confirm database password – Re-enter password for confirmation The publish database is password protected. To access a publish database, users will be required to enter the appropriate password. 476 Affymetrix® GeneChip® Operating Software User’s Guide Data Device: - Server device path – Full path location for the Device to reside on the GCOS server (for example, D:\GeneChipDBDevices\) - Database size – Small or Large (in this example, select Small) The server device path directory specifies the file location on the actual publish database server. If the publish database is being created on a remote server, make sure the path specified is available on that server. An Oracle® publish database is much larger than the default SQL Server database. Verify that there is adequate space on the server before creating an Oracle database. The Small Oracle publish database requires 8GB of hard disk space. The Large Oracle publish database requires 17 GB of hard disk space. These database sizes correspond to publishing approximately 500 and 1000 experiments respectively. Creating an Oracle database will take a while. 18. Click Close after the database is created. You only need to test creating a publish database once from a client. You need not repeat the test on additional clients, as a publish database is quite large. IMPORT TAB 19. Click the Import tab. The Import function copies or imports experiment data created in GCOS and moves the data to a process database on the workstation or GCOS server. appendix A | Client Testing 477 20. On the menu bar, select Tools → File Path. Set the path for the location of the files for import. For example, C:\GeneChip\TempData (Figure A.3). Figure A.3 Select Import File Path window 21. Click OK. 22. Highlight the Expression Folder. From the menu bar, select Import → Create Sample. The Create Expression Sample window appears (Figure A.4). 478 Affymetrix® GeneChip® Operating Software User’s Guide Figure A.4 Create Expression Sample window 23. Enter the following information: - Sample Name: ImportSample - Sample Project: MySampleProject - Sample Type: MySampleType - Description: <optional> - Comments: <optional> The fields in blue are required fields. 24. Click OK. GCOS Manager can import any data for which a library file has been installed. To preserve the experiment information, all data in the sample name, project, and array type should match the data in the experiment file. 25. Select the .exp file and drag it to the sample in the Data to import pane (Figure A.5). appendix A | Client Testing 479 Figure A.5 Importing .exp data The .exp file brings all associated files with the same prefix when placed on the sample (Figure A.6). 480 Affymetrix® GeneChip® Operating Software User’s Guide Figure A.6 Importing .exp brings associated files 26. If there are additional .cel or .chp files associated with this experiment, select the CEL or CHP tab and drag the appropriate file to the sample in the Data to import pane. 27. When all of the files for the import are associated with a sample, select Import → Start Import from the menu bar; or Right-click any file in the Data To Import pane and select Start Import from the shortcut menu that appears. 28. If the import fails, check the import status in status pane. Double-click Import in Task View and review the error message. appendix A | Client Testing 481 USERSET TAB 29. Click the Userset tab. In this tab you can create a userset that defines values for the usermodifiable expression algorithm parameters. You can select the userset by name to analyze experiments using the same set of algorithm parameter settings. Figure A.7 Upper right pane of the Userset tab 30. Enter a name for the set in the Create Set Name field (enter MySet). 31. Select the Probe Array Type (Hu6800) in the Probe Array Types Used field and move it by pressing >>. (Only one probe array type can be moved at a time.) 482 Affymetrix® GeneChip® Operating Software User’s Guide 32. Click Create Set. The userset is created and the userset name is added to the right hand side of the pane. TEMPLATE TAB Template attributes cannot be deleted, only deactivated. A template can be deleted (right-click the template and select Delete from the shortcut menu that appears). 33. Click the Template tab. In this tab the administrator can add fields to the Experiment Template and the Sample Template that are used in GCOS. 34. To create an Experiment Template, highlight the Experiment Templates Folder. 35. Select Template → Create from the menu bar. (If it is grayed out, highlight the Experiment Templates Folder again, or rightclick the Experiment Template Folder and select Create from the shortcut menu that appears) (Figure A.8). appendix A | Client Testing Figure A.8 Template Tab window The Create Template box appears (Figure A.9). Figure A.9 Create Template box 483 484 Affymetrix® GeneChip® Operating Software User’s Guide 36. Enter the name of the template (for example, exp_temp). Click OK. The experiment template attributes window appears in the upper right pane (Figure A.10). Figure A.10 Experiment Template Attributes window 37. Double-click in the Name field to get a blinking cursor. 38. Enter Comments in the Name field. 39. Select String in the Type field. 40. Select No in the Required field. appendix A | Client Testing 485 Figure A.11 Attributes and values entered 41. Click Apply. The Exp_Temp experiment template is added to the Experiment Templates list. (You must select the Experiment Templates folder to see the newly added template.) 42. To create a Sample Template, highlight the Sample Templates Folder. 43. Select Template → Create from the menu bar. (If it is grayed out, highlight the Sample Templates folder again, or right-click the Sample Template folder and select Create from the shortcut menu that appears.) (Figure A.12) 486 Affymetrix® GeneChip® Operating Software User’s Guide Figure A.12 Sample Template window The Create Template window appears (Figure A.13). Figure A.13 Create Template window 44. Enter the name of the template (for example, Sample_Temp). Click OK. The sample template attributes window appears (Figure A.14). appendix A | Client Testing Figure A.14 Sample Template Attributes window 45. Double-click in the Name field to get a blinking cursor. 46. Enter Additional Comments in the Name field. 47. Select String in the Type field. 48. Select No in the Required field. 487 488 Affymetrix® GeneChip® Operating Software User’s Guide Figure A.15 Attributes and Values Entered 49. Click Apply. The Experiment and Sample templates are created and appear in the left hand pane (Figure A.16). You must select the Experiment or Sample template folder to see the list of available templates. appendix A | Client Testing 489 Experiment and sample templates added Figure A.16 Experiment and Sample Templates Added ROLES TAB The roles tab is only available when using GCOS Manager with a registered GCOS server. 50. Click the Roles tab. The Roles tab is used to set up security for the end-users. Therefore, the functions of the Roles tab are solely for the use of the system administrator and the tab only appears for users who have been granted the Maintain Roles privilege. 51. Click GCOS Server Administrator from the left hand pane under the name of the GCOS server. The function of the GCOS Server Administrator is displayed. All privileges are granted for the administrator (Figure A.17). 490 Affymetrix® GeneChip® Operating Software User’s Guide Figure A.17 Administrator Privileges window 52. Click GCOS Server User from the left hand pane under the GCOS server name. The function of the GCOS Server User is displayed. By default, all privileges are granted for the User except for the last five privileges (Figure A.18). Figure A.18 Default User Privileges 53. Under the GCOSServer3 database, expand the GCOS Server Administrator group and GCOS Server User group. appendix A | Client Testing 491 The domain name\Global Group appears and displays the group members (Figure A.19). Figure A.19 Roles Tab window 54. Click the right hand pane to expand the domain and the list of Global Groups. 55. Expand the GCOS Server Global Group. 56. Drag a specific user to the GCOS Server User or GCOS Server Administrator group. This allows individual users or Global Groups access to certain functions on the GCOS server. 57. Click any existing role in the left pane. 58. In the Create Role dialog box, type a name for a new role. 59. Click OK. The new role appears in the left pane under the server. 492 Affymetrix® GeneChip® Operating Software User’s Guide Figure A.20 Roles Tab window EVENT VIEWER AND SERVER TASKS 60. To display the Event Viewer for the GCOS server, select Tools → Server Event Viewer from the menu bar. The Event Viewer for the GCOS server is displayed (Figure A.21). You can view the System, Security and Application Log. appendix A | Client Testing 493 61. Close the Event Viewer. 62. To view the Server Tasks, select Tools → Server Tasks from the menu bar. This monitors and displays the status of any published data and grows as more data are published. Figure A.21 Server Tasks window The task list is populated after experiment data are published. GCOS 1.4 Testing PREPARING AND SCANNING AN EXPERIMENT 63. Select File → New Experiment from the menu bar or click the Experiments button . The Sample and Experiments window is displayed (Figure A.22). 494 Affymetrix® GeneChip® Operating Software User’s Guide Figure A.22 Sample and Experiments window 64. Enter the following information for Experiment 1 (Figure A.23): - Sample Name: MySample - Sample Type: SampleType - Project: SampleProject - Experiment Name: TestExp - Probe Array Type: Hu6800 appendix A | Client Testing 495 Figure A.23 Experiment 1 input 65. Save the experiment information by selecting File → Save from the menu bar or click the Save button . 66. Enter the following information for Experiment 2 (Figure A.24): - Sample Name: MySample - Sample Type: SampleType - Project: SampleProject - Experiment Name: TestExp_2 496 Affymetrix® GeneChip® Operating Software User’s Guide - ProbeArray Type: Hu6800 67. Select the Analysis Set (MySet) that was created in GCOS Manager. (Only analysis sets for the same probe array type appear.) 68. Select the Publish Database (MyPub) that was created in GCOS Manager. 69. Select Publish Intensities. Figure A.24 Experiment 2 input appendix A | Client Testing 70. Save the experiment information by selecting File → Save from the menu bar or click the Save button 497 . 71. A dialog box appears prompting for the publish database password. Enter the password and click Login. 72. From the Sample Template drop-down list, select Sample_Temp. 73. From the Experiment Template drop-down list, select Exp_Temp. 74. Enter the following information for Experiment 3 (Figure A.25): - Sample Name: MySample - Sample Type: SampleType - Project: SampleProject - More Comments: This is a test. - Experiment Name: TestExp_3 - ProbeArray Type: Hu6800 - Comments: Additional comments 75. Set Analysis Set and Publish Database to [No analysis] and [No publish] respectively. 76. De-select Publish intensities. 498 Affymetrix® GeneChip® Operating Software User’s Guide Figure A.25 Experiment 3 input 77. Save the experimental information by selecting File → Save from the menu bar or click the Save button . appendix A | Client Testing 499 HYBRIDIZING AN EXPERIMENT 78. To hybridize an experiment, select Run → Fluidics from the menu bar or click Instrument Control in the shortcut bar and click the Fluidics button . 79. Select Module 1 (Figure A.26). Figure A.26 Fluidics Station window 80. In the Experiment drop-down box, select TestExp. 81. In the Protocol drop-down box, select the protocol to run the experiment on. (Select the appropriate fluidics protocol for the probe array type.) (Figure A.27). 500 Affymetrix® GeneChip® Operating Software User’s Guide Figure A.27 Select Protocol 82. Click Run. The Fluidics Station window displays the Current Stage of the hybridization (Figure A.28). This takes a few minutes. appendix A | Client Testing Figure A.28 Hybridizing experiment Figure A.29 Hybridization complete 501 502 Affymetrix® GeneChip® Operating Software User’s Guide Hybridizing done is displayed when complete (Figure A.29). The experiment is ready to be scanned. 83. Click each module to verify that the hybridization is complete. 84. Click Close to exit the Fluidics station window. 85. For TestExp_2, select the GeneChip Software shortcut bar and click the Workflow Monitor icon Figure A.30 Workflow monitor window . 503 appendix A | Client Testing 86. If you want to skip the hybridization step and place the experiment in scan status, right-click TestExp_2 and select Advance to Scan from the shortcut menu that appears (Figure A.30). SCANNING AN EXPERIMENT 87. To scan an experiment, select Run → Start Scanner from the menu bar, or click Instrument Control in the Instrument Control shortcut bar and then the Start Scanner button . Figure A.31 Scanner dialog box 88. Select TestExp from the Experiment Name drop-down list. 504 Affymetrix® GeneChip® Operating Software User’s Guide Figure A.32 Scanner dialog box, TestExp selected for scan 89. Click Start to begin scanning the chip. Figure A.33 Scanner prompt Scanning a chip through one scan takes approximately 5 minutes. Only one chip can be scanned at a time. 90. Load the chip into the scanner. After the scan is completed, GCOS generates a .cel. 91. Scan the second experiment: from the menu bar select Run → Start Scanner or click the Instrument control shortcut bar and click the Start Scanner button . appendix A | Client Testing 505 92. From the Experiment Name drop-down list, select TestExp_2. 93. Click Start to begin scanning the probe array. After the scan is completed, this experiment has the program automatically create the .cel and the .chp and publishes them into the MyPub database. This is done because Analysis Set and Publish Database fields were selected during the experiment setup. 94. Do not scan TestExp_3. RUNNING AN ANALYSIS 95. To generate a .chp, select the TestExp.CEL. 96. Right-click TestExp.CEL file and select Analyze (Figure A.34). 506 Affymetrix® GeneChip® Operating Software User’s Guide Figure A.34 Analyze Experiment window The status log displays that it is running an analysis for the file. For example, Running analysis for \\<servername>\GeneChip\Affy_Data\Data\TestExp.CHP (Figure A.35). Figure A.35 Status log appendix A | Client Testing 507 97. The status log displays when the analysis is completed (i.e. Analysis completed for \\<servername>\GeneChip\Affy_Data\Data\TestExp.CHP). The .chp file is displayed. If an error message is displayed, make a note of the error message and follow the suggestions in the error message. If problems persist, see Appendix K, Troubleshooting, on page 715. Figure A.36 Analysis output file (.chp) displayed 508 Affymetrix® GeneChip® Operating Software User’s Guide PUBLISHING 98. To Publish an experiment, select Run → Publish from the menu bar or click the Publish button in the GeneChip Software shortcut bar . The Publish window appears (Figure A.37). Figure A.37 Publish window appendix A | Client Testing 509 99. To add data to be published, select Publish → Add Item from the menu bar or click the Add button in the publish window area. The Open window appears and displays experiments available for publishing (Figure A.38). Figure A.38 Open window 100.Highlight the TestExp experiment and click OK. 101.To publish the cell intensity data, select Publish → Options → Publish Intensities from the menu bar. The Publish Database Login window appears. 102.Enter the password for the publish database and click Login. 510 Affymetrix® GeneChip® Operating Software User’s Guide Publish intensities: YES Figure A.39 Publish window with intensities selected 103.To begin the publishing process, select Publish → Publish from the menu bar or click the Publish button in the publish window. appendix A | Client Testing Figure A.40 Publish status window 104.The task window shows the status of the publishing process. Each task contains items that are in a particular task. 511 512 Affymetrix® GeneChip® Operating Software User’s Guide Figure A.41 Publish task item window RUNNING REPORTS 105.To configure the report settings, select Tools → Report Settings → Expression Report from the menu bar, or click the Report Settings in the shortcut bar and click Expression Report . The Expression Report window appears (Figure A.42). appendix A | Client Testing 513 Figure A.42 Expression Report window 106.Select File → Report from the menu bar and select the .chp to generate the report; or Right-click a .chp in the data file tree, and select Report (use the TestExp.CHP file). SAMPLE HISTORY 107.To view the sample history window (Figure A.43), select Run → Sample History from the menu bar; or Click the Sample History button in the shortcut bar. 514 Affymetrix® GeneChip® Operating Software User’s Guide Figure A.43 Sample history window 108.Click a sample. The list of files pertaining to the sample appears in the right hand pane (Figure A.44). appendix A | Client Testing 515 Figure A.44 Sample history window, selected file 109.To view more history information, click the EXP icon and information in the results window is displayed (Figure A.25). 516 Affymetrix® GeneChip® Operating Software User’s Guide Figure A.45 History for the selected file 110.To view history information, click the .dat, .cel, or .chp, click the file. The history information is displayed in the bottom windowpane. appendix A | Client Testing 517 WORKFLOW MONITOR 111.To view the workflow monitor, select Run → Workflow Monitor from the menu bar; or Click the Workflow Monitor icon in the GeneChip Software shortcut bar. 112.To filter information, select Tools → Filters… from the menu bar to display the Filters window. 113.Information can be filtered on the following items: - Assay Type - Sample Name - Experiment - Sample Project - Probe Array Type - Sample Type - User - Date 114.Click OK after any filter selections have been made. 115.The Workflow Monitor shows the current stage of the experiment. The experiment could be in the following stage status: - Experiment Setup - Hybridization - Scan - Grid Alignment - Cell Intensity Analysis - Probe Array Analysis 116.Click the bottom tabs to see the stage where experiments currently stand. 518 Affymetrix® GeneChip® Operating Software User’s Guide Data Mining Tool 3.1 Testing 1. Is your GCOS server using a SQL Server or Oracle® database? 2. If using SQL Server, go to step 5. If using Oracle, go to step 3. 3. If using Oracle, verify that Oracle client utilities are installed. 4. Verify that the alias or aliases (if using a remote GCOS server) are created. When you open the Data Mining Tool (DMT) for the first time a message appears stating “Unable to Determine Publish Database Name”. Click OK. This message appears because a publish database has not yet been registered. 5. Start Data Mining Tool (Start → Programs → Affymetrix → Data Mining Tool). 6. Select Edit → Register Database from the menu bar. The Register Database dialog box appears (Figure A.46). Figure A.46 Register Database dialog box, publish database on GCOS server (Oracle® dialog box, top; Microsoft® SQL Server dialog box, bottom) 7. If using an Oracle database, go to Step 8. If using a Microsoft® SQL Server database go to Step 10. appendix A | Client Testing 519 8. Enter the server’s alias name in the Oracle Alias box. 9. Enter the Publish Database name, then go to Step 12. 10. Enter the Server Name, then click List Databases to display the publish databases for the server in the Publish Database dropdown list. 11. Select a database from the Publish Database drop-down list. 12. Click Register. DMT prompts for the Publish Database Password. 13. Enter the Password and click Login. The database is available to DMT. 14. Select Edit → Select Data Source from the menu bar. 15. Select the Oracle Publish DSN (MyPub, or the name of the DSN created). 16. Select Data → New → Expression Data Mining Tool from the menu bar. 520 Affymetrix® GeneChip® Operating Software User’s Guide Figure A.47 Data Mining Tool window 17. Verify that the data published appears in the DMT window. 18. From the menu bar, select Query → Run Query to execute query or click the Query icon . The query results window is displayed (Figure A.48). appendix A | Client Testing Figure A.48 Query Results window 19. Pivot the query results data by selecting Query → Run Pivot from the menu bar or click the Pivot button . Figure A.49 Pivot running 20. Graph the results by selecting Graph → Scatter... from the menu bar or click the Scatter Graph button . 521 522 Affymetrix® GeneChip® Operating Software User’s Guide Figure A.50 Scatter Graph dialog box 21. Move the analysis files to the X-Axis and the Y-Axis. Click OK appendix A | Client Testing 523 Figure A.51 Scatter Graph GCOS Manager Additional Testing This additional testing allows the system administrator to delete, archive, unarchive, export or take ownership of data. 22. Start GCOS Manager 1.4 (Start → Programs → Affymetrix → GCOS Manager). 23. Expand the Samples list. 24. Expand the Expression Sample list. EXPORT AN EXPERIMENT 25. Select Tools → File Path from the menu bar, or click the File Path button . 26. Select a path location to export the files to (for example, x:\temp). 524 Affymetrix® GeneChip® Operating Software User’s Guide 27. Click the sample named MySample and select Process → File View. 28. In the right hand pane, select TestExp and right-click to select Export, or go to the menu bar and select Process → Export after selecting the TestExp (Figure A.52). Figure A.52 Export window The task window displays the export process. It exports the .dat, .cel, and .chp (Figure A.53). Figure A.53 Status window 29. Once completed, the status returns to Available. If it fails, the status returns ‘Failed.’ 30. To view the error, double-click the Export icon in the Local Task pane . A text file appears. View the error message and try to resolve it. Try exporting again. appendix A | Client Testing 525 ARCHIVE AND UNARCHIVE 31. Click the Process Tab. 32. Expand the TestExp list in the right hand pane. 33. Select the TestExp.CEL file. 34. Right-click and select Archive (Figure A.54), or select Process → Archive from the menu bar. Figure A.54 Archive window The Select Archive Path Location window appears (Figure A.55). Figure A.55 Archive Path Location window 35. Select the path location to archive the files to (for example, c:\temp). Click OK. 526 Affymetrix® GeneChip® Operating Software User’s Guide The archive process begins. All files are archived from the parent down. If there are any files below, it archives them as well. In this example, the .cel and .chp are archived. The archive process is completed when the status is Available. Figure A.56 Status window 36. Click MySample in the Samples list and select Process → Refresh from the menu bar. 37. Expand TestExp in the right hand pane. The files are archived and are displayed in gray. 38. To unarchive, right-click the archived file and select Unarchive; or Click the archived file and select Process → Unarchive from the menu bar. The Unarchive process begins. PUBLISH DATABASE DATA DELETION 39. To delete files from the Publish database, select the Publish tab. 40. Expand the Probe Array Type list. A list of probe array types is displayed of published experiments. 41. Click Hu6800. The published experiments is displayed. appendix A | Client Testing 527 42. Click TestExp. This expands the list to display the .cel and .chp files (Figure A.57). Figure A.57 Publish Tab 43. Select the TestExp.CHP file (the last file), right-click and select delete or select Publish → Delete from the menu bar. The confirmation message to delete appears (Figure A.58). Figure A.58 Confirmation to delete selected Publish data 44. Click Yes. A message appears informing that the file is being deleted. The deleted .chp is removed from the data tree. 45. If the experiment remains in the process database, the data can be republished. 528 Affymetrix® GeneChip® Operating Software User’s Guide PROCESS DATABASE DATA DELETION 46. To delete data from the process database, select the Process tab. 47. Expand the Expression Samples list. 48. Expand MySample. 49. Right-click TestExp and select Delete from the shortcut menu that appears; or Click TestExp and select Process → Delete from the menu bar. A confirmation window appears (Figure A.59). Figure A.59 Confirmation to delete specific experiment selected 50. Click Yes. After deletion, the experiment is removed from the list. 51. Client testing completed. Appendix B Installing Data Mining Tool 3.1 Appendix B 531 Installing Data Mining Tool 3.1 The installation of Data Mining Tool 3.1 does not remove any previous versions of Data Mining Tool. If the DMT is installed in the same directory, it overwrites the previous versions’ files. Data Mining Tool 3.1 This section guides you through the installation of Data Mining Tool 3.1. Listed below is an overview of the steps needed to complete the installation: MICROSOFT® SQL SERVER GCOS SERVER USERS Obtain the name of the GCOS server from your IT personnel if not known (this is needed during installation). 2. Install Data Mining Tool 3.1. 1. ORACLE® GCOS SERVER USERS Install Oracle® Client Utilities on the workstation (Oracle Client Utilities must be the same version installed on the GCOS server). 2. Install SQL* Loader (for better performance). 3. Create an Oracle® Alias. For more information, refer to the Affymetrix® GeneChip® Operating Software Server Installation and Administration Guide. 4. Install Data Mining Tool 3.1. The following are detailed installation instructions for installing DMT. 1. The screen captures depicted in this section may not exactly match the windows displayed on your screen. You must be logged in as administrator to install the DMT 3.1 software. 532 Affymetrix® GeneChip® Operating Software User’s Guide To install DMT: Log in as an administrator. 2. Insert the Affymetrix® DMT 3.1 CD-ROM. 3. If the autorun feature does not start the program: 1. Click Start → Run. B. Type <cd drive letter>:\setup.exe. C. Click OK. The Affymetrix Software Setup window appears, followed by the Welcome window (Figure B.1). A. Figure B.1 Welcome window Click Next. 5. Several consecutive Software License Agreement windows appear. Review the contents in each and click Yes to accept the terms of the agreement. The Customer Information window appears (Figure B.2). 4. appendix B | Installing Data Mining Tool 3.1 533 Figure B.2 Customer Information window 6. Enter your Name, Company and Serial Number. The serial number is located on the Affymetrix® Software Product Registration card. If you do not have a serial number, contact Affymetrix Technical Support. If you are upgrading from a previous version, the Serial Number field populates automatically. 7. Click Next. The Choose Destination Location window appears (Figure B.3). 534 Affymetrix® GeneChip® Operating Software User’s Guide Figure B.3 Choose Destination Location window 8. Click Next. The Select Database Compatibility window appears (Figure B.4). appendix B | Installing Data Mining Tool 3.1 535 Figure B.4 Select Database Compatibility window 9. Select the database that DMT will connect with. - Affymetrix® GCOS Server - if connecting to a GCOS server - Affymetrix® GCOS - if connecting to the local MSDE database 10. Click Next. The Select Database Type window appears (Figure B.5). 536 Affymetrix® GeneChip® Operating Software User’s Guide Figure B.5 Select Database Type window 11. Select the database used on the GCOS server, either SQL Server or Oracle. If you do not know the type of database you are using with the GCOS server, please contact your IT personnel or DBA. 12. Click Next. The Enter Information window appears (Figure B.6). appendix B | Installing Data Mining Tool 3.1 537 Figure B.6 Enter Information windows for the Microsoft® SQL Server database (left) or the Oracle® database (right) 13. In the Enter Information window complete one of the following; - If SQL Server is selected, enter the SQL Server Name (usually the name of the GCOS server). - If Oracle® is selected, enter the Oracle Alias Name. 14. Click Next. The Start Copying Files window appears. 15. In the Start Copying Files window, verify the information and click Next. Program files are copied and the system configures the registry. Database connectivity is verified. For Oracle® systems: If a warning message regarding SQL Loader appears, continue the DMT install until complete. Then, install SQL Loader (part of Oracle) for better DMT performance. After SQL Loader is installed, re-install DMT. The Setup Complete window appears. 538 Affymetrix® GeneChip® Operating Software User’s Guide 16. Select Yes, I want to restart my computer now and click Finish. Appendix C GCOS Instrument Installation Appendix C 541 GCOS Instrument Installation The instrument installation needs to be done only on an instrument system. The instrument installation needs to be run if there has been a change to any of the drivers, otherwise upgrading the software to GCOS is the only installation upgrade required. The instrument installation is for Microsoft® Windows® 2000 Workstation only. This appendix contains the following sections: • The Affymetrix® Scanner 3000 with Fluidics Station Installation (see below) • Fluidics Station Installation (only) (see page 548) • GeneChip® Scanner 3000 Installation (only) (see page 553) For information on installing and using the GeneChip Scanner 3000 with Autoloader, see Appendix L, Using the GeneChip® AutoLoader, on page 731. The Affymetrix® Scanner 3000 with Fluidics Station Installation Follow this section if you have the GeneChip® Scanner 3000. When you install the fluidics station software, confirm the presence of the correct Sealevel card. If you cannot confirm the presence of a Sealevel card, contact Affymetrix technical support. 1. On an instrument workstation that will be controlling fluidics stations, there must be a Sealevel card for communication between 542 Affymetrix® GeneChip® Operating Software User’s Guide the workstation and the fluidics stations. Review Figure C.1 to confirm that a Sealevel card is installed in your system. A single 25 pin connector 25 pin cable adaptor Figure C.1 Confirming the Sealevel card setup: note the 25 pin connector and the 25 pin cable adaptor Launch Microsoft® Windows® 2000 Explorer. 3. Browse to the Instrument CD. 4. Double-click setup.exe within the Instrument CD. The Welcome window appears. 2. Click Next. 6. Several consecutive Software License Agreement windows appear. Click Yes in each window to accept the terms of the agreement. The Choose Destination Location window appears (Figure C.2). 5. appendix C | GCOS Instrument Installation 543 Figure C.2 Choose Destination Location window 7. Click Browse and select the Destination to install the instrument driver (select the same location where you installed GCOS). 8. Click Next. The Select Components window appears (Figure C.3). 544 Affymetrix® GeneChip® Operating Software User’s Guide Figure C.3 Select both the Scanner Files and Fluidics Station and Sealevel card files 9. Select both the Affymetrix Scanner Files option and the Fluidics Station Files option (Figure C.3). Highlight the Affymetrix Scanner Files option and click Change... to select the type of scanner. The following dialog box opens and displays the choice of scanners, Affymetrix® GCS 3000 Scanner and GeneArray® 2500 Scanner (Figure C.4). appendix C | GCOS Instrument Installation 545 Figure C.4 Select the Affymetrix scanner. Select one scanner but not both. the Affymetrix® GCS 3000 Scanner and click Continue. If you click Continue with both scanners selected, the software will display the following message. 10. Select 11. Click Next and enter the Port # for the COM serial port for the Sealevel serial card. Enter 2 (Figure C.5). If the workstation is the Dell GX110, select COM Port 3. 546 Affymetrix® GeneChip® Operating Software User’s Guide Figure C.5 Select the Port Number for the Sealevel card. 12. Click Next. The Start Copying Files window appears (Figure C.6). This is a summary of the information selected. appendix C | GCOS Instrument Installation 547 Figure C.6 Start Copying Files Window 13. Review the information and click Next to continue. Program files and device drivers are copied to your system, and the Setup Complete window then appears (Figure C.7). 548 Affymetrix® GeneChip® Operating Software User’s Guide Figure C.7 Install Complete window 14. Click Finish. Fluidics Station Installation (only) When you install the fluidics station software, confirm the presence of the correct Sealevel card. If you cannot confirm the presence of a Sealevel card, contact Affymetrix technical support. 1. On an instrument workstation that will be controlling fluidics stations, there must be a Sealevel card for communication between the workstation and the fluidics stations. Review Figure C.8 to confirm that a Sealevel card is installed in your system. appendix C | GCOS Instrument Installation 549 A single 25 pin connector 25 pin cable adaptor Figure C.8 Confirming the Sealevel card setup: note the 25 pin connector and the 25 pin cable adaptor 2. 3. 4. 5. 6. Launch Microsoft® Windows® 2000 Explorer. Browse to the Instrument CD. Double-click setup.exe within the Instrument CD. The Welcome window appears. Click Next. Several consecutive Software License Agreement windows appear. Click Yes in each window to accept the terms of the agreement. The Choose Destination Location window appears (Figure C.9). 550 Affymetrix® GeneChip® Operating Software User’s Guide Figure C.9 Choose Destination Location window Click Browse and select the Destination to install the instrument driver (select the same location where you installed Microarray Suite). 8. Click Next. The Select Components window appears (Figure C.10). 7. appendix C | GCOS Instrument Installation 551 Figure C.10 Select only the Fluidic Station and Sealevel card files. Select the Fluidics Station Files option (Figure C.10). This should be the only option selected. 10. Click Next. 11. Enter the Port # for the COM serial port for the Sealevel serial card. Enter 2 (Figure C.11). 9. If the workstation is the GX110, GX260 or GX270, select COM Port 3. 552 Affymetrix® GeneChip® Operating Software User’s Guide Figure C.11 Select the Port Number (Sealevel card only) 12. Click Next. The Start Copying Files window appears (Figure C.12). This is a summary of the information that you selected. Figure C.12 Start Copying Files Window appendix C | GCOS Instrument Installation 553 13. Review the information and click Next to continue. Program files and device drivers are copied to your system, and the Install Complete window appears (Figure C.13). Figure C.13 Install Complete window 14. Click Finish. GeneChip® Scanner 3000 Installation (only) The instrument installation is for Microsoft® Windows® 2000 Workstation only. Launch Microsoft® Windows® 2000 Explorer. 2. Browse to the Instrument CD. 3. Double-click setup.exe within the Instrument CD. The Welcome window appears. 4. Click Next. 1. 554 5. Affymetrix® GeneChip® Operating Software User’s Guide Several consecutive Software License Agreement windows appear. Click Yes in each window to accept the terms of the agreement. The Choose Destination Location window appears (Figure C.14). Figure C.14 Choose Destination Location window Click Browse and select the Destination to install the instrument driver (select the same location where you installed Microarray Suite). 7. Click Next. The Select Components window appears (Figure C.15). 6. appendix C | GCOS Instrument Installation 555 Figure C.15 Select Affymetrix Scanner files only for scanner installation 8. Select only the Affymetrix Scanner Files option. Highlight the Affymetrix Scanner Files option and click Change... to select the type of scanner. The following dialog box opens and displays the choice of scanners, Affymetrix or GeneArray® (Figure C.16). 556 Affymetrix® GeneChip® Operating Software User’s Guide Figure C.16 Select the Affymetrix scanner. Select one scanner but not both. 9. Select the Affymetrix Scanner files and click Continue. If you click Continue with both scanners selected, the software will display the following message. 10. Click Next. The Start Copying Files window appears (Figure C.17). This is a summary of the information that you selected. appendix C | GCOS Instrument Installation Figure C.17 Start Copying Files Window 11. Review the information and click Next to continue. The Install Complete window appears (Figure C.18). 557 558 Affymetrix® GeneChip® Operating Software User’s Guide Figure C.18 Install Complete window 12. Click Finish. Appendix D Using the GeneChip® Scanner 3000 Appendix D 561 Using the GeneChip® Scanner 3000 Affymetrix has designed a new scanner expressly for scanning GeneChip® probe arrays. The GeneChip® Scanner 3000 requires little maintenance. The instrument must be kept clean and free of dust. Dust buildup can degrade performance. Wipe the exterior surfaces clean using a mild dish detergent solution in water. Do not use ammonia based cleaners or organic solvents, such as alcohol or acetone, to clean the system because they may damage the exterior surfaces. Figure D.1 The Affymetrix® GeneChip® Scanner 3000 Do not remove the cover of the scanner. Use the scanner only as instructed in this User Guide. Do not attempt to service the instrument. 562 Affymetrix® GeneChip® Operating Software User’s Guide This appendix contains the following sections: • Introduction (see below) • Safe Operation (see page 564) • Connections and Indicator Lights (see page 569) • Using the Barcode Reader (see page 573) • Scanner 3000 Specifications (see page 576) • Troubleshooting (see page 577) • CE Mark Declaration of Conformity (see page 583) • Regulatory (see page 584) Introduction The Affymetrix® GeneChip® Scanner 3000 is a wide-field, epifluorescent, near-confocal microscope. The scanner uses a 532 nm solid-state laser to excite probe array fluorophores. This in turn produces an emission wavelength appropriate for the probe array being scanned, which is automatically specified in the scan parameters for the selected probe array. As the surface of the probe array is scanned, a photomultiplier tube collects and converts the fluorescent emissions into an electrical signal. An analog-digital converter in the scanner converts this signal into corresponding numeric values representative of fluorescent intensities. These digital intensity values are collected from discrete areas on the array surface and are stored on the computer workstation as pixels that comprise the image data file (the .dat file). For more information on image data, see Chapter 9, Working with Images, on page 141.Affymetrix’ patented Flying Objective™ technology represents a radical departure from conventional laser scanners. The optical system comprises a scan arm that rapidly oscillates from side to side scanning the entire width of the probe array in a continuous arc while the probe array is advanced in front of the objective. The acquired image of the array is returned to the computer software as a set of arcs. The software then geometrically corrects these arcs to form a linear image of the array (Figure D.2). appendix D | Using the GeneChip® Scanner 3000 563 Figure D.2 Schematic of the scanner design The laser source excites the hybridized fluorophores and the photomultiplier system simultaneously captures the resulting fluorescent intensities. The optical components direct the fluorescent beam back through the objective lens, through a dichroic mirror and to the PMT. An analog-digital converter transforms the PMT output into 65536 levels of intensity. Each level of intensity is stored in the software as a 16 bit number (216=65536). The scanner is equipped with an IEC 320 compliant power entry module located at the rear of scanner (Figure D.3). The scanner is equipped with an RJ-45 interface connector compatible with 10/100 Base T Ethernet for communications with the host workstation. 564 Affymetrix® GeneChip® Operating Software User’s Guide The GCOS application controls the scanner. The scan process times for each type of GeneChip® probe array are outlined in the next table. Affymetrix measures the process time from the “Start Scan” command to the return of the cartridge to the instrument entrance port along with the display of the completed scan image on the workstation monitor. Probe Array Type Process Time (typical) 49 5min 15sec 64 4min 30sec 100 3min 30sec 169 3min 30sec 400 2min 45sec After the scanner has completed a scan, GCOS displays a picture of the image in the image window. The software displays the fluorescent intensity values from each pixel within the probe array feature in a grayscale or pseudocolor mode and superimposes a grid on the image to delineate the probe cells. GCOS analyzes the image and derives a single intensity value for each probe cell on an array. This data is automatically generated and saved to the cell intensity file. See Chapter 9, Working with Images, on page 141. The file containing the completed analysis of the probe .cel files to provide the appropriate present, absent or marginal calls. Safe Operation To ensure safe operation of the GeneChip® Scanner 3000: • Read this section and Scanning a Probe Array–GeneChip® Scanner 3000, on page 121 completely before operating the instrument. appendix D | Using the GeneChip® Scanner 3000 565 • Do not attempt to service this instrument. Any attempt at unauthorized service may damage the instrument and/or void the warranty. • The instrument weight is approximately 63 pounds (28.6 Kg). Do not place it on an unstable cart, stand, or table. Failure to properly support the instrument may cause serious damage or injury and may void the warranty. Heavy object. Two people are required to lift the scanner. • The instrument must be surrounded by adequate airspace. Slots and openings in the instrument and the electronics compartment covers are for ventilation. Do not block or cover them. • Never push an object into the instrument ventilation slots; equipment damage or injury may result. Do not set liquids on top of the instrument. • The instrument has an AC receptacle with a safety ground appropriate for the country of destination. The plug is designed to connect only to a 3-prong ground receptacle. This safety feature should not be compromised in any way. If the instrument AC plug does not mate with the available power source receptacle, consult a licensed electrician to install one that does. When to Contact Affymetrix Under any of the following conditions, unplug the instrument from the power source and contact technical Support: • When the power cord is damaged or frayed. • If any liquid has been spilled into the instrument. • If the instrument has been penetrated by water. • If, after service or calibration, the instrument does not perform in accordance with the capabilities stated in the specifications. 566 Affymetrix® GeneChip® Operating Software User’s Guide • If the instrument has been dropped or otherwise damaged. If the instrument must be returned for repair, call Affymetrix® Technical Support. Do not remove the cover of the scanner. Use the scanner only as instructed in this User Guide. Do not attempt to service the instrument. Only qualified service technicians can open and service the scanner. There are no customer serviceable parts. Removing the case exposes the customer to laser and electrical shock hazards. You can learn more about safety in the following sections: • Laser Safety (see below) • Electrical Safety (see page 567) LASER SAFETY The laser is equipped with an automatic shutter that inhibits its output beam and ensures safe operation under conditions encountered in normal operation. The instrument covers, probe array cartridge access port, and protective shutters ensure that during instrument operation no directed or stray laser light leaves the instrument. The GeneChip scanner 3000 is a Class I laser product when the laser is enclosed in scanner case. The laser itself is a Class IIIB laser product. appendix D | Using the GeneChip® Scanner 3000 567 DANGER Laser radiation when open. Avoid direct exposure to beam. The lasers can cause serious injury if the instrument is not operated in accordance with instructions in this user guide. CAUTION Use laser safety glasses when servicing DO NOT STARE INTO BEAM. The green laser is a 532nm solid-state laser. This is a Class IIIb laser and has visible outputs greater than 5mw but no more than 500mw. It must never be operated in an exposed manner. Any object in the direct path of the laser beam may be damaged. Eyes and skin can be seriously damaged by direct exposure to, specular reflections from, or diffuse reflections from this laser. If improperly used, a laser of this type can cause fires. When used according to the instructions in this manual and when all covers are in place, the GeneChip® Scanner 3000 is classified as a Class I Laser Device per 21 CFR 1040. Always take note of laser safety labels; they indicate areas where exposure to laser beams may be hazardous. ELECTRICAL SAFETY The scanner will automatically handle any input voltage from 100 to 240 VAC nominal, 50 to 60 Hz 568 Affymetrix® GeneChip® Operating Software User’s Guide The scanner’s power supply will autodetect the input voltage source and configure itself. CAUTION CAUTION The power supply cord is used as the main disconnect device. Ensure that the socket outlet is located and installed near the equipment and is easily accessible. ATTENTION Le cordon d’alimentation est utilisé comme interrupteur general. La prise de courant doit être située ou installée a proximité du materiel et être facile d’accés. ACHTUNG Zur sicheren Trennung des Gerätes vom Netz ist der Netzstecker zu ziehen. Vergewissern Sie sich, daß die Steckdose leicht zugänglich ist. Maintenance The GeneChip® Scanner 3000 requires little maintenance. The instrument must be kept clean and free of dust. Dust buildup can degrade performance. Wipe the exterior surfaces clean using a mild dish detergent solution in water. Do not use ammonia based cleaners or organic solvents, such as alcohol or acetone, to clean the system because they may damage the exterior surfaces. appendix D | Using the GeneChip® Scanner 3000 569 Connections and Indicator Lights The GeneChip® Scanner 3000 has the following connections on the back of the unit (Figure D.3): • AC power connection • Ethernet connection If you must move the scanner, disconnect all cables first. You can learn more about the connections and lights in: • Connecting the Scanner (see below) • Indicator Lights and On/Off Button (see page 570) CONNECTING THE SCANNER Connect the 3-pronged electric power plug to the workstation. 2. Connect the dedicated Ethernet cable from the scanner to the Ethernet port that is designated on the workstation. The port is located near the bottom of the workstation. 3. You can connect your company’s network cable to the indicated port near the center of the workstation. When you start GCOS, the software will make the proper communication connections. 1. Do not confuse your company’s network connections with the dedicated Ethernet port of the scanner-workstation. The proper scanner connection is located near the bottom of the workstation. This 10/100 Base T Ethernet communications port is dedicated to the scanner-workstation interface. You cannot connect the scanner to your company’s Ethernet communications network. You can, however, connect the workstation’s second Ethernet port to the your company’s Ethernet network. 570 Affymetrix® GeneChip® Operating Software User’s Guide Figure D.3 Scanner Connections- left: rear of scanner -right: rear of computer The reset button is the scanner’s circuit breaker. The breaker switch will be tripped whenever the scanner experiences an electrical fault condition. Press to reset. If you cannot reset this switch, contact Affymetrix technical support. INDICATOR LIGHTS AND ON/OFF BUTTON The front panel has the following button and indicators (Figure D.4). appendix D | Using the GeneChip® Scanner 3000 571 Blue Indicator Light Yellow light Green light I/O (on/off) button Figure D.4 Scanner indicator lights and on/off (I/O) button I/O (on/off) button in the center. 2. Blue indicator light, running vertical at front center, indicates that the scanner is on. 3. Green and yellow light both on indicate scanner boot up in progress. 1. 4. Yellow light 572 Affymetrix® GeneChip® Operating Software User’s Guide On = Idle, laser is warming up (laser not ready, green off) B. Off = System ready, no errors (Green on) C. Flashing = Error 5. Green light A. A. On = System is ready to scan (yellow off) B. Flashing = Scan in progress Summary of Indicator Lights The table below summarizes the light conditions and their meaning. Table D.1 Indicator lights Condition Green Light Yellow Light Blue Indicator Light Meaning Initial boot up Off Off On Initial power up; embedded PC takes control Scanner boot up On On On Embedded PC takes control of scanner boot up Laser warm up Off On On Software enabled and laser is warming up System ready On Off On User communication with scanner enabled Error Off Flashing On Fatal error, reboot scanner and software Scanning enabled Flashing Off On Scanning in progress appendix D | Using the GeneChip® Scanner 3000 573 During the initial power up (CPU boot up), both the green and yellow lights will be off. During the scanner boot up, both the green and yellow lights will be on. Using the Barcode Reader If your scanner is equipped with a barcode reader, follow this section for instructions on its use. 1. Open the GCOS software to the Experiment Information window and place the cursor in the barcode reference field of the GCOS Experiment Information window (Figure D.5). You should open a new experiment and place the cursor into the barcode field before triggering the reader. Otherwise, the barcode will be misinterpreted as some other kind of input. You are required to open a new experiment for each barcode and must enter certain other information before the experiment can be saved. See Table L.4 on page 759 for a summary of information that you can enter in the Experiment Information Window. 574 Affymetrix® GeneChip® Operating Software User’s Guide Figure D.5 Experiment Information Window with cursor in the barcode field 2. Hold a GeneChip probe array cartridge in front of the barcode reader and squeeze the trigger for approximately four seconds until you hear a beep (Figure D.6). appendix D | Using the GeneChip® Scanner 3000 575 Figure D.6 Reading the cartridge barcode 3. The reader reads and sends the barcode to the GCOS Experiment Information window, Barcode field (Figure D.7). 576 Affymetrix® GeneChip® Operating Software User’s Guide Figure D.7 Experiment Information Window with barcode After the software adds the barcode, save the experiment. 5. Repeat steps 1 to 4 until all of the probe array cartridges have been read. 4. Scanner 3000 Specifications Item Weight Dimensions Parameter Value Shipping approx 78 pounds (35.4 Kg) Free-standing 63 pounds (28.6 Kg) Width ~13.25 in. Depth ~27.5 in. Height ~18.25 in. appendix D | Using the GeneChip® Scanner 3000 Item Parameter 577 Value Power Voltage Current Line Frequency 100 - 240 V ~ 4-2A 50 - 60 Hz Working Environment Temperature 59°F-85°F (15°C-30°C) Humidity 10-90% Non-condensing Clearance 2 in. (5 cm) on side, back and top Pollution Degree 2 Installation Category II Altitude <2000m Electrical Supply Provide voltage, frequency or power rating per unit label Main Supply Voltage Fluctuations Are not to exceed ±10% of the nominal supply voltage If you use the scanner in a manner not specified in this user guide, you may impair the protection provided by the equipment. Troubleshooting Problem No image when scanning Intermittent problems scanning Possible Cause Corrective Action Power off or cable loose Check all connections and power. Loss of laser power Contact technical support. Loose cable Check all rear connections. 578 Affymetrix® GeneChip® Operating Software User’s Guide Problem Scanner fails with probe array inside Possible Cause Power failure Corrective Action Manually extract probe array. Check all connections to scanner. Turn scanner on, restart software. You can learn more about troubleshooting in: • Issues Relating to the Scanner’s Operation (see below) • Manually Removing a Lodged Probe Array Cartridge (see page 579) ISSUES RELATING TO THE SCANNER’S OPERATION The GeneChip® Scanner 3000 is a new type of scanner. In using the scanner you may encounter some issues, or problems, that may require your intervention. Below is a list of these issues. Issue Explanation If communications are interrupted during a scan (by a faulty cable connection or power being lost at the scanner, for example) GCOS will properly note the failure and present a message “Cannot connect to Scanner.” However, there are two issues to note. First, GCOS will report such a failure only after a network time-out of about 30 seconds. Second, rarely, if communications have been lost, GCOS and the Scanner may not be able to automatically restore communications once the problem is rectified, and both may become unresponsive. To restore proper operation, verify that the scanner is on, that communication cables are properly connected, and close and restart Microsoft® Windows® then restart GCOS. If the system remains unresponsive, disconnect and reconnect power to the scanner, restart the scanner normally, close and restart Microsoft® Windows® and GCOS. Repeated attempts to send If communications cannot be re-established, please follow the commands (Start, Turn Laser recommendations of item 1 On, etc.) from GCOS to the Scanner while GCOS is reporting the scanner “Offline” may result in GCOS becoming unresponsive until communications are restored appendix D | Using the GeneChip® Scanner 3000 Issue 579 Explanation If the Scanner experiences multiple auto-focus failures, the system may enter an unresponsive state. Follow the recommendations of item 1 to restore communications and correct operation. Laser warm-up lasts for ten minutes, during which time the “Turn Laser On” button will remain unchanged and GCOS will display the status message “Warm-up”. Simply note that this is normal operation. Occasionally, when Reducing the graphics acceleration parameter in Microsoft® Windows® observing DAT images in desktop Properties will eliminate this problem. GCOS, the user may notice, at specific zoom levels, that grid lines do not properly appear. If no chip is inserted and a scan started. The scanner will attempt go through the first parts of the auto-focus routine and then report “Failed to find chrome border”. The scanner should be in the park mode to eject the chip. Auto focus will fail if salt deposits accumulate on the array. Use Tough-Spots® to prevent leaks in the GeneChip® probe array. See the quick reference card, p/n 08-0076. MANUALLY REMOVING A LODGED PROBE ARRAY CARTRIDGE In the event that a probe array becomes lodged in the chip transport mechanism, follow the procedure outlined below. 580 Affymetrix® GeneChip® Operating Software User’s Guide 1. 2. Turn off the scanner. Insert a paper clip or small Allen wrench into the rescue hole on top of the scanner and press to partially lift the cartridge loading door. 3. Using your fingers, gently lift the front edge of the door. As you lift the front edge, lift the back edge approximately 1/4” to open the door straight up to expose the rescue screw in the front. appendix D | Using the GeneChip® Scanner 3000 4. 5. 581 Using a standard (-) screwdriver, turn the rescue screw clockwise to raise the chip transport mechanism. Continue to turn the screw until the probe array cartridge ascends sufficiently to grab it. Note that the screw is fine pitched and requires a number of turns. Stop if you encounter screw resistance. Do not over torque. 582 6. 7. 8. When the cartridge has ascended sufficiently, remove it. Rescrew the chip transport mechanism until it descends completely, or until you encounter resistance. Do not over torque. Close the door. Affymetrix® GeneChip® Operating Software User’s Guide appendix D | Using the GeneChip® Scanner 3000 CE Mark Declaration of Conformity We, Affymetrix, Inc. 4G Crosby Drive, Bedford, Massachusetts Declare under sole responsibility that the Affymetrix® GeneChip® Scanner 3000 conforms with the relevant provisions of the following standards or other normative documents: EU EMC Directive 89/336/EEC: EN 61326:1998 Equipment for Measurement, Control and Laboratory Use, EMC Requirements EN 55011:1998 Industrial, scientific and medical (ISM) radio-frequency equipment Radio disturbance characteristics - Limits and methods of measurement EN 61000-3-2:2001 Limits for harmonic current emissions (equipment input current up to and including 16 A per phase) EN 61000-3-3:1995 Limitation of voltage changes, voltage fluctuations and flicker in public low-voltage supply systems, for equipment with rated current less than or equal to 16 A per phase and not subject to conditional connection EN 61000-4-2:1995 Electrostatic discharge immunity. EN 61000-4-3:1995 Radiated, radio frequency, electromagnetic field immunity. EN 61000-4-4:1988 Electrical fast transient/burst immunity. EN 61000-4-5:1995 Surge immunity. EN 61000-4-6:1996 Immunity to conducted disturbances induced by radio frequency fields. EN 61000-4-11:1994 Voltage dips, short interruptions, and voltage variations immunity. EU Low Voltage Directive 73/23/EEC EN 61010-1:2001 Safety requirements for electrical equipment for measurement, control, and laboratory use -- Part 1: General requirements EN 60825-1:1994+ A2:2001 Safety of laser products -- Part 1: Equipment classification, requirements and user's guide 583 584 Affymetrix® GeneChip® Operating Software User’s Guide Regulatory This device complies with Part 15, Subpart B, Class B of the FCC rules. Operation is subject to the following two conditions: (1) This device may not cause harmful interference, and (2) this device must accept any interference received, including interference that may cause undesirable operation. This device complies with FDA performance standards for laser products except for deviations pursuant to Laser Notice No. 50, dated July 26, 2001. Regulatory Agency Certification AL 02 12 39816 004 92AA See CE Mark Declaration of Conformity, on page 583. Compliant with directive 2002/96/IC (WEEE) Class I Laser Device 21 CFR 1040.10 and 1040.11 For Research Use Only. Not for use in diagnostic procedures. Appendix E Configuring the GCOS Services Appendix E 587 Configuring the GCOS Services GCOS provides services that help you automate your data management: • GCOS Analysis Service performs cell intensity analyses. If you are using GCOS with a networked GCOS Server, you can run the Analysis Service on the workstation or the server. • GCOS Transfer Service lets you automate the archiving of DAT files. The GCOS Transfer Service cannot be used when working with a GCOS Server and when analysis is set to be done on the remote server. Use GCOS Manager to archive the data. This appendix describes: • The requirements for using the Analysis and Transfer services (see below) • How to configure the GCOS Service settings (see page 589) • How to select the GCOS Service options (see page 597) • How to set the Start/Stop Service policies so that the services will run (see page 601) • How to set the Act as Part of the Operating System policies so that the services will run (see page 612) Requirements for Using the GCOS Services In order to use the GCOS Analysis and Transfer services you will need to: • Have a valid domain user and password set up with the necessary privileges (see Domain User Requirements, below). • For analysis on the client: The service has to be configured on the client to run as: (i) a user in the GCOS administrator role on GCOS server. 588 Affymetrix® GeneChip® Operating Software User’s Guide (ii) with privileges to Start/Stop services as part of the user's local system policies, i.e., has the “Log on as service” privilege. • For analysis on the server: The service has to be configured on the GCOS Server. The service has to be configured on the server to run as: (i) a user in the GCOS administrator role on GCOS server. (ii) with privileges to Start/Stop services as part of the user's local system policies, i.e., has the “Log on as service” privilege. • For archiving data to a Universal Naming Convention (UNC) location, the transfer service has to be configured to run as a user that has access to the UNC path. By default, the installation sets the services to run under the local system account. This default is suitable for access to local machine resources only and will prevent the data transfer and analysis services from accessing UNC paths and other network resources. On a GCOS Server, the services should not be configured to run under the SYSTEM account if file security has been turned ON or if the GCOS client workstation has Windows XP Service Pack 2. If a client workstation has Windows XP Service Pack2, then the GCOS Services on the GCOS server should be configured to run as an authenticated domain user. If the service runs under the local SYSTEM account then, the GCOS client will fail to receive status messages about completion of CEL file generation after completion of the scan. DOMAIN USER REQUIREMENTS If you wish to allow the transfer and analysis service to access network resources and/or GCOS Server resources, you need to supply a valid domain user and password that meets the following requirements: • Has privileges sufficient to access the network resources (shares, folders, files), that all users on the machine will require appendix E | Configuring the GCOS Services 589 • Has privileges sufficient to access the process database objects and be a member of a role that will allow access for all users on the machine, when the machine is acting as the client of a GCOS Server • Has privileges to Start/Stop services as part of the user's local system policies. • On a Windows 2000 client, the user should also be part of the “Act as local operating system” policy. For more information, see Setting the Act As Part of the Operating System Policy, on page 612. A user who is in the 'Administrator' role on the server meets these requirements. Configuring the GCOS Service Settings After you have set up the domain user with the necessary privileges and policies, you need to set the GCOS Service settings. The analysis and transfer service use the same settings. Any change to one service is applied automatically to the other service. 590 Affymetrix® GeneChip® Operating Software User’s Guide Start GCOS UI and open Defaults dialog box Is Client configured to work with server? Yes Does user want to analyze CEL on local workstation? No Go to the server and change analysis Svc settings No Yes No Run as System Does user want to archive to UNC path? Yes Enter a domain user who has access to UNC On the client open dialog to change Svc settings If archiving, enter a domain user who has access to UNC Figure E.1 Flow chart for setting the GCOS Service Options You can configure the settings for: • A stand-alone workstation system (see below) • A system configured to work with a GCOS Server (see page 593) • A GCOS Server (see page 595) Refer to the flow chart (Figure E.1) for an overview of the GCOS services options. appendix E | Configuring the GCOS Services 591 CONFIGURING THE GCOS SERVICE SETTINGS ON A STAND ALONE SYSTEM (NO GCOS SERVER) To set the GCOS Service Setting on a stand-alone workstation (i.e., GCOS Server name is empty in the Database tab of the Defaults dialog box): 1. Click the Defaults button in the shortcut window; Or select Tools → Defaults from the menu bar. The Defaults dialog box opens (Figure E.2). Figure E.2 Defaults dialog box, Analysis Settings tab Click the Archive Settings or Analysis Settings tab of the dialog box. 3. Click the GCOS Service Settings button . The GCOS Service Settings dialog box appears (Figure E.3). 2. 592 Affymetrix® GeneChip® Operating Software User’s Guide Figure E.3 GCOS Service Settings dialog box The dialog box has the following controls: 4. Domain Domain on the network to which the GCOS Server is assigned. User The network user ID assigned to the user. Password User’s password. Run As Local System When this is selected, the service will run under the NT SYSTEM account. Apply Applies the entered changes. Cancel Closes the dialog box without applying the changes. Select Run As Local System to use the databases installed on the workstation; The service can run under the Microsoft® Windows® SYSTEM account if archiving is OFF. If archiving is ON, the service must be configured to run under the user credential that has access to the UNC path where data is to be archived. Select Run as Local System if you do not plan on auto-archiving DAT files immediately after scan to a networked drive. appendix E | Configuring the GCOS Services 5. 593 Click Apply. The selected options are applied. CONFIGURING THE GCOS SERVICE SETTINGS ON A CLIENT CONFIGURED TO WORK WITH GCOS SERVER To set the GCOS Service Settings on a client configured with GCOS Server: 1. Click the Defaults button in the shortcut window or select Tools → Defaults from the menu bar. The Defaults dialog box opens (Figure E.4). Figure E.4 Defaults dialog box, Analysis Settings tab 2. Click the Archive Settings or Analysis Settings tab of the dialog box. 3. Click the GCOS Service Settings button The GCOS Service Settings dialog box appears (Figure E.5). . 594 Affymetrix® GeneChip® Operating Software User’s Guide Figure E.5 GCOS Service Settings dialog box The dialog box controls are described on The dialog box has the following controls: (see page 592). 4. Select the Domain/User you wish to use from the drop-down list and enter the password for that user. The analysis /transfer service must be reconfigured to run as a user who has admin rights on the local machine and is in the GCOS Administrator's role on the GCOS Server. The list is populated automatically with a list of users who have the above mentioned rights. For more information about the requirements for the User, see Requirements for Using the GCOS Services, on page 587. 5. Click Apply. The selected options are applied. If you see the following error message (Figure E.6): appendix E | Configuring the GCOS Services 595 Figure E.6 Error message Review the requirements for using the GCOS Services (see page 587). B. Change the Start/Stop Services policies and other settings on your system as necessary (see page 601). A. CONFIGURING THE GCOS SERVICE SETTINGS ON A GCOS SERVER After you have set up the domain user with the necessary privileges and policies, you need to set the GCOS Service settings. To set the GCOS Service Settings on the GCOS Server: 1. Click the Microsoft® Windows® Start button Settings → Control Panels. The Control Panel window opens (Figure E.7). , then select 596 Affymetrix® GeneChip® Operating Software User’s Guide Figure E.7 Control Panel 2. Double-click the GeneChip Analysis and Transfer Icon. The GCOS Service Settings dialog box opens (Figure E.8). Figure E.8 GCOS Service Settings dialog box The dialog box controls are described on (see page 592). appendix E | Configuring the GCOS Services 3. 597 Select the Domain/User you wish to use from the drop-down list and enter the password for that user. If file security is ON, the service has to run under a user who has admin rights on the GCOS Server and is in the GCOS Administrator's role on the GCOS Server. The list is populated automatically with a list of users who have the above mentioned rights. For more information about the requirements for the User, see Requirements for Using the GCOS Services, on page 587. 4. Click Apply. The selected options are applied. Selecting the GCOS Services Options After you have configured the GCOS Service settings, you can select the GCOS options to: • Perform CEL Intensity analyses on the workstation or GCOS Server using the Analysis Service (see below). • Archive the DAT files after scanning using the Transfer Service (see page 599). The GCOS Transfer Service cannot be used when working with a GCOS Server and when analysis is set to be done on the remote server. Use GCOS Manager to archive the data. Any changes applied to the transfer service will be applied automatically to the analysis service. 598 Affymetrix® GeneChip® Operating Software User’s Guide CELL INTENSITY ANALYSIS OPTIONS If you are using GCOS with the GCOS Server, you can perform the analysis of DAT files to produce CEL files on the GCOS Server or the instrument workstation using the GCOS Analysis Service. You must have a domain user with the proper privileges and policies set up to use the cell intensity analysis options. For more information about the requirements, see Requirements for Using the GCOS Services, on page 587. To change the Analysis Service options: In the Settings shortcut bar, click the Defaults button Select Tools → Defaults from the menu bar. The Defaults dialog box appears. 2. Click the Analysis Settings tab (Figure E.9). 1. Cell intensity analysis options Figure E.9 Defaults dialog box, Analysis Settings tab ; or appendix E | Configuring the GCOS Services 599 The Analysis settings are active only when GCOS is being used with the GCOS Server option. 3. Select the desired option from the radio buttons: - Analyze intensity data on local workstation. - Analyze intensity data on GCOS server. If the analysis / transfer service on the client is running under the windows local SYSTEM account, then the “Analyze data on local workstation” radio button is disabled. Click OK. The selected changes are applied. You can track the progress of the intensity data analyses and file archiving in the CEL Analysis and Data Transfer window (see page 133). The other settings of the Analysis Settings tab are described in Analysis Settings, on page 335. 4. SETTING ARCHIVING OPTIONS FOR DAT FILES DAT files may be stored locally or to a network location using the GCOS Transfer Service. A JPG format file lets you view the image for basic QC purposes. The GCOS Transfer Service cannot be used when working with a GCOS Server and when analysis is set to be done on the remote server. Use GCOS Manager to archive the data. 600 Affymetrix® GeneChip® Operating Software User’s Guide To change the Transfer Service options: 1. In the Settings shortcut bar, click the Defaults button Select Tools → Defaults from the menu bar. The Defaults dialog box appears. 2. Click the Archive Settings (Figure E.10). ; or Figure E.10 Defaults dialog box, Archive Settings tab Select the Archive data after scan checkbox to archive the DAT files. 4. Enter a location in the Archive data location box; or Click the Browse button and select a directory for the archived files. The software will not allow a UNC network location if the transfer service is running under the local SYSTEM account. 3. If the service is configured to run as a user who does not have access to a networked location, the auto-archive will fail. appendix E | Configuring the GCOS Services 5. 601 Click OK. The selected changes are applied. Experiments that make use of automation must bypass the analysis service and data transfer service. All analysis in experiments that make use of automation must occur on the GCOS server when working in server mode. Automation can only be used in conjunction with expression arrays. You can track the progress of the intensity data analyses and file archiving in the CEL Analysis and Data Transfer window (see page 133). You can view the JPG file in the Image Window (see The Image Viewer with JPG Files, on page 217). The archived data can be unarchived and made available using the Unarchive command in GCOS Manager. For more information, see Unarchiving Data, on page 361. Setting the Start/Stop Service Policy Both the user running the control applet and the specified 'Run As' user must have Start/Stop services as part of the user's local system policies. You will see a warning when trying to configure the system if you do not have these policies set correctly (Figure E.11). 602 Affymetrix® GeneChip® Operating Software User’s Guide Figure E.11 Error message You will need to set the Start/Stopping Service policy so that your domain user is in the group with these privileges, as explained in this section. If domain-level policy settings are defined, they override the local policy settings. You will need to get the domain administrator to change the domain-level policy settings in this case. You do this in two sets of steps: Check to see if you have administrator privileges on the computer and get them added if necessary (see below). 2. Add the Administrators group to the Start/Stop Services policy (see Adding the Administrators Group to the Start/Stop Service Policy, on page 604). After you have done these steps, you can set the GCOS Service options and configure the GCOS software (see Configuring the GCOS Service Settings, on page 589). 1. CHECKING ADMINISTRATOR PRIVILEGES To check to see if you are in the administrators group on the computer: 1. Right-click on the My Computer icon and select Manage from the pop-up menu. The Computer Management window opens (Figure E.12). appendix E | Configuring the GCOS Services 603 Figure E.12 Computer Management window The window has a data tree on the left side with different objects. Some parent objects may have child objects. The left side displays a list of the child objects or other contents for the object selected in the data tree. 2. Select System Tools → Local Users and Groups → Groups in the tree. A list of groups appears on the Name box on the right side of the window (Figure E.12). 3. Click Administrators in the list. The Administrators Properties dialog box opens (Figure E.13). 604 Affymetrix® GeneChip® Operating Software User’s Guide Figure E.13 Administrators Properties dialog box 4. Make sure your domain and user name are in the Members list. If not, see your local CIS personnel to get your account added. ADDING THE ADMINISTRATORS GROUP TO THE START/STOP SERVICE POLICY After making sure that you are in the Administrators group, you can add the Administrators group to the Start/Stop Service policy. 1. Click the Microsoft® Windows® Start button select Settings → Control Panels. The Control Panels window opens (Figure E.14). , then appendix E | Configuring the GCOS Services Figure E.14 Control Panel window, Administrative Tools icon selected 2. Double-click the Administrative Tools icon. The Administrative Tools window opens (Figure E.15). 605 606 Affymetrix® GeneChip® Operating Software User’s Guide Figure E.15 Administrative Tools window 3. Double-click on the Local Security Policy icon. The Local Security Policy window opens (Figure E.16). Figure E.16 Local Security Settings window appendix E | Configuring the GCOS Services 607 The data tree on the left side of the window displays a list of objects. An object may itself have child objects. The right side of the window displays child objects or other content for the object selected in the data tree. 4. In the Security Settings tree, select Local Policies → User Rights Assignment. A list of user’s rights policies are displayed in the right-hand side of the window (Figure E.16) with the following information: Policy Unique ID for the policy. Local Setting Users or groups assigned to the local policy settings. Effective Setting Users or groups assigned to the domain-level policy settings (overrides the local policy settings). If domain-level policy settings are defined and displayed in the Effective Settings, they override the local policy settings. You will need to get the domain administrator to change the domain-level policy settings in this case. 5. Double-click on the Log On as a Service policy The Local Security Policy Setting dialog box for Log On as a Service opens (Figure E.17). 608 Affymetrix® GeneChip® Operating Software User’s Guide Figure E.17 Local Security Policy Setting dialog box This dialog box lists the users and groups assigned to that policy. If domain-level policy settings are defined, they override the local policy settings. You will need to get the domain administrator to change the domain-level policy settings in this case. 6. Click the Add button. The Select Users or Groups dialog box opens (Figure E.18). appendix E | Configuring the GCOS Services 609 Figure E.18 Select Users or Groups dialog box The Look In drop-down box displays a list of domains. The Name box displays a list of the available users and groups in the selected domain. The bottom box displays users and groups selected for adding to a policy. 7. Select the Administrators group. 8. Click the Add button in the Select Users or Groups dialog box. The Administrators group is displayed in the lower box (Figure E.19). 610 Affymetrix® GeneChip® Operating Software User’s Guide Figure E.19 Select Users or Groups, administrator’s group selected 9. Click the OK button in the Select Users or Groups dialog box. The Select Users or Groups dialog box closes, and the Administrators group is displayed in the Local Security Policy Settings dialog box (Figure E.20). appendix E | Configuring the GCOS Services 611 Figure E.20 Local Security Policy Setting, Administrators group added 10. Click the OK button on the Local Security Policy Setting dialog box. 11. The dialog box closes, and the Administrators group is displayed in the Local Security Settings window, Log on as a Service policy (Figure E.21). 612 Affymetrix® GeneChip® Operating Software User’s Guide Figure E.21 Local Security Settings dialog box, Administrators group added to Log On as a Service policy 12. Close the Local Security Settings dialog box. Setting the Act As Part of the Operating System Policy If you are installing GCOS 1.4 on a computer with the Windows 2000 operating system, you will also need to set Act as Part of the Operating System policy so that your domain user is in the group with these privileges. Both the user running the control applet and the specified 'Run As' user must have Act as Part of the Operating System services as part of the user's local system policies. You will see a warning when trying to configure the system if you do not have these policies set correctly (Figure E.22). appendix E | Configuring the GCOS Services 613 Figure E.22 Error message You will need to set the Act as Part of the Operating system policy so that your domain user is in the group with these privileges, as explained in this section. If domain-level policy settings are defined, they override the local policy settings. You will need to get the domain administrator to change the domain-level policy settings in this case. You do this in two sets of steps: Check to see if you have administrator privileges on the computer and get them added if necessary (see below). 2. Add the Administrators group to the Act as Part of the Operating System policy (see Setting the Act As Part of the Operating System Policy, on page 612 After you have done these steps, you can set the GCOS Service options and configure the GCOS software (see Configuring the GCOS Service Settings, on page 589). 1. CHECKING ADMINISTRATOR PRIVILEGES To check to see if you are in the administrators group on the computer: 1. Right-click on the My Computer icon and select Manage from the pop-up menu. The Computer Management window opens (Figure E.23). 614 Affymetrix® GeneChip® Operating Software User’s Guide Figure E.23 Computer Management window The window has a data tree on the left side with different objects. Some parent objects may have child objects. The left side displays a list of the child objects or other contents for the object selected in the data tree. 2. Select System Tools → Local Users and Groups → Groups in the tree. A list of groups appears on the Name box on the right side of the window (Figure E.23). 3. Click Administrators in the list. The Administrators Properties dialog box opens (Figure E.24). appendix E | Configuring the GCOS Services 615 Figure E.24 Administrators Properties dialog box 4. Make sure your domain and user name are in the Members list. If not, see your local CIS personnel to get your account added. ADDING THE ADMINISTRATORS GROUP TO THE ACT AS PART OF THE OPERATING SYSTEM POLICY (WINDOWS 2000 ONLY) After making sure that you are in the Administrators group, you can add the Administrators group to the Act as Part of the Operating System policy. 1. Click the Microsoft® Windows® Start button select Settings → Control Panels. The Control Panels window opens (Figure E.25). , then 616 Affymetrix® GeneChip® Operating Software User’s Guide Figure E.25 Control Panel window, Administrative Tools icon selected 2. Double-click the Administrative Tools icon. The Administrative Tools window opens (Figure E.26). appendix E | Configuring the GCOS Services Figure E.26 Administrative Tools window 3. Double-click on the Local Security Policy icon. The Local Security Policy window opens (Figure E.27). Figure E.27 Local Security Settings window 617 618 Affymetrix® GeneChip® Operating Software User’s Guide The data tree on the left side of the window displays a list of objects. An object may itself have child objects. The right side of the window displays child objects or other content for the object selected in the data tree. 4. In the Security Settings tree, select Local Policies → User Rights Assignment. A list of user’s rights policies are displayed in the right-hand side of the window with the following information (Figure E.27): Policy Unique ID for the policy. Local Setting Users or groups assigned to the local policy settings. Effective Setting Users or groups assigned to the domain-level policy settings (overrides the local policy settings). If domain-level policy settings are defined and displayed in the Effective Settings, they override the local policy settings. You will need to get the domain administrator to change the domain-level policy settings in this case. 5. In the Local Security Settings dialog box, double-click on the Act as part of the operating system policy (Figure E.28). appendix E | Configuring the GCOS Services 619 Figure E.28 Local Security Settings, Act as part of the operating system policy selected The Local Security Policy Setting dialog box for Act as part of the operating system opens (Figure E.29). 620 Affymetrix® GeneChip® Operating Software User’s Guide Figure E.29 Local Security Policy Setting dialog box, Act as part of the operating system policy selected This dialog box lists the users and groups assigned to that policy. If domain-level policy settings are defined, they override the local policy settings. You will need to get the domain administrator to change the domain-level policy settings in this case. 6. Click the Add button. The Select Users or Groups dialog box opens (Figure E.30). appendix E | Configuring the GCOS Services 621 Figure E.30 Select Users or Groups dialog box The Look In drop-down box displays a list of domains. The Name box displays a list of the available users and groups in the selected domain. The bottom box displays users and groups selected for adding to a policy. 7. Select the Administrators group. 8. Click the Add button in the Select Users or Groups dialog box. The Administrators group is displayed in the lower box (Figure E.31). 622 Affymetrix® GeneChip® Operating Software User’s Guide Figure E.31 Select Users or Groups, administrator’s group selected 9. Click the OK button in the Select Users or Groups dialog box. The Select Users or Groups dialog box closes, and the Administrators group is displayed in the Local Security Policy Settings dialog box (Figure E.32). appendix E | Configuring the GCOS Services 623 Figure E.32 Local Security Policy Setting, Act as part of the operating system policy, Administrators group added 10. Click the OK button on the Local Security Policy Setting dialog box. 11. The dialog box closes, and the Administrators group is displayed in the Local Security Settings window, Act as part of the operating system policy (Figure E.33). 624 Affymetrix® GeneChip® Operating Software User’s Guide Figure E.33 Local Security Settings dialog box, Administrators group added to Act as part of the operating system policy 12. Close the Local Security Settings dialog box. Appendix F Expression Algorithm, Expression Analysis Metrics & Settings Appendix F 627 Expression Algorithm, Expression Analysis Metrics & Settings GCOS and versions of Microarray Suite higher than 5.0 run the Statistical expression algorithm. Previous versions of Microarray Suite (lower than 5.0) run the Empirical expression algorithm. This appendix contains the following sections: • Expression Analysis Algorithm (see below) • Expression Analysis Metrics (see page 636) • Expression Analysis Settings (see page 644) • Cell Summary Report Algorithm (see page 679) Expression Analysis Algorithm Sections in the Algorithm Description: • Notation (see below) • Algorithm Output Definitions (see page 628) • Background Subtraction (see page 629) • Single-Array Expression Analysis (see page 630) • Comparison Expression Analysis (see page 633) • References (see page 635) NOTATION A GeneChip® probe array consists of a number of cells (square-shaped areas on the array) and each contains many copies of a unique probe. Probes are tiled in probe pairs consisting of a perfect match (PM) and a mismatch (MM). The sequence of the PM and MM are the same, except for a base substitution in the middle of the MM probe sequence. A probe set includes a series of probe pairs and represents an expressed transcript (Figure F.1). 628 Affymetrix® GeneChip® Operating Software User’s Guide Figure F.1 Probe set that includes 10 probe pairs ALGORITHM OUTPUT DEFINITIONS Signal A measure of the abundance of a transcript. Stat Pairs The number of probe pairs for a particular probe set on the array. Stat Pairs Used = Pairs - Masked probe pairs - Saturated MM probe pairs This is the number of pairs used by the Statistical Expression algorithm to make the detection call in an single-array analysis. Detection The call in an single-array analysis that indicates if the transcript was Present (P), Absent (A), Marginal (M), or No call (NC). Detection p-value p-value that indicates the significance level of the Detection call. Stat Common Pairs The intersection of the probe pairs from the baseline and experiment that are used by the Statistical Expression algorithm to make the Change call in a comparison analysis. Change The call that indicates the change in transcript level between a baseline and an experiment array. Change p-value p-value that indicates the significance level of the Change call. Signal Log Ratio The change in expression level for a transcript between a baseline and an experiment array. This change is expressed as the log2 ratio. Signal Log Ratio low The lower limit of the Log Ratio within a 95% confidence range. appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 629 Signal Log Ratio high The upper limit of the Signal Log Ratio within a 95% confidence range. BACKGROUND SUBTRACTION The first step in the analysis is to correct for background across the entire array. The calculated background establishes an intensity floor that is subtracted from all intensity values. Raw cell intensities → Background-adjusted intensities The algorithm: • divides the array into equally spaced zones (Figure F.2) • assigns an average background to the center of each zone • computes the distance from each cell to the center of every zone • computes a weighting factor (the reciprocal of the sum of a constant and the square of the distance from the cell to the zone center) • computes the background of each cell by applying the weighting factor to the zone average (the average background assigned to the center of each zone) 630 Affymetrix® GeneChip® Operating Software User’s Guide Figure F.2 Array zones for computing background; the arrow color indicates the relative weights SINGLE-ARRAY EXPRESSION ANALYSIS Detection The Detection call answers the question: Is the transcript of a particular probe set reliably detected by the probe array? We want an answer of Absent or Present. In this context, Absent means the expression level is below the threshold of detection. In the case of uncertainty, we can get a Marginal call. Detection call results are easy to filter and interpret. For example, we may only want to look at genes whose transcripts are Present in a particular experiment. An additional advantage is that the Detection and the Signal values are calculated using independent algorithms that add additional information to the results. Raw cell intensities → Absent, Present, or Marginal Detection call plus p-values appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 631 A No Call detection result occurs if all of the probe pairs of a probe set are excluded from the analysis. A probe pair is excluded if the PM or MM is masked or if the MM is saturated. Discrimination Value The algorithm computes a discrimination value that is used as a filter to remove from further consideration all probe sets with insignificant differences between PM and MM. discrimination value = (PM - MM)/(PM + MM) The median of the discrimination ratios of all the probe pairs of a probe set is compared to a user-modifiable parameter τ (default = 0.015), and produces an intermediate call. Increasing τ can reduce the number of false Present calls, but may also reduce the number of true Present calls. Making the Call A one-sided Wilcoxon’s Signed Rank test is used to calculate a p-value that reflects the significance of the differences between PM and MM. The p-value or statistical significance of a result is the probability that the observed change in a sample occurred by pure chance. For example, a p-value of 0.05 means there are five chances in 100 that the results are not significant. The lower the p-value, the greater the probability that the results are significant. To make a call, the p-value for a probe set is examined on an axis with two user-definable thresholds, α1 and α2 (defaults α1 = 0.04 and α2 = 0.06). For p-values between zero and 0.5, α1and α2 define the thresholds for the calls (Figure F.3). See also Table F.1. The result is reported as the Detection call that is associated with the calculated p-value. 632 Affymetrix® GeneChip® Operating Software User’s Guide Decreasing the significance level α1 can reduce the number of false detected calls and reduce the number of true detected calls. Increasing the significance level α2 can reduce the number of false undetected calls and reduce the number of true undetected calls. Figure F.3 User-modifiable thresholds, α1 and α2 Table F.1 Statistical algorithm Detection call rules Computed Detection p-values Detection Call p < α1 Present α1 ≤ p < α2 p ≥ α2 Marginal (at the limit of detection) Absent Calculating the Signal The signal represents the amount of transcript in solution. Background-adjusted cell intensities → Probe set signal For each PM intensity, a matching MM probe provides a reference background hybridization intensity. If the MM value is less than the appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 633 PM value, the algorithm uses the MM value directly. However, if the MM value is larger than the PM value, the algorithm creates an adjusted MM value based on the average difference intensity between log2 PM and log2 MM, or if that measure is too small, some fraction of PM. The adjusted MM values are used to calculate the log2 PM - log adjusted MM for each probe pair. The Signal for a probe set is calculated as the one-step biweight estimate of the combined differences of all the probe pairs in the probe set. COMPARISON EXPRESSION ANALYSIS A comparison analysis compares the expression levels of the transcripts on one array to those on another. By directly comparing matching cells on two arrays, any inherent differences in hybridization efficiency will cancel out. As a result, this is an accurate and sensitive method of determining changes in expression levels. Differences between PM and MM and Differences between PM and Background (Experiment and Baseline) → Change call and p-value The algorithm computes a primary normalization factor and two additional normalization factors that straddle the primary normalization factor. The spread between the normalization factors is determined by the perturbation parameter (d). To determine the p-values, a signed rank analysis is carried out on the PM and MM differences for each probe pair in a probe set from the two arrays in the comparison. The resulting p-values are used to make the change calls. To make a call, the p-value for a probe set is examined on an axis with four thresholds (Figure F.4). For p-values between zero and 1.0, γ1and γ2 define the thresholds for the calls. The result is reported as the Change call that is associated with the calculated p-value (see Table F.2). The output p-value is the critical pvalue. The critical p-value is the p-value that determines the Change call. 634 Affymetrix® GeneChip® Operating Software User’s Guide The critical p-value, p, is defined by the following: • p = max(p0, p1, p2), if p0 < 0.5, p1 < 0.5 and p2 < 0.5 • p = min(p0, p1, p2), if p0 > 0.5, p1 > 0.5 and p2 > 0.5 • p = 0.5, otherwise Figure F.4 User-modifiable parameters, γ1 and γ2 Table F.2 Statistical algorithm Change call rules Computed Change p-values p0 < γ 1 p1 < γ 1 p2 < γ 1 p0 < γ 2 p1 < γ 2 p2 < γ 2 Else p0 > 1-γ2 p1 > 1-γ2 p2 > 1-γ2 p0 > 1-γ1 p1 > 1-γ1 p2 > 1-γ1 Change call Increasing Marginally increasing No change Marginally decreasing Decreasing appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 635 You may adjust γ1 and γ2 by specifying different values for: γ1L, γ1H, γ2L, γ2H. See Expression Analysis Settings, on page 644. Decreasing γ1 can reduce the number of false Increase and Decrease calls, but can also reduce the number of true Increase and Decrease calls. Increasing γ2 can reduce the number of false No-Change calls, but can also reduce the number of true No-Change calls. Increasing the perturbation parameter (d) can increase the number of true NoChange calls, but can also increase the number of false No-Change calls. A No Call occurs if all of the probe pairs of a probe set are excluded from the analysis. A probe pair is excluded if the PM or MM in the experiment or baseline is masked or saturated. Signal Log Ratio Probes inherently hybridize with slightly different affinities. Relative expression values compare identical probes in the baseline and experiment. As a result, probe specific effects are canceled out. Adjusted cell intensities (Baseline and Experiment) → Signal Log Ratio, Signal Log Ratio low, and Signal Log Ratio high The Signal Log Ratio calculation is an extension of the signal calculation. The discrimination of the log2 ratio is used to correct for outlier probes. The one-step biweight method is used to compute the average log2 ratio of the probe set. The upper and lower limits of the 95th confidence interval are reported as Signal Log Ratio high and Signal Log Ratio low. REFERENCES Hoaglin, D.C., Mosteller, R., Tukey, J.W. 2000. Understanding Robust and Exploratory Data Analysis, John Wiley & Sons, New York. 2. Hollander, M., Wolfe, D.A. 1999. Nonparametric Statistical Methods (second edition), John Wiley & Sons, New York. 1. 636 3. Affymetrix® GeneChip® Operating Software User’s Guide Liu, W.M., Mei, R., Bartell, D.M., Di, X., Webster, T.Q., Ryder, T. 2001. Rank-based algorithms for analysis of microarrays, Proceedings SPIE, 4266. Wilcoxon, F. 1945. Individual comparisons by ranking methods. Biometrics.1:80-83. 5. Roderick, J.A., Little, D., Rubin, B. 1987. Statistical Analysis With Missing Data. John Wiley & Sons, New York. 4. Expression Analysis Metrics GCOS and versions of Microarray Suite higher than 5.0 run the Statistical expression algorithm. Versions of Microarray Suite (lower than 5.0) run the Empirical expression algorithm. The metrics for these algorithms are described in the following sections: • Statistical Expression Algorithm, on page 636 • Empirical Expression Algorithm, on page 638 STATISTICAL EXPRESSION ALGORITHM Single-Array Results Signal A measure of the abundance of a transcript. Detection The call that indicates whether the transcript is detected (P, Present), undetected (A, Absent), or at the limit of detection (M, Marginal). Detection p-value p-value that indicates the significance of the Detection call. Stat Pairs The number of probe pairs for a particular probe set on an array. appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 637 Stat Pairs Used The number of probe pairs per probe set used in the analysis. This may be the total number of probes per probe set on the probe array or the number of probe pairs in a pre-designated subset (for example, probe pairs specified by a probe mask file and/or a masked image). Stat Pairs Used = total probe pairs per probe set – (probe pairs masked in a mask file) – (probe pairs masked in the image) – (saturated MM probe pairs). Comparison Results Signal Log Ratio The change in the expression level of a transcript between a baseline and an experiment array. This change is expressed as the log2 ratio. A log2 ratio of 1 is equal to a fold change of 2. Signal Log Ratio Low The lower limit of the Log Ratio within a 95% confidence interval. Signal Log Ratio High The upper limit of the Log Ratio within a 95% confidence interval. Change The call that indicates the change in the transcript level between a baseline and experiment (Increase (I), Marginal Increase (MI), No Change (NC), Marginal Decrease (MD), Decrease (D)) Change p-value p-value that indicates the significance of the Change call. Stat Common Pairs The intersection of the probe pairs from the baseline and experiment that are used by the Expression algorithm to make the Change call in a comparison analysis. 638 Affymetrix® GeneChip® Operating Software User’s Guide EMPIRICAL EXPRESSION ALGORITHM Versions of Microarray Suite lower than 5.0 run the Empirical Expression algorithm. Single-Array Analysis Results Positive & Negative The number of probe pairs scored positive or negative. A probe pair is called positive if the intensity of the PM probe cell is significantly greater than that of the corresponding MM probe cell. A probe pair is called negative if the intensity of the MM probe cell is significantly greater than that of the corresponding PM probe cell. To evaluate the intensity, the algorithm calculates the ratio and difference associated with each probe pair and compares these values to the Statistical Difference Threshold (SDT) and the Statistical Ratio Threshold (SRT). A probe pair is Positive if: PM – MM > SDT and PM/MM > SRT A probe pair is Negative if: MM – PM > SDT and MM/PM > SRT The SDT is a function of the noise (Q) and is calculated by the software: SDT = Q * SDTmult. The SDTmult and the SRT are usermodifiable parameters. The SDTmult is set at 2.0 for the standard staining protocol or 4.0 for the antibody amplification protocol. (Refer to the Expression Analysis Technical Manual or the HuSNP® Mapping Assay User Manual). The default SRT value is 1.5. Increasing the SDTmult and SRT increases analysis stringency, reducing these thresholds decreases analysis stringency. The number of positive and negative probe pairs is determined for every probe set and are used to derive parameters that describe probe set performance. Pairs The number of probe pairs for a particular probe set on an array. appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 639 Pair Used The number of probe pairs per probe set used in the analysis. This may be the total number of probes per probe set on the probe array or the number of probe pairs in a pre-designated subset (for example, probe pairs specified by a probe mask file and/or a masked image). Pairs Used = total probe pairs per probe set – (probe pairs masked in a mask file) – (probe pairs masked in the image). Pairs in Avg A trimmed probe set that excludes probes with extremely intense or weak signal from the analysis. If 8 or fewer probe pairs are used, Pairs in Avg = Pairs Used (or the number of probe pairs per probe set minus any that are masked). Super scoring is performed if more than 8 probe pairs are used. Superscoring is a process that excludes probe pairs from calculation of the Avg Diff and Log Avg Ratio if they are outside a given intensity range. Microarray Suite calculates the mean and standard deviation of the intensity differences (PM – MM) for an entire probe set (excluding the highest and lowest values). Those values within a set number of standard deviations (STP) are included in the calculation of the Avg Diff or Log Avg Ratio. The STP is a user-modifiable parameter with a default value = 3. Pos Fraction # positive probe pairs/# probe pairs used Log Avg Describes the hybridization performance of a probe set and is determined by calculating the ratio of the PM/MM intensities for each probe pair in a probe set, taking the logs of the resulting values, and averaging them for the probe set: Log Avg = 10 x {[Σ log (PM/MM)] / Pairs in Avg} 640 Affymetrix® GeneChip® Operating Software User’s Guide Log Avg = 0 indicates random cross hybridization. The higher the Log Avg, the more confidence the transcript is present. Pos/Neg The ratio of Positive probe pairs to Negative probe pairs in a probe set (# Positive probe pairs / # Negative probe pairs). Avg Diff ∑ ( PM – M M ) A vg D iff = -----------------------------------Pairs in A vg This parameter serves as a relative indicator of the level of expression of a transcript. It is used to determine the change in the hybridization intensity of a given probe set between two different experiments. The Avg Diff is calculated by taking the difference between the PM and MM of every probe pair (excluding the probe pairs where PM – MM is outside the STP standard deviation of the mean of PM-MM) in a probe set and averaging the differences for the entire probe set. The Avg Diff cannot be used to compare the hybridization intensity levels of two different probe sets on the same array. Absolute Call Each transcript in an single-array analysis has three possible Absolute Call outcomes: Present (P), Absent (A), or Marginal (M). The Absolute call is derived from the Pos/Neg, Positive Fraction, and Log Avg Absolute call metrics. Each Absolute call metric is weighted and entered into a decision matrix to determine the status of the transcript. appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 641 Comparison Analysis Results Increase A probe pair is considered to increase if the intensity difference between the PM and MM probe cells in the experimental sample is significantly higher than in the baseline sample. Two criteria must be met for a probe pair to show a significant increase: (PM – MM)exp – (PM – MM)base > Change Threshold (CT), and [(PM – MM)exp – (PM – MM) base] / max [Q/2, min(|PM – MM|exp |PM – MM|base)] > Percent Change Threshold/100 Decrease A probe pair is considered to decrease if the intensity difference between the PM and MM probe cells in the experimental sample is significantly lower than in the baseline sample. Two criteria must be met for a probe pair to show a significant decrease: (PM – MM) base – (PM – MM) exp > Change Threshold (CT), and [(PM – MM)base – (PM – MM) exp] / max [Q/2, min(|PM – MM|exp, |PM – MM|base)] > Percent Change Threshold/100 The software calculates the Change Threshold (CT) using the SDT (Statistical Difference Threshold) of both the experimental and baseline data. Alternatively, the user may define the CT by entering a value for the CT Multiplier (in the Parameters tab of the Expression Analysis Settings dialog box), which is multiplied by the noise (Q) of the baseline or experimental data, whichever is greater. The Percent Change Threshold is a user-specified value (also set in the Parameters tab of the Expression Analysis Settings dialog box). Inc Ratio For each transcript: # Increased probed pairs / # probe Pairs Used Dec Ratio For each transcript: # Decreased Probe pairs / # probe Pairs Used 642 Affymetrix® GeneChip® Operating Software User’s Guide Pos Change Positive probe pairsexp - # Positive probe pairsbaseline Neg Change Negative probe pairsexp - # Negative probe pairsbaseline Inc/Dec For each transcript: the # increased probe pairs / # decreased probe pairs DPos-DNeg Ratio (Positive Change – Negative Change)/# probe Pairs Used The DPos – DNeg Ratio and Log Avg Ratio Change are usually positive when a transcript changes from a very low to a relatively high expression level and are typically negative when the expression level changes from a high to a very low or undetectable level. Both metrics may have values close to zero if the transcript is present in both the baseline and experimental samples despite an increase or decrease in the level of the transcript. Log Avg Ratio Change Log Avgexp – Log Avgbase The difference between the Log Avg Ratio of the baseline and experimental probe array data (in a comparison analysis) for each transcript. The Log Avg Ratios are recomputed for each for each probe set based on probe pairs used in both the baseline and experimental probe arrays (the recomputed values are not displayed by the software). Difference Call Each transcript in a comparison analysis has five possible Difference Call outcomes: (1) Increase (I), (2) Marginally Increase (MI), or (3) Decrease (D), (4) Marginally Decrease (MD), and (5) No Change (NC). The difference call is derived from the comparison metrics: Max [Increase/Total, Decrease/Total], Increase/Decrease Ratio, Log appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 643 Average Ratio Change, and Dpos – Dneg Ratio. Each comparison metric is weighted and entered into a decision matrix to determine the status of the transcript. Avg Diff Change The Avg Diff values are recomputed for each probe set based on probe pairs used in both the baseline and experimental probe arrays (the recomputed values are not displayed by the software). Avg Diff Change = Avg Diffexp – Avg Diffbaseline B=A An asterisk (*) in this column indicates the transcript is called absent (A) in the baseline. Fold Change The Fold Change indicates the relative change in the expression levels between the experiment and baseline targets. The Fold Change for a transcript is a positive number when the expression level in the experiment increases compared to the baseline and is a negative number when the expression level in the experiment declines. The Fold Change (FC) is calculated as: ⎧ +1 if ( AvgDiff ≥ AvgDiff )⎫ ⎛ ⎞ ⎪ exp base ⎪ ( AvgDiffChange ) FC = ⎜ ----------------------------------------------------------------------------------------------------------------------------⎟ + ⎨ ⎬ < AvgDiff ) ⎝ max [ min (AvgDiff base,AvgDiff exp), Q M × Q C ]⎠ ⎪ -1 if ( AvgDiff exp base ⎪⎭ ⎩ The normalized or scaled Avg Diff values are recomputed in both the experimental and baseline data sets to include only probe pairs used in both the baseline and experiment arrays. Then the Avg Diff Change is calculated as: Avg Diff Change = Avg Diffexp - Avg Diffbase QC = max(Qexp, Qbase) QM = 2.1 for a 50µm feature or 2.8 for a 24µm feature If the noise (Q) of the experiment or baseline array is greater than the Avg Diff of the transcript (the baseline or experimental data), the Fold Change is calculated over the noise and is an approximation [a tilde 644 Affymetrix® GeneChip® Operating Software User’s Guide character (~) precedes the approximated Fold Change value in the *.chp file]. Sort Score The Sort Score is a ranking based on the Fold Change and the Avg Diff Change. The higher the Fold Change and the Avg Diff Change, the higher the Sort Score. Expression Analysis Settings The expression analysis settings are user-modifiable variables with defaults empirically determined by Affymetrix. They are organized by the tabs of the Expression Analysis Settings dialog box: • Scaling Tab, on page 645 • Normalization Tab, on page 655 • Probe Mask Tab, on page 665 • Baseline Tab, on page 674 • Parameters Tab, on page 677 The Expression algorithm relies on these settings to derive biologically meaningful results from the hybridization intensity data. To view the expression analysis settings: 1. Click Analysis Settings in the shortcut bar, then click Expression ; or Select Tools → Analysis Settings → Expression from the menu bar. The Expression Analysis Settings dialog box appears (Figure F.5). appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 645 Figure F.5 Expression Analysis Settings, Scaling tab, User Defined scaling option selected Make a selection from the Probe Array Type drop-down list. The settings are specific for the selected probe array type and do not affect the settings for other types of probe arrays. 3. Click a tab to view the different types of expression analysis settings for the selected probe array type. 2. SCALING TAB Scaling is a mathematical technique applied to the data from several different probe arrays (of the same type) to minimize discrepancies due to variables such as sample preparation, hybridization conditions, staining, or probe array lot. The Scale Factor is also applied to the noise value. GCOS offers three types of scaling: User Defined, All Probe Sets, and Selected Probe Sets. To view the scaling settings: • Click the Scaling tab in the Expression Analysis Settings dialog box (Figure F.5). 646 Affymetrix® GeneChip® Operating Software User’s Guide • Make a selection from the Probe Array Type drop-down list. User Defined Scaling The User Defined scaling option multiplies the signal of each probe set on the array by a user-specified scale factor. 1. In the Scaling tab, choose the User Defined option (Figure F.6). Figure F.6 Expression Analysis Settings, Scaling tab, User Defined scaling option with no scaling specified (scale factor = 1) 2. Enter a number in the Scale Factor box. A scale factor of one is equivalent to no scaling. 3. Click OK to close the Expression Analysis Settings dialog box. All Probe Sets Scaling The All Probe Sets scaling option adjusts the trimmed mean signal of a probe array to a user-specified target signal value. appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 647 The single-array analysis results (*.chp) from different experiments (probe arrays of the same type) that are scaled to the same target signal using the All Probe Sets scaling option may be directly compared. 1. In the Scaling tab, choose the All Probe Sets scaling option (Figure F.7). Figure F.7 Expression Analysis Settings, Scaling tab, All Probe Sets scaling option selected Enter a target signal value. 3. Click OK to close the Expression Analysis Settings dialog box. GCOS examines all of the probe sets on the array to compute the trimmed mean signal and derive a scale factor for the array so that: Target Signal = Scale Factor x Trimmed Mean Signalprobe array The scale factor standardizes the trimmed mean signal of the array to the target signal (Figure F.8). 2. 648 Affymetrix® GeneChip® Operating Software User’s Guide Figure F.8 Scale factors (SF) standardize the average signal of each experiment to a userspecified target signal Selected Probe Sets Scaling The Selected Probe Sets scaling option adjusts the trimmed mean signal of selected probe sets on a probe array to a user-specified target signal value. You can directly compare the single-array analysis results (*.chp) of different experiments (probe arrays of the same type) if the same probe sets have been scaled to the same target signal in each experiment using the Selected Probe Sets scaling option. For this scaling option, GCOS utilizes user-selected probe sets (specified by a Scale Factor mask file) to calculate the trimmed mean signal and derive the scale factor for the probe array so that: Target Signal = Scale Factor x Trimmed Mean Signalselected probe sets The scale factor standardizes the trimmed mean signal of an array to the target signal (Figure F.8). Selected Probe Sets scaling does not change the single-array call because the software also multiplies the intensity of each probe set and the probe array noise by the scale factor. appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 649 Creating a Scale Factor Mask File 1. In the Scaling tab, choose the Selected Probe Sets option (Figure F.9). Figure F.9 Expression Analysis Settings, Scaling tab, Selected Probe Sets scaling option 2. Enter a Target Signal value, then click Create/Open Probe Set Mask. The Probe Set Mask File dialog box displays existing mask files (Figure F.10). Mask files (*.msk) include Scale Factor, Normalization, and Probe mask files. Each type of mask file has a different function and is created in a separate tab of the Expression Analysis Settings dialog box. 650 Affymetrix® GeneChip® Operating Software User’s Guide Figure F.10 Probe Set Mask Definition dialog box 3. Enter a name for the scale factor mask file in the File name box, then click Open. The Probe Set Mask Definition dialog box appears (Figure F.11). Figure F.11 Probe Set Mask Definition dialog box 4. To select probe sets for the mask file: A. Highlight the desired probe set names in the Exclude list. B. Click Include. This adds the selected probe set names to the Include list for the scale factor mask file. appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 5. 651 To remove a probe set name(s) from the Include list: Highlight the name(s) in the Include list. B. Click Exclude. Include All automatically moves all of the probe set names from the Exclude list to the Include list. Exclude All automatically Moves all of the probe set names from the Include list to the Exclude list. A. 6. Click OK when finished to create the scale factor mask file and close the Probe Set Mask Definition dialog box. Selecting a Scale Factor Mask File 1. In the Scaling tab, choose the Selected Probe Sets option (Figure F.12). Figure F.12 Expression Analysis Settings, Scaling tab, Selected Probe Sets option 2. Click Browse (Figure F.12). The Probe Set Mask File dialog box appears (Figure F.13). 652 Affymetrix® GeneChip® Operating Software User’s Guide Figure F.13 Probe Set Mask File dialog box Mask files (*.msk) are specific for the probe array type. GCOS will not open a mask file that is incompatible with the currently selected probe array type. 3. Double-click the desired *.msk file. The Expression Analysis Settings dialog box displays the currently selected *.msk file name in the Scale Factor Mask File box (Figure F.14). appendix F | Expression Algorithm, Expression Analysis Metrics & Settings Figure F.14 Expression Analysis Settings, Scaling tab, scale factor mask file selected Editing a Scale Factor Mask File 1. In the Scaling tab, choose the Selected Probe Sets option (Figure F.15). 653 654 Affymetrix® GeneChip® Operating Software User’s Guide Figure F.15 Expression Analysis Settings, Scaling tab, Selected Probe Sets option 2. Click Create/Open Probe Set Mask. The Probe Set Mask File dialog box appears (Figure F.16). Figure F.16 Probe Set Mask File dialog box 3. Double-click the desired *.msk file. The Probe Set Mask Definition dialog box appears (Figure F.17). appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 655 The Include list displays the probe sets included in the scale factor mask file. The Exclude list displays probe sets not included in the scale factor mask file. Figure F.17 Probe Set Mask Definition dialog box 4. To remove a probe set(s) from the *.msk file: Highlight the probe set name(s) in the Include list. B. Click Exclude. 5. To add a probe set(s) to the *.msk file, highlight the probe set name(s) in the Exclude list, then click Include. Include All automatically moves all of the probe set names from the Exclude list to the Include list. Exclude All automatically moves all of the probe set names from the Include list to the Exclude list. 6. Click OK when finished to close the Probe Set Mask Definition dialog box. A. NORMALIZATION TAB Normalization is a mathematical technique similar to scaling that enables comparison analysis of an experiment and baseline array. GCOS offers three types of normalization: User Defined, All Probe Sets, or Selected Probe Sets normalization. All Probe Sets or Selected 656 Affymetrix® GeneChip® Operating Software User’s Guide Probe Sets normalization minimizes discrepancies between an experiment and baseline array due to variables such as sample preparation, hybridization conditions, staining, or probe array lot. User Defined Normalization User Defined normalization multiplies the signal of each probe set on an array by a user-specified normalization value. 1. In the Normalization tab, choose the User Defined option (Figure F.18). Figure F.18 Expression Analysis Settings, Normalization tab, User Defined normalization option with no normalization specified (Normalization Value = 1) 2. Enter a value in the Normalization Value box. A normalization factor of one is equivalent to no normalization. 3. Click OK to close the Expression Analysis Settings dialog box. appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 657 All Probe Sets Normalization All Probe Sets normalization adjusts or normalizes the trimmed mean signal of the experiment to the trimmed mean signal of the baseline (Figure F.19). Figure F.19 Normalization Value normalizes the average signal of the experiment to the average signal of the baseline 1. In the Normalization tab, choose the All Probe Sets normalization option (Figure F.20). 658 Affymetrix® GeneChip® Operating Software User’s Guide Figure F.20 Expression Analysis Settings, Normalization tab, All Probe Sets normalization option 2. Click OK to close the Expression Analysis Settings dialog box. The software examines all probe sets on the experiment or baseline array to compute a trimmed mean signal for the experiment and a trimmed mean signal for the baseline. It computes a normalization value so that: Trimmed Mean Signalbaseline = (Normalization Value) x (Trimmed Mean Signalexperiment) Selected Probe Sets Normalization Selected Probe Sets normalization adjusts or normalizes the trimmed mean signal of the experiment to the trimmed mean signal of the baseline (Figure F.19). For this normalization option, GCOS utilizes user-selected probe sets (specified by a normalization mask file) to compute the trimmed mean signal of the experiment and baseline, and derive a normalization value so that: appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 659 Trimmed Mean Signalbaseline selected probe sets = Normalization Value x Trimmed Mean Signalexperiment selected probe sets Creating a Normalization Mask File 1. In the Normalization tab, choose the Selected Probe Sets option (Figure F.21). Figure F.21 Expression Analysis Settings, Normalization tab, Selected Probe Sets option 2. Click Create/Open Probe Set Mask. The Probe Set Mask File dialog box appears (Figure F.22). 660 Affymetrix® GeneChip® Operating Software User’s Guide Figure F.22 Probe Set Mask File dialog box The mask files (*.msk) include Scale Factor, Normalization, or Probe mask files. Each type of mask file has a different function and is created in a separate tab of the Expression Analysis Settings dialog box. 3. Enter a name for the new normalization mask file in the File name box and click Open. The Probe Set Mask Definition dialog box appears (Figure F.23). Figure F.23 Probe Set Mask Definition dialog box appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 661 Highlight the desired probe set names in the Exclude list. Click Include to add them to the Include list for the new *.msk file. 5. To remove a probe set name(s) from the Include list, highlight the name(s) in the Include list, then click Exclude. Include All automatically moves all of the probe set names from the Exclude list to the Include list. Exclude All automatically moves all of the probe set names from the Include list to the Exclude list. 4. 6. Click OK when finished to create the normalization mask file and close the Probe Set Mask Definition dialog box. Selecting a Normalization Mask File 1. In the Normalization tab, click Browse (Figure F.24). The Probe Set Mask File dialog box appears (Figure F.25). Figure F.24 Expression Analysis Settings, Normalization tab, Selected Probe Sets option 662 Affymetrix® GeneChip® Operating Software User’s Guide Figure F.25 Probe Set Mask File dialog box Mask files are specific for the probe array type. GCOS will not open a mask file that is incompatible with the currently selected probe array type. 2. Double-click the desired *.msk file. The Probe Set Mask File dialog box closes and the Expression Analysis Settings dialog box displays the selected *.msk in the Normalization Mask File box (Figure F.26). appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 663 Figure F.26 Expression Analysis Settings, Normalization tab, normalization mask file selected Editing a Normalization Mask File 1. In the Normalization tab, choose the Selected Probe Sets option (Figure F.27). 664 Affymetrix® GeneChip® Operating Software User’s Guide Figure F.27 Expression Analysis Settings, Normalization tab, Selected Probe Sets option 2. Click Create/Open Probe Set Mask (Figure F.27). The Probe Set Mask File dialog box appears (Figure F.28). Figure F.28 Probe Set Mask File dialog box 3. Double-click the desired *.msk file. The Probe Set Mask Definition dialog box appears (Figure F.29). appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 665 The Include list displays the probe sets included in the *.msk file. The Exclude list displays the probe sets not included in the *.msk file. Figure F.29 Probe Set Mask Definition dialog box 4. To remove a probe set(s) from the *.msk file: Highlight the probe set name(s) in the Include list. B. Click Exclude. 5. To add a probe set(s) to the *.msk file: A. Highlight the probe set name(s) in the Exclude list B. Click Include. Include All automatically moves all of the probe set names from the Exclude list to the Include list. Exclude All automatically moves all of the probe set names from the Include list to the Exclude list. 6. Click OK when finished to close the Probe Set Mask Definition dialog box. A. PROBE MASK TAB User-selected probe pairs may be excluded or masked from an expression analysis. In the Probe Mask tab (Figure F.30), you can 666 Affymetrix® GeneChip® Operating Software User’s Guide create a probe mask file that specifies the probe pairs to exclude from an analysis. Figure F.30 Expression Analysis Settings, Probe Mask tab Creating a Probe Mask 1. In the Probe Mask tab, click Create/Open Probe Mask (Figure F.30). The Probe Mask File dialog box appears (Figure F.31). appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 667 Figure F.31 Probe Mask File dialog box The *.msk files include Scale Factor, Normalization, or Probe mask files. Each type of mask file has a different function and is created in a separate tab of the Expression Analysis Settings dialog box. 2. Enter a name for the new probe mask file in the File name box and click Open. The Probe Mask Definition dialog box appears (Figure F.32) and displays the probe array type and the probe mask name. 668 Affymetrix® GeneChip® Operating Software User’s Guide Figure F.32 Probe Mask Definition dialog box Select the desired probe set name from the Probe Sets drop-down list and enter the desired probe pair numbers in the Probe Pairs field (following the example format: 1-4,7,15,19). These probe pairs will be omitted from the analysis. 4. Click the box below the Probe Sets drop-down list to add the probe pairs to the probe mask (Figure F.33). 3. appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 669 Figure F.33 Probe pairs selected for the probe mask file Repeat Step 3 and Step 4 to specify additional probe pairs for the probe mask. 6. Click OK when finished adding probe pairs to the probe mask. 5. Selecting a Probe Mask 1. In the Probe Mask tab, choose the Use Probe Mask File option (Figure F.34). 670 Affymetrix® GeneChip® Operating Software User’s Guide Figure F.34 Expression Analysis Settings, Probe Mask tab 2. Click Browse. The Probe Mask File dialog box appears (Figure F.35). Figure F.35 Probe Mask File dialog box 3. Double-click the desired probe mask. The Probe Mask tab displays the selected probe mask (Figure F.36). appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 671 Mask files are specific for the probe array type. GCOS will not open a mask file that is incompatible with the currently selected probe array type. Figure F.36 Expression Analysis Settings, Probe Mask tab Editing a Probe Mask 1. In the Probe Mask tab, click Create/Open Probe Mask (Figure F.37). The Probe Mask File dialog box appears (Figure F.38). 672 Affymetrix® GeneChip® Operating Software User’s Guide Figure F.37 Expression Analysis Settings Figure F.38 Probe Mask File dialog box 2. Click the desired probe mask file and click OK (or double-click the file name). The Probe Mask Definition dialog box displays the probe pairs in the selected probe mask (Figure F.39). appendix F | Expression Algorithm, Expression Analysis Metrics & Settings Figure F.39 Probe Mask Definition dialog box 3. Click the probe set/probe pair entry you wish to edit. The Probe Sets and Probe Pairs box automatically display the selection (Figure F.40). 673 674 Affymetrix® GeneChip® Operating Software User’s Guide Figure F.40 Edit entries in the Probe Pairs box and click the lower box (left) to update the probe pair list (right) 4. Edit or delete the entry in the Probe Pairs field, then click the lower field to update the list of probe pairs (Figure F.40). 5. Click OK when finished to close the Probe Mask Definition dialog box. BASELINE TAB In the baseline tab, the user selects the analysis output file (*.chp) that will serve as the baseline in a comparison expression analysis (Figure F.41). Do not choose the Use Baseline Comparison Data option for an single-array analysis. 1. In the Baseline tab, choose the Use Baseline Comparison Data option (Figure F.41). appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 675 Figure F.41 Expression Analysis Settings, Baseline tab 2. Click Browse. The Baseline Comparison File dialog box appears (Figure F.42). The baseline file must be derived from the same type of probe array selected in the Probe Array Type drop-down list in the Expression Analysis Settings dialog box (Figure F.41). 676 Affymetrix® GeneChip® Operating Software User’s Guide Figure F.42 Baseline Comparison File dialog box 3. Double-click the baseline *.chp and click Open. The Baseline tab displays the selected baseline file for comparison analyses (Figure F.43). Figure F.43 Expression Analysis Settings, Baseline tab appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 677 PARAMETERS TAB The Parameters tab displays user-modifiable Statistical Expression algorithm parameters (Figure F.44). Table F.3 describes the parameters. Versions of Microarray Suite lower than 5.0 run the Empirical Expression algorithm. (for more information about the Empirical Expression algorithm, see Expression Analysis Metrics, on page 636.) Figure F.44 Expression Analysis Settings, Parameters tab The parameter default values were determined through extensive empirical testing at Affymetrix. Changing a parameter value affects the: • algorithm output (for more information about the Statistical and Empirical Expression algorithm outputs, see Appendix F, Expression Analysis Metrics, on page 636) • assay sensitivity (ability to make true Detection or Change calls) 678 Affymetrix® GeneChip® Operating Software User’s Guide • assay specificity (ability to minimize false Detection or Change calls) Table F.3 User-modifiable Statistical Expression algorithm parameters Parameter Alpha1 (α1) Alpha2 (α2) Tau (τ) Description Effect of Changing Parameter The p-value threshold that defines Present and Marginal Detection calls. If: p < α1 the call is Present α1 < p < α2 the call is Marginal Decreasing α1 may reduce the number of false Present calls (increases specificity) and the number of true Present calls (decreases sensitivity). The p-value threshold that defines Marginal and Absent Detection calls. If: α1 < p < α2 the call is Marginal p > α2 the call is Absent Increasing α2 may reduce the number of false Absent calls (increases sensitivity) and the number of true Absent calls (decreases specificity). Threshold for the probe set discrimination score. If the discrimination score < τ, the difference between the PM and MM cells in the probe set is considered insignificant. The algorithm does not make a call for the probe set. Gamma1L (γ1L) The lower limit of γ1 which is the p-value threshold that defines Increase and Marginal Increase Change calls. Gamma1H (γ1H) The upper limit of γ1 which is the p-value threshold that defines Increase and Marginal Increase Change calls. Gamma2L (γ2L) The lower limit of γ2 which is the p-value threshold that defines Marginal Increase and No Change calls. Gamma2H (γ2H) The upper limit of γ2 which is the p-value threshold that defines marginal, No Change and Change calls. Perturbation (d) Determines the spread between the three normalization factors the algorithm computes for an experiment and baseline in a comparison analysis. Increasing τ may reduce the number of false Present calls (increases specificity) and the number of true Present calls (decreases sensitivity). Decreasing γ1L and γ1H decreases γ1. Decreasing γ1 may reduce the number of false Increase and Decrease calls (increases specificity) and may also reduce the number of true Increase and Decrease calls (decreases sensitivity). Increasing γ2L and γ2H increases γ2. Increasing γ2 may reduce the number of false No Change calls (increases specificity) and may also reduce the number of true No Change calls (decreases sensitivity). Increasing d may increase the number of true No Change calls (increases sensitivity) and may also increase the number of false No Change calls (decreases specificity). appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 679 Changing a Parameter Value In the Parameters tab and make a selection from the Probe Array Type drop-down list. 2. Click the parameter you wish to change. The parameter box displays the current value (Figure F.45). 1. Figure F.45 Expression Analysis Settings, Parameters tab Enter a new value for the parameter in the parameter box. 4. Click OK to close the Expression Analysis Settings dialog box. 3. Cell Summary Report Algorithm The Cell Summary report algorithm is useful in identifying gridding, scanning, and library file issues. The Cell Summary report uses the control features on the chip. Control features are cells with special probes; the corresponding targets are spiked into the sample cocktail. The resulting patterns of bright and dark cells are used in grid alignment and other processes. 680 Affymetrix® GeneChip® Operating Software User’s Guide Different algorithms are used for: • Expression, Genotyping and other non-resequencing chips (see below) • Resequencing chips (see page 679) The algorithm input files are: • .GRC files available on the library CD or the Affymetrix web site. • Image CEL files. Algorithm for Non-Resequencing Chips For non-resequencing chips (expression, genotyping, and others), the algorithm uses three types of features for image data evaluation: • OligioB2 • OligoB1 • NonSynthesized Refer to the relevant manual for the GeneChip array for more information about these features. For an ideal experiment: • The nonsynthesized features would have dim intensities. • One of the feature types (either the oligoB2 OR oligoB1) would be bright because it was spiked into the sample cocktail. • The other feature type (oligoB2 OR oligoB1) would be dim because it was NOT spiked into the sample cocktail. The algorithm uses the following steps: 1. The algorithm determines whether the OligioB1 or OligoB2 is overall brighter for this image (the cell file) using the medians of the OligoB1 vs. OligoB2 features. 2. A dim threshold and a bright threshold are determined based on the medians described above. The threshold is the midpoint between the logarithms of the medians. appendix F | Expression Algorithm, Expression Analysis Metrics & Settings 681 3. Features whose intensity is beyond the thresholds are flagged as having unusual brightness (or dimness). These features are observed due to: - Misaligned grid - Debris - Probe death 4. The flagged cells can be masked from the image using the cell summary options. When the image data is opened the flagged cells are visible as masked out data. The cell summary report is saved with a suffix of “_cell_summary.rpt.” The data in the output file is described in The Cell Summary Report, on page 178. Algorithm for Resequencing Chips For Resequencing chips, the algorithm uses three types of features for image data evaluation: • Non-synthesized features. • OligoB2 checker features (oligoB2 features arranged in a checkerboard pattern). • OligoB2 Extended features (oligoB2 features arranged in a Resequencing pattern). Refer to the relevant manual for the GeneChip array for more information about these features. 682 Affymetrix® GeneChip® Operating Software User’s Guide Appendix G GCOS Data Types Appendix G 685 GCOS Data Types Probe Information (Library) Data The probe information or library data includes the probe array design characteristics, probe utilization and content, and scanning and analysis parameters. Library data are unique to each probe array type. Library data includes user-defined mask files (see Table G.1). The default path for the library directory is <servername>:\GeneChip\Affy_Data\Library. Table G.1 User-defined mask files Mask File Name File Extension Description Probe Mask .msk A user-specified list of probe pairs that are excluded from an analysis. Cross Hybridization Probe Mask .msk A type of probe mask that specifies probe pairs that include a PM or MM probe cell whose intensity exceeds a user-specified limit. These probe pairs will be excluded from the analysis. Hybridization Probe Mask .msk A type of probe mask that specifies probe pairs where PM – MM < Difference Threshold or PM/MM < Ratio Threshold. These probe pairs will be excluded from the analysis. Spike Probe Mask .msk A type of probe mask that specifies probe pairs where: (PM – MM)spike – (PM – MM)unspike < Difference Threshold or (PM-MM)spike/ (PM – MM)unspike < 1 + Ratio Threshold. These probe pairs will be excluded from the analysis. Fluidics Protocol Data The fluidics protocol data include the instrument control instructions used by the GeneChip® fluidics station. Fluidics protocols are written to a directory that is specified during GCOS installation. The default 686 Affymetrix® GeneChip® Operating Software User’s Guide path for the fluidics protocols is <servername>:\GeneChip\Affy_Data\Protocol. Experiment Data During experiment setup, you enter information to register the sample and define the experiment. The software generates the other experiment data (image, cell intensities, probe analysis data) as the analysis proceeds. Experiment data are written to a directory that is specified during GCOS installation. The default path for experiment data is <servername>:\GeneChip\Affy_Data\Data\. GCOS stores the experiment data in the process database, a relational database that maintains associations between experiments, samples, and the experiment data generated during analysis. Table G.2 explains the different types of experiment data in the process database. Table G.2 Experiment data files Experiment Data Name Data Description Experiment Information about the experiment name, sample, and probe array type. The experiment name also provides the default name for subsequent data that are generated during experiment analysis. Image Data Image of the scanned probe array (.dat file) and information about the experiment & sample that the image pertains to. Cell Intensities Single intensity value for each probe cell delineated by the grid (.cel file calculated by the Cell Analysis algorithm) and information about the: 1) image that the cell intensities were derived from, and 2) experiment and sample that the image pertains to. Probe Analysis (chip data) Output generated from the analysis of a probe array (.chp file) and information about the: 1) cell intensities for the analysis, 2) image that the cell intensities were derived from, and 3) experiment and sample that the image pertains to. appendix G | GCOS Data Types 687 Table G.2 Experiment data files Experiment Data Name Data Description Report Report generated from the analysis output file (.chp) and information about the: 1) analysis output file (.chp) the report was derived from, 2) cell intensities for the analysis, 3) image that the cell intensities were derived from, and 4) experiment and sample that the image pertains to Data A standard format for text files. GCOS exports text in this file format. A standard format for Excel files. GCOS export text in this file format. 688 Affymetrix® GeneChip® Operating Software User’s Guide Appendix H Base Codes and Amino Acid Abbreviations Appendix H 691 Base Codes and Amino Acid Abbreviations IUPAC Base Codes Table H.1 Base Codes IUPAC Code Group Base(s) A A Adenine C C Cytosine G G Guanine T T Thymine M A or C aMino R A or G puRine W A or T (U) Weak interaction (2 H bonds) Y C or T (U) pYrimidine S C or G Strong interaction (3 H bonds) K G or T(U) Keto V A or C or G not-T or not-U (since V follows U) H A or C or T(U) not-G (since H follows G) D A or G or T(U) not-C (since D follows C) B C or G or T(U) not-A (since B follows A) N A, C, G or T(U) aNy Amino Acid Abbreviations 692 Affymetrix® GeneChip® Operating Software User’s Guide Table H.2 Amino acid abbreviations One Letter Three Letter Amino Acid A Ala Alanine B Asx Asparagine or Aspartic acid C Cys Cysteine D Asp Aspartic acid E Glu Glutamic acid F Phe Phenylalanine G Gly Glycine H Hi Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan Y Tyr Tyrosine Z Glx Glutamine or Glutamic acid Appendix I Toolbars, Hot Keys, & Windowpanes Appendix I 695 Toolbars, Hot Keys, & Windowpanes This appendix reviews toolbar button functions, hot keys, and explains how to resize windowpanes or columns. This appendix contains the following sections: • Toolbars (see below) • Hot Keys (see page 702) • Working with Windowpanes & Columns (see page 705) • Resizing or Hiding Columns in the EAW (see page 706) Toolbars You can display toolbars with text labels (Figure I.1). To display the toolbar button labels, select View → Toolbar → Text Labels from the menu bar. GCOS TOOLBARS Main Toolbar Figure I.1 Main toolbar Table I.1 Main toolbar Menu Bar Command File → Open File → Save Toolbar Button Function Displays the Open dialog box so that data (for example, probe analysis data or image data) may be opened. Saves the open image, experiment, or report. 696 Affymetrix® GeneChip® Operating Software User’s Guide Table I.1 Main toolbar Menu Bar Command Toolbar Button Function File → Print Displays the Print dialog box. Window → Data Tree Displays or hides the data tree. Window → Shortcut Bar Displays or hides the shortcut bar. Window → Status Log Displays or hides the status log. Run → Experiment Info Displays experiment window. Run → Fluidics Displays the Station Selection dialog box for the Affymetrix® Fluidics Station 250/450. Run → Scanner Displays the Scanner dialog box. Run → Stop Scanner Stops a scan in progress. Run → Analysis Runs an analysis on the open *.dat or *.cel data. Edit → Image Settings Displays the Image Settings dialog box. Help → Contents Displays GCOS help. Expression Analysis Window (EAW) Toolbar Figure I.2 EAW toolbar appendix I | Toolbars, Hot Keys, & Windowpanes 697 Table I.2 EAW toolbar button functions Menu Bar Command EAW Toolbar Button Function Edit → Find Displays the Find Probe Set dialog box. Edit → Sort Displays the Sort dialog box. View → Hide Selected Hides selected probe set(s) in the metrics or pivot table. View → Hide Unselected Hides unselected probe set(s) in the metrics or pivot table. View → Unhide All Displays all probe sets previously hidden in the metrics or pivot table. Analysis → Options Displays the Analysis Options dialog box. Graph → Clear Selected Graphs Clears selected graphs from the graph pane in the EAW. Graph → Scatter Correlation Graph Displays the Scatter Graph dialog box. Graph → Series Graph Displays the Select Series Graph Parameter dialog box. Graph → Intensity Bar Graph Plots the Intensity Bar Graph for the selected probe set(s). Graph → Measured Images Displays the hybridization intensity image data (.dat) for the selected probe set(s). Graph → Lasso Points Changes the cursor to a drawing tool that enables the user to draw a circle around points in a scatter graph. 698 Affymetrix® GeneChip® Operating Software User’s Guide Batch Analysis Toolbar Figure I.3 Batch analysis toolbar Table I.3 Batch analysis toolbar button functions Menu Bar Command Edit → Add Item Edit → Remove Item Batch Analysis Toolbar Button Function Displays the Open dialog box to select and add *.cel files to the Batch Analysis window. Removes the selected *.cel files from the Batch Analysis window. Edit → Start Analysis Starts the batch analysis. Edit → Stop Analysis Stops the batch analysis. View → Options Displays the Batch Analysis Options dialog box. Report Toolbar Figure I.4 Report window toolbar appendix I | Toolbars, Hot Keys, & Windowpanes Table I.4 Report toolbar button functions Menu Bar Command Report Toolbar Button Edit → Cut Function Removes a highlighted selection from the report. Edit → Copy Copies a highlighted selection in the report to the system clipboard. Edit → Paste Pastes a copied or cut selection at the current insertion point. Edit → Find Displays the Find dialog box. Edit → Find Next Performs a text search for the item specified in the Find dialog box. Publish Toolbar Figure I.5 Publish window toolbar Table I.5 Publish toolbar button functions Menu Bar Command Publish Window Toolbar Button Function Publish → Add Item Displays the Open dialog box to select and add experiment data (task items) to the publish task. Publish → Remove Item Removes the selected task item(s) from the publish task. 699 700 Affymetrix® GeneChip® Operating Software User’s Guide Table I.5 Publish toolbar button functions Menu Bar Command Publish Window Toolbar Button Function Publish → Publish Send the task to the GCOS server to be published during the time specified by the GCOS Server administrator. Publish → Monitor → Cancel Publish Cancels a task (changes the task status from WAIT to CANCELED). Publish → Monitor → Restart Publish Reinstates a canceled task to be published during the time specified by the GCOS Server administrator (changes the task status from CANCELED to WAIT). GCOS MANAGER TOOLBAR Figure I.6 GCOS Manager toolbar Table I.6 GCOS Manager toolbar button functions Menu Bar Command GCOS Manager Toolbar Button Function Process → Print Experiment Prints experiment information. Tools → Register GCOS Server Opens the Register GCOS Server dialog box Tools → Server Event Viewer Opens the Server Event Viewer appendix I | Toolbars, Hot Keys, & Windowpanes Table I.6 GCOS Manager toolbar button functions Menu Bar Command GCOS Manager Toolbar Button Function Tools → Server Tasks Displays the Task List for the active server. Tools → File Path Displays the Select Export File Path dialog box Tools → Find Opens the Find dialog box Tools → Filter Opens the Sample Filters dialog box Tools → Help Topics Opens GCOS Manager online help. GCOS ADMINISTRATOR TOOLBAR & ICONS Figure I.7 GCOS Administrator toolbar Table I.7 GCOS Administrator toolbar button functions Menu Bar Command GCOS Administrator Toolbar Button Function Opens the Administrator Password dialog box so that a system administrator password or publish database password can be created and updated. 701 702 Affymetrix® GeneChip® Operating Software User’s Guide Table I.7 GCOS Administrator toolbar button functions Menu Bar Command GCOS Administrator Toolbar Button Function Refreshes the data tree in the copy or space management view. Refresh the data tree to view new data or databases added to the system during the current session. Tools → Register GCOS Server Opens the Register GCOS Server box. Tools → Server Tasks Opens the Sample Filters dialog box. Tools → Help Topics Opens GCOS Manager online help. Table I.8 GCOS Administrator Icons Process Database or LIMS System Database (yellow) Project Sample Publish Database (gray) Experiment Information Filter Hot Keys appendix I | Toolbars, Hot Keys, & Windowpanes Table I.9 GCOS hot key descriptions Menu Bar Command Hot Key File → Open Ctrl + O File → Print Ctrl + P Edit → Copy Ctrl + C File → Save Ctrl + S Edit → Image Settings S Edit → Delete Del Edit → Unmask All Cells Shift + U View → Grid G View → Probe Cell Data C View → Corner → Upper Left F5 View → Corner → Upper Right F6 View → Corner → Lower Left F7 View → Corner → Lower Right F8 View → Image → Measured M View → Image → Difference D View → Image → Average A View → Next Highlight N View → Previous Highlight P View → Clear Highlights E View → Probe Tiling T 703 704 Affymetrix® GeneChip® Operating Software User’s Guide Table I.9 GCOS hot key descriptions Menu Bar Command Hot Key 1, 2, 3, etc. (toggle between open image windows) I (Image window zoom in) Select an area in the image window with the cursor and press I (Zooms in on the selected area) O (Image window zoom out) Shift+O (Image window full zoom out) L (intensity autoscale) Edit → Mask Cells X Edit → Unmask Cells U Help F1 Scroll up one page Page Up Scroll down one page Page Down Scroll left one page Ctrl + Page Up Scroll right one page Ctrl + Page Down Scroll up 1/10 page Up arrow Scroll down 1/10 page Down arrow Scroll left 1/10 page Left arrow Scroll right 1/10 page Right arrow appendix I | Toolbars, Hot Keys, & Windowpanes Table I.10 GCOS Manager hot key descriptions Menu Bar Command Hot Key View → Refresh F5 Working with Windowpanes & Columns You can resize windowpanes. 1. Place the mouse pointer over a windowpane border so that it changes to a double arrow (Figure I.8). Figure I.8 Resize windowpanes horizontally or vertically 2. Drag the border to resize the windowpane. 705 706 Affymetrix® GeneChip® Operating Software User’s Guide Resizing or Hiding Columns in the EAW You can resize or hide columns in the Expression Analysis window (EAW). GCOS stores the column settings for future sessions on a per user basis (identified by the logon name) so that one user’s settings do not affect the settings of another. Position the mouse over the left or right cell border in the column header so that it changes to a double arrow . 2. Drag the cell border to resize the width of the column. 3. To hide a column, drag the left or right cell border of the column header until the column width is reduced to zero; or Right-click the column header in the EAW and select Hide Column from the shortcut menu that appears. 1. Appendix J Database Management & Data Source Name Descriptions Appendix J 709 Database Management & Data Source Name Descriptions This appendix contains the following sections: • Performance Tuning for SQL Server Databases (see below) • Client Tuning (see page 711) Performance Tuning for SQL Server Databases Microsoft® SQL Server automatically tunes many of the server configuration options, therefore requiring little, if any, tuning by a database administrator. Although these configuration options can be modified by the database administrator, it is generally recommended that they are left at their default values, allowing SQL Server to automatically tune itself based on run-time conditions. However, if necessary, the following components can be configured to optimize server performance: • SQL Server Memory • I/O subsystem • Microsoft® Windows® options To improve performance of querying, it is recommended that the SQL Server database is in three devices: data, index and log. With this separation, reading and writing of data is improved which helps decrease amount of time it takes to query data. DATA DEVICE The data device stores the experimental data, published data, and other data associated with GCOS. These data are accessed by GCOS and all applications that work on GCOS. The name of this device is <publish database name>_DAT.mdf. To move the locations of data devices from one disk to another, use the Space Manager tool in GCOS Administrator. 710 Affymetrix® GeneChip® Operating Software User’s Guide To improve performance, the Data Device file should be placed on a dedicated physical disk. INDEX DEVICE The Index Device is available for publish databases. The Index Device provides quick access to data and can enforce uniqueness on the rows in a table. With this device, it allows searches in a database program to find data in a table without scanning the entire table. This device is a list of values in a table with the storage locations of rows in the table that contain a value. The name of this device is <publish database name>_INDEX_mdf. To move the locations of data devices from one disk to another, use the Space Manager tool in GCOS Administrator. Basic Configuration The index files can be located with the Data Device files, however, performance may be gained by placing the Index Device file on a different disk with low activity. Advanced Configuration Place the Index Device file on a dedicated physical disk. If a remote AADM server is used, place the Data Device file on a dedicated physical disk. LOG DEVICE The Log Device holds the information used to recover the database. There must be at least one log file for each database. To move the locations of log devices from one disk to another, use the Space Manager tool in GCOS Administrator. appendix J | Database Management & Data Source Name Descriptions 711 Basic Configuration The Log Device file can be placed where there is space. Advanced Configuration Place the Log Device file on a dedicated physical disk. If a remote AADM server is used, place the Data Device file on a dedicated physical disk. ADDITIONAL PERFORMANCE TUNING INFORMATION These are other suggestions that can help improve the performance of your system. • Separate all devices SQL Server uses. For example, place the data device, index device, and log device each on their own individual physical disk. The separation of these files improves the performance by allowing the drive heads to perform read only or write only operations during the majority of the publishing operation. • Use a remote AADM server for publishing and place the data device, index device, and log device each on their own individual physical disk. • Placing both the index device and log device on the same disk can slow performance since there is a lot of read and write activity being performed. Client Tuning As part of the software requirement for the client, it is recommended to install SQL*Loader on the client running Data Mining Tool if connecting to an Oracle® GCOS server. Also, a registry setting can be added on the client machine when preforming a large pivot. 712 Affymetrix® GeneChip® Operating Software User’s Guide SQL*LOADER If your GCOS server uses an Oracle® database, it is advisable to install SQL*Loader on the client workstation. Contact your Database Administrator to install SQL*Loader. SQL*Loader is included on the Oracle® installation CD. SQL*Loader is used to bulk loading probe set lists and annotations that users save into the database. With SQL*Loader installed on the client, the process of saving large amounts of data at time is improved. Without SQL*Loader installed on the client, it may take a long time to save the information. The same version of SQL*Loader must be installed on the workstation as on the GCOS server. Appendix K Troubleshooting Appendix K 715 Troubleshooting This section of the manual helps you troubleshoot problems that may be encountered during installation or administration of the server and the different software products on the clients. This will also help you with some basic troubleshooting techniques and what information should be gathered to help Technical Support work towards providing a resolution to a potential problem that you are experiencing. This appendix contains the following sections: • Troubleshooting Questions (see below) • GCOS 1.4 Troubleshooting (see page 716) • GCOS Manager 1.4 Troubleshooting (see page 721) • Library Files Troubleshooting (see page 727) Troubleshooting Questions Some basic tests may quickly reveal the cause of the problems. It is important to isolate the problem down to a particular area. Some questions to answer before calling Technical Support for help: Is it a client or a server problem? If server, has anything changed? Addition of software? Addition of network functions? Restoring of databases? Check the Event log. Check the SQL Server log (if using a Microsoft® SQL Server database). Which database is being accessed? Is there enough free space available? Can you reproduce the problem? What steps were taken? What is the exact error message? Does it happen with a particular user or all users? What has changed on the client? 716 Affymetrix® GeneChip® Operating Software User’s Guide Is the user logged onto the correct domain? Does the user have permissions to access GCOS Server? Can you write to the publish database? Can you access other databases? Can you reproduce the problem? What steps were taken? What is the exact error message? GCOS 1.4 Troubleshooting This section provides tips on troubleshooting problems in the following GCOS features: • Installation (see below) • Installing Pre-GCOS Version Libraries (LIMS 3.0 Libraries) on the Client (see page 717) • Publishing (see page 718) • Licensing (see page 720) • Workflow (see page 720) INSTALLATION • I am unable to install the software. Verify that you have administrative privileges in order to install the software. • Error connecting to GCOS Server database. Check the connectivity. Check the server is running. • When installing GCOS 1.4 the installation tries to copy files, however, the error message “cannot move error - 145” appears. What should I do? Verify that all Affymetrix applications are closed and not running. Log in as another user and, again, try installing the application. It may be the user’s profile is corrupted. appendix K | Troubleshooting 717 • The GCOS software shows the following dialog box when installing the software or MSDE fails to install. Figure K.1 Stop message GCOS requires MSDE. In order to install MSDE, the “Server” service also known as the “lanmanmanger” service needs to be installed and running. GCOS will attempt to start the service if it has stopped. To see if the “Server” service is installed, right-click “My Computer” and select “Manage”. Navigate to the “Services and Applications” Node”. Select Services and see if the Server service is installed. To install the Server service the workstation must have a Network Interface Card (NIC) or a Microsoft loop-back adapter. Once the NIC has been installed, the “Server” service can be installed by adding the “File and Printer Sharing for Microsoft Networks” to the Local Area Connection properties. Refer to www.microsoft.com or contact your system administrator for details on installing the server service. INSTALLING PRE-GCOS VERSION LIBRARIES (LIMS 3.0 LIBRARIES) ON THE CLIENT In order to install LIMS 3.0 version libraries (pre-June 2003 libraries) on a GCOS client, you must complete the following four steps. 1. Run ToggleLIMS3LibrarySupport.exe file by double-clicking the file. (The ToggleLIMS3LibrarySupport.exe file is located in the 718 Affymetrix® GeneChip® Operating Software User’s Guide directory where GOCS was installed.) This enables the older library installer and temporarily disables GCOS. 2. Install the old version library. 3. Run the ToggleLIMS3LibrarySupport.exe file by double-clicking the file again. This enables GCOS and disables the old library installer. GCOS cannot be run until this step is completed. 4. Run the library migrate program (from the subdirectory 'Migrate' contained in the GCOS Target directory, or a new version library install CD). PUBLISHING • Unable to publish data. The server task window displays that it is in the queue and says “wait”. Verify that the publishing service is active in the server task window of GCOS 1.4. If the service is stopped, contact the system administrator to start the gcdoservice. • GCOS shows “Publishing Stopped” when a publish window is launched. Check if CGCDOService is started. • Unable to publish the .chp file. The user is able to publish any .exp and .cel files, however, when publishing the .chp file, the error message “Failed to publish x.chp: SQL Command Failure.” This is possibly due to a probe array type that has been installed and published. However, a new probe array with the same name containing new probe information was installed. The probe set names were changed and therefore do not match and that is why the .chp file with the new probe array information cannot be published. Workaround: Delete the data that is published from that probe array type. Reanalyze that existing .chp files and publish again. • Unable to Publish to an Oracle® GCOS Server system and the Oracle® Publish database was created successfully. appendix K | Troubleshooting 719 Check the registry setting: HKLM\Software\ODBC\ODBC.INI on the GCOS server. Locate the Oracle ODBC DSN created <servername>_ORCL_GATC. Verify the values: String Value Name HOST_OS HOST_SQLLOAD Oracle® 8.1.7 Value WinNT4 sqlldr Note: String values are case-sensitive For the Publish database: <publish database name>_AffyPub: String Value Name Value PWD <encrypted password> UID <publish database name> Alias <name of the alias> Note: The name of the alias created points to the GATC publish server Do the temporary files get created? *.ctl on the root of the c: drive for the Oracle database. The temporary files on the c:\ drive for SQL Server are s*.* (short file names). There must be enough free space to create these temporary files (at least 400MB). • “Failed to read <name of experiment> from GCOS Server for the .dat file” message appears when publishing. There is no .dat file. The experiment needs to be scanned and is currently in the scan stage. • “Failed to read <name of experiment> from GCOS Server for Chip.” message appears when publishing. There is no object file. The experiment is currently in the hybridization queue and needs to be hybridized and scanned. Publish Task Queue should be purged from time to time. As the task queue grows, especially above 10,000 entries, GCOS will appear to hang temporarily when placing jobs in the publish 720 Affymetrix® GeneChip® Operating Software User’s Guide queue. You may want to backup the text of this log prior to purging. LICENSING • Upon Launching GCOS 1.4, the license window appears. I enter the license, however the window keeps appearing. Verify that when entering the license, the license number is entered exactly as seen. The license is case sensitive. Ensure that you do not enter any additional spaces. WORKFLOW • Failure in GetSampleBy name command error. Verify that you did not enter any illegal characters used when creating a sample or experiment. Some illegal characters include: < >:;\”^|*/? • Cannot connect to Affymetrix GCOS Server. Verify that the network is up. Also, verify that the server is online. On the client side, verify that the registry key values are correct. • Experiments don't show up in the data tree. Verify your filter is not filtering out everything, especially the date range. • When trying to reanalyze an analysis after an upgrade in GCOS, the error message “Base call failed for \\<servername>\gcgcosserver\data\\<file.chp> - Invalid base call algorithm. Verify base call default settings for this Probe Array Type or verify Tools\Defaults\File locations settings” appears. One indication of this error message is that the library file update on the server may not have updated properly. appendix K | Troubleshooting 721 GCOS Manager 1.4 Troubleshooting This section provides tips on troubleshooting problems in the following GCOS Manager features: • Installation (see below) • Delete (see page 722) • Archiving (see page 722) • Importing (see page 723) • Usersets (see page 723) • Templates (see page 724) • Exporting (see page 724) • Publish (see page 724) • GCOS Manager FAQs (see page 725) INSTALLATION • When trying to register a GCOS server or click a tab in GCOS Manager, the error message “GCOS Server Component not installed” appears. What does this mean? This means that there is a permission or access problem, or may occur where the shared pool size is too small. Verify that the user has permissions to access the server. The logon name is part of the GCOS Server Global Group. Verify that the user is registering a server with the GCOS Server components installed. Verify that the database services are started (either SQL or Oracle®). • The Roles tab in GCOS Manager is missing. The Roles tab is unavailable when you work with GCOS connected to the local MSDE database server. The global group may have the wrong domain name prefix in the GCOS Server Admin database table 'USERS'. This will happen if 722 Affymetrix® GeneChip® Operating Software User’s Guide your MIS changes the name of the domain. As users begin to migrate to Win2K from NT, some administrators might change the domain name. If they do, you will need to change the domain name in the 'USERS' table. • Slow connection when trying to connect to the server with one of the client applications (i.e.: GCOS Manager). What is going on? If users experience slow connection to the server, verify that the Domain Controller and DNS are properly configured and registered with other Domain Controllers within the network. DELETE • Unable to delete data from the process database. Verify the error message. Error message displayed “Failed to read experiment: xxx.EXP” It is possible that the data was migrated in from a beta version and the character limit exceeded 25 characters. In this case, the entire sample must be deleted in order to remove the file in question. • When trying to delete data from a Publish database, it displays “failed” When viewing the log, the message “IpublishData – Failure in CheckForOpenConnections: cannot delete database with open connections (2)” What does this mean? If another user has the database open (using DMT or publishing data), data cannot be deleted. This is because the tables are locked and cannot be accessed. Wait until the users are done publishing and not using DMT. ARCHIVING • Unable to archive. Error message “Archive File Copy Error”. Verify that the path location specified for archiving has enough free space to archive the file(s) selected. appendix K | Troubleshooting 723 IMPORTING • Unable to import. I get a copy file error message in the text log. Verify that the permissions are set properly. There is enough free space to import the files. If the data is on a CD, try copying it to the local hard drive and import again. • Trying to import and the error message “Cannot modify experiment <name of experiment>. It is waiting to be hybridized, scanned, or it’s image file requires grid alignment.” The experiment that is selected to be imported cannot be imported because this experiment is already on the queue and is waiting to be completed by one of the above steps. • Trying to import data and it fails. Verify that the .exp and .dat files exist with the same name. Verify that the files are a valid size. If the data is on a jaz disk, CDROM, or other media, try copying it to the local hard drive and import again. USERSETS • In GCOS Manager, the user is unable to create a userset. Verify that the information entered is correct. Verify that the userset parameters are valid in GCOS Manager. Verify that the library files have been installed or updated. • In GCOS Manager, I click the Usersets tab and get the message “Failed to load probe arrays -?|” This means that there have not been any probe arrays (library files) installed. 724 Affymetrix® GeneChip® Operating Software User’s Guide TEMPLATES • Unable to create a template. Verify that there is not any other template with the same name. Verify that the template was saved. EXPORTING • Unable to export. Error message “CopyFile Error=112”. Verify that the path location specified for exporting has enough free space to write the .exp, .dat, .cel and .chp files. PUBLISH • Unable to create new Publish database. Verify that the username and password entered are correct. Verify that the path location specified exists. Verify that the path location specified has enough hard disk space. Verify that the open cursors set to 200 (Oracle databases only). Verify that the publish database name does not start with a number. All publish databases and database passwords must begin with a letter. • Creating a SQL Server Publish database. The error message “Device too small” or “Device too large” is displayed. What should the creation size be? The size of creating the publish database is between 128 MB and 1024 MB. • Trying to create an Oracle® publish database and it fails. Verify that the username and password entered is correct. The password for the username gatc is ‘gatc’ (all lowercase). Verify that the path exists. Verify that the open cursors is set to 200. Verify there is enough hard disk space. • Creating an Oracle® database and it fails. The log shows that there are not enough cursors. What does this mean? appendix K | Troubleshooting 725 Verify that the INIT.ORA contains the open_cursors =200 (It needs enough cursors to create a database, otherwise all cursors are locked and in use). • Unable to delete Publish data from the publish AADM database. The publish data selected to be deleted may be in use by another user. Other users may have the Data Mining Tool application open and connected to that publish AADM database. Therefore, in order to delete the data, the other user must exit out of DMT. • After removing a publish database from the database application, the database name still appears in the publish tab. Why? The system DSN still exists in the ODBC Control Panel. Remove the DSN and it will not appear in the application. GCOS MANAGER FAQS This section of the manual lists frequently asked questions (FAQs) regarding Affymetrix GCOS Manager. If you still need assistance after reviewing these questions and answers, please contact Affymetrix Technical Support. For support contact information, see Technical Support, on page 6. Q: I want to view information about other expression samples. I try to select another sample by clicking its icon, but the software doesn’t change to a different sample. What should I do? A: In GCOS Manager, click the text representing the sample or experiment name rather than the icon. Q: How can I change the “export to” or “import from” location? A: On the menu bar, click the icon. If you are in the Process, Publish, or Find tabs, this will select a target location for exported files. If you are in the Import tab, this will select a location to import files. 726 Affymetrix® GeneChip® Operating Software User’s Guide Q: How do I use the Find command to search the Publish database? A: The Find command only works with the Process database. Q: I know some of my samples (projects or experiments) are in the process database. However, when I use the Find command, the results produce an empty set. Why does this happen? A: In the Find window, the Select Object tab, the Advanced Filter tab, and the Data Filter tab are additive. Therefore, if you select experiments with the text string “xxxAssay” using the Select Object tab and then limit the search to Project X using the Advanced Filter tab, the Find command searches for experiments with the text string “xxxx” that are members of Project X. Q: I want to delete (assume ownership, export, or archive) multiple data items (experiments or samples) at one time. However I cannot select multiple data items at one time in the process database. How can I accomplish this? A: You can SHIFT-select or Ctrl-select multiple data items at one time in the Find window. Most of the functions that are available in the Process tab are also available in the Find window. Q: I want to archive a sample but the software will not let me choose that function. How do I archive a sample? A: Experiments or samples cannot be archived. A list of available functions in the Find window is listed below: Analysis data: archive, assume ownership, delete, and export Experiments: assume ownership, delete, export, and rename Samples: assume ownership, delete, and rename Q: How do I get out of the Find window and back to the process database? A: Simply close Find window or go to the Microsoft® Windows® menu and select “GCOS server manager.” Q: Can I view property information about files in the publish database? A: No, this information is not available. But you can view property information about files in the process database in the Publish or Process tab. appendix K | Troubleshooting 727 Q: I cannot find my image data (.dat) under the Import tab. I have confirmed that the .dat file exists on the local drive and that I have the import location mapped correctly. Why doesn't GCOS Manager let me import my image data (.dat)? A: GCOS Manager imports .exp data and the associated image data (.dat) with the same name. Make certain that the folder with the image data (.dat) includes the .exp data with the same name. As a last resort, open the image data (.dat) in Affymetrix® GCOS. If an .exp does not exist for the image data (.dat), GCOS will automatically create one. Q: When I try to open a Sample folder, only a few samples are displayed and I know that there should be more in the database. What happened to the samples? A: The Sample filter remains active during successive sessions. Reset Sample Filter parameters. See Filtering Data, on page 71. Library Files Troubleshooting INSTALLATION • During the library file installation on the server, it shows that the ask is “not responding” in task manager. What is wrong? The library file installation is responding. During this time, it is writing the gene descriptions and sequence information into the databases. This will take a while depending on the number of probe arrays selected. Do not stop or cancel the task. • After an upgrade, the library files update installation is run. The installation completes very fast. Is this correct? Yes. The update copies the *.cif file in the X:\GeneChipDB\Library directory and adds new parameters to the database. Descriptions and sequences are not installed. 728 Affymetrix® GeneChip® Operating Software User’s Guide Appendix L Using the GeneChip® AutoLoader Appendix L 731 Using the GeneChip® AutoLoader The GeneChip® Autoloader is a new addition to the GeneChip® Scanner 3000, designed expressly for scanning multiple GeneChip® probe arrays. The Autoloader can scan up to 48 probe arrays automatically without operator presence. Figure L.1 The Affymetrix® GeneChip® Scanner 3000 with AutoLoader This appendix contains the following sections: • Setting Up the AutoLoader (see below) • Installing and Configuring the E-mail System (see page 739) • Operating the Scanner-AutoLoader (see page 746) 732 Affymetrix® GeneChip® Operating Software User’s Guide • Quick Reference Walkthrough (see page 747) • The AutoLoader Run (see page 751) • Cleaning and Maintenance (see page 787) • Troubleshooting (see page 787) • GeneChip® Scanner 3000 - AutoLoader Specifications (see page 794) • CE Mark Declaration of Conformity (see page 795) • Regulatory (see page 796) Setting Up the AutoLoader The GeneChip® Scanner 3000 with an AutoLoader has the following connections on the unit (Figure L.3): • AC power connection • Ethernet connection • Keyboard/barcode reader connection If you must move the scanner, disconnect all cables first. • The instrument weight is approximately 100 pounds (45.36 Kg). Do not place it on an unstable cart, stand, or table. Failure to properly support the instrument may cause serious damage or injury and may void the warranty. Heavy object. Two people are required to lift the scannerAutoLoader. See the following sections for more information on setting up the Autoloader: • Connecting the Scanner and Workstation (see below) • Indicator Lights and On/Off Button (see page 737) appendix L | Using the GeneChip® AutoLoader 733 CONNECTING THE SCANNER AND WORKSTATION 1. Couple the barcode reader to the keyboard using the coupling block (Figure L.2). Connect the coupling block to the workstation’s keyboard port (Figure L.3). Cable to Keyboard Cable to Barcode Reader Cable to Workstation Keyboard Receptacle Barcode readerKeyboard Coupling Block Figure L.2 Coupling the barcode reader to the keyboard Connect the 3-pronged electric power plug to the workstation (Figure L.3). 3. Connect the dedicated Ethernet cable from the scanner to the Ethernet port that is designated on the workstation. The port is located near the bottom of the workstation (Figure L.3). 4. You can connect your company’s network cable to the indicated port near the center of the workstation (Figure L.3). When you start GCOS, the software will make the proper communication connections. 5. Connect the cabling to the scanner with AutoLoader (Figure L.4) 2. 734 Affymetrix® GeneChip® Operating Software User’s Guide Do not confuse your company’s network connections with the dedicated Ethernet port of the scanner-workstation. The proper scanner connection is located near the bottom of the workstation. This 10/100 Base T Ethernet communications port is dedicated to the scanner-workstation interface. You cannot connect the scanner to your company’s Ethernet communications network. You can, however, connect the workstation’s second Ethernet port to the your company’s Ethernet network. 6. On the rear of the AutoLoader, there are two drain tubes: an upper and a lower drain tube. Place the upper drain tube in a beaker or other receptacle to collect the condensation from the AutoLoader (Figure L.4). The reset button is the scanner’s circuit breaker. The breaker switch will be tripped whenever the scanner experiences an electrical fault condition. Press to reset. If you cannot reset this switch, contact Affymetrix technical support. appendix L | Using the GeneChip® AutoLoader 3-Pronged Electric Power Plug KeyboardBarcode Coupler Block Network Connection Barcode Reader Connection Keyboard Cable AutoLoader Connection Figure L.3 Workstation rear cable connections 735 736 Affymetrix® GeneChip® Operating Software User’s Guide Circuit Breaker Reset Button Cable Receptac to Works Power Cable Upper Drain Tube Lower Drain Tube Figure L.4 Scanner with AutoLoader rear connections appendix L | Using the GeneChip® AutoLoader 737 INDICATOR LIGHTS AND ON/OFF BUTTON The front panel has the following button and indicators (Figure L.5). Blue Autoloader Indicator Light Blue Scanner Indicator Light Green light I/O (on/off) button Yellow light Figure L.5 The AutoLoader indicator lights I/O (on/off) button in the center. 2. Blue indicator light on scanner body, running vertical at front center. This light extends to the bottom of the AutoLoader and is always on when the scanner is on. 1. 738 Affymetrix® GeneChip® Operating Software User’s Guide Blue indicator light on AutoLoader, running vertical at front center. This light appears to be merely an extension of the scanner light. However, when on, it indicates that the AutoLoader door is closed and locked. It will turn off when the door is unlocked. 4. Green and yellow light both on indicate scanner boot up in progress. 3. 5. Yellow light On = Idle, laser is warming up (laser not ready, green off) B. Off = System ready, no errors (Green on) C. Flashing = Error 6. Green light A. On = System is ready to scan (yellow off) B. Flashing = Scan in progress A. Summary of Indicator Lights The table below summarizes the light conditions and their meaning. Table L.1 Indicator lights Condition Green Light Yellow Light Blue Scanner Indicator Light Blue AutoLoader Indicator Light Meaning Initial boot up Off Off On Off Initial power up; embedded PC takes control Scanner boot up On On On Off Embedded PC takes control of scanner boot up Laser warm up Off On On Off Software enabled and laser is warming up System ready On Off On Off Scanner ready for use and AutoLoader door is unlocked waiting to receive a carousel appendix L | Using the GeneChip® AutoLoader 739 Table L.1 Indicator lights Condition Green Light Yellow Light Blue Scanner Indicator Light Blue AutoLoader Indicator Light Meaning Error Off Flashing On Off Fatal error, reboot scanner and software, AutoLoader door is unlocked to remove carousel if necessary Scanning Flashing Off On On Scanning is in progress and AutoLoader door locked Scanning Flashing Off On Off Scanning is in progress and AutoLoader door is unlocked Installing and Configuring the E-mail System In the event of an AutoLoader error condition, you can enable the software to send an e-mail containing error information using Microsoft Exchange server or SMTP. The user sets up the e-mail system just once. See the following sections for information on installing and configuring the E-mail system: • E-mail System Installation (see below) • E-mail Messages (see page 744) E-MAIL SYSTEM INSTALLATION The instrument installation software is on a separate CD. Use this CD and the following procedure to install the instrument control software. Launch Microsoft® Windows® 2000 Explorer. 2. Double-click setup.exe within the Instrument folder. The Welcome window appears. 3. Click Next. 1. 740 Affymetrix® GeneChip® Operating Software User’s Guide Several consecutive Software License Agreement windows appear. 4. Click Yes in each window to accept the terms of the agreement. 5. The Choose Destination Location window appears. 6. Click Browse and select the Destination to install the instrument driver (select the same location where you installed GCOS). 7. Click Next. The Select Components window appears (Figure L.6). Figure L.6 The Select Components window, only the Affymetrix Scanner files box checked 8. Remove the check from the Fluidics station and Sealevel card files box. Select only the Affymetrix Scanner Files option. Highlight the Affymetrix Scanner Files option and click Change... to select the type of scanner. The following dialog box opens and displays the choice of scanners, Affymetrix® GCS 3000 Scanner and GeneArray® 2500 Scanner (Figure L.7). appendix L | Using the GeneChip® AutoLoader 741 Figure L.7 The Select Sub-components window, only the Affymetrix Scanner files box checked Select the Affymetrix® GCS 3000 Scanner and click Continue. 10. Click Next. The Email Option window appears (Figure L.8). 9. Using Microsoft® Exchange If you are using Microsoft Outlook with Microsoft Exchange, you must configure Outlook XP to work with the Exchange server. Each user can set up an individual distribution list, and all users share the same name for each of their own individual distribution lists. Thus there can be two cases in sending e-mail: - There is a single e-mail contact for everybody - There is a common distribution list name, and every user must set up a list with that name. Configure it by the following steps. 742 Affymetrix® GeneChip® Operating Software User’s Guide Figure L.8 The Email Option window with Microsoft® Exchange Server checked 1. Select the Microsoft Exchange Server. Enter an e-mail address or a distribution list that has been set up in Outlook. To set up a distribution list see the section below. When an e-mail is sent during a normal run, you must be logged into the workstation and have an account on the exchange server. Setting up Outlook for a distribution list Let us call the name of the distribution list AutoLoaderGroup. Launch Microsoft Outlook. 2. On the File menu, point to New, and then click Distribution List. 3. In the Name box, type the name that is used in the Instrument Install (e.g. AutoLoaderGroup). 4. Click Select Members. 1. appendix L | Using the GeneChip® AutoLoader 743 In “Show names from the list,” choose the address book that contains the e-mail addresses you want in your distribution list. 6. In the “Type name or select from list box,” type a name you want to include, or select a name from the list below, and then click Members. Do this for each person you want to add to the distribution list, and then click OK. If you want to add a longer description of the distribution list, click the Notes tab, and then type the text. 5. 7. You save the distribution list in your Contacts folder by the name you give it. All e-mail generated during an AutoLoader run will be sent to the people in the distribution list Each user, who plans to run the AutoLoader through his/her user account, should create a distribution list. Configuring SMTP If you are using SMTP, configure it as follows. Figure L.9 The e-mail contact configuration window with SMTP configured 744 Affymetrix® GeneChip® Operating Software User’s Guide Enter the IP address of the SMTP server and the port number at which SMTP transactions occur. 2. Enter an e-mail address or comma separated list of e-mail addresses to which the e-mail must be sent. 1. No E-mail Options Select this option to disable the e-mail system. Place a check in box None. E-MAIL MESSAGES If the e-mail system is enabled, the instrument control software sends an e-mail alert for conditions that may occur during an AutoLoader run. For a list of serious errors, see AutoLoader Error Messages, on page 789. In case of a fatal error: 1. The software sends an e-mail to the specified set of e-mail addresses or to the Outlook distribution list. The software provides you with the ability to send an e-mail without your intervention. 3. The software uses extended MAPI to send an e-mail. 4. Each e-mail message contains the following information: 2. Date and Time B. Scanner ID C. All experiment information displayed in the status window. Table L.2 lists the relevant e-mail messages. A. appendix L | Using the GeneChip® AutoLoader 745 Table L.2 E-mail Message Conditions. These will appear on the scan status field of the status screen at the bottom of the GCOS GUI such as the Experiment Information Window. (See Troubleshooting, on page 787 for recommended action). Condition Detected By Action/Mitigation Carousel Home Error Carousel Home not detected after more than one carousel rotation Log error, stop run, notify user via GUI and e-mail Grip Home Error Grip Home not detected after more than full actuator travel, a mechanical error Log error, stop run, notify user via GUI and e-mail Feeder Fail Cartridge not detected in scanner or AutoLoader during load or unload Log error, stop run, notify user via GUI and e-mail Load Request Error, Cartridge in Scanner Cartridge already in scanner when software directs AutoLoader to load a cartridge Log error, stop run, notify user via GUI and e-mail Unload Error, Cartridge in AutoLoader Cartridge detected already in AutoLoader when software directs AutoLoader to unload a cartridge (Autoloader cannot unload the cartridge.) Log error, stop run, notify user via GUI and e-mail Cooling Over Temperature Cooling set point not attained within 1 hour of activation Log error, disable cooling, notify user via GUI and e-mail, continue AutoLoader run Cooling Under Temperature Cooling temperature < 5ºC Log error, disable cooling, notify user via GUI and e-mail, continue AutoLoader run Door Opened Door was opened in the middle of a scan causing current chip to be rescanned Log error and e-mail 746 Affymetrix® GeneChip® Operating Software User’s Guide Table L.2 (Continued) E-mail Message Conditions. These will appear on the scan status field of the status screen at the bottom of the GCOS GUI such as the Experiment Information Window. (See Troubleshooting, on page 787 for recommended action). Condition Detected By Action/Mitigation Power failure/ restore Power was lost/or restored to the workstation and /or AutoLoader If a UPS is attached to the workstation, an email will be sent Network disconnect A network disconnect between workstation and scanner was detected Log error and e-mail user End of Run Scanner stops the AutoLoader run upon encountering a chip previously scanned or after scanning 48 chips Notify user via GUI and e-mail that the run has completed Operating the Scanner-AutoLoader This chapter describes how to use GCOS to control the Affymetrix® GeneChip® Scanner and AutoLoader (Figure L.1). appendix L | Using the GeneChip® AutoLoader 747 define experiment (*.exp) Process probe array in fluidics station Scan probe array (save image to *.dat) Compute cell intensity data (save intensity data to *.cel) Analyze *.cel (save results to *.chp) Generate report (*.rpt) Figure L.10 Assay & analysis flow chart Quick Reference Walkthrough The table below illustrates the steps required to scan a set of probe arrays. 748 Step 1. Enter and save the experiment information in the Experiment Information Window. For more information, see The Experiment Information Window, on page 758. 2. Hybridize the probe arrays. For more information, see the GeneChip® Expression Analysis Technical Manual and relevant package inserts. Affymetrix® GeneChip® Operating Software User’s Guide Description appendix L | Using the GeneChip® AutoLoader Step 3. Wash the probe arrays. For more information, see the GeneChip® Fluidics Station 450/250 User’s Guide and relevant package inserts. 4. Apply Tough-Spots® For more information, see Using Tough-Spots® to Prevent Leaks, on page 763. Description 749 750 Step 5. Load probe array cartridges into carousel. For more information, see Loading Cartridges into the Carousel, on page 767. 6. Load carousel into the AutoLoader. Set properly by turning it to set flush with housing. Close the door. For more information, see Loading the Carousel into the AutoLoader, on page 769. Affymetrix® GeneChip® Operating Software User’s Guide Description appendix L | Using the GeneChip® AutoLoader Step 7. 751 Description Start the AutoLoader run. For more information, see Scanning a Probe Array in Automode, on page 771. • If you want to scan the probe arrays without waiting for them to have warmed up, check box. • If you want to rescan currently loaded probe arrays that had been scanned in a previous run, check Allow rescans box. The .dat file from the previous run will not be overwritten. The AutoLoader Run This section shows you how to scan multiple GeneChip® probe arrays using the new Affymetrix® GeneChip® Scanner 3000 equipped with the AutoLoader. Figure L.11 Affymetrix® GeneChip® probe array cartridge: note the location of the flange. The AutoLoader will accept the cartridge in only one orientation. 752 Affymetrix® GeneChip® Operating Software User’s Guide The Autoloader operation is described in more detail in the following sections: • Quick Reference Guide to Using the AutoLoader (see below) • The Experiment Information Window (see page 758) • Turning on the Scanner-AutoLoader (see page 762) • Using Tough-Spots® to Prevent Leaks (see page 763) • Using the Barcode Reader (see page 765) • Loading Cartridges into the Carousel (see page 767) • Loading the Carousel into the AutoLoader (see page 769) • Scanning a Probe Array in Automode (see page 771) • Scanning a Probe Array in Manual Mode (see page 777) • Stopping an AutoLoader Run (see page 782) • Shutting Down the Scanner (see page 783) • Using GCOS without a Scanner. (see page 783) • Using GCOS with the AutoLoader Disabled. (see page 784) • Turning on the Laser at GCOS Launch (see page 786) QUICK REFERENCE GUIDE TO USING THE AUTOLOADER The following table can help you find specific procedures based on the type of task you are looking for. If you want to: Then do this: Use the AutoLoader in automode 1. Click Tools → Defaults. 2. Clear the check box Enable Manual Mode. 3. Click OK (Figure L.21). appendix L | Using the GeneChip® AutoLoader If you want to: Then do this: Use the AutoLoader in manual mode 1. Click Tools → Defaults. 753 2. Check Enable Manual Mode. 3. Click OK (Figure L.24). Turn on the laser immediately when you launch GCOS 1. Click Tools → Defaults. 2. Check Turn on Laser at startup. 3. Click OK (Figure L.30). Add an experiment 1. Open the GCOS software, and click Experiments. Information window. to open the Experiment 2. Enter and save the experiment information in the Experiment Information Window. 754 Affymetrix® GeneChip® Operating Software User’s Guide If you want to: Then do this: Add a barcode 1. Open the GCOS software to the Experiment Information window. • Note: A new experiment must be opened for each barcode. 2. Place cursor in the barcode field. 3. Hold a GeneChip probe array cartridge in front of the barcode reader and squeeze the trigger for up to four seconds until you hear a beep. The reader reads and sends the barcode to the GCOS Experiment Information window, Barcode field. The software automatically fills other fields. 4. After the software adds the barcode, save the experiment. 5. Repeat steps 1 to 4 until you have read all of the probe array cartridges. appendix L | Using the GeneChip® AutoLoader If you want to: Then do this: Start a scanning run 1. Load the carousel and place the carousel into the AutoLoader. 2. Click the Start button . • If the arrays are at room temperature, or you want to scan the probe arrays without waiting for them to warm up, check the appropriate box. • If you want to allow the probe arrays to be rescanned, check the appropriate box. (The rescan function is handy if you want to rescan a currently loaded probe array that had been scanned in a previous run.) 3. Click OK. The AutoLoader homes and performs an inventory of the probe arrays and the scanning run begins. 755 756 If you want to: Affymetrix® GeneChip® Operating Software User’s Guide Then do this: Add a probe array during a scan 1. Click the Add button or select Run → Add Chips. • If you want to unlock the door and immediately begin adding cartridges, click the Add Now button. The blue indicator light on the front of the AutoLoader will go out signifying that the door is unlocked and you can load more cartridges. The cartridge being scanned, if any, will be rescanned, and the previously created .dat file WILL BE OVERWRITTEN. • If you want to wait for the scan to complete before adding a probe array, click the Add after Scan button. The current scan will continue until completion, then the door will unlock and you can load more cartridges. The Autoloader blue indicator light will go out to signify that the door is unlocked. • In both cases, after you have added your cartridges and closed the door, the following window will appear. Click OK. 2. Click the Resume button or Run → Resume. The door then locks and the blue indicator light turns on. Use GCOS without a scanner 1. Click Tools → Defaults. 2. Clear the check box Scanner Installed. 3. Click OK (Figure L.28). Disable AutoLoader and use the scanner only 1. Click Tools → Defaults. 2. Check Disable AutoLoader. 3. Click OK (Figure L.29). appendix L | Using the GeneChip® AutoLoader If you want to: Stop a scan 757 Then do this: 1. Click the Stop button . 2. When the following window appears, click OK. Caution: If you stop the scanner while a probe array is in the process of scanning, you will lose all scan information from that probe array. If you rescan the array, it may be affected due to uneven photo-bleaching. This could potentially make the data from the array difficult to compare to other array data. Terminating a scan run The AutoLoader run will terminate under certain normal circumstances. Table L.2 outlines under what conditions a scan run will or will not terminate. Table L.3 Summary of scan run termination conditions The scan run will terminate if: The scan run will not terminate if: You press the Stop button. Caution: If you stop the scanner while a probe array is in the process, you will lose all scan information from that probe array. If you rescan the array, it may be affected due to uneven photo-bleaching. This could potentially make the data from the array difficult to compare to other array data. You check Allow Rescans box. When the AutoLoader encounters a probe array that was previously scanned in an earlier run, it will rescan that probe array and will create additional .dat files (.dat1, .dat2, etc.). The AutoLoader detects a probe array with the same barcode in the current run, i.e., in the currently loaded carousel. The probe array will not be rescanned. You clear the Allow Rescans box. When the AutoLoader encounters a probe array that was previously scanned in an earlier run, it will log the probe array but will not rescan it. It will continue the run. 758 Affymetrix® GeneChip® Operating Software User’s Guide Table L.3 (Continued) Summary of scan run termination conditions The scan run will terminate if: The scan run will not terminate if: The AutoLoader detects 48 scanned cartridges in the same run. You click the Add Now button. The AutoLoader door will unlock to accept new probe array cartridges. The scan in progress will complete. When you close the door and continue, the AutoLoader will home, take inventory and move to the same probe array that was in the process of being scanned when the door was opened. It will discard the earlier .dat file and rescan that probe array. Note: this has nothing to do with the Allow Rescans check box. You click the Add After Scan button. The AutoLoader door will wait until the scan in progress is complete then unlock the door. When you close the door and continue, the AutoLoader will home, take inventory and move to the next probe array from that which was in the process of being scanned when the door was opened. THE EXPERIMENT INFORMATION WINDOW The Experiment Information window (EIW) provides you with the means for generating and saving a database of your experimental data (Figure L.15). Before you can advance to the next experiment, you must first save the current experiment. Certain information has to be entered before the experiment can be saved. Table L.4 lists the available fields and their definitions and whether or not they are required in order to save the experimental data. For more information, see Registering a Sample & Defining an Experiment, on page 80. appendix L | Using the GeneChip® AutoLoader 759 Table L.4 Definitions of the experiment data fields in the Experiment Information Window Field Definition Required for save Sample Template Dropdown box for templates associated with samples No Sample Name The name of the sample. Yes Sample Type The type of sample. This is an attribute of the sample that describes its type. Example: blood, cell line, tissue, etc. Yes Sample Project The name of the project associated with the sample. Yes Sample owner Read-only field showing the owner of the sample in the database. Yes Experiment Template Dropdown box for experiment templates No Experiment Name The name of the experiment. Yes Probe Array Type This is the probe array type associated with the experiment, and automatically filled in when you enter the barcode. Yes Barcode User entered barcode Yes (No if AutoLoader is in manual mode) Expiration Date Array Expiration date embedded in Affymetrix barcode, and automatically filled in when you enter the barcode. No Probe array Lot number Array lot number embedded in Affymetrix barcode, and automatically filled in when you enter the barcode. No Experiment owner Read-only field showing experiment owner Yes 760 Affymetrix® GeneChip® Operating Software User’s Guide Table L.4 (Continued) Definitions of the experiment data fields in the Experiment Information Window Field Definition Required for save User-set Drop down list of user-sets in the database used for storing analysis parameters No Publish Database Drop down list of names of the publish databases. Used when selecting automation No Publish Intensities Checkbox to be selected if publishing intensities to the database after completing the analysis No Scan Status Window GCOS software provides you with the status of all scans, the status of the AutoLoader door and status of the current scan. This window is located at the bottom of user interface (Figure L.12). Below (Table L.5) describes the status fields. appendix L | Using the GeneChip® AutoLoader 761 Status window Figure L.12 The scan status window Table L.5 The GCOS Status Fields and Descriptions Status Field Description Position Position occupied by a given cartridge in the AutoLoader carousel Experiment Name The experiment name associated with the scan associated with a given cartridge position Probe Array Type The probe array type for the scan associated with a given cartridge position Barcode ID The unique identifier in the barcode for the scan associated with a given cartridge position User Name of the user (experiment owner) for the scan associated with a given cartridge position Time & Date The date and time when scan started and completed 762 Affymetrix® GeneChip® Operating Software User’s Guide Table L.5 (Continued) The GCOS Status Fields and Descriptions Status Field Description Scan Status The status of the scan. (Autofocus, scanning). This field displays all scanner status strings associated with the scan and retrieved from the scanner. The message strings that may appear in this field are listed below. Note: Not all of these messages will appear in each AutoLoader run. Status Field Strings Autofocus Scan Status - % of lines scanned Scan Complete status Grid alignment errors Autofocus Errors The experiment XXX has already been scanned Chip load failures Invalid barcode errors Experiment does not exist errors AutoLoader door open errors AutoLoader Door (viewed in status bar) The status of the AutoLoader door Number of Cartridges (viewed in status bar) Number of cartridges in the AutoLoader carousel as determined by inventory History (viewed as a log) A running history of the last 99 arrays scanned and the information on the current array (if any) being scanned TURNING ON THE SCANNER-AUTOLOADER Press the on/off (I/O) switch on the front panel. The scanner’s onboard computer will boot up. The bootup process takes a few of minutes. appendix L | Using the GeneChip® AutoLoader 763 During this time both the yellow and green light will be on. The scanner enters the laser warm-up state. During this warm-up time, the green light will turn off and the yellow light will remain on. You must wait 10 minutes for the laser to stabilize. USING TOUGH-SPOTS® TO PREVENT LEAKS Tough-Spots® are chemically inert polyvinyl labels that adhere to all plastics. Affymetrix recommends using 3/8-inch circle diameter Tough-Spots to prevent leakage from the cartridge septa. Before loading the probe array cartridge, follow this procedure to prevent the leaking of fluids from the cartridge during scanning. Even if you have already applied Tough-Spots to the cartridge prior to hybridization or after washing, you must remove the old Tough-Spots and apply new ones before you load them into the AutoLoader. Affymetrix recommends the use of Tough-Spots® obtained from Affymetrix P/N 64-0158 or from USA Scientific, Inc. P.O. Box 3565 Ocala, FL 34478 (800)LABTIPS P/N 9185-0000 To reduce the risk of leakage, do not use excessively large pipette tips to pierce the septa. 1. On the back of the probe array cartridge, clean excess fluid from around septa (Figure L.13). 764 Affymetrix® GeneChip® Operating Software User’s Guide Figure L.13 The GeneChip® probe array cartridge 2. Carefully apply one Tough-Spot over each of the two septa. Press to ensure that the spots remain flat. If a Tough-Spot does not apply smoothly; that is, if you observe bumps, bubbles, tears or curled edges, do not attempt to smooth them out. Remove the spot and apply a new one (Figure L.14). Tough-SpotsTM Figure L.14 Applying Tough-Spots® to cartridge septa appendix L | Using the GeneChip® AutoLoader 765 USING THE BARCODE READER If you have not already done this during the initial experiment setup, you must now add a new barcode for each experiment. 1. Open the GCOS software to the Experiment Information window and place the cursor in the barcode reference field of the GCOS Experiment Information window (Figure L.15). You should open a new experiment and place the cursor into the barcode field before triggering the reader. Otherwise, the barcode will be misinterpreted as some other kind of input. A new experiment has to be opened for each barcode, and certain other information has to be entered before the experiment can be saved. Figure L.15 Experiment Information Window with cursor in the barcode field 766 2. Affymetrix® GeneChip® Operating Software User’s Guide Hold a GeneChip probe array cartridge in front of the barcode reader and squeeze the trigger for up to four seconds until you hear a beep (Figure L.16). Figure L.16 Reading the cartridge barcode 3. The reader reads and sends the barcode to the GCOS Experiment Information window, Barcode field (Figure L.17). The software also automatically fills in the “Probe Array Type,” “Probe Array Lot,” and “Expiration Date” fields. These are readonly fields. appendix L | Using the GeneChip® AutoLoader 767 Figure L.17 After the software adds the barcode, save the experiment. 5. Repeat steps 1 to 4 until all of the probe array cartridges have been read. 4. LOADING CARTRIDGES INTO THE CAROUSEL 1. Load your cartridges into the carousel (up to 48). Note that only one orientation is possible (Figure L.18). Cartridges should be loaded into the carousel starting at position #1. Additional cartridges need not be contiguous. A run will stop after 48 cartridges OR when the same barcode is read within the same run. 768 Affymetrix® GeneChip® Operating Software User’s Guide Figure L.18 Loading the cartridge into the chip carousel, note that each slot is numbered., 1 through 48, and each cartridge can fit in only one orientation appendix L | Using the GeneChip® AutoLoader 769 LOADING THE CAROUSEL INTO THE AUTOLOADER 1. Load the carousel into the AutoLoader by inserting the carousel into the AutoLoader and turning the carousel until the alignment pin seats into the alignment hole (Figure L.19) Alignment Hole Carousel Mounting Key Flat Alignment Key in the Autoloader Alignment Pin Figure L.19 Loading the cartridge carousel into the AutoLoader 770 2. Affymetrix® GeneChip® Operating Software User’s Guide Turn the carousel clockwise until the carousel mounting key flat seats gently into the AutoLoader alignment key. You may have to turn the carousel several times before it will seat into the alignment pin and alignment key. When seated properly, the carousel will be flush with the AutoLoader housing. Close the AutoLoader door (Figure L.20). Figure L.20 Inserting and turning the carousel; the carousel should be seated and flush with housing. appendix L | Using the GeneChip® AutoLoader 771 The seating of the key flat is confirmed by a gentle falling of the carousel into the key. SCANNING A PROBE ARRAY IN AUTOMODE If all probe arrays have valid, associated barcodes, you can run the AutoLoader in automode. If there exists an identical barcode within the database from an earlier AutoLoader run, and you want to rescan the current probe array with that same barcode, check the Allow Rescans box (Figure L.22). This will create additional .dat files. The original .dat file WILL NOT BE OVERWRITTEN. However, if the AutoLoader encounters the same barcode within the same run, the run will terminate. 1. Set the default settings. Click Tools → Defaults (Figure L.21). B. Clear the check box of Enable Manual Mode. C. Click OK. A. 772 Affymetrix® GeneChip® Operating Software User’s Guide Figure L.21 Defaults window showing “Enable Manual Mode” with the check box cleared 2. Click the Start button in the Instrument Control shortcut bar or the main toolbar; or Select Run → Start Scanner from the menu bar. The Start window appears (Figure L.22). Figure L.22 The Start Scanner window in automode If you have not already loaded your probe array cartridges, do so now. appendix L | Using the GeneChip® AutoLoader 773 If you want to skip the ten minute warm-up period before scanning the first probe array, keep the default check in the Arrays at room temperature box. 3. Click OK in the GeneChip Scanner dialog box to start the run. - The AutoLoader blue indicator light will light up signifying that the AutoLoader door is now locked. - The carousel automatically homes itself and performs inventory to determine the number and position of cartridges present. - The scanning run begins. During the scan, the green light will flash, and the yellow light will be off. - The AutoLoader completes the autofocus operation before scanning each of the probe arrays. This takes approximately two to three minutes. The scanner cannot be stopped during this period. - The run will stop automatically when the last cartridge is scanned. - At the completion of each scan, the GCOS software will attempt grid alignment. If it is successful, the scan data will be automatically advanced to the Grid Alignment processing state. - If the View Scan In Progress feature is enabled (select View → Scan in Progress from the menu bar), the Image window automatically opens in the main display area when a scan starts. It displays the fluorescence intensity of the probe array at approximately 200 lines at a time as the scan progresses. - To enable (or disable) this option, select View from the menu bar and place (or remove) a check mark next to Scan in Progress. After the scan is completed, GCOS: A. saves the image data to an image data file (*.dat) (displayed in the main display area) 774 Affymetrix® GeneChip® Operating Software User’s Guide aligns a grid on the *.dat to identify the probe cells C. automatically computes probe cell intensities and saves the data to the cell intensity file (*.cel) D. ejects the probe array. The AutoLoader will skip probe array cartridges if the AutoLoader encounters: B. A. cartridges with unreadable or invalid barcodes or without barcodes cartridges with barcodes that are not associated with an experiment C. cartridges that have been previously scanned if the Allow Rescans checkbox is cleared (Figure L.22). B. AUTOROTATION The AutoLoader is equipped with a heater to warm up the cartridges prior to scanning in order to reduce condensation and fogging of the probe array cartridge window. The autorotation routine is used for temperature stability but only after the AutoLoader run is complete or during a power failure as described below. Autorotation occurs during a power failure only if the uninterrupted power supply (UPS) is included as an accessory. The UPS provides power to the scanner/AutoLoader during a power failure. If the power fails during the scan of an array, that scan is completed and then the system turns off the heater and enters the autorotation mode to conserve power and cool the chips in the carousel. The system will also attempt to send an e-mail to notify the user of the power failure. During an AutoLoader run, the carousel is rotating as the chips are processed to introduce the next chip to the scanner, so autorotation is not needed. After the AutoLoader run is complete the heater is turned off and the carousel is rotated to get even cooling of the chips. appendix L | Using the GeneChip® AutoLoader 775 Adding Cartridges during a run The software provides you with a button that will allow you to unlock the AutoLoader door and add additional probe array cartridges while in the middle of an AutoLoader run. You have two choices: to add cartridges immediately even while a scan is in progress (in which case the .dat file is discarded and replaced with a newly collected one after the AutoLoader run is resumed), or, as a second choice, to add cartridges after a scan has completed (in which case the .dat file is saved). The Add Chips button is enabled only after the AutoLoader has started a run in automatic mode. Click the Add Chips button , or select Run → Add Chips from the menu bar. The software displays a dialog box with three buttons (1) Add Now (2) Add after scan is complete (3) Cancel (Figure L.23). Figure L.23 The Add Chips selection buttons 1. If you click the Add Now button: A. The blue AutoLoader indicator light will turn off signifying that the AutoLoader door is now unlocked. Open the door and add probe array cartridges. If a scan is in progress, the software will continue to record the scan. However, to avoid the possibility of generating a corrupt 776 Affymetrix® GeneChip® Operating Software User’s Guide .dat file, it will discard that scan that was in progress when the door was opened. The Resume button procedure. is available only for the Add Chips B. After you have added your cartridges, close the door and the following window appears. C. Click OK and then click the Resume button or select Run → Resume. The blue AutoLoader indicator light turns on signifying that the door is locked. The software homes the carousel and inventories the number of present probe arrays. The carousel then moves to that last previously scanned probe array (when the door was opened) and continues the scanning run by rescanning that probe array cartridge. The cartridge is rescanned and a new.dat file overwrites the previous .dat file. Do not load a cartridge into the same carousel position that was occupied by the cartridge that had just been scanned, i.e., do not replace cartridges. 2. If you click the Add after scan is complete button, A. The AutoLoader will wait until the current probe array cartridge has undergone the autofocus and scan procedures before unlocking the door to allow you to add probe array cartridges. appendix L | Using the GeneChip® AutoLoader 777 The blue AutoLoader indicator light will turn off signifying that the AutoLoader door is now unlocked. C. Open the door and add probe array cartridges. D. After you have added your cartridges, close the door and the following window appears. B. The GCOS status bar will also display a “waiting to start” status. Click OK and then click the Resume button or select Run → Resume. The blue AutoLoader indicator light turns on signifying that the door is locked. The software homes the carousel and takes inventory of the probe array cartridges present. The carousel then proceeds to the next cartridge position following the previously scanned probe array. The AutoLoader continues the run from that cartridge position. 3. If you click the Cancel button, the AutoLoader will continue the AutoLoader run. E. SCANNING A PROBE ARRAY IN MANUAL MODE In the AutoLoader Automode, each probe array cartridge requires a valid barcode in order to be scanned. The manual mode feature allows you to scan one probe array at a time without the requirement of a barcode. This is useful if you must scan probe arrays that have invalid or absent barcodes. 1. Set the default settings. A. Click Tools → Defaults (Figure L.24). Check Enable Manual Mode. C. Click OK. B. 778 Affymetrix® GeneChip® Operating Software User’s Guide Figure L.24 Enable Manual Mode checked 2. Click the Start button in the Instrument Control shortcut bar or the main toolbar; or Select Run → Start AutoLoader from the menu bar (Figure L.25). appendix L | Using the GeneChip® AutoLoader 779 Figure L.25 The Scanner control window The scanner Start window appears. At this point, you can choose from a number of options: You can click Start without selecting an experiment. If your cartridge has a valid barcode, the software will get the barcode from the cartridge in the AutoLoader and select the correct experiment. 2. If your cartridge has a valid barcode, you can scan the barcode on the probe array cartridge in to the barcode field. The software will retrieve the experiment associated with the array. 3. If your cartridge does not have a valid barcode, you can manually select an experiment as described below. 1. A. The Experiment Name drop-down list displays the experiments (*.exp) in the current directory that have not been scanned (no *.dat file containing scan data exists for the experiments). 780 Affymetrix® GeneChip® Operating Software User’s Guide B. To include scanned experiments in the Experiment Name drop-down list, choose the Include Scanned Experiments option. If filters have been applied in GCOS Server mode, the drop-down list may not include all experiments. Click Filter to open the Filters dialog box and view or specify filters. Select the experiment name of the probe array to be scanned from the Experiment Name drop-down list. - The Probe Array Type field automatically displays the probe array type that was entered during experiment setup. GCOS sets the Number of Scans per probe array type (one for the GeneChip® Scanner 3000). - You may change the number of scans in the Scanner dialog box (Figure L.25). Multiple scans are statistically averaged to create a single image data file (*.dat). C. Increasing the number of scans increases the scan time as well as the amount of fluorophore bleaching and may result in lower fluorescence intensities. Click Start. The Scanner dialog box appears (Figure L.26). D. appendix L | Using the GeneChip® AutoLoader 781 Figure L.26 The start scanner window in manual mode - You can check Array at room temperature if you want to skip the ten-minute warm-up period before scanning the probe array. The default has the box already checked. You can eject or load a cartridge by clicking on the Load/Eject button at any time except during the time the scanner is engaged in the autofocus operation or the scanning run. 4. Load the probe array cartridge into slot number 1. The cartridge slot at position number 1 is the only slot available in Manual Mode. 5. Click OK in the Start Scanner box (Figure L.26) to start the autofocus routine. This takes approximately two to three minutes. The scanner cannot be stopped during this period. During the scan, the green light will flash, and the yellow light will be off. If the View Scan In Progress feature is enabled (select View → Scan in Progress from the menu bar), the Image window 782 Affymetrix® GeneChip® Operating Software User’s Guide automatically opens in the main display area when a scan starts. It displays the fluorescence intensity of the probe array at approximately 200 lines at a time as the scan progresses. To enable (or disable) this option, select View from the menu bar and place (or remove) a check in the box next to Scan in Progress. After the scan is completed, GCOS: saves the image data to an image data file (*.dat) (displayed in the main display area) B. aligns a grid on the *.dat to identify the probe cells C. automatically computes probe cell intensities and saves the data to the cell intensity file (*.cel) A. D. ejects the probe array. Ejecting a probe array cartridge The probe array cartridge will automatically eject after a run; however, if you must eject a cartridge, click the Load/Eject button (Figure L.25). This command is only available in manual mode. This completes the manual mode section. STOPPING AN AUTOLOADER RUN The Stop button is only available after you have clicked the Start button. Click this button if you want to abort a scan or run in progress. If you stop the scanner while a probe array is in the process, you will lose all scan information from that probe array. If you rescan the array, it may be affected due to uneven photo-bleaching. This could potentially make the data from the array difficult to compare to other array data. 1. Click the STOP toolbar button from the menu bar. or select Run → Stop Scanner appendix L | Using the GeneChip® AutoLoader 2. 783 At the prompt, click Yes to stop the scanner or No to elect not to stop the AutoLoader run (Figure L.27). Figure L.27 Stop AutoLoader prompt 3. A window will display the message “The scanner will not stop until autofocus has finished.” Click OK. After you stop a scan, the scanner will automatically eject the cartridge. SHUTTING DOWN THE SCANNER Close the GCOS software. This is the best way to shut off the laser. 2. Press the I/O button on the front panel to turn off the instrument. 1. USING GCOS WITHOUT A SCANNER. In the event that you would like to use GCOS and do not have or do not want to attach a scanner, you should clear the check box of the Scanner Installed option in the Defaults window. This will prevent the software from continually attempting to make contact with the scanner. Click Tools → Defaults. 2. Clear the check box of Scanner Installed (Figure L.28). 3. Click OK. 1. 784 Affymetrix® GeneChip® Operating Software User’s Guide Figure L.28 Scanner Installed with the check box cleared USING GCOS WITH THE AUTOLOADER DISABLED. If you have a working scanner but the AutoLoader is not operating, you can still use the scanner, but you must check the Disable AutoLoader option in the Defaults window. This will disable the AutoLoader and enable you to use the scanner alone as you would in manual mode. Click Tools → Defaults. 2. Check Disable AutoLoader. 3. Click OK (Figure L.29). 1. appendix L | Using the GeneChip® AutoLoader 785 Figure L.29 Disable AutoLoader checked A window will appear asking that you reboot the scanner. 4. Press the scanner front panel I/O button once to turn off the scanner (Figure L.5). Wait a few moments, the press the I/O button to turn on the scanner. Open the AutoLoader door. 6. Manually load a probe array cartridge into the slot. 7. Close the AutoLoader door. 8. Scan the probe array in the same manner as the AutoLoader in manual mode. See Scanning a Probe Array in Manual Mode, on page 777. 5. 786 9. Affymetrix® GeneChip® Operating Software User’s Guide After scanning the probe array, you must manually remove the probe array cartridge from the AutoLoader. See Steps 7 though 13 in Manually Removing a Lodged Probe Array Cartridge, on page 790. TURNING ON THE LASER AT GCOS LAUNCH You have two options as to how to control the actuation of the laser. You can have the GCOS software turn on the laser at the launch of the GCOS software. This allows you to begin the ten-minute warmup period immediately. Or you can wait until you are about to run a series of probe array cartridges to turn on the laser. You still wait ten minutes for the laser to warm up. 1. Click Tools → Defaults (Figure L.30). If you want to turn on the laser immediately upon launching the GCOS software, check Turn on Laser at startup. B. If you want to wait until you are ready to run a scanning procedure, before turning on the laser, clear the check box for Turn on Laser at startup. 2. Click OK. A. appendix L | Using the GeneChip® AutoLoader 787 Figure L.30 Turn on Laser at startup checked Cleaning and Maintenance The AutoLoader requires little in the way of customer maintenance. The instrument must be kept clean and free of dust. Dust buildup can degrade performance. Wipe the exterior surfaces clean using a mild dish detergent solution in water. Do not use ammonia based cleaners or organic solvents, such as alcohol or acetone, to clean the system because they may damage the exterior surfaces. Clean the carousel by hand using warm water and, if necessary, mild detergent. Troubleshooting Troubleshooting tips are given in the table below and in the following sections: • AutoLoader Error Messages (see page 789) 788 Affymetrix® GeneChip® Operating Software User’s Guide • Manually Removing a Lodged Probe Array Cartridge (see page 790) Problem Possible Cause No image when scanning • Power off or cable loose Corrective Action Check all connections and power. • Loss of laser power Contact technical support. • Image display disabled Enable image display Intermittent problems scanning • Loose cable Check all rear connections. Scanner fails with probe array inside • Power failure Manually extract probe array. Check all connections to scanner. Turn scanner on, restart software. Carousel does not automatically home • Check for stuck cartridge • Carousel not seated on D ring • Alignment Pin not engaged in Carousel • Door is open or ajar • Door is open when blue LED is off. Carousel does not rotate • Door is open or ajar • System is warming up, chip in heater • Carousel not seated on D ring • Alignment Pin not engaged in Carousel • Laser in Scanner is warming up. GCOS has Start grayed out in this case Carousel misses next chip • Cartridge UP sensor not working, call technical support. Stuck cartridge AutoLoader freezes up See Manually Removing a Lodged Probe Array Cartridge, on page 790 • Door is open or ajar appendix L | Using the GeneChip® AutoLoader Problem AutoLoader overheats Possible Cause 789 Corrective Action • Heater Failure Call technical support. • TE failure Call technical support. • TE hot fans vent blocked Autofocus routine fails to conclude • Try to rescan chip. • Check for salt on chrome border. If still error, call technical support. The cartridge does not descend into scanner. • Carousel not seated correctly • Door is open or ajar • Heater is waiting until chip is at temperature. AUTOLOADER ERROR MESSAGES The following error messages indicate a serious malfunction of the scanner with AutoLoader. Your probe arrays, or the data generated from them, may be at risk. You should shut down the AutoLoader and remove the carousel. Do not continue to use the AutoLoader in Automode. Call Affymetrix Technical Support. However, if the AutoLoader appears to be operating normally, you can continue to use the AutoLoader in Manual Mode. See Scanning a Probe Array in Manual Mode, on page 777. HEATER_LOW “Warning: The warming chamber temperature is low. Refer to the troubleshooting guide.” COLD_CHAMBER_LOW “Warning: The cold chamber temperature is low. Refer to the troubleshooting guide.” COOL_HOTSIDE_HIGH “Warning: The cooler hot-side temperature is high. Refer to the troubleshooting guide.” Note: Before calling technical support, check around the ventilation vents to ensure that nothing is blocking them. 790 Affymetrix® GeneChip® Operating Software User’s Guide COLD_CHAMBER_HIGH “Warning: The cold chamber temperature is high. Refer to the troubleshooting guide.” Note: Before calling technical support, check the AutoLoader door to ensure that it is not open. HEATER_HIGH “Warning: The warming chamber temperature is high. Refer to the troubleshooting guide.” MANUALLY REMOVING A LODGED PROBE ARRAY CARTRIDGE In the event that a probe array becomes lodged in the chip transport mechanism, follow the procedure outlined below. 1. Turn the AutoLoader off and remove the power cord from the back of the unit. 2. Open the AutoLoader door on top of the unit. 3. Remove the carousel from the system. (Keep cartridges in carousel at proper temperature while recovering the cartridge still in the scanner). appendix L | Using the GeneChip® AutoLoader 791 4. Remove the hole plug, which is just in front of the cartridge slot in the base piece of insulation. In the photo to the left, the screwdriver is inserted into this hole. 5. Using a standard, flat (-) screwdriver, (13-0257) gently slide it down through the hole making sure not to damage the shaft and spring that are protruding into the hole. When the screwdriver stops, it should be in contact with the scanner Y stage screw. Slowly turn the screwdriver until you feel it engaging the slot on the screw of the scanner Y stage. 6. Slowly turn the screw clockwise until it hits a hard stop and cannot turn further. (Do not try to turn it further or use excessive force because it will break the Y stage in the scanner). The Y stage has now ascended to its maximum position. 792 7. Using your fingers, slowly slide the slot pin, which is sticking through the slot in the base piece of insulation, to the right until it stops. You should see the little pinch rollers near the cartridge slot close a little as you do this. Affymetrix® GeneChip® Operating Software User’s Guide Pinch Rollers Slot Pin appendix L | Using the GeneChip® AutoLoader 8. Insert a 3/16” hex driver (13-0255) into the hole that is located on the front of the AutoLoader housing on the left. You should feel it engage a coupling. 9. Turn the hex driver counter clock wise until you see the cartridge appear through its opening. (The cartridge should stay up if you stop turning the hex driver). If you don't see the cartridge after turning the hex driver ten seconds go to step 11. 10. Grab and hold the cartridge with your fingers. Using your other hand slowly slide the slot pin (Step 7) back to the left. This should open up the pinch rollers. Pull the cartridge out. 11. If you do not see the cartridge after turning the hex driver for 10 seconds, stop. 793 794 Affymetrix® GeneChip® Operating Software User’s Guide 12. Using tool (13-0256)with the hook down and toward the back, slide it vertically down against the front of the cartridge opening, about 1.5 inches. (There is a small groove made for this tool in the middle of the front cartridge guide). 13. Pull the top finger grip of the tool toward the front of the unit, and then pull it up while still putting pressure towards the front. The cartridge should come up with the tool. When you see it, grab the cartridge and pull it out of the unit. 14. If you cannot get the cartridge out after doing this procedure, call for Affymetrix technical support. 15. Put the hole plug back into the hole in the base piece of insulation. 16. Plug the scanner back in and turn it on. 17. Load the carousel after the scanner boots up. 18. If cartridges continue to become lodged in the AutoLoader, you should call technical support. GeneChip® Scanner 3000 - AutoLoader Specifications Item Weight Dimensions Parameter Value Shipping approx 115 pounds (52.2 Kg) Free-standing approx 100 pounds (45.4 Kg) Width ~13.25 in. Depth ~21.25 in. Height ~32 in. Power See Scanner 3000 Specifications, on page 576. Working Environment Temperature 59°F-85°F (15°C-30°C) appendix L | Using the GeneChip® AutoLoader Item Parameter 795 Value Humidity 10-90% Non-condensing Clearance 2 in. (5 cm) on side, back 12.5 in. on top Pollution Degree 2 Installation Category II Altitude <2000m Electrical Supply See Scanner 3000 Specifications, on page 576. Main Supply Voltage Fluctuations See Scanner 3000 Specifications, on page 576. CE Mark Declaration of Conformity We, Affymetrix, Inc. 4G Crosby Drive, Bedford, Massachusetts Declare under sole responsibility that the Affymetrix® GeneChip® Scanner 3000-AutoLoader conforms with the relevant provisions of the following standards or other normative documents: EU EMC Directive 89/336/EEC: EN 61326:1998 Equipment for Measurement, Control and Laboratory Use, EMC Requirements EN 55011:1998 Industrial, scientific and medical (ISM) radio-frequency equipment - Radio disturbance characteristics - Limits and methods of measurement EN 61000-3-2:2001 Limits for harmonic current emissions (equipment input current up to and including 16 A per phase) EN 61000-3-3:1995 Limitation of voltage changes, voltage fluctuations and flicker in public low-voltage supply systems, for equipment with rated current less than or equal to 16 A per phase and not subject to conditional connection EN 61000-4-2:1995 Electrostatic discharge immunity. 796 Affymetrix® GeneChip® Operating Software User’s Guide EN 61000-4-3:1995 Radiated, radio frequency, electromagnetic field immunity. EN 61000-4-4:1988 Electrical fast transient/burst immunity. EN 61000-4-5:1995 Surge immunity. EN 61000-4-6:1996 Immunity to conducted disturbances induced by radio frequency fields. EN 61000-4-11:1994 Voltage dips, short interruptions, and voltage variations immunity. EU Low Voltage Directive 73/23/EEC EN 61010-1:2001 Safety requirements for electrical equipment for measurement, control, and laboratory use -- Part 1: General requirements EN 60825-1:1994+ A2:2001 Safety of laser products -- Part 1: Equipment classification, requirements and user's guide Regulatory This device complies with Part 15 of FCC Rules. Operation is subject to the following two conditions: (1) This device may not cause harmful interference, and (2) This device must accept any interference received, including interference that may cause undesired operation. This device complies with FDA performance standards for laser products except for deviations pursuant to Laser Notice No. 50, dated July 26, 2001. This Class A digital apparatus meets all requirements of the Canadian Interference-Causing Equipment Regulation. Cet appareil numérique de la classe A respecte toutes les exigences du Règlement sur le matériel broullier du Canada. appendix L | Using the GeneChip® AutoLoader Regulatory Agency 797 Certification AL 02 12 39816 004 92AA See GeneChip® Scanner 3000 - AutoLoader Specifications, on page 794. Compliant with directive 2002/96/EC (WEEE) Class I Laser Device 21 CFR 1040.10 and 1040.11 For Research Use Only. Not for use in diagnostic procedures. 798 Affymetrix® GeneChip® Operating Software User’s Guide Appendix M Data Transfer Tool Guide Appendix M 801 Data Transfer Tool Guide The Affymetrix® Data Transfer Tool (DTT) provides a convenient way to transfer data in and out of GCOS for archiving and sharing. The DTT provides the following functionality: • Ability to exclude DAT files • Ability to set a variety of span file sizes to save DTT Archives onto removable media • Greater Backward Compatibility • Filter data from DTT Archive or flat files for transferring IN • Added file types for quicker archiving and unpacking • Detection and resolution of conflicts with templates, samples, and usersets when using DTT Archive/Flat Files This appendix provides information about using DTT. Using the Data Transfer Tool To start the Data Transfer Tool: • Click the Microsoft® Windows® Start button and select Programs → Affymetrix → Data Transfer Tool. At start up, the Welcome window displays the initial options (Figure M.1). When working with GCOS 1.2 and higher software, the Data Transfer Tool can be invoked from the file menus of the applications. 802 Affymetrix® GeneChip® Operating Software User’s Guide Figure M.1 Welcome Screen for DTT on computer with GCOS installed GCOS SYSTEM WELCOME SCREEN When used with a GCOS system, the DTT allows you to transfer the following types of data: • GCOS Data • Mining Data stored in an AADM database Before transferring data IN from a Mining (AADM) DTT Archive or CAB file, you must create a new, empty mining (AADM) database in GCOS Manager. For more information, refer to the GCOS Manager documentation. appendix M | Data Transfer Tool Guide 803 You can transfer data in different formats: • DTT Archive files • Flat Files • CAB files • MAS files (transfer IN only) For more information about the different input and output data formats, see DTT Options Dialog Box for GCOS System, on page 804. In the Welcome page, you can: • Select Transfer Data In or Transfer Data Out operations • Open the DTT Options page to select input and output data formats (see page 804) • Review information about your system: - Affymetrix system installed on your computer - The system version - System data location: The GCOS system (Local or Server) to which the data will be transferred. When using DTT with GCOS, use GCOS to switch between Local and Server mode. Use the buttons at the bottom to: Show Log Show the log for all transfer operations. Back Return to the previous step (disabled in this screen). Next Go to the next step. Cancel Close the dialog box. Show/Hide Help Display or conceal the Help page. To select the operation: 1. Select whether you want to: - Transfer data IN to GCOS. - Transfer data OUT of GCOS. 804 Affymetrix® GeneChip® Operating Software User’s Guide If you want to specify a file type for your transfer, click DTT Options. The DTT Options Page opens (see below). 3. Click Next. The appropriate page opens. 2. DTT Options Dialog Box for GCOS System Click the appropriate tab to: • Select file types for Transfer IN (see below) • Select file types for Transfer OUT (see page 806) DTT Options Page: Transfer IN Tab for GCOS System The Transfer In tab allows you to choose between the following data input formats (Figure M.2): • DTT Archive/Flat Files: GCOS Process Data or Publish Database contents (DTT Archive format only) Select this option to transfer in data from DTT Archive files or flat files. The flat files or DTT Archive file must have the associated XML file(s) with the GCOS project and sample information. The DAT/CEL/CHP data will be transferred into GCOS under the appropriate project/sample and the data files will be converted to the appropriate GCOS format. appendix M | Data Transfer Tool Guide 805 Figure M.2 GCOS System Data Transfer Options dialog box, Transfer IN Tab When using DTT Archive files stored on CDs, it is highly recommended that users copy all contents of the CD to a local drive and then import the contents. Before transferring Mining data IN, you must create a new, empty mining (AADM) database in GCOS Manager. For more information, refer to the GCOS Manager documentation. • CAB Files: GCOS Process Data or Publish Database contents Select this option to transfer in data from CAB files. The DAT, CEL and CHP data contained in the CAB file will be transferred to GCOS into the appropriate project/sample in GCOS. The data will be transferred into GCOS under the appropriate project/sample and files will be converted to the appropriate GCOS format. 806 Affymetrix® GeneChip® Operating Software User’s Guide • Migration of Microarray Suite Data Select this option to transfer in DAT/CEL/CHP data that do not have the XML file with the GCOS project and sample information. A project and sample name will have to be provided to successfully transfer this data into GCOS. Other information required for transfer IN will be regenerated using the information available in the DAT/CEL headers. The data will be inserted under the user provided project and sample name. To select the input format: 1. Select the radio button for the file type you wish to use. - DTT Archive Files - Flat Files - CAB Files 2. Click OK in the Data Transfer Options dialog box. DTT Options Page: Transfer OUT Tab for GCOS System The Transfer Out tab allows you to choose between the following data output formats (Figure M.3): • DTT Archive (GCOS Process Data and Publish Database contents) Select this option to transfer data out to flat files which are then packaged into DTT Archive file(s). The DTT Archive file(s) contain DAT, CEL, and CHP data files, along with an XML file with information on the GCOS sample and project under which the experiment was created. Use this format to preserve all GCOS information when transferring the data. DTT Archive file options will allow the DTT Archive file to be broken into smaller span files if required. DTT Archive files get a .DTT extension. • Flat Files (GCOS Process Data only) Select this option to transfer data out to flat files that consist of DAT, CEL, and CHP data files, along with an XML file with information on the GCOS sample and project under which the appendix M | Data Transfer Tool Guide 807 experiment appears. Use this option to preserve all the GCOS information when transferring the data from one GCOS system to another. The XML files generated using this option have to be transferred with the DAT, CEL and CHP data. • CAB (GCOS Process Data and Publish Database contents) Select this option to transfer data out to CAB files that contain the DAT, CEL, and CHP data files, along with a text file with information on the GCOS sample and project under which the experiment appears. Use this option when CAB files are required and it is necessary to preserve all the GCOS information when transferring the data across GCOS systems. CAB files get a .CAB extension. Figure M.3 GCOS System Data Transfer Options dialog box, Transfer Out tab To select the output file format: 1. Select the radio button for the file format you wish to use. - DTT Archive Files 808 Affymetrix® GeneChip® Operating Software User’s Guide If this file type is selected, you can choose a span file size from the drop-down list to match the capacity of the storage media you are using. - Flat Files - CAB Files If you select CAB, you will see a dialog box (Figure M.4): Figure M.4 Data Transfer Options dialog box, Transfer Out tab Select the I understand DTT Archive is a better option checkbox. B. Click Yes. A. 2. Click OK in the Data Transfer Options dialog box. TRANSFERRING DATA IN You can use DTT to transfer Affymetrix data into the following types of systems: • GCOS System: Transfer data into the Process or Publish databases. You can transfer the following types of data into a GCOS system: - GCOS Process data into the GCOS database - Mining Data into an empty Publish database - MAS 5.x data into the GCOS database appendix M | Data Transfer Tool Guide 809 Before transferring data IN from a Mining (AADM) DTT Archive or CAB file, you must create a new, empty mining (AADM) database in GCOS Manager. For more information, refer to the GCOS Manager documentation. The system type is automatically detected by DTT. You can transfer data archived in the following file types: • DTT Archive files (see page 809) • Flat files (GCOS Process data only) (see page 809) • CAB files: - Into a GCOS system (see page 828) • MAS 5.x files (GCOS Process data only): Transfer individual files (EXP, DAT, CEL, CHP) and experiment information generated using MAS 5.x software. If the EXP file is missing, the Data Transfer Tool will try and create an experiment file with the available information. For more information about importing MAS 5.x files, see page 828. You must select the file type for transfer on the Welcome screen. Transferring DTT Archive/Flat File Data IN to a Computer with GCOS You must select the file type for transfer on the Welcome screen. To transfer data in DTT Archive or Flat File format into a computer with GCOS: 1. Select data types and data for input (see page 810). 810 Affymetrix® GeneChip® Operating Software User’s Guide When using the Flat File format, you can only transfer GCOS Process data. Review selected data (see page 817). 3. Resolve conflicts with (GCOS Systems only): - Templates (see page 821) - Samples (see page 822) - Usersets (see page 824) 2. 4. Check that conflicts have been resolved and view the progress of the transfer (see page 827). Transfer DTT Archive/Flat File Data IN to GCOS: Setup The Setup screen allows you to select DTT Archive and Flat File data for transfer in to a computer with: • GCOS System You must select the file type for transfer on the Welcome screen. When using DTT Archive files stored on CDs, it is highly recommended that users copy all contents of the CDs to a local drive and then import the contents. appendix M | Data Transfer Tool Guide 811 Figure M.5 Transfer DTT Archive/Flat File Data IN to GCOS System: Setup To set up to transfer data IN to the system: Step 1: Select the input data location A. Click the Browse button and use the dialog box to select a directory with the DTT Archive/Flat Files for transfer. Select the Remember checkbox to make this directory the default directory for GCOS. If you select a directory containing a large number of DTT Archive files, the display of the file list in the Contents of Folder list may be delayed. 812 Affymetrix® GeneChip® Operating Software User’s Guide Step 2: Select Data Type A. Select the Data Type for input. Transfer GCOS Data: transfer archived Process data. 2) Transfer Mining Data: transfer archived Mining data (only available when used with a GCOS system). 1) Before transferring data IN from a Mining (AADM) DTT Archive file, you must create a new, empty mining (AADM) database in GCOS Manager. For more information, refer to the GCOS Manager documentation. Step 3: Select DTT Archive/Flat data The available DTT Archive or Flat Files are displayed in the contents list with the following information: File Name Unique ID assigned to the file. Date Date when archive was created. A. Select the DTT Archive or Flat Files from the list: ❑ Click Select All to select all archives and flat data ❑ Click Clear All to clear all selections ❑ Click on File Properties to display the File Properties box for the selected DTT Archive or Flat Files (see page 817). Step 4: Select Experiments to Restore Experiments in the selected DTT Archives or Flat Files are displayed in the Experiment list with the following information: Exp Experiment ID Proj Project ID Sample Sample associated with the experiment. Archive/XML Archive/XML file containing the experiment information A. Select the experiments for transfer in the Experiment List. appendix M | Data Transfer Tool Guide 813 Use the controls below the box to: Exclude DAT data Exclude all DAT files from the transfer process. Clear All Clear all selections. Experiment Information Display the Experiment Information box for selected files (see page 814). Filter Open the Filters dialog box (see page 815). B. Click Next. ❑ If you are transferring GCOS data, the Review window open (see page 817). ❑ If you are transferring Mining data, the Select a database dialog box opens (Figure M.6). Figure M.6 Select a Database dialog box This dialog box displays the available empty Mining (AADM) databases. Select a database to store the Mining (AADM) data from the list in the box. b. Click OK. A dialog box asks for the password to the mining database. a. 814 Affymetrix® GeneChip® Operating Software User’s Guide Enter the password and click OK. The Review window opens (see page 817). c. Experiment Information Figure M.7 DTT Archive Experiment Information The Experiment Information dialog box (Figure M.7) displays the following information: Data Type Type of data (dat, cel, chp). Data Name Identifier for the file. Experiment Unique ID for the associated experiment. Number of DAT files Number of DAT files in the selected archive. Number of CEL files Number of CEL files in the selected archive. Number of CHP Files Number of CHP files in the selected archive. Close button Click to close the Experiment Information box. appendix M | Data Transfer Tool Guide Transfer In Filters Dialog Box To filter displayed experiments: 1. Click the Filter button in the Setup screen (Figure M.8). Filter button Figure M.8 Transfer DTT Archive/Flat File Data IN to GCOS System: Setup The Transfer In Filters dialog box opens (Figure M.9). 815 816 Affymetrix® GeneChip® Operating Software User’s Guide Figure M.9 Transfer In Filters dialog box 2. Select the parameters for the experiments you are interested in from the following: Assay Type Type of assay performed. Probe Array Type: Particular probe array design used in the experiment. User: Person who created or imported the files. Project Category assigned in GCOS. Used to group a collection of samples. Sample Identifier for the sample, assigned in GCOS. Use Use to filter using date ranges. Experiment Date Select to filter on date experiment was created. 3. DAT File Date Select to filter on date DAT file associated with that experiment was created. From Date Start of date range. To Date: End of date range. Click Apply Filters to display data with the selected parameters; or Click Close to close the dialog box without applying the filters. appendix M | Data Transfer Tool Guide 817 Properties Page The Properties page displays additional data about the DTT Archive or Flat File selected in the Contents list, including (Figure M.10): • The machine on which the file was created. • The number of experiments in the file. • The number of span files associated with the file. • Comments entered by the user when the file was created. To close the Properties page: • Click OK. Figure M.10 File Properties page Transfer DTT Archive/Flat Files IN to GCOS: Review Data This screen displays a list of the files selected for transfer with indications if there are problems (Figure M.11). 818 Affymetrix® GeneChip® Operating Software User’s Guide Figure M.11 Data Transfer IN to GCOS System: Review Data (DTT Archive/Flat Files) appendix M | Data Transfer Tool Guide 819 To review the data for transfer: 1. Review the list of files for conflicts or other problems. The Selected Item list displays the following information: Data Type • Project • Sample • Experiment • DAT data • CEL data • CHP data • GRD data • JPG Data with conflict indicators: Data cannot be transferred due to conflict with template, sample, or userset or other problem. Data can be transferred. Transfer with warning: Data can be transferred but problems exist (may overwrite existing data, etc.). Data Name ID for the file. New Data Name Displays the new name selected for the template, sample, or userset during conflict resolution. Conflict Description of the conflict (also displayed in the Error Description box). 2. Click in an item row to see the full description of the problem with a file in the Error Description box. For more information about the error messages, see Error Messages and Troubleshooting Guide, on page 854. Click the Conflict Resolution Wizard button to resolve conflicts using the wizards, if necessary (see page 820). 3. Click Next. The Transfer In Status window opens (see page 827). 820 Affymetrix® GeneChip® Operating Software User’s Guide Conflict Resolution Wizard Use the Conflict Resolution Wizard to resolve problems with: • Sample and Experiment Templates (see page 821) Templates are predefined forms used to register samples and define experiments in GCOS; a template has a set of attributes used to provide information about the sample or experiment. A template conflict arises when there are discrepancies in the number or name of attributes used in the GCOS template and the DTT Archive/ Flat File template. For more information about template conflicts, see Template Issues, on page 854. • Samples (see page 822) Samples are created in GCOS, given attributes, and assigned to a project and sample type. A sample conflict arises when the same sample name exists in GCOS and in the DTT Archive/Flat file, but the samples are assigned to different project names, sample types, or other attributes. For more information about sample conflicts, see Sample Issues, on page 857. • Usersets (see page 824) Usersets are a set of predefined parameter settings for the analysis algorithm. A userset conflict arises when there is discrepancy in the parameters used in the GCOS userset and the DTT Archive/ Flat File userset. For more information about userset conflicts, see Userset Issues, on page 859. The Conflict Resolution Wizard allows you to resolve these conflicts by renaming the template, sample, or userset for the files being transferred into GCOS using DTT. You may need to resolve conflicts in more than one category to transfer the data. appendix M | Data Transfer Tool Guide 821 Resolve Templates Tab The Resolve Templates tab allows you to resolve conflicts in templates (Figure M.12). A typical template conflict involves discrepancies in the attributes for the template in the GCOS database as opposed to the template used for the data to be imported. Figure M.12 Conflict Resolution Wizard, Resolve Templates tab To resolve a conflict in the templates: A. View the list in the Error Information box. The Error information box displays a list of the Sample and Experiment templates with conflicts with the following information: Template Name with conflict indicators: Data cannot be transferred due to conflict with template, sample, or userset or other problem. 822 Affymetrix® GeneChip® Operating Software User’s Guide Data can be transferred. Transfer with warning: Data can be transferred but problems exist (may overwrite existing data, etc.). Template Type Can be Experiment Template or Sample Template. Error Information Additional information about the error (also displayed in the box above the OK button. For more information about template conflicts, see Template Issues, on page 854. Step 1: Template names can be changed using prefix/suffix or clicking on template name. A. Change the template name by: ❑ Clicking on the template name in the Error Information box and editing the name. ❑ Using the Prefix and Suffix boxes: Enter a prefix or suffix in the appropriate box. b. Click Rename All. All templates in the list are renamed. c. Click Revert All to undo the changes if necessary. Revert All undo the changes in the template names. Step 2: Click Resolve button to see if the conflicts are resolved. a. A. Click the Resolve button and see if the error indicators in the list disappear. B. Click OK to accept the changes. Resolve Samples Tab The Resolve Samples tab allows you to resolve conflicts between samples in the database and in the files for transfer by renaming the sample name for the file to be transferred (Figure M.13). appendix M | Data Transfer Tool Guide 823 Figure M.13 Conflict Resolution Wizard, Resolve Samples tab To resolve a conflict in the samples: 1. View the list in the Error Information box. Error information displays a list of the samples with conflicts with the following information: Sample Name With conflict indicators: Data cannot be transferred due to conflict with template, sample, or userset or other problem. Data can be transferred. Transfer with warning: Data can be transferred but problems exist (may overwrite existing data, etc.). Error Information Additional information about the error (also displayed in the box above the OK button. 824 Affymetrix® GeneChip® Operating Software User’s Guide For more information about sample conflicts, see Sample Issues, on page 857. Step 1: Change Sample names using prefix/suffix or clicking on template name. A. Change the Sample name by: ❑ ❑ Clicking on the sample name in the Error Information box and editing the name. Using the Prefix and Suffix boxes: Enter a prefix or suffix in the appropriate box. b. Click Rename All. All samples in the list are renamed. c. Click Revert All to undo the changes if necessary. Revert All undo the changes in the template names. Step 2: Click Resolve button to see if the conflicts are resolved. a. Click the Resolve button and see if the error indicators in the list disappear. B. Click OK to record the changes. A. Resolve Usersets Tab The Resolve Usersets tab allows you to resolve conflicts in usersets (Figure M.14). A typical userset conflict involves discrepancies in the parameters and values for the userset in the GCOS database as opposed to the userset used for the data to be imported. appendix M | Data Transfer Tool Guide 825 Figure M.14 Conflict Resolution Wizard, Resolve Sample tab To resolve a conflict in the userset: 1. View the list in the Error Information box. The Error information box displays a list of the Usersets with conflicts with the following information: Userset Name With conflict indicators: Data cannot be transferred due to conflict with template, sample, or userset or other problem. Data can be transferred. Transfer with warning: Data can be transferred but problems exist (may overwrite existing data, etc.). Error Information Additional information about the error (also displayed in the box above the OK button. 826 Affymetrix® GeneChip® Operating Software User’s Guide For more information about userset conflicts, see Userset Issues, on page 859. Step 1: Userset names can be changed using prefix/suffix or clicking on template name. A. Change the Userset name by: ❑ ❑ Clicking on the Userset name in the Error Information box and editing the name. Using the Prefix and Suffix boxes: Enter a prefix or suffix in the appropriate box. b. Click Rename All. All usersets in the list are renamed. c. Click Revert All to undo the changes if necessary. Step 2: Click Resolve button to see if the conflicts are resolved. a. Click the Resolve button and see if the error indicators in the list disappear. B. Click OK to record the changes. A. appendix M | Data Transfer Tool Guide 827 Transfer DTT Archive/Flat File data In to GCOS: Transfer Data The Transfer Data page displays the transfers in progress or completed during this session, with basic information about the transfer (Figure M.15). Figure M.15 Transfer Status page (DTT Archive or flat files into GCOS system) 828 Affymetrix® GeneChip® Operating Software User’s Guide The Selected Items box lists the data selected for transfer: Data Type • Project • Sample • Experiment • DAT data • CEL data • CHP data • GRD data • JPG data with conflict indicators: Data cannot be transferred due to conflict with template, sample, or userset or other problem. Data can be transferred. Transfer with warning: Data can be transferred but problems exist (may overwrite existing data, etc.). Data Name ID for the file. Message Status of the transfer operation. The lower box displays the contents of the transfer log for this session. Transferring CAB or MAS 5.x Files IN to a GCOS System You can use DTT to transfer GCOS data, Mining data, and MAS 5.x data in CAB or MAS 5.x format into a GCOS System. Before transferring data IN from a Mining (AADM) CAB file, you must create a new, empty mining (AADM) database in GCOS Manager. For more information, refer to the GCOS Manager documentation. appendix M | Data Transfer Tool Guide 829 To transfer data in CAB or MAS 5.x format: Make initial setup for transfer (see below). 2. Select data for transfer (see page 832). 3. Track progress of transfer (see page 836). 1. You must select the file type for transfer on the Welcome screen. Transfer CAB or MAS 5.x Files IN to GCOS System: Setup Figure M.16 Transfer CAB files IN to GCOS system: Setup 830 Affymetrix® GeneChip® Operating Software User’s Guide To perform initial setup: Figure M.17 Transfer MAS 5.x Data IN to GCOS System: Setup You must select the file type (CAB or MAS 5.x) for transfer on the Welcome screen. Step 1: Select the input data location A. Click the Browse button and use the dialog box to select a directory where the CAB or Microarray Suite 5.x data files are located. B. Select the file type for input: You cannot select a file type on the Setup screen when importing CAB files. appendix M | Data Transfer Tool Guide 831 The following options are used only to import MAS 5.x flat files (.cel, .chp, and .dat): ❑ DAT - CEL - CHP: Import image data (*.DAT). When specifying the image data, the associated experiment information from the EXP file, the cell intensity data (CEL) and the probe analysis data (CHP) is automatically imported. ❑ CEL - CHP: Import cell intensity data (*.CEL) and probe analysis data (*.CHP). When specifying the cell intensity data (CEL), the associated experiment information in the EXP file, the DAT file if it is present, and probe analysis data (CHP) is automatically imported with the cel intensity data. If the DAT file is missing, it will be marked as archived in the GCOS system. Step 2: Select sample parameters Not applicable when transferring CAB file data. When importing MAS 5.x data: A. Select options for creating samples on import: ❑ Automatically create a sample for each experiment imported. ❑ Create a single sample for all experiment files being imported. Enter a Sample Name or select from the drop-down list. Only active if placing all experiments under a single sample. C. Enter a Sample Type or select from the drop-down list. D. Enter a Project Name or select from the drop-down list. Step 3: Data will be stored in the following location B. A. Review the GCOS database location (Local or GCOS Server) and change if necessary. 832 Affymetrix® GeneChip® Operating Software User’s Guide To change the default destination, open GCOS and select the appropriate system (GCOS server or local system). The data transfer tool must be closed and reopened for settings to take effect. You can only use the DTT to transfer MAS 5.x data into a GCOS Workstation. You cannot use the DTT to transfer MAS 5.x data into a GCOS Server. To transfer MAS 5.x data into a GCOS Server, use the import functionality in GCOS Manager application. B. Click Next. The Select Data screen opens (see page 832). Transfer CAB or MAS 5.x Files IN to GCOS System: Select Data Figure M.18 Select Data: GCOS CAB file appendix M | Data Transfer Tool Guide 833 To select a CAB file: 1. Select the CAB file that contains the data to be transferred from the Select CAB files box. To select adjacent items, press and hold the Shift key while you click the first and last item in the selection. To select non-adjacent items, press and hold the Ctrl key while you click the items. You cannot select GCOS data and Mining data files for transfer in the same operation. This box lists the CAB files in the directory specified in Step 1 of the Setup page. GCOS and Mining CAB files are labeled. If you select a CAB file with GCOS data, you can review the contents of the file in the Data: (EXPs, DATs, CELs, CHPs only) box. CAB files with version 4.0.0 will not contain EXP files. A version 4.0.0 CAB file containing only experiments will not have its contents listed. To see the version of the CAB file see Step 2. The CAB version is listed in the CAB file properties. Keep all span files together when transferring CAB files into GCOS. 2. Click Properties for more information about the selected CAB file. The Properties page opens. 3. Click Start to begin the transfer. Follow the directions in the notice boxes that appear. - If you are transferring GCOS data, the Transfer Data window opens (see page 836). - If you are transferring Mining data, the Select a database dialog box opens (Figure M.19). 834 Affymetrix® GeneChip® Operating Software User’s Guide Figure M.19 Select database dialog box This dialog box displays the available empty Mining (AADM) databases. Select a database to store the Mining (AADM) data from the list in the box. 2) Click OK. A dialog box asks for the password to the mining database. 1) 3) Enter the password and click OK. You can track the progress of the transfer in the Transfer Data page. appendix M | Data Transfer Tool Guide 835 To select MAS 5.x files: Figure M.20 Select Data: MAS 5.x file Select the MAS 5.x files that you wish to transfer from the Select files box. To select adjacent items, press and hold the Shift key while you click the first and last item in the selection. To select non-adjacent items, press and hold the Ctrl key while you click the items. To select all files in the box, click Select All. To clear all selections, click Clear All. 2. Click Start to begin the transfer. Follow the directions in the notice boxes that appear. The Transfer Data window opens. 1. 836 Affymetrix® GeneChip® Operating Software User’s Guide Properties Page Figure M.21 Properties Page The Properties page (Figure M.21) displays additional data about a GCOS CAB file selected in the Select page, including: • The machine on which the CAB file was created. • The number of experiments in the GCOS CAB file. • The number of span files associated with the CAB file. Span files are automatically created when the CAB file size exceeds 2 GB. Keep all span files together when transferring CAB files into GCOS. • Comments entered by the user when the CAB file was created. To close the Properties page: • Click OK. Affymetrix Data Transfer Tool: Transfer Data The Transfer Data page displays the transfers in progress or completed during this session, with basic information about the transfer. The progress bar (displayed only for transfers with CAB files) shows the progress of the transfer taking place. appendix M | Data Transfer Tool Guide 837 Figure M.22 Transfer Data, transferring Mining Data from CAB file IN to GCOS TRANSFERRING DATA OUT You can use the DTT to transfer the following types of data out of GCOS: • GCOS Process data • Mining Data You can transfer data out of GCOS in the following formats: • DTT Archive (see page 838) • Flat File (GCOS Process data only) (see page 838) • CAB files (see page 846) 838 Affymetrix® GeneChip® Operating Software User’s Guide For more information about selecting file types for data transfer, see DTT Options Dialog Box for GCOS System, on page 804. You cannot use DTT to transfer data out of a computer unless GCOS is installed. You must select the file type for transfer on the Welcome screen. Transferring Data OUT in DTT Archive and Flat Files To transfer data OUT in DTT Archive and Flat Files: Set up the transfer (see page 839). 2. Select the actual data to be transferred (see page 841). 3. Review the data that will be transferred to the CAB file (see page 844). 4. Observe the progress of the transfer on the Transfer Data page screen (see page 844). 1. You must select the file type for transfer on the Welcome screen. appendix M | Data Transfer Tool Guide 839 Data Transfer Out of System: Setup (DTT Archive and Flat Files) Figure M.23 Data Transfer OUT of System: Setup (DTT Archive/Flat Files) You must select the file type for transfer (DTT Archive or Flat File) on the Welcome screen. To set up a data transfer OUT: Step 1: Select Data Type A. Select the data type for transfer: ❑ Transfer GCOS Data: transfer data from GCOS Process Database. ❑ Transfer Mining Data: transfer data from Mining (AADM) database (option not available when using Flat File format). 840 Affymetrix® GeneChip® Operating Software User’s Guide Selecting Transfer Mining Data will export the entire contents of the selected Mining (AADM) database. Step 2: Select the filters These controls are active only if you are transferring GCOS data. A. Select the filters for the data displayed in the Data Select screen: Assay Type Type of assay performed Probe Array Type: Particular probe array design used in the experiment. User: Person who created or imported the files. Project Category assigned in GCOS. Used to group a collection of samples. Sample Identifier for the sample, assigned in GCOS. Select Date Filter Use to filter using date ranges. Experiment Date Select to filter on date experiment was created. DAT File Date Select to filter on date DAT file associated with that experiment was created. From Date Start of date range. To Date: End of date range. Only data that matches the parameters selected here is displayed in the Select Data screen (Figure M.24). Step 3: Data will be retrieved from the following location A. Review the location of the data source and change if necessary. To change the location of the data source (GCOS local or GCOS Server) open GCOS and change the option in the default settings for the application. The Data Transfer Tool must be closed and reopened for settings to take effect. appendix M | Data Transfer Tool Guide B. 841 Click Next. The Select Data page opens (see below). Data Transfer Out of System: Select Data (DTT Archive and Flat Files) Figure M.24 Data Transfer OUT of System: Select Data (GCOS data) To transfer GCOS data: Step 1: Select Data to transfer OUT Select the type of data to display using the Display All dropdown box. B. Select the data in the Select Data box. ❑ Click Clear All to clear previous selections. ❑ Click Select All to select all data shown on the screen. A. 842 Affymetrix® GeneChip® Operating Software User’s Guide The data copied to the archive file set is different for some of the data selections: ❑ Project: For each selected project, the sample, experiment, and all DAT, CEL, and CHP data, is copied to the archive file set with the Project data. ❑ Sample: For each selected sample, the parent project of the sample and all experiment(s), DAT, CEL, and CHP data under the sample, is copied to the archive file set with the Sample data. ❑ Experiment: For each selected experiment, the parent project and sample of the experiment and all DAT, CEL, and CHP data under the experiment, is copied to the archive file set with the Experiment data. ❑ DAT Data: For each selected DAT, the parent project, sample, and experiment of the DAT data is copied to the archive file set file with the DAT data. CEL and CHP data is not copied. ❑ CEL Data: For each selected CEL, the parent project, sample, experiment, and DAT dat of the CEL data, is copied to the archive file set with the CEL data. CHP data is not copied. ❑ CHP Data: For each selected CHP, the parent project, sample, experiment, DAT, and CEL data is copied to the archive file set with the CHP data. Step 2: Include DAT Data Choose whether to include or exclude the DAT data (option not available for transferring mining (AADM) data). Step 3: DTT Archive/Flat File Information A. A. Enter a location, file name, and comments for the archive file set that will be created. appendix M | Data Transfer Tool Guide 843 This step is disabled when transferring data to Flat Files. B. Click Review to review the data that will be transferred into the archive file set. C. Click Start to begin the transfer. Follow the directions in the notice boxes that appear. Figure M.25 Data Transfer OUT of System: Select Data (Mining (AADM) data) To transfer Mining Data: Certain options are disabled in this screen when transferring Mining (AADM) data. 844 Affymetrix® GeneChip® Operating Software User’s Guide Step 1: Select Data to Transfer OUT The Display All drop-down list is disabled when working with Mining Data. Select the Mining (AADM) database in the Select Data box. Step 2: Include DAT Data A. The Step 2: Include DAT Data option is not active when transferring Mining (AADM) data. Step 3: DTT Archive/Flat File Information A. Enter a location, file name, and comments for the DTT Archive file or the XML file for the flat files to be created. B. Click Review. The Enter Password DB opens (Figure M.26). Figure M.26 Enter Password dialog box C. Enter the password for the selected database and click OK. The Data Transfer Out of System: Review Data window opens (see page 844). Transferring Data Out of System: Review Data (DTT Archive and Flat File) The Review Data page allows you to review the data selected for the DTT Archive or Flat File. appendix M | Data Transfer Tool Guide 845 To review the data chosen for transfer: 1. Review the Selected Items list: Data Type or Mining Database • Project • Sample • Experiment • DAT data • CEL data • CHP data • GRD data • JPG Data with conflict indicators: Data cannot be transferred due to conflict with template, sample, or userset or other problem. Data can be transferred. Transfer with warning: Data can be transferred but problems exist (may overwrite existing data, etc.). 2. Data Name Data type (see list above). Message Error or warning message Size Estimate (MB) Estimate of the necessary disc space in megabytes. Error Description Details of the detected problem, if any. Click Next to continue the transfer The Transfer Data page opens. Affymetrix Data Transfer Tool: Transfer Data (DTT Archive and Flat Files) The Transfer Data page displays the transfers in progress or completed during this session, with basic information about the transfer. The Selected Items box lists the data selected for transfer: 846 Affymetrix® GeneChip® Operating Software User’s Guide Data Type • Project • Sample • Experiment • DAT data • CEL data • CHP data • GRD data • JPG Data with conflict indicators: Data cannot be transferred due to conflict with template, sample, or userset or other problem. Data can be transferred. Transfer with warning: Data can be transferred but problems exist (may overwrite existing data, etc.). Data Name ID for the file. Message Status of the transfer operation. The lower box displays the contents of the transfer log for this session. Transferring Data OUT in CAB Files To transfer data OUT of GCOS to a CAB file: Set up the transfer (see page 847). 2. Select the actual data to be transferred (see page 849). 1. Review the data that will be transferred to the CAB file (see page 852). 4. Observe the progress of the transfer on the Transfer Data page screen (see page 836). 3. You must select the file type for transfer on the Welcome screen. appendix M | Data Transfer Tool Guide 847 Data Transfer Out of System: Setup (CAB) Figure M.27 Data Transfer OUT of System: Setup (CAB) You must select the file type for transfer on the Welcome screen. To set up a data transfer OUT into CAB format: Step 1: Select Data type A. Select the data type for transfer: ❑ Transfer GCOS Data: transfer data from GCOS Process Database. ❑ Transfer Mining Data: transfer data from Mining (AADM) database. 848 Affymetrix® GeneChip® Operating Software User’s Guide Selecting Transfer Mining Data will export the entire contents of the selected Mining (AADM) database. Step 2: Select the Filters A. Select the filters for data displayed in the next screen. These controls are active only if you are transferring GCOS data. You can filter using the following parameters: Assay Type Type of assay performed. Probe Array Type Particular probe array design used in the experiment. User Person who created or imported the files. Project Category assigned in GCOS. Used to group a collection of samples. Sample Identifier for the sample, assigned in GCOS. Date Data created during a range of dates: From Start date. To End date. Step 3: Data will be retrieved from the following location Review the data location. To change the location of the data source (GCOS local or GCOS Server) open GCOS and change the option in the default settings for the application. The Data Transfer Tool must be closed and reopened for settings to take effect. B. Click Next. The Select Data page opens. A. appendix M | Data Transfer Tool Guide 849 Data Transfer out of System: Select Data (CAB) Figure M.28 Data Transfer OUT of System: Select Data (CAB) To select GCOS Data for transfer: Step 1: Select Data to transfer OUT Select the type of data to display using the Display All dropdown box. B. Select the data in the Select Data box. ❑ Click Clear All to clear previous selections. ❑ Click Select All to select all data shown on the screen. The data copied to the CAB file is different for some of the data selections: A. 850 Affymetrix® GeneChip® Operating Software User’s Guide ❑ ❑ ❑ ❑ ❑ ❑ Project: For each selected project, the sample, experiment, and all DAT, CEL, and CHP data, is copied to the CAB file with the Project data. Sample: For each selected sample, the parent project of the sample and all experiment(s), DAT, CEL, and CHP data under the sample, is copied to the CAB file with the Sample data. Experiment: For each selected experiment, the parent project and sample of the experiment and all DAT, CEL, and CHP data under the experiment, is copied to the CAB file with the Experiment data. DAT Data: For each selected DAT, the parent project, sample, and experiment of the DAT data is copied to the CAB file with the DAT data. CEL and CHP data is not copied. CEL Data: For each selected CEL, the parent project, sample, experiment, and DAT dat of the CEL data, is copied to the CAB file with the CEL data. CHP data is not copied. CHP Data: For each selected CHP, the parent project, sample, experiment, DAT, and CEL data is copied to the CAB file with the CHP data. Step 2: Include DAT Data Choose whether to include or exclude the DAT data (option not available for transferring mining (AADM) data). Step 3: CAB Information A. A. Enter a location, file name, and comments for the CAB file that will be created. B. Click Review to review the data that will be transferred into the CAB file. appendix M | Data Transfer Tool Guide 851 To select Mining Data for transfer: Selecting Transfer Mining Data will export the entire contents of the selected Mining (AADM) database. Certain options are disabled in this screen when transferring Mining (AADM) data. Step 1: Select Data to Transfer OUT The Display All dropdown list is disabled when working with Mining Data. Select the Mining (AADM) database in the Select Data box. Step 2: Include DAT Data A. The Step 2: Include DAT Data option is not active when transferring Mining (AADM) data. Step 3: CAB Information A. Enter a location, file name, and comments for the CAB file that will be created in Step 3. B. Click Review The Enter Password DB opens (Figure M.29). Figure M.29 Enter Password dialog box 852 Affymetrix® GeneChip® Operating Software User’s Guide C. Enter the password for the selected database and click OK. The Data Transfer Out of System: Review Data window opens (see below). Transferring Data Out of System: Review Data (CAB) Figure M.30 Data Transfer OUT of System: Review Data (CAB) The Review Data page allows you to review the data selected for the CAB file. When transferring GCOS data, the page lists any of the following data types that have been selected: • Project • Sample • Experiment appendix M | Data Transfer Tool Guide 853 • DAT data • CEL data • CHP data • GRD data • JPG data When transferring mining data, the page lists the selected mining (AADM) databases. • Click Start to begin the transfer and go to the Transfer Data page (see page 836). 854 Affymetrix® GeneChip® Operating Software User’s Guide Error Messages and Troubleshooting Guide Error messages appear in DTT when there is a conflict of some sort between the data being imported and the existing contents of the GCOS databases. Many of these conflicts can be resolved using the Conflict Resolution Wizard (see Conflict Resolution Wizard, on page 820). Other problems may require other action to resolve. Error messages describe: • Template Issues (see below) • Sample Issues (see page 857) • Userset Issues (see page 859) • Miscellaneous Issues (see page 860) TEMPLATE ISSUES Templates are predefined forms used to register samples and define experiments in GCOS; a template has a set of attributes used to provide information about the sample or experiment. A template conflict arises when there are discrepancies in the number or name of attributes used in the template in GCOS and the template in the DTT Archive/Flat File. For more information about resolving template conflicts, see Resolve Templates Tab, on page 821. appendix M | Data Transfer Tool Guide 855 Table M.1 Template issues Error Message Why the Transfer IN is flagged as an error How to resolve the error Sample Template [<Name of sample template>] cannot be restored because the template already exists in GCOS but has different template attributes. Extra attribute(s): <Name of attribute> The sample template to be transferred IN with the sample already exists in GCOS. The sample template attributes are different. The extra attribute in the sample to be transferred IN is called <Name of attribute> This message will also apply to experiment templates. Use the Conflict Resolution Wizard to rename the template to be transferred in. As an alternative, add the attribute that is displayed in the error message into the template already in GCOS. Sample Template [<Name of template>] cannot be restored because the template already exists in GCOS but has different template attributes. Data type mismatch for attribute(s): <Name of attribute> The sample to be transferred In and the sample in GCOS use a template with identical names and attributes. However, the data type (string, integer, float, integer etc.) for the listed attribute is different when comparing the two templates. This message will also apply to experiment templates. Use the Conflict Resolution Wizard to rename the template to be transferred in. Sample Template [<Name of template>] cannot be restored because the template already exists in GCOS but has different template attributes. Controlled values mismatch for attribute(s): <Name of attribute> The sample to be transferred In and the sample in GCOS use a template with identical names and attributes. Use the Conflict Resolution Wizard to rename the template to be transferred in. However, the listed attribute is a controlled attribute that uses different control values when compared with the controlled values used by the template attribute in GCOS. This message will also apply to experiment templates. 856 Affymetrix® GeneChip® Operating Software User’s Guide Table M.1 Template issues Error Message Why the Transfer IN is flagged as an error How to resolve the error Warning: Sample Template [<Name of template>] will not be restored because the template already exists in GCOS but the template attributes are in different order. Restored sample will use the existing template in the GCOS. This is only a warning. The attribute order used by the incoming template is different than the attribute order of the template already in GCOS. No further action is required. Warning: Sample Template [<Name of sample template>] already exists in GCOS but the template in the DTT Archive contains more template attributes. The extra attributes will be inserted into the existing template in GCOS. Extra attribute(s): <Name of attribute>. This is a warning or an error based on the user rights. It indicates that the template to be transferred in with the data already exists in GCOS. The template in GCOS has fewer attributes than the one being transferred IN. The GCOS template will be updated with the new attributes if the user has rights to update the template in the database. On a GCOS Server, this may show up as an error if the user does not have rights to the update the template. This message will also apply to experiment templates. When this is displayed as a warning, no further action is required. Failed to restore experiment [<Name of experiment>] because there are errors in restoring the associated experiment template [<Name of experiment template>]. This error is displayed when the software fails to transfer IN the experiment template. All experiments using this template and all children of the experiment(s) will fail to transfer IN. Use the Conflict Resolution Wizard to rename the template to be transferred in. This message will also apply to experiment templates. This message will shows up as an error when transferring data IN to a GCOS Server and if the user does not have permission to maintain templates. To resolve this, use the conflict wizard to rename the template and bring it in as a new template. Alternately, speak with your GCOS Server administrator to get the appropriate permission to maintain templates on the server. This can be done by the administrator using the roles tab in GCOS Manager. appendix M | Data Transfer Tool Guide 857 SAMPLE ISSUES Samples are created in GCOS, given attributes, and assigned to a project and sample type. A sample conflict arises when the same sample name exists in GCOS and in the DTT Archive/Flat file, but the samples are assigned to different project names, sample types, or other attributes. For more information about resolving sample conflicts, see Resolve Samples Tab, on page 822. Table M.2 Sample issues Error Message Why the Transfer IN is flagged as an error How to resolve the error The sample already exists in GCOS but has different sample types. The sample type in the DTT Archive is [<sample type value of Sample to transfer IN>] but the one in GCOS is [<sample type value of sample in GCOS>]. The “sample type” field for sample associated with the experiment to be transferred IN has a different value than the one used by the sample with the same name in GCOS. Use the Conflict Resolution Wizard to rename the sample used by the experiment to be transferred in. As an alternative, open up the experiment in GCOS UI and change the sample type value to be the same as that used by the sample to be transferred IN. The sample already exists in GCOS but has different project names. The project name in the DTT Archive is [<Project value of sample to transfer IN>] but the one in GCOS is [<Project value of sample in GCOS>]. The “Project” field for sample associated with the experiment to be transferred IN has a different value than the one used by the sample with the same name in GCOS. Use the Conflict Resolution Wizard to rename the sample used by the experiment to be transferred IN. As an alternative, open up the experiment in GCOS UI and change the “Project” attribute value to be the same as that used by the Sample to be transferred IN. 858 Affymetrix® GeneChip® Operating Software User’s Guide Table M.2 Sample issues Error Message Why the Transfer IN is flagged as an error How to resolve the error The sample already exists in GCOS but has different project names and sample types. Project name is [<Project value of sample to transfer IN>] and sample type is [<sample type value of Sample to transfer IN>] for the sample in the DTT Archive but [<Project value of sample in GCOS>] and [<sample type value of sample in GCOS>] in GCOS respectively. The “sample type” and “Project” value for the sample associated with the experiment to be transferred IN has a different value than the one used by the sample with the same name in GCOS. Use the Conflict Resolution Wizard to rename the sample used by the experiment to be transferred in. As an alternative, open up the experiment in GCOS UI and change the “sample type” and “Project” value to be the same as that used by the sample to be transferred IN The sample already exists in GCOS but has different sample attribute value. The attribute that have different values is <name of attribute>. A sample using the same name as the one to be transferred IN already exists in GCOS and uses the same template as the sample to be transferred IN. The sample template attribute value in the sample to be transferred IN does not match up with the sample template attribute value already in GCOS. Use the Conflict Resolution Wizard to rename the sample used by the experiment to be transferred in. The sample already exists in GCOS but the two sample use templates with different names. A sample using the same name as the one to be transferred IN already exists in GCOS. The two samples use a different sample template. Use the Conflict Resolution Wizard to rename the sample used by the experiment to be transferred in. The sample already exists in GCOS but has different number of sample attributes. The sample in the DTT Archive has 3 attributes but the one in GCOS has 4. A sample using the same name as the one to be transferred IN already exists in GCOS. The two samples use templates with identical names. However the number of template attributes used by the sample in GCOS is different than the sample in the DTT Archive. Use the Conflict Resolution Wizard to rename the sample used by the experiment to be transferred in. appendix M | Data Transfer Tool Guide 859 USERSET ISSUES Usersets are a set of predefined parameter settings for the analysis algorithm. A userset conflict arises when there is discrepancy in the number and values of parameters used in the GCOS userset and the userset in the DTT Archive/Flat File. For more information about resolving userset conflicts, see Resolve Usersets Tab, on page 824. Table M.3 Userset Issues Error Message Why the Transfer IN is flagged as an error How to resolve the error Userset [<Name of the userset>] cannot be transferred IN because the user does not have the appropriate privilege to create/ update usersets. Please use the roles tab in GCOS Manager to get the necessary privilege. This is an error that will be displayed when transferring IN data onto a GCOS Server and the user does not have permission to transfer in Usersets. This message will shows up as an error when transferring data IN to a GCOS Server and if the user does not have permission to maintain userset. To resolve this, use the conflict wizard to rename the userset and bring it in as a new userset. As an alternative, speak with your GCOS Server administrator to get the appropriate permission to maintain usersets on the server. This can be done by the administrator using the roles tab in GCOS Manager. Userset [<Name of userset>] cannot be restored because the userset already exists in GCOS but has different parameter sets and/or attributes mismatched. <Name of parameter> This is an error that will be displayed when the user tries to transfer in a userset that already exists in the GCOS database. The userset parameters for the array do not match. Use the Conflict Resolution Wizard to rename the userset to be transferred in. 860 Affymetrix® GeneChip® Operating Software User’s Guide Table M.3 Userset Issues Error Message Why the Transfer IN is flagged as an error How to resolve the error Warning: Userset [<Name of userset>] already exists in GCOS but the userset in the DTT Archive contains more parameter sets. The extra parameter set will be inserted into the existing userset in GCOS. <Name of parameterset> This is a warning or an error based on create/update permissions. The message will be displayed when the user tries to transfer in a userset that contains more parametersets than the userset in GCOS. The extra userset will be inserted into GCOS if the user has appropriate permissions. When this is displayed as a warning, no further action is required. This message will shows up as an error when transferring data IN to a GCOS Server and if the user does not have permission to maintain usersets. Speak with your GCOS Server administrator to get the appropriate permission to create/maintain usersets on the server. This can be done by the administrator using the roles tab in GCOS Manager. This userset uses multiple probe arrays one of which is <Name of userset>. Probe array type <[Name of userset not installed in GCOS]> is not installed on the system. Please reinstall this probe array on your system and retry the transfer IN. This is an error when the user is trying to transfer IN an experiment that uses a userset and the incoming Userset has multiple probe arrays, one of which is not installed on the system. The software will not transfer IN the experiment if this probe array has not been installed on the system. Resolve by installing the library for the probe array type the name of which is given in the error message. MISCELLANEOUS ISSUES The following error messages appear for miscellaneous issues that can cause problems transferring data IN to GCOS. appendix M | Data Transfer Tool Guide 861 Table M.4 Miscellaneous Issues Error Message Why the Transfer IN is flagged as an error How to resolve the error Probe array type [<name of probe array>] is not installed on the system. Experiment [<Name of experiment>] cannot be restored. This message is displayed when trying to transfer IN an experiment for which the probe array does not exist on the local system. Install the library file for the probe array specified in the error message. Experiment already exists in GCOS database This is an error message displayed when an experiment name in GCOS conflicts with an experiment to be transferred IN. GCOS can have only 1 experiment with the same name in the database. Use GCOS Manager to delete the experiment in GCOS to transfer IN the experiment from the archive. Failed to restore experiment [<Name of experiment>] because there are errors in restoring the associated experiment template [<Name of experiment template>]. This error is displayed when the software fails to transfer IN the experiment template. All experiments using this template and all children of the experiment(s) will fail to transfer IN. Use the Conflict Resolution Wizard to rename the template to be transferred in. Hybridization protocol script [<Name of script>] is not installed on the system. The protocol parameter will not be restored. This is a warning indicating that the parameters from the wash and stain script will not be inserted into the GCOS database because the fluidics script has not been installed using the script installer. The wash and stain scripts can be installed using the appropriate fluidics script installer. Once the script is installed, the parameters will be restored when the experiment is transferred IN. 862 Affymetrix® GeneChip® Operating Software User’s Guide Table M.4 Miscellaneous Issues Error Message Why the Transfer IN is flagged as an error How to resolve the error Failed to transfer data file [<Name of file>] which already exists in the GCOS database. This message is displayed if the data file is associated with an experiment in the database. The experiment name in GCOS database is different than the experiment name in the data to be transferred IN. However, the children of the experiment to be transferred IN, clash with datafiles with the same name in the GCOS database. It will not be possible to transfer IN the data files without first deleting the data files in GCOS. Use GCOS Manager to delete the files in the database if they are no longer required. Data file [<Name of file>] cannot be transferred because the associated parent data file [<Name of parent>] cannot be transferred. This message will be displayed for all children of a GCOS object that will has an error. E.g. If an experiment has an error icon next to it, all children of the experiment (DAT, CEL and CHP) will have this error message. If a CHP file uses a userset and the userset cannot be transferred IN, the CHP file will fail to transfer IN. Use the Conflict Resolution Wizard to resolve templates, samples and usersets. If the parent is an experiment, the error is displayed if the experiment already exists in the database. Warning: Data file [<Name of data file>] already exists in GCOS data folder. The data file will be overwritten. This is a warning. This indicates that there is a file in the GCOS data directory that will be overwritten. This applies to JPG and GRD files. It will also apply the DAT, CEL and CHP files when the files are not associated with any experiment in the GCOS database. Move the files out of the data directory if they need to be preserved. appendix M | Data Transfer Tool Guide 863 Table M.4 Miscellaneous Issues Error Message Why the Transfer IN is flagged as an error Warning: Experiment file [<Name of EXP file>] already exists in the <Name of directory>. The experiment file will be overwritten. This message will show up on MAS 5.x systems when the experiment to be transferred IN already exists. How to resolve the error Move the experiment file out of the data directory if they need to be preserved. Parsing Failures and Character Issues The DTT Archive and Flat File formats use XML files for the experiment information that DTT uses to import the data. When the XML file contains characters that can’t be parsed by the XML parser, DTT gives the following error message (Figure M.31): Figure M.31 XML Parsing Error message When the error appears, you need to create a DTTConfig.CSV mapping file that provides the correct ASCII decimal code for the character, so DTT can use the mapping file to parse the XML file. To create the DTTConfig.CSV mapping file: 1. Determine which character is causing the problem in the XML file. The most likely source of the invalid character is the experiment name, sample name, or one of the sample/experiment template attribute values. If you have used a non-US English symbol, then the non-US english symbol would need to be changed to an ASCII representation. 864 Affymetrix® GeneChip® Operating Software User’s Guide GCOS has only been validated on a U.S.English environment. 2. Determine the ASCII decimal character code for that character. You can use the Symbol dialog box to find the ASCII decimal character code: Open a document in Microsoft® Word. B. Select Insert → Symbol from the Word menu bar. The Symbol dialog box opens (Figure M.32). A. Figure M.32 Symbol dialog box in Microsoft Word Select Normal Text for the font D. Select the symbol causing the problem in the list. E. Select ASCII (decimal) in the From drop-down box. C. Note the ASCII character code 3. Create the DTTConfig.CSV character mapping file. F. appendix M | Data Transfer Tool Guide 865 The DTTConfig.CSV file (Figure M.33) is a comma separated value file. Figure M.33 .CSV file It contains a line for each character that needs the special mapping to ASCII code in the format: symbol, ASCII decimal code. You can use any text-editing program, such as Notepad, to create the file. 4. Place the .CSV file in the same directory as the DTT executable. DTT will use the character mappings to parse the XML files. DTT 1.1 can parse the following characters without a .CSV file: &, <, >,°, ®, £, ¥ GCOS Data Compatibility Table GCOS software is backwards compatible. This means data generated with previous versions of GCOS or MAS 5.x software can be read with the latest version of GCOS. Data in CAB files is converted only when transferring data generated on GCOS systems into a system with Microarray Suite 5.x or when transferring data generated on GCOS 1.2 or later versions into GCOS 1.0/1.1/1.1.1. The data compatibility table gives additional information about the types of data transfers that are possible. 866 Affymetrix® GeneChip® Operating Software User’s Guide Any experiments using probe array types that require sub-gridding on DAT images will not be restored to GCOS versions 1.2 and lower. This includes Microarray Suite Software. Table M.5 Data compatibility table Target system Data format in CAB file Comments Microarray Suite 5.0 The CEL and CHP data formats were updated in GCOS 1.x. CEL and CHP Data generated on GCOS 1.x systems will be converted to the MAS 5.0 format. The software will copy the DAT data if they were transferred to the CAB file during the CAB creation process. Data generated on a GeneArray® 2500 scanner can be transferred to a MAS 5.0 system. Data acquired from all other scanners will be rejected. Only expression and universal assay data can be transferred into a system with MAS 5.0. CEL data for the associated CHP data is required to convert CHP data from a GCOS system to a MAS 5.x system. The Data Transfer tool is not supported on Microsoft® Windows® NT SP6a systems. Microarray Suite 5.1 The CEL and CHP data formats were updated in GCOS 1.x. CEL and CHP Data generated on GCOS systems will be converted to appropriate formats. The software will copy the DAT data if they were transferred to the CAB file during the CAB creation process. Data generated using the GCS 3000 High Resolution scanner cannot be transferred to a MAS 5.1 system. CEL data for the associated CHP data is required to convert CHP data from a GCOS system to a MAS 5.x system. Only expression and universal assay data can be transferred into a system with MAS 5.1. appendix M | Data Transfer Tool Guide 867 Table M.5 Data compatibility table Target system Data format in CAB file Comments GCOS 1.0/1.1/1.1.1 The CHP data format was updated going from GCOS 1.0/ 1.1/1.1.1 to GCOS 1.2 or later. CHP data generated on GCOS 1.2 or later will be converted to the appropriate GCOS 1.0/1.1/1.1.1 format. GCOS software is backwards compatible. All data which was generated on a Microarray Suite 5.x system will be copied without any conversion. Expression, Mapping, Universal and Resequencing data can be transferred between the two GCOS systems. CHP data generated using the new Dynamic Model Mapping algorithm cannot be transferred to a GCOS 1.0/ 1.1/1.1.1 system because the software does not support the new algorithm. CEL data for the associated CHP data is required to transfer CHP data from a GCOS 1.2 or later system to a GCOS 1.0 or MAS 5.x system. Data from GCOS 1.3 and higher systems that use sub-grids cannot be transferred to a GCOS 1.0/ 1.1/1.1.1/1.2 system GCOS 1.3 New algorithms to support data that require sub-grids were introduced in GCOS 1.3. In addition when a DAT file is created, a JPG representation of the same file is created automatically. These files cannot be transferred to a GCOS system with version less than 1.3. GCOS 1.4 The CHP data format for CustomSeq arrays was updated going into GCOS 1.4. This data will lose new fields if transferred back to older systems that do not support the resequencing format. System without any Affymetrix Software. When transferring CAB data into systems without any Affymetrix software, all data is converted into GCOS 1.1.1 format. Before extracting CHP data from the CAB files, it is necessary to copy the library (*CDF) files into the target directory. 868 Affymetrix® GeneChip® Operating Software User’s Guide Index 871 Index Symbols indicator lights 737 manual mode 777 Microsoft Exchange 741 regulatory 796 scanning a probe array 751– 786 setting up 732 SMTP 743 specifications 794 stopping a scan 782 troubleshooting 787 .CSV file 863 for character mapping 864 A Act as Part of the Operating System policy 612 Affymetrix Technical Support 6 algorithm Alignment 147 Cell Summary Report 679 Expression 45 empirical 638 statistical 636 expression 627 Alignment algorithm 147 alignment, grid 147 alignment, main grid 154 alignment, subgrid 162 alpha1 678 alpha2 678 amino acid codes 691 Analysis and Transfer Status window 133 analysis information 253 automode 771 avg diff empirical analysis 640 avg diff change empirical analysis 643 B=A empirical analysis 643 background subtraction 629 barcode 754 barcodes 80, 83, 109, 124, 125 baseline data 674 batch analysis renaming results 306–308 resuming 309 running 309 selecting baseline 303 selecting userset 302 analysis information table 243 exporting 244 Analysis Service 598 analysis settings 335 archiving data 359–361 attributes 439 activate in template 440 deactivate in template 440 edit in template 441 insert in template 440 Batch Analysis window 298 removing data 308 toolbar 698 batch file exporting 309 importing 310 bookmarks creating 213 editing 214 viewing 214 AutoLoader 731–797 AC power connection 732 disabled 784 email system 739 Ethernet connection 732 computing 195 image settings 196–204 recalculating 196 Cell Summary Report 178 algorithm 679 for Expression arrays 181 for Mapping arrays 181 for Resequencing arrays 186 generating 178 change 628 change p-value 628 client software B exporting 244 call 631 carousel 767, 769 CE Mark Declaration of Conformity 583 cell coordinates 329 cell intensity data 44 C CAB files (DTT) 807 testing DMT 518 GCOS Manager 471, 523 troubleshooting GCOS 716 GCOS Manager 721 codes amino acid 691 IUPAC base 691 columns resizing, hiding, or reordering 706 comparison analysis results empirical algorithm 641 statistical algorithm 637 comparison expression analysis 229–230, 235–242, 633 selecting baseline 236 configure data storage 55 fluidics station 74 scanner 75 872 controls Affymetrix® GeneChip® Operating Software User’s Guide defaults See expression report controls. CustomSeq for Resequencing arrays aligning subgrids 172 D data archiving 359–361 exporting 368 filtering 71–73 opening 69 unarchiving 361 viewing information 69 Data Mining Tool analysis settings 335 image settings 328–333 Defaults dialog box Database tab 55 descriptions 251 detection 628, 630 detection p-value 628 difference call empirical analysis 642 discrimination value 631 documentation conventions used 3 online 5 domain user requirements for GCOS Services 588 installing 531–538 data ownership 363 assign 363 assume 363 DPos-DNeg ratio empirical analysis 642 DTT 801 error messages 854 data storage configure 55 DTT Archive file format 806 Data Transfer Tool see DTT data tree 64 DTT CAB files 807 DTT flat files format 806 display options 66 shortcut menu 66 data types 68 experiment data 686 fluidics protocols 685 probe information 685 database change password 461 gene information 47 process 47 publish 47 database (workstation) relocate 466 database setting 55 dec ratio empirical analysis 641 decrease empirical analysis 641 E editing experiment information 85 expression report 192, 291 empirical algorithm metrics 638 Entrez 252 error message DTT 854 error messages 789 experiment defining 80 renaming 67 experiment data publishing 46 experiment information editing 85 viewing 85 Experiment Information Window 758 experiment setup 41 exporting data 368 analysis information 244 batch file 309 metrics table 246–249 expression algorithm 627 expression analysis 45 analysis information 253 analysis information table 243 exporting 244 comparison 229–230, 235– 242 selecting baseline 236 graphs intensity bar graph 279 scatter graph 260 series graph 274, 279 measured images 257 metrics table 245, 246 report 283–289 settings 231 single-array 226–228, 232– 234 expression analysis settings baseline 674 normalization 655 probe masks scaling 645 user-modifiable parameters 677 Expression Analysis window (EAW) 46, 242 expression report 283–289 controls 290 editing 192, 291 find feature 192, 291 generating 178, 284 parameters 181, 186, 287– 289 Index printing 343 settings 180, 285 Publish tab 381 Roles tab 441 starting 347 Template tab 425 troubleshooting 721 Userset tab 415 F filter samples process tab 380 filters 71–73 find feature 253 expression report 192, 291 probe set information 253 GCOS Server checking database setting 55 GCOS server register 369, 460 unregister 371, 460 view events 371 view publish tasks 372 flat files (DTT) 806 fluidics protocol bypassing steps 112 editing 113 resuming 111 running 110 setting up 109–110 GCOS server tasks cancel or restart 376 GCOS Services 56 Configuring 587 requirements for using 587 Start/Stop Service policies 601, 612 fluidics station configuring 74 editing ID 115 priming 106–109 four-color arrays 130 GCOS Transfer Service 599 gene information database 47 GeneChip assay overview 38–47 G Gamma 678 GCOS installing new users 9–15 installing library files (new install) 19–25 installing library files (upgraded install) 25–32 starting 53 troubleshooting 716 user interface 61–67 GCOS Administrator Space Manager 464 starting 455 GCOS Analysis Service 598 GCOS Manager functions 350 Import tab 396 Process tab 356 GeneChip AutoLoader 731 graphs intensity bar graph 279 scatter graph 260–274 series graph 274, 279 grid 333 aligning 154 aligning main grid 154 aligning subgrid 162 viewing 151 grid alignment 147 H highlights 202, 331 hybridization 43 873 I image data calculating average intensity 205 image settings 196–204 probe cell intensity cell image view 205 measured image view 205 image data file 773, 782 image settings 196–204, 328– 333 cell coordinates 329 color 329 current image only 197 global 198 grid 333 highlights 202, 331 intensity 328 intensity range 199 pixel coordinates 200, 329 Image window 141 opening multiple windows 143 task bar 146 import tab filter samples 397 windowpanes 396–400 importing data 400–414 associate data and samples 408–413 remove a sample for import 407 specify sample for import 402–408 start import 414 importing files Batch Analysis Window 310 inc ratio empirical analysis 641 inc/dec empirical analysis 642 874 installing GCOS new users 9–15 library files (new GCOS install) 19–25 library files (upgraded GCOS install) 25–32 instrument installation 541 intensity 328 intensity bar graph 279–283 clearing 283 options 280 plotting 280 intensity range 199 Internet Browser window 266 invalid character in XML file used with Data Transfer Tool 863 invalid characters 863 IUPAC base codes 691 J Affymetrix® GeneChip® Operating Software User’s Guide mask file 659 masks See probe mask. measured images expression probe array 257 metrics empirical algorithm 638 statistical expression algorithm 636 metrics table 245, 246 exporting 246–249 MIAME sample template 427 Microsoft SQL Server database 47, 389 MSDE database 47, 389 neg change empirical analysis 642 NetAffx 252 normalization 655–665 all probe sets 657 selected probe sets 658 user defined 656 normalization mask file creating 659 editing 663 selecting 661 L library files 685 new GCOS install 19–25 upgraded GCOS install 25– 32 license troubleshooting 720 log avg online documentation 5 Oracle database 47 Oracle publish database 393 outliers masking 216 unmasking 217 viewing 215 empirical analysis 639 empirical analysis 642 aligning 154 display options 250 exporting 252 pixel coordinates 200, 329 pos change empirical analysis 642 pos fraction empirical analysis 639 pos/neg empirical analysis 640 cell intensity data 341 image data 341 report 343 privilege settings functions defined 447 probe array highlighting tiles 211 hybridization 43 notation 627 scanning 43, 751–786 tiling information 208 probe array type delete 366, 385 cell image view 205 measured image view 205 probe cells calculating average intensity 205 data 205–207 viewing data 209 probe mask 665–674 creating 666 editing 671 selecting 669 P main grid change 461 perturbation 678 pivot table 249 probe cell intensity O log avg ratio change M empirical analysis 639 password printing N JPG files created during DAT file archiving 217 viewing 217 pairs in avg pair used empirical analysis 639 Index probe set descriptions 251 obtaining information 252 probe set information finding 253 hiding 256 reordering or removing 255 sorting 254 process data delete 364 R automatic backup 462 find feature 376–380 process tab GCOS server 369 register GCOS server 460 renaming 67 experiment, sample, project 67 renaming feature 67 report printing 343 reports expression analysis 283–289 roles filter samples 380 functions 358 windowpanes 356–358 create 444–448 define privileges 446–448 delete 451 delete a user 450 naming 444 role membership 448 project 67 renaming 67 publish database 47 accessing 384 capacity 389 create 388–396 Oracle on GCOS server 393 SQL on GCOS server 391 workstation 389 locations and sizes 389 selecting 316 publish tab windowpanes 381– 383 published data delete 386 publishing 46 canceling a task 322 intensity data 320 overwriting data 321 publishing a task 321 restarting a task 322 specifying a task 316 scanner register process database 47 875 roles tab windowpanes 443– 444 S sample registering 41, 80 renaming 67 sample history data view 91 displaying 91 process view 91, 93–95 scale factor 646 scale factor mask file creating 649 editing 653 selecting 651 scaling 645–655 all probe sets 646 selected probe sets 648 user defined 646 configuring 75 GeneChip Scanner 3000 561 CE Mark Declaration of Conformity 583 laser safety 566 regulatory 584 scanning a probe array 121–130 shutdown 133 shutting down 133 specifications 577 stopping a scan 132 troubleshooting 577 scanning 43, 751–786 scatter graph options 269 plotting 261 selecting points 267 series graph 274–279 options 277 plotting 275 shortcut bars 61–64 GeneChip Software 61 Instrument Control 63 Settings 64 Signal 628 signal calculation 632 signal log ratio 628, 635 high 629 low 628 single-array analysis 226–228, 630 single-array analysis results statistical algorithm 636 sort score 644 sorting probe set information 254 Space Manager 464 SQL server 2000 publish database 391 Start/Stop Service policies for GCOS Services 601, 612 876 stat common pairs 628 stat pairs 628 statistical expression algorithm 287–289, 636 status log 67, 234 subgrid alignment 162 Affymetrix® GeneChip® Operating Software User’s Guide transfer data into GCOS 808 out of GCOS 837 Transfer Service 599 troubleshooting 715, 787 autoloader 787 GCOS 716 GCOS Manager 721 CustomSeq for Resequencing arrays 172 subgrids 148 U T unarchiving data 361 unregister GCOS server 460 user interface 61–67 userset task bar 146 tau 678 technical support 6 template tab windowpanes create 417–420 delete 424 modify 421–424 426–427 templates activate attributes 440 attributes 439 create 428–437 deactivate attributes 440 deactivate or activate 438 edit 437 edit attributes 441 insert attributes 440 MIAME 427 rename 438 testing DMT 518 GCOS Manager 471, 523 userset tab windowpanes 415– 417 W windowpanes resizing 705 windows Expression Analysis window (EAW) 46, 242 GCOS Administrator 456 GCOS Manager 349 Image window 141 Internet Browser 266 workflow monitor 39, 95 tiles highlighting 211 tiling information 208 toolbars Batch Analysis Window 698 main 695 publish 699 report 698 TOUGH-SPOTS™ 122 Tough-Spots™ 763 cell intensity analysis tab 100 grid alignment tab 98 hybridization tab 98 probe array analysis 100 publish tab 100 scan tab 98 workstation database property information 466 relocate 466 space information 466
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