Agilent Genomic Workbench 6.5 Methylation

Agilent Genomic Workbench 6.5 Methylation
Agilent
Genomic Workbench 6.5
Methylation (CH3) Analysis
User Guide
For Research Use Only. Not for use in diagnostic procedures.
Agilent Technologies
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Methylation (CH3) Analysis Guide
In This Guide...
This guide describes how to use the Methylation (CH3)
application of Agilent Genomic Workbench Standard Edition
6.5 to apply algorithms that help identify methylated regions.
1
Getting Started
This chapter gives instructions on how to start the
application and enter license information. It also gives an
overview of how to analyze CH3 data.
2
Importing, Managing, and Exporting CH3 Data and Other Content
This chapter describes how to import, organize, manage, and
export CH3 data and other content in Agilent Genomic
Workbench.
3
Displaying CH3 Data and Other Content
This chapter shows you how to display log ratio data from
imported feature extraction data files and analysis results, as
well as gene list and track content, in the Genomic Viewer. It
also gives you instructions on how to change parameters to
display the data and content the way you prefer.
4
Setting Up Methylation (CH3) Analysis
This chapter gives instructions on how to set up the analysis
functions for Methylation (CH3) experiments. These include
the Preprocessing, Analysis and Reports tabs.
5
Methylation (CH3) Analysis Reference
This chapter describes the tabs, commands, shortcut menus
and dialog boxes specific to Agilent Genomic Workbench
methylation (CH3) data analysis. The chapter also includes
information on the format of the reports created by the
program.
Methylation (CH3) Analysis Guide
3
6
Statistical Algorithms
This chapter provides implementation details of the
algorithms used in the CH3 application of Agilent Genomic
Workbench. The CH3 application algorithms facilitate the
statistical analysis of methylated genomic regions. The first
section presents the main methylation detection algorithm.
The next section describes the display options available for
methylation analysis.
4
Methylation (CH3) Analysis Guide
Contents
1
Getting Started
13
What is the CH3 Application?
15
Using Agilent Genomic Workbench on a Mac
16
Entering a License and Starting the CH3 Application 17
To enter your license for analyzing Methylation (CH3) data interactively
To start the CH3 application 19
Transferring Data from the eArray Web Site
18
20
Using Main Window Components to Display/Analyze Data 21
What are the main window components? 21
What can you do with the main components for display of data and
results? 23
Switching Applications
26
Using Tabs and Command Ribbons
Tabs 27
Commands 28
27
Using the Navigator to Search for Data
To search the Navigator 32
29
Using the Genomic Viewer to Display Data 34
What is the Genomic Viewer? 34
To change the size of and detach panes from the Agilent Genomic Workbench
main window 36
To maximize and reattach panes to the Agilent Genomic Workbench main
window 37
General Instructions for Displaying Microarray Data/Results
38
General Instructions for Setting Options for CH3 Interactive Analysis
Getting Help
Methylation (CH3) Analysis Guide
39
40
5
Contents
To get help within Agilent Genomic Workbench 40
To contact Agilent Technical Support 41
To learn about Agilent products and services 41
2
Importing, Managing, and Exporting CH3 Data and Other Content
43
Importing Files 44
To select a different location for data files 45
To import Agilent GEML design files 46
To import Axon design files 47
To import Agilent FE or Axon data files 47
To import a UDF file 49
To import a genome build 52
To import tracks 53
To import array attributes 54
To import an experiment file 55
To import a probe file 55
To import a genome 56
To transfer catalog and workgroup data 56
Working with Experiments to Organize Imported Data 57
To display the array designs and data in the program 57
To create a new experiment 58
To add arrays to an experiment 60
To change the order of arrays in an experiment 61
To change the display names for arrays in an experiment 62
To rename an array in an experiment 63
To remove arrays from an experiment 63
To select or remove calibration array(s) 64
To show or hide array attributes in an experiment 65
To display or edit array attributes in an experiment 65
To display or edit the attribute values of a specific array 66
Managing Content 68
To display a list of the content stored in the program
6
68
Methylation (CH3) Analysis Guide
Contents
To find specific content items in the Navigator 70
To display the properties of a specific design 70
To download a design from eArray 71
To update probe annotation in design files 71
To rename an array in the Design Data pane 72
To remove data or design files from the program 72
To create a gene list 74
To import a gene list 75
To display the genes in a gene list 75
To add one gene list to another 76
To rename a gene list 76
To delete gene list(s) 76
To display the details of a track 77
To combine tracks 77
To rename a track 78
To delete tracks 79
Exporting and Saving Content 80
To export array attributes 80
To export experiments 81
To export a gene list 82
To export tracks 82
To copy what you see in the main window 83
To copy the list of array colors for an experiment 83
To save data and design information from an experiment
3
Displaying CH3 Data and Other Content
84
85
Selecting an Experiment 86
To select an experiment 86
To select or deselect arrays in the experiment 87
To change the display color of an array 89
To share an experiment 90
To remove a user from a shared experiment 90
Methylation (CH3) Analysis Guide
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Contents
Displaying Array Data 91
To display the scatter plots 91
To show or hide data in the scatter plots 92
To customize scatter plot ranges and colors 92
To change scatter plot appearance 93
To print the scatter plot 95
To create custom scales for Views 95
To locate and display data (or results) within the Views
95
Displaying Content (Gene Lists/Tracks) 98
To show gene lists in Gene View 98
To select gene list display color 99
To display a gene list as a table 99
To change the appearance of genes in Gene View 100
To show tracks in Gene View 100
To change the appearance of tracks 101
To show track information in reports 102
To limit data to the genomic boundaries of the track 103
To display a track in UCSC Browser 103
To upload a track to UCSC Browser 103
To change the graphical display to a different genome build
Searching for Probe and Gene Information 106
To search Tab View for specific probe information 106
To search an experiment using a query 107
To search for probes in eArray 107
To search for probes in a chromosomal location 108
To search an experiment using a query 108
To search for probes in eArray 109
To search for probes in a chromosomal location 109
To search the Web for information on probes in Tab View
To create a custom Web search link 110
To update or delete a custom Web search link 111
8
105
110
Methylation (CH3) Analysis Guide
Contents
4
Setting Up Methylation (CH3) Analysis
Working with Methylation Options
113
114
Changing Preprocessing and Analysis options 115
To combine (fuse) arrays 115
To set up a moving average (Log Ratio) calculation to smooth the data 117
To set up a moving average (ZScore) calculation to smooth the data 118
To apply methylation (CH3) event detection 119
To apply BATMAN (Bayesian Tool for Methylation Analysis) 120
Displaying results and generating reports
To display results of analysis 121
To upload a track to UCSC 123
To save a result 124
To restore a saved result to the display
To generate a probe report 125
To generate a Batman report 128
5
Methylation (CH3) Analysis Reference
121
125
131
Agilent Genomic Workbench CH3 Application Main Window
Switch Application Menu
132
133
Command Ribbons 134
Home command ribbon 134
Preprocessing command ribbon 137
Analysis command ribbon 139
Reports command ribbon 142
View command ribbon 143
Help command ribbon 144
Navigator 146
Search pane 149
Design Data pane – icons, special text, and buttons 151
Design Data pane – actions and shortcut menus 152
Experiment pane – icons, special text, and buttons 157
Methylation (CH3) Analysis Guide
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Contents
Experiment pane — actions and shortcut menus 158
My Entity List pane – Icons, buttons, and special text 164
My Entity List pane – Actions and shortcut menus 164
My Networks pane 167
Tasks pane 167
Tasks pane – Icons, buttons, and special text 167
Tasks pane – Actions and shortcut menus 168
Genomic Viewer 169
Genome View 169
Chromosome View 171
Gene View 173
The View Cursor 177
Tab View 178
Status Bar
182
Dialog Boxes 183
Add Gene List <name> to 183
Agilent Feature Extraction Importer 184
Array Set 186
Batman Parameter Setup 187
Batman Report Dialog 188
Catalog and Workgroup Data 190
Combine Tracks 191
Configure Coloring Ranges and Shades 193
Confirm Overwrite 196
Create Experiment 197
Create Gene List 199
Create Query 201
Customize Search Link 203
Design Properties 204
Edit Array Color 207
Edit Array Order 208
Experiment Properties 209
10
Methylation (CH3) Analysis Guide
Contents
Export 211
Export Array Attributes 212
Export Experiments 216
Export Tracks 217
Find in column 218
Gene List 220
Go To Gene/Genomic Location 221
Import 222
Import (experiments) 224
Import GEML design files 225
Import Genome Build 226
Import Track 227
Microarray Properties 229
New Condition 232
Probe Methylation Status Setup 233
Probe Methylation Report Dialog 234
Sample Attributes 235
Scroll to Column 236
Select Color 237
Select data type for experiments 240
Select Experiment for Creating Query 241
Select Users 242
Set genome build and species for Axon design files 243
Sharing for enterprise 245
Show/Hide Columns 246
Simple HD Probe Search 247
Track 254
UDF Import Summary 256
Universal Data Importer - Map Column Headers 257
Upload Track to UCSC 260
User Preferences 262
View Preferences 268
Report Format
Methylation (CH3) Analysis Guide
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11
Contents
6
Statistical Algorithms
275
Overview of Methylation Detection and Visualization Algorithms
Methylation detection and measurement algorithms 276
Visualization algorithms 277
Methylation Detection and Measurement Algorithms
Methylation status detection algorithm 278
Bayesian tool for methylation analysis (BATMAN)
Visualization Algorithms 293
Moving average (linear smoothing)
Triangular smoothing 294
12
278
288
293
Appendix 297
Methylation calling – the beta distribution
References
276
297
298
Methylation (CH3) Analysis Guide
Agilent Genomic Workbench 6.5 – Methylation (CH3) Analysis
User Guide
1
Getting Started
What is the CH3 Application? 15
Using Agilent Genomic Workbench on a Mac 16
Entering a License and Starting the CH3 Application 17
Transferring Data from the eArray Web Site 20
Using Main Window Components to Display/Analyze Data 21
Switching Applications 26
Using Tabs and Command Ribbons 27
Using the Navigator to Search for Data 29
Using the Genomic Viewer to Display Data 34
General Instructions for Displaying Microarray Data/Results 38
General Instructions for Setting Options for CH3 Interactive Analysis 39
Getting Help 40
This chapter gives an overview of the Methylation (CH3) application of
Agilent Genomic Workbench. It shows you how to start the application
and find help, and gives general instructions on how to get started with
analyzing CH3 data.
Before or after you import extracted data into the program, you can
assign identification information and attributes to the samples through the
Sample Manager tab. See the Sample Manager User Guide.
To display or analyze imported CGH data, you organize the data files into
logical units called experiments. Experiments are used to define the data
you want to display or analyze using Agilent Genomic Workbench. You can
then use the Preprocessing, Analysis, Discovery and Reports tabs of the
program to interactively analyze the data in the experiment for
aberrations. For detailed steps on how to create an experiment, see the
Data Viewing User Guide.
Agilent Technologies
13
1
14
Getting Started
Methylation (CH3) Analysis Guide
Getting Started
What is the CH3 Application?
1
What is the CH3 Application?
The CH3 (methylation) application is a framework used to identify
methylation events in your samples. CH3 microarray analysis can help
identify methylated regions isolated using affinity based methods such as
methylated DNA immunoprecipitation. The software helps you:
• Identify molecular events associated with DNA methylation
• Find and validate gene regulation and regulatory networks by
creating high- resolution, genome- wide methylation profiles
• Show modes of action of compounds and target genes by
understanding the relation of DNA methylation to transcriptional
control
With the CH3 application, you can:
• Import data from the Agilent Feature Extraction and Axon programs,
and import UDF files
• Use an intuitive graphical interface to display data and annotations
in the context of an organism’s genome, at several simultaneous
levels of detail
• Use a Z- score or BATMAN (Bayesian Tool for Methylation Analysis)
algorithm to calculate the probability that probes are methylated or
unmethylated
• Compare the moving average of your log ratio and Z- score data
within the boundaries of CpG Island tracks
You cannot use workflow mode for methylation event detection.
Methylation (CH3) Analysis Guide
15
1
Getting Started
Using Agilent Genomic Workbench on a Mac
Using Agilent Genomic Workbench on a Mac
The content of this User Guide applies to both the Windows and Mac
versions of Agilent Genomic Workbench. Both of these versions have the
same features. However, when you use the Mac version of the program,
please note the following:
Windows command
Equivalent Mac command
Right-click
• Command-click (
-click)
• On Macs with trackpads, other options are available. On
certain machines, you place two fingers on the trackpad
while you press the button below the trackpad. See the user
guide for your specific machine.
• If you have a third-party mouse that has more than one
button, you may be able to use one of the buttons as a right
mouse button.
Control-click
Control-click (Same as the Windows command)
Shift-click
Shift-click (Same as the Windows command)
(Close button)
16
(Close button)
Methylation (CH3) Analysis Guide
Getting Started
Entering a License and Starting the CH3 Application
1
Entering a License and Starting the CH3 Application
This section describes how to enter your license for the CH3 application
to begin analyzing CH3 data.
When you start Agilent Genomic Workbench for the first time, the
program opens in the Home tab, with the Open Application tab
displayed. From this tab, you can click any of the application areas, enter
license information, or click Help to open the User Guide for that
application.
Figure 1
Open Application tab for CH3
Methylation (CH3) Analysis Guide
17
1
Getting Started
To enter your license for analyzing Methylation (CH3) data interactively
To enter your license for analyzing Methylation (CH3) data
interactively
1 Click the Open Application tab if it is not already displayed.
2 Click License next to the description of DNA Analytics (CH3 Module).
The License tab of the User Preferences dialog box appears.
Figure 2
License tab of the User Preferences dialog box
There are two ways to provide the license information:
Use a Server Location
1 Unzip the license .txt file into a folder on your server, to which the
program has access.
2 Copy the path for that folder to the Clipboard.
3 In the User Preferences License tab, click Server Location.
18
Methylation (CH3) Analysis Guide
Getting Started
To start the CH3 application
1
4 Paste the license folder path into the field below Server Location. (To
paste the license for both Windows and Mac computers, hold down the
ctrl key and press V.)
5 Click Apply, or click OK to apply the license and close the dialog box.
Enter a Text License
1 Find the folder that contains the CH3 application license .txt file.
2 Double- click the license name to open the file in Notepad (or open the
file in another text editor), and copy the text displayed to the
Clipboard.
3 In the User Preferences License tab, click Text License.
4 Paste the license information into the License text box. (To paste the
license for both Windows and Mac computers, hold down the ctrl key
and press V.)
5 Click Apply.
6 If you have no other licenses, click OK.
OR
If you have another license, click the arrow from the Select Analysis
Application list, select the application and repeat steps 1- 5.
To start the CH3 application
• In the Open Application tab, click the icon next to DNA Analytics
(CH3 Module)
.
The CH3 application starts and the Genomic Viewer is displayed.
Methylation (CH3) Analysis Guide
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1
Getting Started
Transferring Data from the eArray Web Site
Transferring Data from the eArray Web Site
When you install Agilent Genomic Workbench, a set of “core data” is
transferred to your database. This includes administrative data, control
grids, and the names (only) of Catalog and workgroup probe groups, bait
groups, microarray designs, and libraries.
Additional data from the eArray Web site are required in order to perform
eArrayXD functions. This content is downloaded from the eArray Web site
using the Data command from the Home tab. For example, to search for
expression probes from the Agilent Catalog, you must first transfer the
Catalog expression probe data from the eArray Web site to your server.
See “Catalog and Workgroup Data” on page 190. For more information on
how to download data from the eArray Web site, see the eArrayXD User
Guide.
In order to import or analyze extracted microarray data using Agilent
Genomic Workbench, you must first download the design files that match
those microarrays from the eArray Web site, or import them. See “To
download a design from eArray” on page 71 and “To import Agilent GEML
design files” on page 46. It is not necessary to transfer the entire catalog
or workgroup data from eArray to analyze extracted microarrays or run
Feature Extraction or analysis workflows.
20
Methylation (CH3) Analysis Guide
Getting Started
Using Main Window Components to Display/Analyze Data
1
Using Main Window Components to Display/Analyze Data
You can use the data viewing capability in Agilent Genomic Workbench
without a license to view data for many types of arrays, including CGH,
ChIP, and Methylation (CH3). You can use the data analysis capability in
Agilent Genomic Workbench only if you have a license for one or more of
the DNA Analytics programs (CGH, ChIP, or Methylation).
What are the main window components?
You use four primary components of the Agilent Genomic Workbench main
window to import, manage, export, display, and analyze extracted data.
• Home tab commands — import, manage and export data
• Navigator — create and fill new experiments with array data
When you make the experiment active, the data appear in the display,
called Genomic Viewer.
• Genomic Viewer — display data and content in four Views: Genomic
View, Chromosome View, Gene View, and Tab View
You use commands in the interactive analysis tabs to perform
preprocessing, analysis, and reporting of data. You can view the results
of data analysis in the Genomic Viewer.
• View tab commands — change appearance of Genomic Viewer display
Figure 3 shows the main window of Agilent Genomic Workbench when the
Genomic Viewer tab is selected, and identifies the names of its
components.
Methylation (CH3) Analysis Guide
21
1
Getting Started
What are the main window components?
Tabs
Command Ribbon
Switch Applications
Genome
View
Genomic
Viewer
Chromosome
View
The View Cursor
Gene
View
Tab View
Status Bar
Navigator
Figure 3
22
Agilent Genomic Workbench main window showing major components for CH3
Methylation (CH3) Analysis Guide
Getting Started
What can you do with the main components for display of data and results?
1
What can you do with the main components for display of data and
results?
See the table below for the parts of the main window you use to display
log ratio data and results.
Table 1
Components of Agilent Genomic Workbench main window for display of data
and results
To do this
Use this part of the main window
Change program to CGH, ChIP,
Methylation (CH3), Expression,
microRNA, or SureSelect Target
Enrichment module
Switch Application button: Click the button and click the
program module you want to open. The window and
options are different for the different program types.
Download design files
Navigator for Design Files: Right-click the name of a
design file and click Download. Icons give the status of a
library or microarray design (update required, draft,
review, completed, or submitted). See the eArrayXD User
Guide for details.
Import or export data
Home tab: Click the Import or Export button to select the
data you want to import or export. See Chapter 2,
“Importing, Managing, and Exporting CH3 Data and Other
Content” for more information.
Select array data to display in the three
graphical views or in the Tab View as a
table
Experiment pane of the Navigator: Create an experiment
with the imported data, select the experiment, and then
select the data within the experiment to display or
analyze. See Chapter 3, “Displaying CH3 Data and Other
Content” for more information.
Methylation (CH3) Analysis Guide
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1
Getting Started
What can you do with the main components for display of data and results?
Table 1
Components of Agilent Genomic Workbench main window for display of data
and results (continued)
To do this
Use this part of the main window
Display array data/results for only a
certain portion of a chromosome
Genome View: Select a chromosome to display in
Chromosome View. You cannot view log ratio data points
here.
Chromosome View: Select a gene region to display in
Gene View. You can display log ratio data points here if
you select Scatter Plot in the View Preferences dialog
box.
Gene View: See the log ratio data next to a selected
region of a chromosome, with associated genes and
track-based annotation. See Chapter 4, “Setting Up
Methylation (CH3) Analysis” for details about these
Views.
Show/Hide or customize the data
points for the scatter plots
Gene View: Move the mouse pointer over Scatter Plot to
display the options. Or, right-click and then click View
Preferences.
Chromosome View: Right-click and then click View
Preferences.
View tab: Click View Preferences.
See Chapter 3, “Displaying CH3 Data and Other Content”
for information on how to do this.
Display array data next to tracks or
gene lists
My Entity List pane of Navigator: Add or select a track or
gene list to have it appear in Gene View.
See Chapter 3, “Displaying CH3 Data and Other Content”
for information on how to do this.
24
Methylation (CH3) Analysis Guide
Getting Started
What can you do with the main components for display of data and results?
Table 1
1
Components of Agilent Genomic Workbench main window for display of data
and results (continued)
To do this
Use this part of the main window
Change the appearance of the display
View Tab: Click View Preferences. From the View
Preferences dialog box, you can change the orientation,
select what type of data to view, and configure scatter
plot options.
Genomic Viewer: Right-click any View except the Tab
View and select View Preferences. In the View
Preferences dialog box, you can select to show or hide the
scatter plots and how to display them, including results.
See Chapter 3, “Displaying CH3 Data and Other Content”
for more information.
Analyze or reanalyze displayed data
Preprocessing Tab: Click this tab to display commands
you use to manipulate the data before you apply the
algorithms.
Analysis Tab: Click this tab to display commands you use
to analyze the data.
Reports Tab: Click this tab top display commands you use
to generate and manage reports.
For more information on what you can do in these tabs,
see “Tabs” on page 27.
Methylation (CH3) Analysis Guide
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Getting Started
Switching Applications
Switching Applications
You can use the Agilent Genomic Workbench to work with a variety of
different data types. Because the requirements for the display of data (and
calculation of results, if using a license) are different for different data
types, you must switch the application for the type of data you want to
display.
The Switch Applications menu, located at the upper right corner of the
Agilent Genomic Workbench window, is used to change the application.
The selected application is marked
. The selected application is also
displayed in the title bar of the Agilent Genomic Workbench main window.
Figure 4
26
Switch Application panel
Methylation (CH3) Analysis Guide
Getting Started
Using Tabs and Command Ribbons
1
Using Tabs and Command Ribbons
Tabs
When you click a tab, groups of commands or single commands appear
that are specific for that tab. The tabs that are displayed change
depending on what licenses you have, and what application is selected
(such as CGH, ChIP, CH3).
Figure 5
Tabs for CH3 interactive analysis
The following table summarizes the capabilities in the interactive tabs of
the CH3 application of Agilent Genomic Workbench:
Tabs
CH3 Capabilities
Preprocessing
Combine (fuse) array designs
Analysis
Calculate a moving average on log ratio data
Calculate a moving average on ZScores generated by probe
methylation algorithm
Apply probe methylation algorithm
Apply BATMAN algorithm
Reports
Generate probe report
Generate BATMAN report
Methylation (CH3) Analysis Guide
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Getting Started
Commands
Commands
The area where commands appear is called a command ribbon. The
command ribbon that appears when you click the Home tab for CH3 is
shown below. The commands that appear in the command ribbon change
depending on what application module is selected, and which tab in that
application module is selected.
Figure 6
Home command ribbon for CH3 interactive analysis
For a complete description of all of the command ribbons and commands
you see in Agilent Genomic Workbench, see “Command Ribbons” on
page 134.
28
Methylation (CH3) Analysis Guide
Getting Started
Using the Navigator to Search for Data
1
Using the Navigator to Search for Data
This section gives you instructions on how to search for design files,
extracted FE data, experiments and other information in the Navigator of
Agilent Genomic Workbench. The Navigator contains different panes when
you select the eArrayXD or Sample Manager tabs. See the User Guides for
those applications for information on the Navigator contents.
Search pane
Design Data pane
Experiment pane
My Entity List pane
My Networks pane
Tasks pane
Figure 7
Navigator panes
The Navigator shows the array data, experiments, and other content
stored in Agilent Genomic Workbench that is available to the logged in
user. It contains the following panes:
Methylation (CH3) Analysis Guide
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Getting Started
Using the Navigator to Search for Data
Pane
Comments
Search
Lets you search within any pane of the Navigator for a specific
item (array or build, for example). You must type the entire array
name or term; otherwise, use asterisks (*) as wild cards for
unspecified strings. For example, type *1234* to find any item that
contains “1234”.
Design Data
Contains microarray data files, organized by design and application
type, and then by genome build.
Shows all probe groups and microarray designs that are available
to you, organized by folders. For the SureSelect Target Enrichment
application type, the program shows all bait groups and libraries. In
general, you can:
• Expand or collapse folders to show or hide content.
• Look at the icon that appears with an item to monitor its status.
• Right-click the name of a folder or item to open a shortcut menu
that lets you take action on the item.
See “Design Data pane – icons, special text, and buttons” on
page 151 and “Design Data pane – actions and shortcut
menus” on page 152.
30
Experiment
Contains Agilent Genomic Workbench experiments. Experiments
are organizational units that contain links to microarray data and
design files. In data analysis modules, experiments also contain
saved results.
My Entity List
Contains gene lists and tracks:
• Gene Lists are collections of genes of interest. You can create
them within the program, import and export them, and apply
them to Gene View and Chromosome View.
• Tracks are collections of annotation or other information that
map to specific genomic locations. You can import, export, and
combine tracks, and display them in Gene View with your array
data and analysis results.
Methylation (CH3) Analysis Guide
Getting Started
Using the Navigator to Search for Data
Pane
Comments
My Networks
Contains the biological networks/pathways that you found using
Network Search or that you create using a literature search in
eArrayXD. For more information, see the eArrayXD Users Guide.
Tasks
Shows the jobs that you have submitted (such as downloading
designs from eArray). Some jobs are completed locally by the
eArrayXD server program. Others are sent to the eArray Web site
for completion. In general, you can:
• Look at the icon that appears with a job to monitor its status.
• Right-click the name of a pending task to open a shortcut menu
that lets you take further action on the job.
See “Tasks pane” on page 167.
Methylation (CH3) Analysis Guide
1
31
1
Getting Started
To search the Navigator
To search the Navigator
You can search one or all of the panes of the Navigator for items that
match a specific search term. Figure 8 shows the search pane of the
Navigator, and identifies a couple of its elements.
Search Term Box
Pane List
Figure 8
Search pane of the Navigator
1 At the top of the Navigator, in the Pane list, select the pane to search.
To search in all panes, select All Panels. If the pane list is not visible,
click
to show it.
2 In the search term box, type the desired search term. The search term
is not case sensitive, but it must contain the complete entry that you
want to find. You can use asterisks (*) to represent one or more
unspecified characters. For example, type *12345* to find any item that
contains “12345”.
3 Click
.
The program searches the selected pane(s) for items that match your
search term. If it finds matching items, the program expands the
appropriate folders, and displays the names of the matching items in
red. The first matching item is highlighted in yellow.
32
Methylation (CH3) Analysis Guide
Getting Started
To search the Navigator
1
4 Do any of the following:
• To highlight the next matching item, if one is available, click
.
• To highlight the previous matching item, click
5 After you complete the search, click
search, as well as your search term.
Methylation (CH3) Analysis Guide
.
to clear the results of the
33
1
Getting Started
Using the Genomic Viewer to Display Data
Using the Genomic Viewer to Display Data
What is the Genomic Viewer?
Genomic Viewer is the graphics and tabular display section of the Agilent
Genomic Workbench main window. In the Genomic Viewer, extracted data
and analysis results are tabulated and displayed next to depictions of the
genome, selected chromosome, and selected genes of the species whose
array data you are analyzing.
There are four main views in the Genomic Viewer, as shown in Figure 9.
• Genome View – A graphical representation of the entire genome for the
selected species. Use this view to select the chromosome to show in the
other views.
• Chromosome View – A graphical representation of the selected
chromosome, displayed with cytobands and a plot area. Click or drag
the mouse to select a region to display in the Gene View.
• Gene View – A more detailed view of the chromosomal region selected
in the Chromosome View.
• Tab View – Displays design annotation and log ratio data related to the
chromosome you select in Chromosome View
For more information on the Genomic Viewer and its views, see
Chapter 5, “Methylation (CH3) Analysis Reference”.
34
Methylation (CH3) Analysis Guide
Getting Started
What is the Genomic Viewer?
1
Chromosome View
Gene View
Genome View
Tab View
Figure 9
Genomic Viewer in vertical orientation
Methylation (CH3) Analysis Guide
35
1
Getting Started
To change the size of and detach panes from the Agilent Genomic Workbench main window
To change the size of and detach panes from the Agilent Genomic
Workbench main window
• To change the size of a pane in the main window, drag one of its inside
borders.
• To detach a pane from the main window and open it in a separate
window, click its Detach button
.
Drag a border to
to resize pane
Click here
to detach View
Figure 10
36
Changing the size of and detaching panes
Methylation (CH3) Analysis Guide
Getting Started
To maximize and reattach panes to the Agilent Genomic Workbench main window
1
To maximize and reattach panes to the Agilent Genomic Workbench
main window
• To display a view full- screen in a separate window, click its Maximize
button.
• To reattach a view in a separate window to the main window, click its
Close button.
Click here
to maximize View
Figure 11
Methylation (CH3) Analysis Guide
Click here to
reattach View
Maximizing and reattaching panes
37
1
Getting Started
General Instructions for Displaying Microarray Data/Results
General Instructions for Displaying Microarray Data/Results
An experiment is
the folder that
holds data from
any array set you
select for the
experiment. The
folder also holds
analysis results.
You set up experiments to display all data and results in the Genomic
Viewer. To set up an experiment you:
• Import data
• Create a new experiment
• Add the imported data to the experiment
• Select the experiment to display data
For step- by- step instructions on how to display data, see the Data
Viewing User Guide.
Change to the
CH3 application
Turn on scatter
plot options
Add data to the new
experiment
Create an
experiment
Select the
experiment
Figure 12
38
Select arrays to
display
Import
design files
Import data files
•
Agilent FE files
•
Axon files
•
UDFs
Review data
(tracks and
genomic
boundaries)
Typical pathway for displaying microarray data/results
Methylation (CH3) Analysis Guide
Getting Started
General Instructions for Setting Options for CH3 Interactive Analysis
1
General Instructions for Setting Options for CH3 Interactive
Analysis
Figure 3 shows the pathway for setting up an experiment and analyzing
data interactively with the CH3 application. After you import data and set
up experiments, you can set up preprocessing and analysis calculations
before you make the experiment active, or apply them afterwards. You can
combine designs, apply an algorithm to show probabilities that genomic
regions are methylated or not and report the results. When you change
each option after experiment activation, the program recalculates the
results. For more information on how to change analysis options, see
Chapter 4, “Setting Up Methylation (CH3) Analysis”.
Set up and select
experiment
Display and save
results
Select /deselect
arrays to include
in reanalysis
Combine
designs
Compare moving
average of log
ratio and Z-score
data with genomic
region tracks
Apply Z-score
and/or BATMAN
algorithms to find
methylation
events
Generate reports
Figure 13
Typical analysis pathway – Interactive mode for CH3 application
Methylation (CH3) Analysis Guide
39
1
Getting Started
Getting Help
Getting Help
To get help within Agilent Genomic Workbench
Agilent Genomic Workbench has several help resources. All help guides
open using Adobe® Acrobat®.
Help Resource
Description/Instructions
Methylation (CH3)
Analysis User Guide
This user guide, which you are now reading, supplies comprehensive
help on all available CH3 tasks. You can access it easily from anywhere
within the program.
1 In any tab of Agilent Genomic Workbench, click the Help tab.
2 On the Help Ribbon, click Application Guide.
Methylation (CH3) Analysis User Guide opens.
Other User Guides
The Help tab in Agilent Genomic Workbench lets you view any of the
available user guides that apply to the currently selected application
type.
1 Set the desired application type. See “Switching Applications” on
page 26.
2 In the Agilent Genomic Workbench tab bar, click Help.
The names of the available user guides appear in the command
ribbon.
3 Click the desired user guide.
The selected user guide opens.
Product Overview Guide
An additional guide gives an overview of the capabilities within Agilent
Genomic Workbench and describes how to start and find help for all of
the programs. In addition, it helps you with system administration and
troubleshooting.
1 In any interactive analysis tab of Agilent Genomic Workbench, click
the Open Application tab.
2 At the upper right corner of the Open Application tab, click Product
Overview.
40
Methylation (CH3) Analysis Guide
Getting Started
To contact Agilent Technical Support
1
To contact Agilent Technical Support
Technical support is available by phone and/or e- mail. A variety of useful
information is also available on the Agilent Technical Support Web site.
Resource
To find technical support contact information
Agilent Technical Support
Web site
1
2
3
4
Contact Agilent Technical
Support by telephone or
e-mail (United States and
Canada)
Telephone: (800-227-9770)
Contact Agilent Technical
Support by telephone or
e-mail (for your country)
1 Go to http://chem.agilent.com.
2 Select Contact Us.
3 Under Worldwide Sales and Support Phone Assistance, click to
select a country, and then click Go. Complete e-mail and telephone
contact information for your country is displayed.
Go to http://chem.agilent.com.
Select a country or area.
Under Quick Links, select Technical Support.
Select from the available links to display support information.
E-mail: informatics_support@agilent.com
To learn about Agilent products and services
To view information about the Life Sciences and Chemical Analysis
products and services that are available from Agilent, go to
www.chem.agilent.com.
Methylation (CH3) Analysis Guide
41
1
42
Getting Started
To learn about Agilent products and services
Methylation (CH3) Analysis Guide
Agilent Genomic Workbench 6.5 – Methylation (CH3) Analysis
User Guide
2
Importing, Managing, and Exporting
CH3 Data and Other Content
Importing Files 44
Working with Experiments to Organize Imported Data 57
Managing Content 68
Exporting and Saving Content 80
This chapter describes how to import, organize, manage, and export
methylation (CH3) data and other content within the user interface of
Agilent Genomic Workbench.
Agilent Technologies
43
2
Importing, Managing, and Exporting CH3 Data and Other Content
Importing Files
Importing Files
You can use the Home tab to import many kinds of files into Agilent
Genomic Workbench. The table below describes the kinds of files you can
import, and the topics in this section that describe how to import them.
The Design Data pane of the Navigator displays all of the content available
for the user who is logged in. Some of this content (such as Catalog
designs not yet downloaded) must be downloaded from the eArray Web
site before it can be used. Some of the content is available for you to use
but not change (red, read- only content), and some of the content you
imported and can change (green). See “Navigator” on page 146 for more
information on the Navigator panes and how to use them.
For information on downloading microarray designs and other content
from the eArray Web site, see the eArrayXD User Guide.
44
Type of file
Description
See these topics
Microarray data files
• Agilent Feature Extraction
(*.txt) data files
• GenePix/Axon (*.gpr) data
files
• Universal Data Files (UDFs)
(*.txt files)
“To import Agilent FE or Axon
data files” on page 47
“To import a UDF file” on
page 49
Microarray design files
• Agilent GEML (*.xml) design
files
• GenePix/Axon (*.gal) design
files
“To import Agilent GEML
design files” on page 46
“To import Axon design
files” on page 47
Genome builds
Agilent-supplied genome
information for human, mouse
and rat genomes
“To import a genome build” on
page 52
Tracks
BED format annotation track
files
“To import tracks” on page 53
Array attributes
.txt files that you have created
yourself or previously exported
from Agilent Genomic
Workbench
“To import array attributes” on
page 54
Methylation (CH3) Analysis Guide
Importing, Managing, and Exporting CH3 Data and Other Content
To select a different location for data files
Type of file
Description
See these topics
Experiments
ZIP format file of experiments
exported from Agilent Genomic
Workbench
“To import an experiment
file” on page 55
Probe file
Microarray design creation
process using an uploaded file
as the source of probes
See the eArrayXD User Guide
for more information.
“To import a probe file” on
page 55.
Custom Genome
ZIP file containing at least one
FASTA format sequence file
See the eArrayXD User Guide
for requirements and
information.
“To import a genome” on
page 56
2
To select a different location for data files
By default, the program stores microarray and experimental data files in
C:\Program Files\Agilent\Agilent Genomic Workbench Standard Edition
<version number>\data. If you want, you can select a different location.
CAUTION
Do not select a location that contains a backup data folder; the current data overwrites
the data in the folder you select.
1 In the Home tab, click User Preferences.
The User Preferences dialog box appears. See “User Preferences” on
page 262.
2 In the Miscellaneous tab, under Data Location, click Browse.
An Open dialog box appears.
3 Select a location, then click Open.
The selected location appears in the User Preferences dialog box, in
Data Location.
4 Click OK.
Methylation (CH3) Analysis Guide
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2
Importing, Managing, and Exporting CH3 Data and Other Content
To import Agilent GEML design files
To import Agilent GEML design files
The Agilent Genomic Workbench database must contain designs that match
the Agilent Feature Extraction data files you want to import. The design
file must be present before any extraction data files are imported. Your
imported GEML files contain array- specific information such as probe
names, annotations, and chromosomal locations, and are associated with a
specific genome build. To import an Agilent GEML file, use the following
procedure.
NOTE
• Catalog design files must be downloaded from the eArray Web site.
• Designs with multiple genome builds are supported for both Catalog and custom designs. For
Catalog designs, the design must first be downloaded from the eArray Web site. You can then
import the design for other genome builds.
1 In the Home tab, click Import > Design Files > GEML File.
The Import Design Files dialog box appears. See “Import” on page 222.
The dialog box shows only *.xml files.
2 To select a file for import, click its name. To select additional files, hold
down the ctrl key while you click their names.
3 Click Open.
The program validates the selected file(s), and the Import GEML Design
Files dialog box appears. See “Import GEML design files” on page 225.
• If a design file passes validation, the Status column shows Valid in
green.
• If the design is an Agilent Catalog design, and is not yet downloaded
from the eArray Web site, the status shows Not Allowed in red. You
must download the file from the eArray Web site.
• If a design file fails validation, Corrupt appears in the Status column
beside it, and the program will not import the file. To remove the
corrupt design from the list, click its Remove button
.
4 Click Start Import.
The program imports the file(s). The files appear as new design folders
in the Data folder of the Navigator with the genome build as a node
within the folder.
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Methylation (CH3) Analysis Guide
Importing, Managing, and Exporting CH3 Data and Other Content
To import Axon design files
2
You can import two design files with the same name, but associated
with different genome builds; for example, Hg17 or Hg18. If you do, the
program creates a single design folder with two nodes, one for each
genome build.
To import Axon design files
You can import Axon (*.gal) microarray design files into Agilent Genomic
Workbench. The program requires the Axon design files that match all
Axon array data files you import.
1 In the Home tab, click Import > Design Files > Axon File.
The Import Axon Design Files dialog box appears. See “Import” on
page 222. The dialog box shows only *.gal files.
2 To select a file for import, click its name. To select additional files, hold
down the ctrl key while you click their names.
3 Click Import.
The program validates the selected file(s), and the Set genome build
and species for Axon design files dialog box appears. See “Set genome
build and species for Axon design files” on page 243.
• If a design file passes validation, the Status column will show Valid in
green.
• If a design file fails validation, Corrupt appears in the Status column
beside it, and the program will not import the file. To remove the
corrupt design from the list, click its Remove button
.
4 For each design file, select the appropriate Species and Genome Build.
5 Click Start Import.
The program imports the file(s). The files appear as new design folders in
the Data folder of the Design Data pane, organized by application (CGH,
ChIP, or methylation, for example).
To import Agilent FE or Axon data files
You can import several types of microarray data files into Agilent Genomic
Workbench:
Methylation (CH3) Analysis Guide
47
2
Importing, Managing, and Exporting CH3 Data and Other Content
To import Agilent FE or Axon data files
• Agilent Feature Extraction (FE) *.txt data files
• Axon (*.gpr) data files
• Universal Data Files (UDFs) (*.txt files) See “To import a UDF file” on
page 49 for instructions on how to import this file type.
In order to import Agilent Feature Extraction files, you must import the
representative GEML array design files first. In order to import Axon data
files, you must import the representative Axon.gal design files first. See
“To import Agilent GEML design files” on page 46 or “To import Axon
design files” on page 47.
1 In the Home tab, do one of the following:
• To import Agilent FE data files, click Import > Array Files > FE
File.
• To import Axon data files, click Import > Array Files > Axon File.
A dialog box appears. Only data files of the appropriate type appear.
See “Import” on page 222.
2 To select a file for import, click its name. To select additional files, hold
down the ctrl key while you click their names.
3 Do one of the following:
• For Agilent FE files, click Open.
• For Axon files, click Import.
In either case, the Agilent Feature Extraction Importer dialog box
appears. “Agilent Feature Extraction Importer” on page 184.
4 Set the following, as needed:
s
48
Setting
Comments
Name
The names of imported arrays are often cryptic. You can give
any array a more meaningful label.
a Double-click the name of the array.
b Edit the name, as desired.
c Press Enter.
Methylation (CH3) Analysis Guide
Importing, Managing, and Exporting CH3 Data and Other Content
To import a UDF file
2
Setting
Comments
Dye Flip
For each array:
• Select Normal if:
• The test samples were labeled with cyanine-5 (red).
• The control samples were labeled with cyanine-3 (green).
• The imported ratio (test/control) should be reported
directly.
• Select Flipped if:
• The test samples were labeled with cyanine-3 (green).
• The control samples were labeled with cyanine-5 (red).
• The imported ratio (control/test) should be reported with
the ratio inverted (test/control).
The program does not combine dye-flip pairs.
Overwrite arrays with
duplicate names
If you mark this option, the program deletes an existing array
data file if it has the same name as one you import.
5 Do one of the following:
• To import the file(s) while you wait, click OK.
• To import the file(s) in the background, click Run in Background.
This lets you continue while the program imports the files.
To import a UDF file
UDF files are plain text files that contain array data in tab- delimited
format. Files must contain the following six columns of information, in
any order. Each column must contain the following column names, as
column headers, or you must “map” the names from the file to these
columns in Agilent Genomic Workbench:
• Probe name
• Chromosome name
• Start position
• Stop position
• Description
• Signal intensity data (The file can contain additional columns, each
with data from an additional array.)
Methylation (CH3) Analysis Guide
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2
Importing, Managing, and Exporting CH3 Data and Other Content
To import a UDF file
When you import a UDF file, the program creates a new design based on
the information you enter during import, and the information in the file
itself. This design contains all of the arrays represented in the file. The
program also creates a new experiment that contains the arrays.
1 In the Home tab, click Import > Array Files > UDF File.
The UDF Files dialog box appears. See “Import” on page 222. Only *.txt
files appear in the dialog box.
2 Select the desired UDF file, then click Open.
The Select data type for experiments dialog box appears. “Select data
type for experiments” on page 240.
3 For each array, set the following, as needed:
Setting
Comments
Experiment Name
By default, the program creates an experiment with the same
name as the imported file. To change the name:
a Double-click the name.
b Edit the name as desired.
c Press Enter.
Data type
• Select the mathematical form of the signal intensity data for
the array. The options are ratio, log2 ratio, log10 ratio, and
ln ratio.
Design type
• Select CH3.
4 Click Continue.
The Universal Data Importer – Map column headers dialog box appears.
The main table in the dialog box contains the first few rows of data
from the file. Column headings that are contained in the first line of
the file appear at the top of the table as a guide. See “Universal Data
Importer - Map Column Headers” on page 257 for more information.
5 Below each column heading, select the label that identifies the content
of the column. Use each label exactly once, except for LogRatio, which
you can use multiple times. Alternatively, in Select Mapping, select a
saved column map.
These options are available:
50
Methylation (CH3) Analysis Guide
Importing, Managing, and Exporting CH3 Data and Other Content
To import a UDF file
Label
Description
ProbeName
The column contains names of probes.
ChrName
The column contains names of chromosomes.
Start
First chromosomal location for the probe.
Stop
Last chromosomal location for the probe.
Description
Text annotation related to the probe.
LogRatio
The column contains array data values that correspond to each probe.
You can use this label more than once.
2
6 Under Species Info, select the species and Genome Build appropriate
to the data in the file.
7 If you expect to import many similar UDFs in the future, follow these
steps to save the column map:
a Under Mapping Info, click Save Mapping As.
An Input dialog box appears.
b Type a name for the column map, then click OK.
The name of the saved map appears in Select Mapping.
In the future, you can select this mapping and apply it to any UDF file
that you import.
8 By default, the program creates a “Virtual Array ID” that becomes the
Array ID attribute for the array(s) in the UDF. To create your own
virtual Array ID, follow these steps:
a Under ArrayID Info, clear Use System Generated ArrayID.
b Double- click the number in Virtual ArrayID, then type the desired
new Virtual Array ID.
For more information on Array IDs, see the Sample Manager User
Guide.
9 Click Import.
The program validates your column mapping. A dialog box appears. If
you need to fix the column map, the dialog box has a list of the
missing column label(s). If the column map is complete, a message asks
if you want to import additional files with the same mapping.
10 Do one of the following:
Methylation (CH3) Analysis Guide
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2
Importing, Managing, and Exporting CH3 Data and Other Content
To import a genome build
• If you want to import additional files with the same column
mapping, follow these steps to include these files in the import:
a Click Yes.
The UDF Files dialog box appears.
b Click the name of a file to select it for import. Hold down the ctrl
key while you click the names of additional files.
c Click Open.
• If you do not want to include additional file(s) in the import, click
No.
The Program imports all requested files, and the UDF Import Summary
dialog box appears. This dialog box shows the imported files, the
number of lines of data that were imported for each file, and the
number of lines that were skipped, if any. If a file name appears in red,
the program may not have imported the file. See “UDF Import
Summary” on page 256.
11 Click OK.
In the Design Data pane, in the appropriate design type folder within
the Data folder, a new design folder appears. The design folder contains
the imported array data.
A new experiment appears in the Experiments folder in the Experiment
pane, that contains the array data. This experiment has the name of the
imported UDF file, unless you changed it during import.
To import a genome build
In general, the program uses the genome build specified in the array
design file, and protects it from changes. If a genome build is not available
in the program, you can import one.
NOTE
Use arrays from a single genome build in an experiment.
1 In the Home tab, click Import > Genome Build.
The Import Genome Build dialog box appears. See “Import Genome
Build” on page 226.
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Methylation (CH3) Analysis Guide
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To import tracks
2
2 Set the following. All are required.
Setting
Instructions
Species
• Type the genome’s species of origin, as you would want it to
appear within the program.
Build Name
• Type the name of the genome build you want to import, as
you would want it to appear within the program.
Refseq File
This file contains information on gene locations for Gene View.
a Click Browse.
A dialog box appears.
b Select the desired file, then click Open.
Cyto-band File
This file contains the graphic information on the cyto-bands
for Genome and Chromosome Views.
a Click Browse.
A dialog box appears.
b Select the desired file, then click Open.
3 Click OK.
To import tracks
You can import BED format track files into Agilent Genomic Workbench.
Track files contain specific features correlated with chromosomal
locations, and apply to a specific genome build of a given species.
1 In the Home tab, click Import > Track.
The Import Track dialog box appears. See “Import Track” on page 227.
2 Set the following. All are required.
Setting
Instructions
Species
• Select the species to which the track applies.
Build Name
• Select the specific genome build of the species to which the
track applies.
Methylation (CH3) Analysis Guide
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2
Importing, Managing, and Exporting CH3 Data and Other Content
To import array attributes
Setting
Instructions
Track Name
• Type a name for the track.
This name identifies the track within the program, including
the name that appears if you include the track in Gene View.
Track File
a Click Browse.
A dialog box appears.
b Select the name of the track (*.bed) file that you want to
import.
c Click Open.
The location of the file appears in Track File.
3 Click OK.
The program imports the track. To display the track in Gene View, and
to manage tracks, see “To show tracks in Gene View” on page 100.
To import array attributes
An array attributes file is a tab- delimited *.txt file that contains a list of
arrays by Array ID, and values for array attributes. Attributes are pieces
of array- specific information, such as the hybridization temperature or the
name of an array set that contains the array.
Although you can import array attributes with this function, the Sample
Manager application lets you import and assign array attributes more
easily. See the Sample Manager User Guide for more information. To
import an array attributes file
1 From the Home tab, click Import and then select ArrayAttributes.
The Import microarray attributes dialog box appears. See “Import” on
page 222.
2 Select the microarray attributes file, then click Import.
The program imports the file. If the ArrayIDs in the file do not match
the ArrayIDs of arrays in the program, a dialog box appears. The dialog
box has a list of the ArrayIDs in the file that do not match. Click No to
stop the import process, or click Yes to continue anyway.
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Methylation (CH3) Analysis Guide
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To import an experiment file
2
To import an experiment file
In Agilent Genomic Workbench, an experiment is a set of links to
microarray data and design files, and any associated results. An Agilent
Genomic Workbench experiment file is a single ZIP file that contains the
design and data files for one or more experiments. You can import
• Experiment files created in Agilent Genomic Workbench on another
computer
• Agilent Genomic Workbench 5.0 and 6.x experiment files
1 In the Home tab, click Import > Experiments.
The Import Experiments dialog box appears. See “Import” on page 222.
2 Select the ZIP file that contains the experiment(s) you want to import,
then click OK.
The program imports the experiment file. Designs appear as new folders
in the Design Data pane, in the applicable design type folder. Array
data appears within the applicable design folder, organized by genome
build. In addition, the experiment(s) appear in the Experiment pane,
containing the appropriate arrays.
Agilent Genomic Workbench experiment files contain all of the design and
array data files for an experiment, but do not include any analysis
parameter settings, array selections, or analysis results. To export the data
and design files from one or more experiments, see “To export
experiments” on page 81.
To import a probe file
1 In the Home tab, on the Command Ribbon, click Import > Probe
Upload.
The Probe Upload dialog box appears.
2 Complete the dialog box, and click Preview. See the eArrayXD User
Guide for more information.
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To import a genome
To import a genome
You can import a user- defined genome for use with the Genomic Tiling or
Bait Tiling tools. When you do, the genome becomes a permanent part of
the database on your Agilent Genomic Workbench server, and is available
to all of the users in your workgroup. For details on Genomic Tiling and
Bait Tiling, and how to use the Import > Custom Genome for Tiling
command, see the eArrayXD User Guide.
To transfer catalog and workgroup data
You can transfer probe data and exon boundary data from the eArray Web
site to your Agilent Genomic Workbench server. You can also transfer
probe data from both the Agilent Catalog and from the folders of your
workgroup. Probe data are available by application type (i.e. Expression,
ChIP, and so on). Exon boundary data apply to all application types. For
more information, see “Catalog and Workgroup Data” on page 190.
1 In the Home tab, click Data.
2 For the selected type of data, click Download.
A Data Download task is submitted and appears in the Tasks folder of
the Navigator.
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Working with Experiments to Organize Imported Data
2
Working with Experiments to Organize Imported Data
This section describes how to organize imported array data and designs
into experiments. Experiments, shown in the Experiment pane of the
Navigator, contain links to specific array data and design files in the
Design Data pane. After you set up an experiment, you can then analyze
selected array data within the experiment.
Because experiments only contain links to the actual data and design
files, any number of experiments can use a given set of files. In the CH3
application, experiments also can contain saved experiment results.
To display the array designs and data in the program
• To display the directory of data in the program, use the Design Data
pane (Figure 14). Double- click a folder to expand or collapse it, or click
the
and
buttons.
Figure 14
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To create a new experiment
In the Design Data pane, the program organizes design files by the
application (CGH, ChIP, or methylation, for example) to which they apply.
It organizes array data files by genome build under the design with which
they are associated.
You can right- click many elements of the Design Data pane to open
shortcut menus. For more information, see “Design Data pane – actions
and shortcut menus” on page 152.
Many icons can appear in the Design Data pane. See “Design Data pane –
icons, special text, and buttons” on page 151 for a complete list.
The Search pane can help you find specific data files or other content. See
“To find specific content items in the Navigator” on page 70.
For more information on working with and managing microarray design
files, see the eArrayXD User Guide.
To create a new experiment
In Agilent Genomic Workbench, experiments are organizational units that
contain links to data and design files. To display or analyze data, you
must first create an experiment and associate the desired data files with
it. Because experiments only contain links to the actual data and design
files, any number of experiments can use a given set of files. In data
analysis applications (CGH, ChIP, or methylation, for example),
experiments can also contain saved experiment results.
1 In the Home tab, click Create Experiment.
The Create Experiment dialog box appears. See “Create Experiment” on
page 197.
2 Type a Name and an optional Description for the experiment.
3 Do one of the following:
• To create an empty experiment, and add data to it later, click OK.
The program creates the experiment. To add arrays to the
experiment later, see “To add arrays to an experiment” on page 60.
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To create a new experiment
2
• To create an experiment and add data to it now, follow these steps:
(You can add or remove data from the experiment later.)
a Click Properties.
The Experiment Properties dialog box appears. See “Experiment
Properties” on page 209.
b Under Select Design, select the design and genome build
associated with the desired array data.
The applicable arrays appear in Array List.
c In Array List, click the name of an array that you want in your
experiment. Hold down the ctrl key while you click the names of
additional arrays.
d Click
.
The program transfers the selected arrays to the Selected Array
List.
The dialog box also shown other options for adding arrays. See
“Experiment Properties” on page 209 for more information.
e Click OK.
The program creates the new experiment, and adds data to it from
the selected arrays.
• To create an experiment and add data to it using the “drag and
drop” method, follow these steps:
a To create an empty experiment, click OK.
The program creates the experiment.
b From the Design Data pane, expand a design to see the build and
array data.
c Drag an array from the Design Data pane and drop it onto the
experiment folder in the Experiment pane.
In all cases, a folder with the name of the new experiment appears in
the Experiment pane of the Navigator.
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To add arrays to an experiment
Figure 15
Experiment pane of the Navigator
To add arrays to an experiment
After you create an experiment, or import one, you can add arrays to it.
When you add arrays to an experiment, you create links between the
experiment and the array data and design files. Because the program does
not move the actual files, multiple experiments can share the same arrays.
1 In the Experiment pane, double- click the Experiments folder to expand
it.
2 Right- click the name of the experiment, then click Show Properties.
The Experiment Properties dialog box appears. See “Experiment
Properties” on page 209.
3 Under Select Design, select the design file and genome build for the
arrays to add.
The arrays for the selected design file and genome build appear in
Array List.
4 In Array List, select the arrays to add to the experiment. To select a
single array, click its name. To select additional arrays, hold down the
ctrl key while you click their names.
5 Click
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To change the order of arrays in an experiment
2
The program transfers the selected arrays to the Selected Array List.
The dialog box also has other options for adding arrays. See
“Experiment Properties” on page 209 for more information.
6 Click OK.
Or, to add array data to an experiment using the “drag and drop” method,
1 From the Design Data pane, expand a design to see the build and array
data.
2 Drag an array from the Design Data pane and drop it onto the
experiment folder in the Experiment pane.
If needed, the program adds appropriate design and genome build folders
to your experiment folder in the Experiment pane. It places the arrays you
selected in the appropriate genome build folder.
NOTE
If you add arrays to a selected experiment, and analysis options are marked (for example,
Probe Methylation or Batman), you need to clear and then mark these options again to
apply the analysis to the arrays.
To change the order of arrays in an experiment
When you select an experiment, a table appears in the Tab View of
Genomic Viewer that contains log ratio values for arrays in the
experiment. See “Tab View” on page 178. You can change the order in
which the arrays appear the table. If you display separate scatter plots in
Gene View and Chromosome View for each array, the array order also
determines the order in which these plots appear. You can use this feature
to organize your arrays more logically, or to make it more convenient to
display certain arrays. It is especially useful if you have many arrays.
1 In the Experiment pane, right- click the name of the desired experiment,
then click Edit Array Order.
The Edit Array Order dialog box appears. See “Edit Array Order” on
page 208.
2 In Design, select the design that contains the arrays whose order you
want to change.
The arrays from the selected design appear in Array Name.
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To change the display names for arrays in an experiment
3 Do any of the following:
• To move an array up in the list, click its name, then click
• To move an array down in the list, click its name, then click
.
.
• To sort the list based on a specific microarray attribute, select the
desired attribute in Order by.
4 Click OK.
To change the display names for arrays in an experiment
You can change the name displayed for arrays in an experiment, based on
array attributes. When you change the display names for arrays in an
experiment, the array names are changed only for the selected experiment.
The display names are unchanged in the Design Data pane and in the
other experiments.
1 Expand the folders in the Experiment pane until you see the
experiment you want to change.
2 Right- click the experiment name, and select Show Properties.
3 In the Experiment Properties dialog box, click Display Name by and
select an attribute to use for display of array names.
Click OK. The names of the arrays in the experiment are changed to the
selected attribute. If the attribute does not exist for an array, the Global
Display Name is displayed.
NOTE
62
To change the name of an array that is displayed throughout Agilent Genomic Workbench,
change its Global Display Name using Sample Manager. For more information, see the
Sample Manager User Guide.
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To rename an array in an experiment
2
To rename an array in an experiment
When you rename an array in an experiment, you change the array’s name
only within the context of the selected experiment. The name of the array
is unchanged in the Design Data pane, and in other experiments.
1 Expand the folders in the Experiment pane until you can see the array
to rename.
2 Right- click the name of the desired array, then click Rename.
An Input dialog box appears.
3 Type the new name for the array, then click OK.
The name of the array in the tab view of the selected experiment is
renamed. The global display name of the array is not changed.
To remove arrays from an experiment
When you remove arrays from an experiment, you only remove the links
between the experiment and the data files. The files are still available in
the program for use in other experiments. To completely remove files from
the program, see “To remove data or design files from the program” on
page 72.
1 In the Experiment pane, expand folders until you can see the desired
experiment, and the array(s) that you want to remove from it.
2 In the Arrays or Calibration Arrays folder of the desired experiment,
click the name of an array to select it for removal. Hold down the ctrl
key while you click the names of additional arrays.
3 Right- click one of the selected array names, then click Delete.
A Confirm dialog box appears.
4 Click Yes.
The program removes the links between the experiment and the
selected array data files. If the removal of arrays leaves a design folder
in the experiment empty, the program removes this folder as well.
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To select or remove calibration array(s)
To select or remove calibration array(s)
After you add an array to an experiment, you can select it as a calibration
array. The program lists calibration arrays within the Calibration Arrays
folder of the experiment with a special icon
. You can also remove the
calibration designation from an array.
To select an array as a calibration array
1 Expand the folders of the Experiment pane until you can see the array
that you want to select as a calibration array.
2 Right- click the name of the desired array, then click Select for
Calibration. To select all of the arrays of a given design in the
experiment as calibration arrays, right- click the genome build folder of
the desired design, then click Set for Calibration.
The program selects the array as a calibration array. In the Calibration
Arrays folder of the applicable genome build and design within the
experiment, the array appears with a special icon
.
To deselect an array from calibration
1 Expand the folders of the Experiment pane until you can see the
desired array. The program lists calibration arrays in the Calibration
Arrays folder(s) of the applicable genome build(s) and design(s) within
each experiment.
2 Right- click the name of the desired array, then click Deselect from
Calibration.
To deselect multiple calibration arrays at once, select all of the desired
arrays. Right- click one of the arrays, then click Deselect from
Calibration. (To select multiple arrays, click the name of one array,
then hold down the ctrl key and click the names of additional arrays. To
select a contiguous block of arrays, click the name of the first array,
then hold down the Shift key and click the name of the last one.)
The program removes the array(s) from calibration, and moves the
arrays to the Arrays folder of the applicable genome build and design
within the experiment. The icons of the arrays change to the standard
(non- calibration) array icon
.
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To show or hide array attributes in an experiment
2
To show or hide array attributes in an experiment
Sample attributes are pieces of information specific to an array, such as
chip barcode or hybridization temperature. You can show or hide
attributes for the arrays in the experiment with the Sample Attributes
dialog box. See “Sample Attributes” on page 235.
1 Right- click the experiment whose attributes you want to show or hide,
or to change.
You see the array attributes and their values that were set up in the
Sample Manager table. See the Sample Manager User Guide.
2 Click Edit Attributes.
3 Click Show/Hide Attributes.
The Show/Hide Columns dialog box appears. See “Show/Hide
Columns” on page 246.
NOTE
You cannot hide the required attributes. These include Array ID, Global Display Name,
Green Sample, Red Sample (for 2-color arrays), and Polarity.
4 Mark the check boxes for the attributes you want to show, or clear the
check boxes for the attributes you want to hide. These changes are
applied globally for the arrays.
5 Click Save.
6 In the Show/Hide Columns dialog box, click Close.
7 Click Close.
NOTE
You cannot create new attributes using this dialog box. To do this, you must use the Sample
Manager tab. See the Sample Manager User Guide.
To display or edit array attributes in an experiment
1 Right- click the experiment whose attributes you want to display or edit.
2 Click Edit Attributes.
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To display or edit the attribute values of a specific array
You see the array attributes and their values that were set up in the
Sample Manager table. See the Sample Manager Guide. See “Sample
Attributes” on page 235.
3 Double- click the cell whose array attribute value you want to change.
NOTE
You cannot change Array ID, Polarity, Extraction Status, or IsMultiPack attributes for
extracted or UDF arrays.
4 Click Save Changes.
5 Click Close.
To display or edit the attribute values of a specific array
Array attributes are pieces of information specific to an array, such as
chip barcode or hybridization temperature. In the Navigator, you can
display or change attributes for each array.
NOTE
Attributes for arrays that are read-only cannot be edited. Also, you cannot edit the required
attributes: Array ID, Global Display Name, Green Sample, Red Sample (for 2-color arrays),
and Polarity.
1 Expand the folders of the Design Data pane or the Experiment pane
until you can see the array of interest.
2 Right- click the name of the array, then click Show Properties.
The Microarray Properties dialog box appears, with a list of array
attributes. See “Microarray Properties” on page 229. You can also edit
the attributes of a specific array from this dialog box. In addition, if the
array is an Agilent array, you can see header and feature information
sent from the Agilent Feature Extraction program.
3 When you are finished, click Close.
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To display or edit the attribute values of a specific array
NOTE
2
You use the Sample Manager tab to organize, create, import, and export array attributes.
See the Sample Manager User Guide.
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Managing Content
Managing Content
This section describes how to create, find, rename, update, combine,
and/or remove content such as data, gene lists, and tracks, stored in
Agilent Genomic Workbench. To display the data, gene list and track
content, see Chapter 3, “Displaying CH3 Data and Other Content”.
To display a list of the content stored in the program
The Design Data and My Entity List panes of the Navigator show the
content stored in Agilent Genomic Workbench. For more information on
the Navigator and its contents, see “Navigator” on page 146.
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To display a list of the content stored in the program
Figure 16
2
Agilent Genomic Workbench Navigator
Design Data pane – Shows all of the design and array data files stored in
the database. For more information, see “To display the array designs and
data in the program” on page 57.
My Entity List pane – Shows the gene lists and tracks stored in the
program. To display the names of gene lists or tracks available in the
program, double- click the names of folders to expand or collapse them, or
click the
or
buttons.
NOTE
Content that is used in eArrayXD, such as the probes and probe groups that you use to
create custom microarray designs, is covered in the eArrayXD User Guide.
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To find specific content items in the Navigator
To find specific content items in the Navigator
At the top of the Navigator is a search pane that can help you find
specific content items. See “Search pane” on page 149.
1 Type a search term in the box at the top of the Navigator. The search
term is not case- sensitive, but it must reflect the entire name of the
content item that you want to find. You can use asterisks (*) as
wildcards to represent a group of unspecified characters. For example,
if you type *1234*, the search will find all items that contain “1234” in
the name.
2 By default, the program searches all panes of the Navigator. To limit
your search to a specific pane, click . In the list that appears, select
the desired pane.
3 Click
.
The program searches the selected pane(s). If it finds item(s) that
match your search term, it expands folders so that the items are visible,
and selects them in red. You may need to scroll down to see all the
search results.
4 To clear the results of a search, click
.
To display the properties of a specific design
Design properties include general information about a design, such as its
name, application type, and associated species. They also include a list of
the names and chromosomal locations of probes.
1 Expand the folders of the Design Data pane until you can see the
genome build folder(s) within the desired design folder.
2 Right- click the desired genome build folder, then click Show Properties.
The Design Properties dialog box appears. See “Design Properties” on
page 204.
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To download a design from eArray
2
To download a design from eArray
In order to analyze extracted microarray data in Agilent Genomic
Workbench, the design for the microarray must be present in the database.
The Design Data pane of the Navigator shows the available design content.
See “Design Data pane – icons, special text, and buttons” on page 151.
1 Expand the Agilent Catalog or workgroup folder until you see the
design you want to download.
2 Right click the design you want to download, and click Download from
eArray.com.
An information box appears that lets you know the download task is
started.
3 Click OK.
The task appears in the Tasks pane of the Navigator. The color of the
circle shows the status of the task. See “Tasks pane – Icons, buttons,
and special text” on page 167.
To update probe annotation in design files
Agilent regularly makes updates to probe annotations on its eArray Web
portal. If you have imported Agilent array designs into Agilent Genomic
Workbench, and you are a registered eArray user, you can download the
updated design files from within Agilent Genomic Workbench. For more
information about eArray, go to https://earray.chem.agilent.com and click
Help.
1 In the Home tab, click User Preferences.
The User Preferences dialog box appears.
2 In the Miscellaneous tab, under eArray User Details, type your eArray
Username and Password. See “User Preferences” on page 262.
3 Click OK.
4 Expand the folders of the Design Data pane until you can see the
design that you want to update.
5 Right- click the desired design, then click Download from eArray. This
option appears only for Agilent designs.
A confirmation dialog box appears.
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To rename an array in the Design Data pane
6 Click Yes.
The program downloads an updated design, if one is available.
To rename an array in the Design Data pane
This topic describes how to rename an array in the Design Data pane,
which changes the Global Display Name for the array. If you rename an
array in this way, and subsequently add the array to an experiment, the
array appears in the experiment with the new name. It also changes the
array name in any experiment to which it is already linked. To rename an
array only within the context of a specific experiment, see “To rename an
array in an experiment” on page 63.
NOTE
You can only rename an array that you imported.
1 Expand the folders of the Design Data pane until you can see the array
you want to rename.
2 Right- click the name of the array, then click Rename.
An Input dialog box appears.
3 Type a new name for the array, then click OK.
The program renames the array.
To remove data or design files from the program
You can delete array design and data files from the program when you are
finished with them.
1 If an array that you want to delete is associated with an experiment,
first delete it from the experiment. See “To remove arrays from an
experiment” on page 63.
2 In the Design Data pane, expand folders until you can see the design
folder or array that you want to delete.
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To remove data or design files from the program
NOTE
2
You cannot delete data or design files that are read-only.
3 Do one of the following:
• For array data files, click the name of the first array, then hold down
the ctrl key while you click the names of additional arrays within the
same design.
• For array design folders, click the name of the first design folder,
then hold down the ctrl key while you click the names of additional
ones. This selects the designs and all array data files within them for
deletion.
4 Right- click the name of a selected design folder or array data file, then
click Delete.
A confirmation dialog box appears.
5 Click Yes.
The program deletes the selected files.
CAUTION
When you delete files, you permanently remove them from Agilent Genomic
Workbench. To restore deleted files, you must import them again.
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To create a gene list
To create a gene list
When you create a gene list, you create a list of the genes in a contiguous
chromosomal region that you define. To create a list of genes in multiple
regions, create multiple gene lists, and combine them. See “To add one
gene list to another” on page 76.
1 Follow these steps to define a chromosomal region for your gene list. If
you know the exact start and end locations of the desired chromosomal
region, skip to step 2.
a In Genome View, select the desired chromosome.
The selected chromosome appears in Chromosome View. See
“Chromosome View” on page 171.
b In Chromosome View, in the plotting area to the right of the
chromosome, drag the pointer over the chromosomal region of
interest.
The program draws a blue box around the region, and displays the
region in greater detail in Gene View.
c In Gene View, adjust the view so only the genes of interest appear.
For a description of the adjustment commands available in Gene
View, see “Gene View” on page 173.
2 Right- click anywhere within the log ratio plotting area in Gene View,
then click Create Gene List.
The Create Gene List dialog box appears. See “Create Gene List” on
page 199.
3 In the dialog box set the Name, Description and Color.
4 In the dialog box select the chromosomal region for the new gene list.
5 Click OK.
The new gene list appears in the Gene List folder in the My Entity List
pane of the Navigator.
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To import a gene list
2
To import a gene list
A gene list file is a plain text (*.txt) file that contains one gene name per
line. When you import a gene list into Agilent Genomic Workbench, it
appears in the Gene List folder in the My Entities List pane. You can use
the gene list to highlight specific genes, or to show or hide the appearance
of genes and data, in Gene and Chromosome Views. See “To show gene
lists in Gene View” on page 98.
1 In the My Entities List pane, double click the Entities folder to expand
it.
2 Right- click the Gene List folder, then click Import Gene List.
An Import dialog box appears. See “Import” on page 222.
3 Select the desired gene list file. To select additional gene list files, hold
down the ctrl key and click their names.
Click OK.
To display the genes in a gene list
You can display the genes in a gene list as a table.
1 Expand the folders in the My Entity List pane until you can see the
desired gene list.
2 Right- click the gene list, then click View In Table.
The Gene List dialog box appears, with a table that contains the names
of the genes in the gene list. You can also use this dialog box to edit
the description of the gene list and its display color. See “Gene List” on
page 220.
You can also create gene lists. For more information, see “To create a
gene list” on page 74.
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To add one gene list to another
To add one gene list to another
You can add one gene list (a source gene list) to another (the target gene
list). The program appends the source gene list to the end of the target
gene list, and leaves the source gene list unchanged.
1 Expand the folders in the My Entity List pane until you can see the
gene lists that you want to combine.
2 Right- click the desired source gene list, then click Add to Gene List.
A dialog box appears. For more information, see “Add Gene List
<name> to” on page 183.
3 In Select target gene list, select the desired target gene list.
4 Click OK.
To rename a gene list
The name of a gene list identifies it within the Gene List folder of the My
Entity List pane. You can rename gene lists.
1 Expand the folders of the My Entity List pane until you can see the
gene list that you want to rename.
2 Right- click the desired gene list, then click Rename.
An Input dialog box appears.
3 Type a new name for the gene list, then click OK.
To delete gene list(s)
1 In the My Entity List pane of the Navigator, click to expand the Gene
List folder.
2 Click the name of a gene list to delete. Hold down the ctrl key while
you click the names of additional gene lists.
3 Right- click one of the selected gene lists, then click Delete.
A confirmation dialog box appears.
4 Click Yes.
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To display the details of a track
2
To display the details of a track
You can display a table that contains the values for a list of track
attributes.
1 In My Entity List pane, expand the Tracks folder to see the track.
2 Right- click the name of the track, then click View Details.
3 Data describing the track appear in a Track table. See “Track” on
page 254.
To combine tracks
You can create a track that contains elements from two or more existing
tracks. The existing tracks must be available in Agilent Genomic
Workbench, and they must be associated with the same genome build.
1 In the My Entities List pane, double- click the Entities folder to expand
it, if necessary.
2 Right- click the Tracks folder, then click Combine Tracks.
The Combine Tracks dialog box appears. See “Combine Tracks” on
page 191.
3 In Name, type a name for the combined track. The program uses this
name to identify the track in the Tracks folder, and to label the track if
it appears in Gene View.
4 Click New Condition.
A new row appears in the Track/Operator list.
5 Under Track, select the first track to combine.
6 Click New Condition, then select another Track/Operator pair. You can
set up as many Track/Operator pairs as you like, but you must set up
at least two. When you add a track, the program automatically assigns
the AND operator to the previous track.
To remove the bottom row from the list, click Delete Condition. To
delete all rows from the list, and erase any entry in Name, click Reset.
7 Under Operator, select one of the following:
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To rename a track
Operator
Comments
AND
Creates a combined track out of 2 tracks that will contain elements that appear
in both tracks
OR
Creates a combined track out of 2 tracks that will contain elements that appear
in either of the tracks
MINUS
Removes the elements of the second track from the first track.
8 Click Save.
Your combined track appears in the Tracks folder of the My Entities
List pane. The Combine Tracks dialog box remains open for you to
create another combined track.
9 Click Close.
To rename a track
The name of a track identifies it both within the Tracks folder of the My
Entity List pane, and in Gene View when you select Show In UI for the
track. You can rename tracks.
1 Expand the folders of the My Entity List pane until you can see the
track that you want to rename.
2 Right- click the desired track, then click Rename.
An Input dialog box appears.
3 Type a new name for the track, then click OK.
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To delete tracks
2
To delete tracks
1 In the My Entity List pane of the Navigator, expand the Tracks folder.
2 Click the name of a track to delete. Hold down the ctrl key while you
click the names of additional tracks.
3 Right- click one of the selected tracks, then click Delete.
A confirmation dialog box appears.
4 Click Yes.
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Exporting and Saving Content
Exporting and Saving Content
This section describes how to export several kinds of files from the
program.
To export array attributes
You can export selected array attributes for any imported arrays. You first
select the arrays and then the attributes for the selected arrays. You can
export array attributes from the Home tab or from the short- cut menu for
an experiment.
1 Click Home > Export > Array Attributes.
OR
In the Experiment pane of the Navigator, right- click an experiment of
interest, and click Export Attributes.
The Export Array Attributes dialog box appears with the Array tab
displayed. See “Export Array Attributes” on page 212.
If you opened this dialog box by right- clicking an experiment, only
those arrays selected for the experiment appear in the Selected Array
List. You can add or subtract from the list.
2 Under Select Design, select the design file and genome build for the
arrays to add.
The arrays for the selected design file and genome build appear in
Array List.
3 In Array List, select the arrays whose attributes you intend to export.
To select a single array, click its name. To select additional arrays, hold
down the ctrl key while you click their names.
4 Click
.
The program moves the selected arrays to the Selected Array List.
5 Click Next to select attributes for the selected arrays.
The Export Array Attributes dialog box appears with the Attribute tab
displayed. See “Attribute Tab” on page 214.
All of the attributes for the arrays are already located in the Selected
Attribute List.
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To export experiments
2
6 Move any attributes you don’t want to export to the Available List.
a In the Selected Attributes List, select those attributes you do not
intend to export.
b Click
.
7 Click OK.
The Export dialog box appears. See “Export” on page 211.
8 Select the folder in which to save the attributes, and click Export.
The attributes are saved to the selected folder as a .txt file.
To export experiments
You can export experiments as a ZIP file to transfer them to another
computer. Exported experiments contain the associated design and array
data files, only. The program does not export information about array
selections, or any analysis parameters or results.
1 In the Home tab, click Export > Experiments.
The Export Experiments dialog box appears. See “Export
Experiments” on page 216.
2 Mark the experiments that you want to export. To export all
experiments, click Select All.
3 Click OK.
An Export dialog box appears. See “Export” on page 211.
4 Select a location and type a name for the exported ZIP file.
5 Click Export.
The program exports all selected experiment(s) together as a single ZIP
file.
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To export a gene list
To export a gene list
You can export a gene list as a text file that contains one gene per line.
1 In the My Entity List pane, in the Gene List folder, right- click the gene
list that you want to export, then click Save As.
A Save As dialog box appears.
2 Select a location and type a name for the file.
3 Click Save.
A message appears when the operation is complete.
4 Click OK.
To export tracks
You can export selected tracks as a BED format track file. You can then
import this file into Agilent Genomic Workbench on another computer, or
into a genome browser that accepts BED format files.
1 In the Home tab, click Export > Tracks.
The Export Tracks dialog box appears. See “Export Tracks” on
page 217.
2 Mark the tracks to export. To select all tracks for export, click Select
All.
3 Click OK.
An Export dialog box appears.
4 Select a location and type a name for the exported track file, then click
Export.
The program exports the track(s) as a single BED format track file.
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To copy what you see in the main window
2
To copy what you see in the main window
You can copy panes of the main window to the clipboard as images, and
then paste them into a new document in another program (such as Word,
or PowerPoint). The images contain only what actually appears on your
screen — regions to which you must scroll are not included.
1 In the View tab, click Copy.
2 In the shortcut menu that appears, click the name of the pane that you
want to copy. You can copy any view, or the Navigator. To copy all of
the panes, click All.
The program copies the selected pane(s) to the clipboard.
NOTE
To adjust how data is displayed in the panes, use the View Preferences dialog box. See
“View Preferences” on page 268 for more information.
3 Open a document in a program that accepts images. In that program,
click Edit > Paste, or the appropriate paste command.
To copy the list of array colors for an experiment
You can copy the list of arrays in an experiment, and the colors assigned
to them, to the clipboard as an image. You then paste the image into a
document in another program such as Word or PowerPoint.
1 In the Experiment pane, expand the Experiments folder.
2 Right- click the name of the desired experiment, then click Edit Array
Color.
The Edit Array Color dialog box appears. See “Edit Array Color” on
page 207.
3 In the dialog box, click Edit > Copy.
The program copies the names of the arrays and their colors to the
clipboard as an image.
4 Open a program that accepts images. Click Edit > Paste, or the
appropriate paste command for the specific program.
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To save data and design information from an experiment
To save data and design information from an experiment
You can save the data and design information from a single design in an
experiment as a tab- delimited text file.
1 In the Experiment pane, expand the Experiments folder until you see
the genome build(s) for the design you want to export.
2 Right- click the name of the desired genome build, then click Save As
Text File.
A dialog box appears.
3 Select a location and type a name for the file, then click Save.
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Agilent Genomic Workbench 6.5 – Methylation (CH3) Analysis
User Guide
3
Displaying CH3 Data and Other Content
Selecting an Experiment 86
Displaying Array Data 91
Displaying Content (Gene Lists/Tracks) 98
Searching for Probe and Gene Information 106
This chapter shows you how to display log ratio data from imported
feature extraction data files, as well as gene and track content, in the
Genomic Viewer. It also has instructions on how to customize the display
of data and content to meet your needs.
To learn about the options for the main window and the dialog boxes for
displaying data, see Chapter 5, “Methylation (CH3) Analysis Reference”.
Agilent Technologies
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Selecting an Experiment
Selecting an Experiment
An experiment is a set of links to microarray data and design files, and
any associated results. You can see a list of the experiments in the
Experiments pane of the Navigator. See “Navigator” on page 146 for more
information.
When you select an experiment and the Preprocessing and Analysis
options have not been turned on or set to apply, the program shows the
log ratio data of selected arrays in the active experiment, if certain
options have been set. See “To locate and display data (or results) within
the Views” on page 95 for more information.
When you select an experiment and Preprocessing and Analysis options
have been turned on or set to apply, the program automatically begins the
analysis of the selected array data with current settings and displays its
results.
This section describes how to select an experiment, select or deselect
arrays for further analysis, and analyze arrays one at a time.
To select an experiment
When you select an experiment, the program begins the analysis with the
current settings. You can either set the Preprocessing and Analysis
parameters before you select the experiment or change the settings one at
a time after the first analysis and reanalyze. Every time you change a
Preprocessing or Analysis setting for an active experiment, the program
recalculates results.
1 If necessary, do one of the following to add the desired experiment to
the Experiment Pane in the Navigator:
• Create a new experiment and add data to it. See “To create a new
experiment” on page 58.
• Import a saved experiment file. See “To import an experiment
file” on page 55.
2 In the Navigator, double- click the name of the experiment.
The Experiment Selection dialog box appears.
3 Click Yes.
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To select or deselect arrays in the experiment
3
In the Experiment pane of the Navigator, the name of the experiment
turns blue. The name also appears in the title bar of the main window.
Tables of data and design information appear in Tab View.
If you have selected to show the results of an algorithm calculation, then
results appear for the first array when you select the experiment, if you
have not selected any other arrays.
You can select or deselect arrays in the experiment before or after you
select the experiment. Every time you select or deselect an array in a
selected experiment or change a setting, the program reanalyzes the new
data set with the changed settings. See “To select or deselect arrays in the
experiment” on page 87.
When you select the experiment after deselecting it or selecting another
one, the experiment is simply restored if the settings in the UI haven’t
changed. If they have changed, the program reanalyzes all of the arrays
assigned when the experiment was last selected.
To select or deselect arrays in the experiment
To include arrays for display and analysis, you select them from the
arrays available, either in an inactive experiment or the selected one.
When you first create an experiment, the program automatically sets the
first array in the experiment for analysis. If you do not select additional
arrays for analysis, only the first one is analyzed when the experiment is
selected.
To select the arrays for analysis before experiment selection:
1 Hold down the Shift key to highlight contiguous arrays or hold down the
ctrl key to highlight noncontiguous arrays.
2 Right- click the highlighted arrays, and click Select.
Even though the selected arrays do not change color, they will change
color after activation.
In the Navigator, the color or an array’s icon has the following meaning
after experiment selection:
Array is not selected.
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To select or deselect arrays in the experiment
Array is selected. The specific color matches the color of the column
headings for the array in Tab View. In addition, the program displays
aberration results and moving averages related to this array in this color.
To configure a custom color for the array, see “To change the display color
of an array” on page 89.
To select or deselect arrays in a selected experiment:
1 In the Navigator, expand the folders of the selected experiment.
2 Click the name of an array you want to include in the display.
To include additional arrays, hold down the ctrl key while you click
their names. To include a contiguous block of arrays, click the name of
the first array in the block, then hold down the Shift key while you click
the name of the last one.
3 Right- click the name of one of the highlighted arrays, then click Select.
4 After you select the arrays, if you have Analysis options selected
(Methylation or Batman, for example), clear the options and then mark
them again. The program re- analyzes the data set within the
experiment and displays the data in Genome, Chromosome, and Gene
Views. You can see the data and results for just the selected arrays in
the Selected Arrays tab in Tab View.
To show analysis results if they do not appear, see “To display results
of analysis” on page 121.
To customize the appearance of the results in Genome, Chromosome,
and Gene Views, see “To change scatter plot appearance” on page 93.
You can also use the headings of columns in Tab View that contain array
data to select and deselect arrays.
• Click a column heading to select that array only.
• Hold down the ctrl key while you click a column heading to select or
deselect an array without changing the status of other arrays.
• Right- click a column heading to open a shortcut menu with options
that let you select or deselect that array, or all arrays.
For more information on Tab View, see “Tab View” on page 178.
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To change the display color of an array
3
To change the display color of an array
The color assigned to an array sets the color of its icon when you select
the array within an experiment. It also changes the colored square in the
array’s column heading in Tab View.
1 In the Experiment pane, in the Experiments folder, expand the folder of
an experiment until you can see the array whose color you want to
edit.
2 Right- click the desired array, then click Edit Array Color.
The Select Color dialog box appears. The dialog box offers three
different ways to choose the desired color. See “Select Color” on
page 237.
3 Select the desired color in one of the following ways:
Dialog box tab
Instructions
Swatches
• Click the desired color swatch.
HSB
(Hue/Saturation/Brightness)
Type or adjust the values in H (Hue), S (Saturation), and B
(Brightness), or alternately, follow these steps:
a Select H, then drag the slider to select a hue based on the
color strip to its right.
b Click an appropriate location in the large color box to the
left of the slider to set the saturation and brightness levels
of the color.
Both the HSB and equivalent RGB values of the color
appear in the dialog box. Note these values; they will be
useful if you need to duplicate this color in the future.
RGB (Red/Green/Blue)
Do any of the following. Note the final RGB Values; they will be
useful if you need to duplicate this color in the future.
• Drag the Red, Green, and Blue sliders.
• Type or adjust values in the boxes to the right of the sliders.
Samples of the color in different contexts appear under Preview. The
upper half of the color sample on the right shows the original color for
comparison.
4 Adjust the color as desired, then click OK.
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To share an experiment
You can also manage all of the colors for all of the arrays in an
experiment. Right- click the desired experiment, then click Edit Array
Color. For more information, see “Edit Array Color” on page 207.
To share an experiment
You can select enterprise users to share the experiment. These users can
change or delete the experiment.
1 In the Experiment pane of the Navigator, right- click on an experiment
for which you are the owner.
2 Select Share.
3 In the Sharing for enterprise dialog box, click Add. See “Sharing for
enterprise” on page 433.
The Select users dialog box opens. See “Select Users” on page 427.
4 Select the user(s) to share the experiment.
5 Click OK to add the users and close the Select Users dialog box.
6 Click OK to close the Sharing for enterprise dialog box.
To remove a user from a shared experiment
1 In the Experiment pane of the Navigator, right- click on an experiment
for which you are the owner.
2 Select Share.
3 In the Sharing for enterprise dialog box, select the user(s) to remove
from the experiment. See “Sharing for enterprise” on page 433.
4 Click Remove.
5 Click OK to close the Sharing for enterprise dialog box.
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Displaying Array Data
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Displaying Array Data
After you select an experiment, you can change how data appear within
the Views or change the appearance of the Views that contain the data (or
results).
To display the scatter plots
By default, display of scatter plots is turned On. If you do not see the
scatter plot(s), do one of the following:
1 From the View tab, click View Preferences. See “View Preferences” on
page 268 for more information.
2 In the View Preferences dialog box, under Data Visibility, select All
views and then mark the box next to Scatter Plot.
OR
1 Right- click in any of the views, and select View Preferences. See “View
Preferences” on page 268 for more information.
2 In the View Preferences dialog box, under Data Visibility, select All
views and then mark the box next to Scatter Plot.
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To show or hide data in the scatter plots
To show or hide data in the scatter plots
1 In the Gene View, move the mouse cursor over the arrow next to
Scatter Plot, and do any of the following:
To do this
Follow these steps
Show or hide the Methylation
Results plot and color the results
by Z-Score values
• To show the data points – Mark the Methylation Results
check box and select Z-Score Values from the list.
• To hide all data points – Clear the Methylation Results
check box.
Show or hide Log Ratio values in
the Log Ratios plot
• To show the data points – Mark the Log Ratios check box
and select Log Ratio Values from the list.
• To hide all data points – Clear the Log Ratios check box.
Show or hide log ratios and
color-code them by Probe Score
values in the Log Ratios plot
• To show the data points – Mark the Log Ratios check box
and select Probe Score Values from the list.
• To hide the data points, Clear the Log Ratios check box.
Change the ranges and colors for
all scatter plots
• Click Configure Color and Ranges to enter ranges and
change colors. See “Configure Coloring Ranges and
Shades” on page 193 for more information.
2 Click X to close the Scatter Plot window.
To customize scatter plot ranges and colors
In order to make it easier to see significant results, you can customize the
display of scatter plot data. For each data type (Z- Score, Log Ratio, probe
score) you can set custom ranges and colors for the display. For channels,
you can set custom colors only.
Add and customize a range
1 In Gene View, move the mouse pointer over Scatter Plot to display the
options.
2 Mark the check box under Methylation Results or Log Ratios.
3 Select a data type from the Color by list.
4 Click Configure Color and Ranges.
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To change scatter plot appearance
3
The Configure Coloring Ranges and Shades dialog box appears where
you set ranges and colors for any of the data types. For more
information, see “Configure Coloring Ranges and Shades” on page 193.
5 In the Configure Coloring ranges and Shades dialog box, click the
Methylation Results or Log Ratios tab and then select the data type to
configure.
6 Enter minimum and maximum numbers to define a range for the data
type.
7 Click Color to open the Select Color dialog box. Use the tabs to select
a color for the range. See “Select Color” on page 237 for more
information.
8 Click OK to close the Select Color dialog box and return to the
Configure Coloring ranges and Shades dialog box.
9 Click Add Range to add the custom range to the range list.
10 When you are done, click OK to close the dialog box.
Edit or remove a range
1 In the Configure Coloring ranges and Shades dialog box, click the
Methylation Results or Log Ratios tab and then select the data type to
configure.
2 In the range list, mark the Edit/Delete box to select the range. You can
mark more than one range.
3 Click Edit Range to change the minimum and maximum values, or to
change the color for the selected range.
4 Click Delete Range to delete the selected range.
5 Click OK to close the dialog box.
To change scatter plot appearance
You use the View Preferences dialog box to change the appearance of the
scatter plots in Chromosome and Gene views.
1 Right- click any part of a scatter plot, then click View Preferences.
Or, click the View tab, and then click View Preferences.
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To change scatter plot appearance
The View Preferences dialog box appears. See “View Preferences” on
page 268.
2 Do any of the following:
To do this
Follow these steps
Show or hide the scatter plot
a In the View tab under Data Visibility, in View, select All
Views.
b Do one of the following:
To show the scatter plot, mark Scatter Plot.
To hide the scatter plot, clear Scatter Plot.
c Click OK.
Change the symbol that
appears for data points
You can select the symbol separately for each design type.
a In the View tab, under Rendering Patterns, select the
desired Design type.
b Under Styles, for each data type, select the desired symbol.
c Click Apply.
Show a separate scatter plot
in Gene and Chromosome
Views for each selected array
a In the View tab, under View Alignment, under Rendering
Style, select Stacked.
b Click Apply.
Show one scatter plot that
contains data for selected
arrays
a In the View tab, under View Alignment, under Rendering
Style, select Overlaid.
b Click Apply.
Enable ToolTips for the
scatter plot in Gene View
ToolTips show information about an individual data point when
you place the pointer over it.
a Click the View tab.
b Under Data Visibility, in View, select Gene View.
c Mark Scatter Tool Tip.
d Click Apply.
3 Click OK.
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To print the scatter plot
3
To print the scatter plot
You can print the scatter plot as it appears in Genome, Chromosome, and
Gene views. Each view selected in the analysis is printed on separate
pages. Chromosomes and genes appear on the printed pages, but tracks do
not.
1 In the Home tab, click Print.
The Print dialog box appears.
2 Set print options, then click OK.
To create custom scales for Views
You can customize the scale used for display in the Chromosome View and
Gene View. Custom scales are applied to both views.
1 Click the View tab and then click View Preferences.
2 In the View Preferences dialog box, under Configure Scales, mark the
box next to Apply for Methylation Results and/or Log Ratios.
3 In Range, type a value to use for the range. The range you type changes
the scale for the display of the selected data.
To locate and display data (or results) within the Views
To look through the data of the selected arrays, do any of the following. In
general, all views are synchronized; if you select a location or region in
one view, the other views move there as well.
To do this
Follow these steps
Select a specific chromosome
to display
• In Genome View, click the chromosome.
All other views switch to the selected chromosome.
Display data in a region of the
selected chromosome
• In Chromosome View, drag the pointer over the region.
Gene View expands (or shrinks) to show only the selected
region. Tab View scrolls to the new cursor location.
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To locate and display data (or results) within the Views
To do this
Follow these steps
Zoom in and out in Gene View • Click
to zoom in.
• Click
to zoom out.
Scroll through the selected
chromosome
• Click
to scroll up.
Re-center Gene View or
Chromosome view
Click anywhere in Chromosome View, or anywhere within the
scatter plot in Gene View.
The location you click becomes the new cursor location.
Move all views to a specific
genomic location
a Click Home > Go To Gene/Genomic location.
A dialog box appears.
b Under Genomic Location, select a Chromosome, and type a
Base Position.
c Click Go.
All views move to the selected location.
Display the location of a
specific gene in the center of
all Views
a Click Home > Go To Gene/Genomic location.
A dialog box appears.
b Under RefSeq by Symbol, either select the gene (if
available) or type the name of the gene.
c Click Go.
All views move to the location of the selected gene.
• Click
to scroll down.
Note: These arrows will appear side by side for horizontal
orientation.
• In Tab View, click any entry in any table, except a column
Display the data selected in
heading.
Tab View in the center of
Chromosome and Gene Views
Chromosome and Gene views: The genetic location of the
selected data appears in the center of Chromosome and
Gene Views.
Scroll to a specific column in
Tab View (for the selected
chromosome)
96
a In Tab View, right-click any column heading, then click
Scroll To Column.
The Scroll to Column dialog box appears. See “Scroll to
Column” on page 236.
b In Select Column, select the column.
c Click OK.
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To locate and display data (or results) within the Views
To do this
Follow these steps
Search for a specific column
entry in Tab View, and move
the cursor there
a In Tab View, right-click any entry except a column heading,
then click Find in column.
The Find in column dialog box appears. See “Find in
column” on page 218.
b Set the search parameters, then click Find Next.
The program searches the column using your search
parameters, and highlights the row of the first entry that
matches. The cursor moves to the location defined in the
highlighted row.
Display the exact
chromosomal location of the
cursor
At the bottom of the main window, look at the first cell of the
Status bar. The location appears as the chromosome followed
by the base position. For more information on the status bar,
see “Status Bar” on page 182.
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Displaying Content (Gene Lists/Tracks)
Displaying Content (Gene Lists/Tracks)
To show gene lists in Gene View
A gene list is a set of genes of interest. Within the program, you can
highlight the genes in the gene list in Gene View, or limit the display of
data, genes, and tracks to the regions selected by a gene list.
You can import gene lists into Agilent Genomic Workbench, and you can
also create them in the program and export them. See “To import a gene
list” on page 75, and “To export a gene list” on page 82.
In Gene View, the names of all genes normally appear in gray. When you
apply a gene list, the program highlights the listed genes in their defined
display color. You can also limit the genes and/or data that appear in
Gene View and Chromosome View to only the listed genes.
1 In the My Entity List pane of the Navigator, expand the Gene List
folder. If the gene list does not appear, create or import it. See “To
create a gene list” on page 74, or “To import a gene list” on page 75.
2 Right- click the gene list, then do one of the following to apply it:
• To show all genes and all data, and highlight the listed genes in their
display color, click Highlight.
• To show only the listed genes and only the data for those genes, click
Show only.
Gene and Chromosome views change accordingly. In the My Entity List
pane of the Navigator, the name of the gene list appears in italics.
To remove the effects of a gene list, right- click the active gene list in
the Navigator, then click Show All.
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To select gene list display color
3
To select gene list display color
In Gene View, the names of all genes normally appear in gray. When you
apply a gene list, the program highlights the listed genes in their defined
display color. You can customize this color.
1 In the My Entity List pane of the Navigator, expand the Gene List
folder.
2 Right- click the name of the gene list whose color you want to change,
then click View in Table.
The Gene List dialog box appears.
3 Under Color, click Color.
A dialog box appears.
4 Select the desired color.
The dialog box offers three different ways to select the color. See “To
select gene list display color” on page 99.
5 Adjust the color as desired, then click OK.
6 In the Gene List dialog box, click OK.
To display a gene list as a table
You can display the description of a gene list and the names of the genes
in it.
1 In the My Entity List pane of the Navigator, in the Gene List folder,
right- click the desired gene list, then click View in Table.
The Gene List dialog box appears. See “Gene List” on page 220. The
names of the genes appear in Gene Names. You can also use this dialog
box to edit the description of the gene list, or to change its display
color. To change the display color, see “To select gene list display
color” on page 99.
2 When you are finished displaying the list, click OK.
You can also export a gene list. See “To export a gene list” on page 82.
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To change the appearance of genes in Gene View
To change the appearance of genes in Gene View
You use the User Preferences dialog box to change the appearance of the
genes in Chromosome and Gene views.
1 Right- click any part of the Gene View, then click User Preferences.
The User Preferences dialog box appears.
2 Click Tracks.
See “Tracks tab” on page 262.
3 Do one or more of the following:
To do this
Follow these steps
Show or hide genes in Gene
View
a Under Visualization Parameters:
To show genes – Under Genes, mark Show Gene Symbols.
To hide genes – Under Genes, clear Show Gene Symbols.
b Click Apply.
Change the display font for
genes (and track annotations)
in Gene View
a In the Gene Symbols tab, under Font, select a new Font,
Font Style, and Font Size.
b Click Apply
Change the display angle for
genes (and track annotations)
in Gene View
a Under Visualization Parameters, under Genes, in
Orientation (Degrees), type a new orientation in degrees.
0º is horizontal.
b Click Apply.
4 Click OK.
To show tracks in Gene View
Tracks contain information for specific genomic locations. A multitude of
tracks from diverse sources is available for many species. You can display
tracks next to genes and microarray data in Gene View.
1 Select and show microarray data. See “To select an experiment” on
page 86.
2 In the My Entity List pane, open the Tracks folder.
3 Right- click the track you want to display, and click Show In UI.
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To change the appearance of tracks
3
Or, you can do this:
1 In Gene View, right- click anywhere within the scatter plot, then click
User Preferences.
The User Preferences dialog box appears. See “User Preferences” on
page 262.
2 Click Tracks.
3 Mark the Show In UI check box of each track.
4 Click OK.
The program displays the selected tracks in Gene View.
To change the appearance of tracks
In the Tracks tab of the User Preferences dialog box, you can change the
appearance of tracks as described in the table below.
To do this
Follow these steps
Include track information in
reports
a In the list of tracks, in the Show in Report column, mark the
check boxes of the desired tracks.
b Click Apply.
Doing this adds a column with the hits from the track file. For
each aberrant interval, it reports the entries from the track file
for that interval in that separate column.
Show or hide annotations in
all tracks
• To show annotations in all tracks: under Tracks, mark Show
Annotations.
• To hide annotations in all tracks: under Tracks, clear Show
Annotations.
Display all selected tracks as
a single track
• Under Tracks, mark Show Overlaid.
The program combines the annotations of all selected
tracks into a single track named Overlaid Track.
• To show tracks individually again, clear Show Overlaid.
Display the parameters and
the list of annotations of a
track
• In the list of tracks, for the desired track, click Details.
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To show track information in reports
To do this
Follow these steps
Change the display font for
a Under Font, select a new Font, Font Style, and Font Size for
track annotations (and genes)
track annotations.
b Click Apply.
The program changes the display font of track annotations
and genes in Gene View.
Change the order in which
tracks appear in Gene View.
The order of tracks in the Gene Symbols tab controls the
left-to-right order of tracks in Gene View.
a Click the name of the track you want to move.
b Do any of the following:
• To move the track up in the list of tracks (and farther left
in Gene View), click its name, then click Up.
• To move the track down in the list of tracks (and farther
right in Gene View), click its name, then click Down.
c Click Apply.
Change the display angle of
• Under Genes, in Orientation, type a new orientation (in
track annotations (and genes)
degrees). 0º is horizontal.
The program changes the display angle of track annotations
and genes in Gene View.
To show track information in reports
1 In the list of tracks, in the Show in Report column, mark the check
boxes of the desired tracks.
2 Click Apply.
Doing this adds a column with the hits from the track file. For each
aberrant interval, it reports the entries from the track file for that interval
in that separate column.
NOTE
102
The annotations that are displayed in tab view are taken from the design file. This may
include names of genes and other annotations. The positions of these genes may also be
supplied as a separate Gene track and may contain unnecessary information.
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To limit data to the genomic boundaries of the track
3
To limit data to the genomic boundaries of the track
1 In the list of tracks in My Entity List, right- click the track whose
boundaries you want to use to limit the display of the data.
2 Mark Genomic Boundaries.
You can remove the boundaries by clearing the check box.
To display a track in UCSC Browser
1 In the My Entity List pane, expand the Tracks folder and find the track
you want to view in the UCSC browser.
2 Right- click the track name, and click Show in UCSC.
The UCSC Browser appears, if you are connected to the Internet
(Figure 17). You may have to enable pop ups or set your other browser
preferences on the UCSC browser Web site.
3 Follow the instructions on the web site for what you want to do.
To upload a track to UCSC Browser
1 Right- click Gene View, and click Upload Track to UCSC.
2 The Upload Track to UCSC dialog box appears.
Complete the dialog to define the track you wish to upload. See
“Upload Track to UCSC” on page 260.
The UCSC Browser appears, if you are connected to the Internet
(Figure 17). You may have to enable pop ups or set your other browser
preferences on the UCSC browser Web site.
3 Follow the instructions on the web site for what you want to do.
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To upload a track to UCSC Browser
Figure 17
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Track displayed in UCSC browser
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To change the graphical display to a different genome build
3
To change the graphical display to a different genome build
The default graphical display for Genome, Chromosome and Gene Views
represents human genome build 18.
• To change the graphical display to a different genome build, select an
experiment whose data are based on a design file of a different genome
build.
The display automatically changes when you select an experiment that
contains a design file with a different genome build, such as human
genome build 17, or a mouse or rat genome build.
If a genome build is not available for the design file you import, you must
import the genome build first. See “To import a genome build” on page 52.
The program will not let you add arrays that belong to one genome build
to an experiment that contains arrays of a different genome build.
See also “To create a new experiment” on page 58, “To add arrays to an
experiment” on page 60, and “To select an experiment” on page 86.
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Searching for Probe and Gene Information
Searching for Probe and Gene Information
To search Tab View for specific probe information
You can find a specific entry in a column of a data table in Tab View. For
more information on Tab View, see “Tab View” on page 178.
1 In Tab View, right- click anywhere in the column you want to search,
then click Find in column.
The Find in column dialog box appears. The search column also
appears in the title bar of the dialog box.
NOTE
The Find in column function works within the selected chromosome.
2 Set the search parameters, as described below.
Parameter
Comments/Instructions
Find in column
• Type the text you want to find (the search term). This can be an entire
entry, or part of one.
Direction
• Select one of these options:
• Up – Search the column upwards from the current cursor location
(the highlighted row of the table).
• Down – Search the column downwards from the current cursor
location (the highlighted row of the table).
Tip: Click a row in Tab View to highlight it.
Conditions
• Mark any of these, as desired:
• Match Case – Find entries that match upper and lower case
characters in the search term.
• Match whole word – Find an entry only if the entire entry matches
the search term.
3 Click Find Next.
If the program finds a match, it highlights the row that contains the
matching entry, and resets the cursor to the corresponding position.
You can click Find Next as many times as you want, and the program
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To search an experiment using a query
3
continues to search for additional matching entries in the column. If it
finds no match, the message: String not found appears in black in the
lower part of the dialog box.
4 When you complete your search, click Cancel to close the dialog box.
To search an experiment using a query
You can create a query to search for samples in an experiment, based on
conditions you select. Query results are returned based on aberration
result, sample attribute, or annotation. Examples of they type of
information that can be found using a query include:
• Return all female (samples) with de novo deletions on
Chromosome 15
• Return any records that have aberrations in a specified region
• Return the FE output file name for the AMADID 039902
1 From the Experiment pane, right- click the experiment folder, and select
Create Query.
The Select experiment for creating query dialog box opens. See “Select
Experiment for Creating Query” on page 426.
2 Mark the experiment(s) for which you want to create the query, then
click OK.
The Create Query dialog box appears. See “Create Query” on page 301.
3 Select a query to use, or create a new query with conditions you want.
See “New Condition” on page 398. Click OK to save the condition and
return to the Create Query dialog box.
4 Create and add the conditions you want to the query.
5 Click Execute to start the query. Results of the query are displayed in
the Results box.
To search for probes in eArray
A High Density (HD) Search from an Agilent HD probe database, retrieves
probes that cover specific regions of the genome of a given species. The
eArray Web site has separate databases that contain HD probes for CGH,
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To search for probes in a chromosomal location
ChIP- on- chip, and methylation microarrays. In a Simple HD Search, you
set the overall density of retrieved probes. You can find more information
about probes in the eArrayXD User Guide.
1 Right- click in the data plot area of Gene View, then click Simple HD
Search. See “Simple HD Probe Search” on page 436.
The eArrayXD tab opens with the Simple HD Probe Search pane. The
Genomic Intervals and Species search criteria reflect the selected region
in the Genomic Viewer.
2 To return to the Gene View, click the Genomic Viewer tab.
To search for probes in a chromosomal location
Use this to find probes in a chromosomal region that you specify.
1 In Gene View, click on a chromosomal location, or hold down the mouse
button and drag a chromosomal region of interest.
2 Right- click in Gene View, and click Chromosomal Location Search.
The Probe Search pane opens, with the selected location and species
pre- set.
3 Complete the dialog with search parameters, and click Search. See the
eArrayXD User Guide for information on how to set the search
parameters.
The results of the search appear in the bottom of the pane.
To search an experiment using a query
You can create a query to search for samples in an experiment, based on
conditions you select. Query results are returned based on attribute
conditions in the query. For example, a query can return the FE output
file name for the AMADID 039902.
1 From the Experiment pane, right- click the experiment folder, and select
Create Query.
The Select experiment for creating query dialog box opens. See “Select
Experiment for Creating Query” on page 241.
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To search for probes in eArray
3
2 Mark the experiment(s) for which you want to create the query, then
click OK.
The Create Query dialog box appears. See “Create Query” on page 201.
3 Select a query to use, or create a new query with conditions you want.
See “New Condition” on page 232. Click OK to save the condition and
return to the Create Query dialog box.
4 Create and add the conditions you want to the query.
5 Click Execute to start the query.
Results of the query are displayed in the Results box.
To search for probes in eArray
A High Density (HD) Search from an Agilent HD probe database, retrieves
probes that cover specific regions of the genome of a given species. The
eArray Web site has separate databases that contain HD probes for CGH,
ChIP- on- chip, and methylation microarrays. In a Simple HD Search, you
set the overall density of retrieved probes. You can find more information
about probes in the eArrayXD User Guide.
1 Right- click in the data plot area of Gene View, then click Simple HD
Search. See “Simple HD Probe Search” on page 247.
The eArrayXD tab opens with the Simple HD Probe Search pane. The
Genomic Intervals and Species search criteria reflect the selected region
in the Genomic Viewer.
2 To return to the Gene View, click the Genomic Viewer tab.
To search for probes in a chromosomal location
Use this to find probes in a chromosomal region that you specify.
1 In Gene View, click on a chromosomal location, or hold down the mouse
button and drag a chromosomal region of interest.
2 Right- click in Gene View, and click Chromosomal Location Search.
The Probe Search pane opens, with the selected location and species
pre- set.
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To search the Web for information on probes in Tab View
3 Complete the dialog with search parameters, and click Search. See the
eArrayXD User Guide for information on how to set the search
parameters.
The results of the search appear in the bottom of the pane.
To search the Web for information on probes in Tab View
You can use any entry in a table in Tab View as the basis for a Web
search.
1 In Tab View, right- click any data table entry other than a column
heading.
2 Click one of the available sites.
If the site of interest does not appear in the shortcut menu, you can
create a custom search link. See “To create a custom Web search link”
below.
The selected site opens in your Internet browser. The program sends
the table entry to the site as a search string.
To create a custom Web search link
If you need to search a different database or site based on data table
entries, you can create your own custom search link. When you right- click
a table entry in Tab View, a shortcut menu opens, and your custom link
appears in it. If you select this link, Agilent Genomic Workbench opens the
site in your Web browser and sends the table entry to the site as a search
string.
1 Right- click any data table entry in Tab View, except a column heading,
then click Customize Link.
The Customize Search link dialog box appears. See “Customize Search
Link” on page 203.
2 Click New.
3 In the Input dialog box, in URL name, type a name for the link.
This name will appear in the shortcut menu that opens when you
right- click a data table entry.
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To update or delete a custom Web search link
3
4 Click OK.
5 In URL, type the complete URL needed to send a search string to the
site. Use <target> as the query string value.
For example, this URL sends selected table entries to Google.com:
http://www.google.com/search?hl=eng&q=<target>
6 Click Update, then click Yes.
To update or delete a custom Web search link
1 Right- click any data table entry in Tab View other than a column
heading, then click Customize Link.
The Customize Search link dialog box appears.
2 In URL Name, select the custom search link to update or delete.
3 Do one of the following:
To do this
Follow these steps
Update a Web search link
a Edit the URL name and the URL as needed.
b Click Update.
A Confirm dialog box appears.
c Click Yes.
Delete a Web search link
• Click Delete.
4 Click Close.
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Agilent Genomic Workbench 6.5 – Methylation (CH3) Analysis
User Guide
4
Setting Up Methylation (CH3) Analysis
Working with Methylation Options 114
Changing Preprocessing and Analysis options 115
Displaying results and generating reports 121
This chapter gives instructions on how to set up the analysis functions for
Methylation (CH3) Analysis experiments. These include the Preprocessing,
Analysis and Reports tabs.
Agilent Technologies
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Working with Methylation Options
Working with Methylation Options
For a detailed description of the Methylation (CH3) analysis tabs and their
commands – Preprocessing, Analysis, Reports – see Chapter 5,
“Methylation (CH3) Analysis Reference”.
Figure 18
Tabs available in the CH3 application
Table 1 describes the sections in this chapter and the tasks you do during
interactive analysis.
Table 2
Methylation analysis topics
Subject
See these topics
Changing Preprocessing
and Analysis options
“To combine (fuse) arrays” on page 115
“To set up a moving average (Log Ratio) calculation to smooth the
data” on page 117
“To set up a moving average (ZScore) calculation to smooth the
data” on page 118
“To apply methylation (CH3) event detection” on page 119
“To apply BATMAN (Bayesian Tool for Methylation Analysis)” on
page 120
Displaying results and
generating reports
“To display results of analysis” on page 121
“To upload a track to UCSC” on page 123
“To save a result” on page 124
“To restore a saved result to the display” on page 125
“To generate a probe report” on page 125
“To generate a Batman report” on page 128
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Changing Preprocessing and Analysis options
4
Changing Preprocessing and Analysis options
To combine (fuse) arrays
If you have two arrays that use different design files, you can combine
(fuse) them into one larger virtual array. You do this to increase the
coverage of the genome in your design. If for example, you have a catalog
array, you can design another array to add probes between the catalog
probes to increase the density of coverage. With the Fuse function, you
can combine the array data to see all the probe data in the display at
once. The program cannot combine arrays from more than two different
design files.
Requirements for fusing arrays:
• Each array has a different design file.
• All of the arrays are of the application type. (For this application,
CH3.)
• None of the arrays are fused arrays.
• The samples you hybridize to the arrays are all aliquots from the
same preparation.
• (Preferred) Hybridization and labeling occur for all samples together
under the same conditions.
1 Create and select a new experiment. See “To create a new
experiment” on page 58 and “To select an experiment” on page 86.
2 Add the arrays to be combined to the experiment. See “To add arrays
to an experiment” on page 60.
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To combine (fuse) arrays
3 Assign the same value to the ArraySet attribute of every array you
want to combine. Follow these steps for each array:
a In the Experiment pane of the Navigator, right- click the name of the
array, then click Show Properties.
The Microarray Properties dialog box appears.
b Next to the ArraySet attribute, under Value, click
A text box appears.
.
c Type a value in the text box. Type the same value for the ArraySet
attribute of every array.
d Click Close.
4 In the Preprocessing tab, under Combine, click Fuse.
The Array Set dialog box lists the arrays to be combined. See “Array
Set” on page 186.
NOTE
Double-check the values in the ArraySet Attribute column of the dialog box. Agilent
Genomic Workbench combines all of the array pairs that have the same value for this
attribute.
5 Set any of these options, as desired:
• Select Normalization – Select None or Centralization. Centralization
adds or subtracts a constant value from each log ratio measurement.
This recenters the log ratio values, and makes sure that the
zero- point reflects the most common ploidy state.
• Remove arrays from experiment after fuse – To delete the initial
individual arrays from the experiment, mark this option so that the
experiment will not contain duplicate data.
6 Click Continue.
The program combines the arrays. The new array appears in the
Experiment pane of the Navigator in a new design folder within the
active experiment. The folder name contains the names of both designs.
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To set up a moving average (Log Ratio) calculation to smooth the data
NOTE
4
• If the original arrays change, you can manually update the combined array. Fuse the
same set of arrays again.
• If you want to combine many arrays, consider importing an array attributes file. See “To
import array attributes” on page 54.
• When you combine (fuse) arrays, the centralization algorithm (if selected) is applied to
the individual arrays before they are combined.
To set up a moving average (Log Ratio) calculation to smooth the
data
With the Moving Average tool, a moving average is computed for each
point in the data set using a sliding window of fixed size, centered on that
point. This algorithm uses the pre- algorithm probe log ratios to calculate a
moving average line plot. See “Visualization algorithms” on page 277.
1 In the Analysis tab, under Moving Average, mark or clear Show to
show or hide the Moving Average Line Plot.
2 In Algorithm, select Linear or Triangular.
• Linear – The linear algorithm calculates a standard, unweighted
average using every probe Log Ratio score within a specified window
size (specified by either a number of adjacent measurements or a
positional range). This average is applied sequentially to every point
by moving the window along the chromosome. See “Moving average
(linear smoothing)” on page 293.
• Triangular – The triangular algorithm calculates a weighted average
using every probe Log Ratio within a specified window size. The
triangular algorithm is more sensitive to localized variations in the
data. See “Triangular smoothing” on page 294.
3 In Line width, select the thickness (in pixels) of the Moving Average
Line Plot.
The range is 1- 5 pixels.
4 In Window, select the size of the moving average window.
Moving averages are computed with windows of sizes based on either a
specific length of base- pairs (5 Kb to 50 Kb or .1 Mb to 50 Mb) or a
fixed number of data points (1 pt. to 60 pt.).
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To set up a moving average (ZScore) calculation to smooth the data
To set up a moving average (ZScore) calculation to smooth the data
The Moving Average (ZScore) applies a moving average to each point in
the combined methylation Z- score data set using a sliding window of fixed
size, centered on that point. This algorithm uses the post algorithm
combined Z- scores to calculate a moving average line plot. See “Overview
of Methylation Detection and Visualization Algorithms” on page 276.
1 In the Analysis tab, in the Moving Average (ZScore) group, mark or
clear Show to show or hide the Moving Average Line Plot.
2 In Algorithm, select Linear or Triangular. See “Visualization
algorithms” on page 277.
Linear – The linear algorithm calculates a standard, unweighted average
using every combined Z- score within a window size (that is defined by
either a number of adjacent measurements or a positional range). This
average is applied sequentially to every point by moving the window
along the chromosome. See “Visualization algorithms” on page 277.
Triangular – The triangular algorithm calculates a weighted average
using every combined Z- score within a specified window size. The
triangular algorithm is more sensitive to localized variations in the data.
See “Triangular smoothing” on page 294.
3 In Line width, select the thickness (in pixels) for the Moving Average
Line Plot.
The range is 1- 5 pixels.
4 In Window, select the size of the moving average window, or type the
size of the window.
Moving averages are computed with windows of sizes based on either a
specific length of base- pairs (5 Kb to 50 Kb or .1 Mb to 50 Mb) or a
fixed number of data points (1 pt. to 60 pt.).
The window can be set manually to as low as .5 Kb (type .5 Kb).
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To apply methylation (CH3) event detection
4
To apply methylation (CH3) event detection
The purpose of a methylation detection algorithm is to identify
methylation sites located within genomic regions such as promoters, CpG
islands, etc. It is used in conjunction with CpG Island tracks that either
already exist in the program’s database or that you import.
1 Set up genomic region tracks (such as CpG islands) in Gene View.
See “To import tracks” on page 53 for instructions on how to import
and display these tracks in Gene View and in the report.
2 Select the experiment now, or after step 3.
See “To select an experiment” on page 86.
3 Click Analysis.
4 Under Probe Methylation Select ZScore, select the algorithm, and then
mark Apply.
The Probe Methylation Status Setup dialog box appears.
See “Probe Methylation Status Setup” on page 233.
5 To select a Tm (melting temperature) mapping file, click Browse and
find the folder containing the file.
6 Select the file, and click Open.
7 Click Continue.
The algorithm is automatically applied if you’ve already selected the
experiment, or is applied when you select it. To learn how the algorithms
work see “Methylation Detection and Measurement Algorithms” on
page 278.
The Z- score or BATMAN score results appear in a second channel of Gene
View next to the log ratio data. You can now calculate a moving average
for the Z- scores. See the next topic.
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To apply BATMAN (Bayesian Tool for Methylation Analysis)
To apply BATMAN (Bayesian Tool for Methylation Analysis)
When BATMAN is selected, a Bayesian tool is used to calculate
methylation results (BATMAN). The BATMAN algorithm reflects the
underlying distribution of CpG dinucleotides by calculating a Gaussian
distribution of methylated sites to give the observed probe signal. The
signal used in BATMAN is either the probe log ratio or the methylation
detection algorithm Z- score. For more information on the BATMAN
algorithm, see “Bayesian tool for methylation analysis (BATMAN)” on
page 288.
1 In the Analysis tab, under Batman, mark the check box to enable the
Batman algorithm.
The Batman Parameter Setup dialog box is displayed. See “Batman
Parameter Setup” on page 187.
2 Complete the Batman Parameter Setup and click Continue to apply the
calculations and show results in the views.
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Displaying results and generating reports
4
Displaying results and generating reports
The section shows you how to make sure the analysis results are displayed
automatically after you select an experiment. To learn more about what
the results mean, see “Visualization algorithms” on page 277.
To display results of analysis
After you set up an experiment and analyze it, the program displays the
results automatically in Genome, Chromosome, and Gene Views if the
display is turned on. Figure 19 and Figure 20 show examples of results
displayed in the Gene View. To customize the way the results appear, see
Chapter 3, “Displaying CH3 Data and Other Content”.
Results for probe methylation and log ratios compared to a CpG Island track
Figure 19
Methylation (CH3) Analysis Guide
Gene View, showing Z-score data next to log ratios and a CpG Island track
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To display results of analysis
Results for moving averages within genomic boundaries of CpG Island track
Figure 20
Gene View, showing moving averages within genomic boundaries of tracks
If the analysis results do not appear in any of the Views
Check these four potential problem areas, in order:
• Make sure you have marked the Apply check box. Under Analysis, mark
Apply for Probe Methylation and/or Batman.
• Make sure you have selected the experiment or selected the arrays of
interest. See “To select an experiment” on page 86.
• Make sure you have turned on the display for the moving averages or
the Z- score algorithm. Do the following:
1 Right- click any of the Views, and click View Preferences.
2 Click the View tab.
3 Under Data Visibility, in View, select All views, then mark any of
the check boxes for the information to display.
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To upload a track to UCSC
4
You can also select a single View.
4 Click OK.
• Make sure you are looking in the correct region of Chromosome View.
See Chapter 3, “Displaying CH3 Data and Other Content” to find out how
to display the Scatter Plot and how to search the View displays to see
results.
To upload a track to UCSC
The program lets you upload methylation results to the UCSC Web site for
further review.
1 Right- click in Gene View and select Upload track to UCSC.
The Upload Track to UCSC dialog box appears. See “Upload Track to
UCSC” on page 260.
2 Complete the dialog box, (mark Methylation Score under Select Track
Source) and click OK.
The track is uploaded to the UCSC Web site (Figure 21). Enable pop
ups or set preferences on the UCSC Web site, if necessary.
3 Follow the instructions on the UCSC Web site to review your data.
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To save a result
Figure 21
Uploaded track in UCSC browser
To save a result
The program lets you save the current result of the active experiment. You
can run many different analyses in the same experiment, and save each
one. Later, you can restore any of your saved results.
If you are saving a result for the first time for the experiment:
1 In the Experiment pane of the Navigator, right- click the experiment,
and click Save Experiment Result, or
Click Home > Save Experiment Result.
A dialog box asks if you want to save the results of the current
experiment.
2 Click Yes.
The Save experiment result dialog box appears.
3 Type a name for the result, then click OK.
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To restore a saved result to the display
4
If you have already saved at least one result for the experiment:
1 In the Experiment pane of the Navigator, expand the folders of the
current experiment.
The currently selected result, if any, appears in blue in the Results
folder.
2 Click Home > Save Experiment Result or
Right- click the experiment, and click Save Experiment Result.
A dialog box asks if you want to select one of the following actions:
• To replace the current result with another saved result, click
Overwrite Current Result.
• To add the current results to the list of experimental results, click
Create New Result.
• To change views to another result without changing the current
result, click Continue Without Saving.
To restore a saved result to the display
1 If necessary, select the experiment that contains the result that you
want to see. See “To select an experiment” on page 86.
2 In the Experiment pane of the Navigator, expand the folder of the
active experiment, then expand its Results folder.
3 Right- click the desired result, then click Restore result.
The restored result appears in Genome, Chromosome, and Gene Views.
To generate a probe report
The probe report includes the following information either for single
chromosomes or the complete genome:
• Log ratio for each event
• Number of probes
• Start and stop of each event
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Setting Up Methylation (CH3) Analysis
To generate a probe report
• Whether the probe is in a CpG island, a promoter, or inside a gene
boundary
• Probable gene for the probe
1 Click Reports, then click Generate Report.
The Probe Methylation Report Setup dialog box appears.
2 To select if you want a report to include the results for the whole
genome or for individual chromosomes, click either Complete Genome
or Per- Chromosome.
3 Click Browse to find the folder for containing the report(s).
4 Type the report name, and click Open.
The report file name has the extension .xls.
5 Click OK.
6 When asked if you want to display the report, click Yes.
If you selected Complete Genome, an Excel spreadsheet appears containing
all the data:
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Setting Up Methylation (CH3) Analysis
To generate a probe report
Figure 22
4
Excel methylation report for Complete Genome
If you selected Per- Chromosome, the text files for the individual
chromosomes appear in the folder you selected.
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Setting Up Methylation (CH3) Analysis
To generate a Batman report
Figure 23
List of text methylation reports for each chromosome
To generate a Batman report
The Batman report includes the following information either for single
chromosomes or for the entire genome:
• Cytoband name
• Chromosome name
• Probe name
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To generate a Batman report
4
• Start and Stop of each event
1 Click Reports and then select Generate Batman Report.
The Batman Report Dialog dialog box opens. See “Batman Report
Dialog” on page 188.
2 To select if you want a report to include the results for the whole
genome or for individual chromosomes, click either Complete Genome
or Per- Chromosome.
3 Click Browse to find the folder for containing the report(s).
4 Type the report name, and click Open.
The report file name has the extension .xls.
5 Click OK.
6 When asked if you want to display the report, click Yes.
If you selected Complete Genome, an Excel spreadsheet appears containing
all the data:
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To generate a Batman report
Figure 24
Excel Batman report for complete genome
If you selected per- chromosome for the report output, the text files for the
individual chromosomes appear in the folder you selected.
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Agilent Genomic Workbench 6.5 – Methylation (CH3) Analysis
User Guide
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Agilent Genomic Workbench CH3 Application Main Window 132
Switch Application Menu 133
Command Ribbons 134
Navigator 146
Genomic Viewer 169
Status Bar 182
Dialog Boxes 183
Report Format 272
This chapter describes the parts of the Agilent Genomic Workbench main
window that you use to import, organize, manage, export and display
array data and other content. It also describes the tab commands,
shortcut menus, and dialog boxes that can appear.
Agilent Technologies
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Agilent Genomic Workbench CH3 Application Main Window
Agilent Genomic Workbench CH3 Application Main Window
The main window of the CH3 Application contains the major components
illustrated in Figure 25.
Tabs
Command Ribbon
Switch Application
Genome View
Cursor
Chromosome View
Gene View
Status Bar
Tab View
Navigator
Figure 25
132
Agilent Genomic Workbench Standard Edition – CH3 application main window (horizontal orientation)
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Switch Application Menu
5
Switch Application Menu
Figure 26
Switch Application menu
The Switch Application menu lets you change to the other data display
and analysis application type in DNA Analytics. Mark the desired
application type.
CGH
(Separate license required) Imports, displays, and analyzes array- based
comparative genomics hybridization (aCGH) data in both an interactive
“analyze as you go” mode, and an automated workflow mode.
ChIP
(Separate license required) Imports, displays, and analyzes ChIP- on- Chip
microarray data in both an interactive “analyze as you go” mode, and an
automated workflow mode.
CH3
(Separate license required) Imports and displays data from
microarray- based studies of genomic methylation patterns.
Expression
Use eArrayXD to create and view probe groups.
microRNA
Use eArrayXD to create and view probe groups.
SureSelect Target
Enrichment
Use the Quality Analyzer function for SureSelect Target Enrichment.
Import, export, and view data, and use eArrayXD to search, and create
Bait Groups. See the SureSelect Quality Analyzer User Guide for more
information.
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Command Ribbons
Command Ribbons
When you click a tab, groups of commands or single commands appear at
the top of the tab. This group of commands is called a command ribbon.
The interactive tabs of the CH3 Application include Home, Preprocessing,
Analysis, Reports, View, and Help. This section provides descriptions of
the commands for each of these tabs, as well as for the Help tab.
Descriptions of the remaining tabs are located in the following guides:
• Sample Manager — Sample Manager User Guide
Figure 27
Tab bar with command ribbon
Home command ribbon
The Home command ribbon displays the functions that let you import,
manage, export and display CH3 data and content for further CH3
analysis.
Figure 28
User Preferences
134
Home command ribbon
Opens the User Preferences dialog box with the following tabs:
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Home command ribbon
Data
Import
5
Tab
Description
Tracks
Opens a dialog box that lets you manage which tracks to display in
Genomic Viewer and how they appear. See “User Preferences” on
page 262.
Miscellaneous
Opens a dialog box where you can select a new location for your
data files and set up access to the eArray web site. See “User
Preferences” on page 262.
License
Opens a dialog box where you can enter an Agilent Genomic
Workbench application license, if you purchase one after using the
unlicensed version. See “User Preferences” on page 262.
Opens the Catalog and Workgroup Data window, where you can choose to
download data from the eArray catalog or from your workgroup. See
“Catalog and Workgroup Data” on page 190. For more information, see the
eArrayXD User Guide.
Opens a menu of file types that you can import:
Option
Description
Array Files
Opens a menu with these options:
• FE File – Opens the Import FE Files dialog box, where you can
select an Agilent Feature Extraction array data file to import.
See “Import” on page 222 and “To import Agilent FE or Axon
data files” on page 47.
• Axon File – Opens the Import Axon Files dialog box, where you
can select Axon (*.gpr) files for import. See “Import” on
page 222 and “To import Agilent FE or Axon data files” on
page 47.
• UDF File – Opens the UDF Files dialog box, where you can select
a Universal Data File (UDF) to import. See “Import” on page 222
and “To import a UDF file” on page 49.
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Home command ribbon
Export
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Option
Description
Design Files
Opens a menu with these options:
• GEML File – Opens the Import Design Files dialog box, where
you can select Agilent GEML-based (*.xml) array design files for
import. See “Import” on page 222 and “To import Agilent GEML
design files” on page 46.
• Axon Design File – Opens the Import Axon Design Files dialog
box, where you can select Axon (*.gal) array design files for
import. See “Import” on page 222 and “To import Axon design
files” on page 47.
Genome Build
Opens the Import Genome Build dialog box, where you can import
Agilent-supplied genome build files. See “Import Genome
Build” on page 226 and “To import a genome build” on page 52.
Array Attributes
Opens the Import microarray attributes dialog box, where you can
select a microarray attributes file. See “Import” on page 222.
Track
Opens the Import Track dialog box, where you can select a BED
format track file for import, and create a display name for the track.
See “Import Track” on page 227 and “To import tracks” on page 53.
Experiments
Opens the Import Experiments dialog box, where you can select a
ZIP format experiment file for import. See “Import
(experiments)” on page 224 and “To import an experiment file” on
page 55.
Probe Upload
Lets you import a file of probe sequences and annotation. For more
information, see the eArrayXD User Guide.
Custom Genome for Tiling
Opens the Import Genome dialog box, where you upload a user
defined genome for use with the Genomic Tiling and Bait Tiling
tools in eArray XD. For more information, see the eArrayXD User
Guide.
Opens a menu that lets you export several kinds of files.
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Home command ribbon
Create
Experiment
5
Option
Description
Experiments
Opens the Export Experiments dialog box, where you can select
one or more experiments for export as a single ZIP file. See “Export
Experiments” on page 216 and “To export experiments” on
page 81.
Tracks
Opens the Export Tracks dialog box, where you can select one or
more tracks to export as a single BED format file. See “Export
Tracks” on page 217 and “To export tracks” on page 82.
Array Attributes
Opens the Export Array Attributes dialog box, where you can select
arrays and their attributes for export. See “Export Array
Attributes” on page 212.
Opens the Create Experiment dialog box, where you can create a new,
empty experiment and add data to it. See “Create Experiment” on
page 197 and “To create a new experiment” on page 58.
Save Experiment
Results
Opens a confirm dialog box. Click Yes and the Save experiment result
dialog box opens where you enter a name and description for the results
to save.
Go to
Gene/Genomic
Location
Moves the cursor to the location in Chromosome and Gene Views that you
select. See “Go To Gene/Genomic Location” on page 221.
Print
Exit
Opens the Print window to print the display.
Closes the program.
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Preprocessing command ribbon
Preprocessing command ribbon
The Preprocessing ribbon has a single command you use to prepare for
CH3 analysis: Fuse.
Figure 29
CH3 Preprocessing command
The Fuse command lets you combine multiple array designs into a larger
virtual combined design. It also lets you combine both intra- and
inter- array replicate probes.
Click to open the Array Set dialog box, where you can select the names of
arrays that are combined, set options, and combine designs together to
form a larger virtual design. See “Array Set” on page 186, and “To
combine (fuse) arrays” on page 115.
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Analysis command ribbon
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Analysis command ribbon
The Analysis ribbon contains the commands you use for CH3 analysis.
Figure 30
CH3 Analysis ribbon
Moving Average (Log Ratio)
Figure 31
Moving Average (Log Ratio) command
The settings under Moving Average(Log Ratio) control the calculation and
display of line plots that represent smoothed log ratio data for each
selected array. These plots can appear in Genome, Chromosome, and Gene
views.
Show
Mark this check box to show moving average line plots, or clear the check
box to hide them. You turn the display of moving average line plots for
specific views on or off from the View tab by selecting View Preferences.
In View Preferences, under Data Visibility, select the desired view, then
mark or clear Moving Average. See “View Preferences” on page 268.
Algorithm
Select one of these options:
• Linear – The linear algorithm calculates a standard, unweighted average
using every Log Ratio score within a defined window size (specified by
either a number of adjacent measurements or a positional range). This
average is applied sequentially to every point by moving the window
along the chromosome.
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Analysis command ribbon
• Triangular – The triangular algorithm calculates a weighted average
using every Log Ratio within a specified window size. The triangular
algorithm is more sensitive to localized variations in the data. See
“Overview of Methylation Detection and Visualization Algorithms” on
page 276.
Line width
Window
Select the desired thickness (in pixels) for the moving average line plots.
You can select a thickness from 1 to 5 pixels.
Select the desired size of the moving average window. You can select
either a specific number of base pairs (5 Kb to 50 Mb), or a specific
number of data points (1 pt to 60 pt). You can also type a value. The
program calculates a moving average for each selected array based on a
window of the given size centered on each point in the array.
Moving Average (ZScore)
Figure 32
Moving Average (ZScore) command
The settings under Moving Average (Z- Score) control the calculation and
display of line plots that represent smoothed Z- score data for each
selected array. These plots can appear in Genome, Chromosome, and Gene
views. You must apply the Probe Methylation algorithm first.
Show
Algorithm
Mark this check box to show moving average line plots for the Z- score
data, or clear the check box to hide them. You enable or disable the
display of moving average line plots for selected views in View
Preferences. See “View Preferences” on page 268.
Select one of these options:
• Linear – The linear algorithm calculates a standard, unweighted average
using every combined Z- score within a selected window size (specified
by either a number of adjacent measurements or a positional range).
This average is applied sequentially to every point by moving the
window along the chromosome.
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Analysis command ribbon
5
• Triangular – The triangular algorithm calculates a weighted average
using every Z- score within a selected window size. The triangular
algorithm is more sensitive to localized variations in the data. See
“Triangular smoothing” on page 294.
Line width
Select the thickness (in pixels) for the moving average line plots. You can
select a thickness from 1 to 5 pixels.
Window
Select the size of the moving average window. You can select either a
number of base pairs (5 Kb to 50 Mb), or a number of data points (1 pt
to 60 pt). You can also type a value. The program calculates a moving
average for each defined array based on a window of the given size,
centered on each point in the array.
Probe Methylation
You apply this command to make Z- score calculations to find out whether
a Methylation detection array probe is methylated or unmethylated. See
“Overview of Methylation Detection and Visualization Algorithms” on
page 276.
Figure 33
Select ZScore
Apply
Probe Methylation command
Select the algorithm to use for calculation of probe methylation score.
Click Apply to display the Probe Methylation Status Setup dialog box to
assign a melting temperature (Tm) mapping file, if necessary, to the
experiment. See “Probe Methylation Status Setup” on page 233.
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Reports command ribbon
Batman
Figure 34
Batman command
Click Apply to display the Batman Parameters Setup dialog box where you
set the parameters for the Batman tool for calculation and display of
methylation data. See “Batman Parameter Setup” on page 187 for more
information.
Reports command ribbon
Figure 35
CH3 Report command
The Reports command ribbon lets you save all the important information
about the Z- score statistics and CpG Island tracks that can help you find
out whether regions of the genome are methylated or not. You use a
spreadsheet program or your Internet browser to open the report files the
program creates. This lets you further display, analyze, and organize the
result.
Generate Probe
Report
142
Opens the Probe Methylation Report Dialog dialog box, where you can
enter a location and a name for the probe report file and select whether
you want individual reports for each chromosome or one report on the
complete genome. See “Probe Methylation Report Dialog” on page 234.
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View command ribbon
5
The Methylation Report makes information about the results of the probe
methylation algorithm available in .xml format (complete genome) or in
.txt format (individual chromosomes). You can use a spreadsheet program
to open all these files. See “Report Format” on page 272 for a description
of the columns in the report.
Generate Batman
Report
Opens the Batman Report Dialog box where you set the parameters and
select a location to save the report.
View command ribbon
The View command ribbon lets you change the display of data/results in
Genomic Viewer.
Figure 36
View command ribbon
View Preferences
Opens the View Preferences dialog box where you customize the display of
data and results in the Genomic Viewer. For more information, see “View
Preferences” on page 268.
Copy
This command opens a menu with the options listed below. In general, the
Copy command copies pane(s) of the main window to the Clipboard as an
image. You can then paste the image into a document in another program.
See “To copy what you see in the main window” on page 83.
Option
Description
All
Copies all panes of the main window to the Clipboard as an image.
Navigator
Copies only the Navigator to the Clipboard as an image.
Tab View
Copies only Tab View to the Clipboard as an image.
Genome view
Copies only Genome View to the Clipboard as an image.
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Help command ribbon
Show
Option
Description
Chromosome view
Copies only Chromosome View to the Clipboard as an image.
Gene view
Copies only Gene View to the Clipboard as an image.
Opens a menu with all available elements of the main window. Mark the
check box for the ones to display in Genomic Viewer.
View In Table
Signal Intensity
Annotation
Mark the check box to see the red and green raw signal intensities of the
log ratio data in the Tab View.
Mark the check box to show annotations in the Tab View.
Cyto band info
View In Gene
View
Mark the check box to display cytobands in the Gene View.
NonUnique Probes
Highlight
Mark the check box to display non unique probes in a different color.
Custom Data
Show
Mark the check box to display custom data in the Genomic Viewer.
Help command ribbon
The Help command ribbon lets you display the available Agilent Genomic
Workbench help guides, and get information about software version,
installation history, and check for software updates. Help guides are
opened in Adobe Reader.
Figure 37
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Help command ribbon for CH3 module
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Help command ribbon
Table 3
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Table of Agilent Genomic Workbench Help
Help Command
Action
Application Guide
Opens the Agilent Genomic Workbench application user guide for the
selected application.
eArrayXD
Opens the eArrayXD User Guide. This guide comprehensive
help on all available eArrayXD tools.
Sample Manager
Opens the Sample Manager User Guide, that shows how to use the
Sample Manager module of Agilent Genomic Workbench to organize
microarrays and edit their attributes.
Feature Extraction
Opens a menu that lets you choose from the following Feature Extraction
help guides:
• Feature Extraction Quick Start Guide – An overview of how to use the
Feature Extraction software to extract and generate QC reports for
Agilent microarrays
• Feature Extraction User Guide – A comprehensive user guide that
explains how to extract and generate QC reports for Agilent microarrays
• Feature Extraction Reference Guide – Contains tables that list default
parameter values and results for Feature Extraction (FE) analyses, and
explanations of how FE uses its algorithms to calculate results.
Quality Tools
Opens the Quality Tools User Guide, that describes how to query, filter, and
evaluate microarray extractions within Agilent Genomic Workbench.
Data Viewing
Opens the Data Viewing User Guide that describes how to import,
organize, manage, export and display data and other content (experiments,
gene lists, tracks) within Agilent Genomic Workbench. It is targeted for
users who have no DNA Analytics application license(s).
About
Opens a message with information about the version number and
copyright of the program.
Installation History
Opens the Installation History dialog box, that shows what versions and
updates were installed.
Check Updates
Checks for available updates to the software. If an update is available, you
are asked if you want to install it. If no update is available, a message
appears to let you know.
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Navigator
Navigator
This section describes the parts of the Navigator, and the shortcut menus
and other functionality available within it.
Search pane
Design Data pane
Experiment pane
My Entity List pane
My Networks pane
Tasks pane
Figure 38
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Navigator panes
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Navigator
5
The Navigator (Figure 38) shows the array data, experiments, and other
content stored in Agilent Genomic Workbench. It contains the following
panes:
Pane
Comments
Search
Lets you search within any pane of the Navigator for a specific
item (array or build, for example). You must type the entire array
name or term; otherwise, use asterisks (*) as wildcards for
unspecified strings. For example, type *1234* to find any item that
contains “1234”. See “Search pane” on page 149.
Design Data
Contains microarray data files, organized by design and application
type, and then by genome build.
Shows all probe groups and microarray designs that are available
to you, organized by folders. For the SureSelect Target Enrichment
application type, the program shows all bait groups and libraries. In
general, you can:
• Expand or collapse folders to show or hide content
• Look at the icon that appears with an item to monitor its status.
• Right-click the name of a folder or item to open a shortcut menu
that lets you take action on the item.
See “Design Data pane – icons, special text, and buttons” on
page 151 and “Design Data pane – actions and shortcut
menus” on page 152.
For Agilent Catalog content, the names of all available items
appear in the AgilentCatalog folder. However, to work with this
content, you must specifically request a download of the actual
data from the eArray Web site.
Experiment
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Contains Agilent Genomic Workbench experiments. Experiments
are organizational units that contain links to microarray data and
design files. In data analysis modules, experiments also contain
saved results.
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Navigator
Pane
Comments
My Entity List
Contains gene lists and tracks:
• Gene Lists are collections of genes of interest. You can create
them within the program, import and export them, and apply
them to Gene View and Chromosome View.
• Tracks are collections of annotation or other information that
map to specific genomic locations. You can import, export, and
combine tracks, and display them in Gene View with your array
data and analysis results.
• See “My Entity List pane – Icons, buttons, and special text” on
page 164.
My Networks
Contains the biological networks/pathways that you found using
Network Search or that you create using a literature search in
eArrayXD. For more information, see the eArrayXD Users Guide.
Tasks
Shows the jobs that you have submitted. Some jobs are completed
locally by the eArrayXD server program. Others are sent to the
eArray Web site for completion. In general, you can:
• Look at the icon that appears with a job to monitor its status.
• Right-click the name of a pending task to open a shortcut menu
that lets you take further action on the job.
See “Tasks pane” on page 167.
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Search pane
5
Search pane
The Search pane lets you find all occurrences of a specific search term in
the Design Data, Experiment, and/or My Entity List panes. See “To find
specific content items in the Navigator” on page 70. It also contains
several buttons that you can use to move, hide, show or resize the
Navigator.
Search term box
Detach
button
Resize
buttons
Search term
clear button
Search button
Scroll buttons
Figure 39
Pane list
button
Navigator – Search pane
Detach button
Click to move the Navigator from the main window of the program and
open it in a new, separate window.
Resize buttons
Click to hide, show, or expand the Navigator.
Search term box
Where you type your desired search term. Search terms are not
case- sensitive, but they must reflect the entire name of an array or other
content item that you want to find. You can use asterisks (*) as wildcards
to represent groups of unspecified characters. For example, type *12345*
to search for any content that contains the string “12345”.
Pane list
Lets you limit a search to a specific pane. Select the name of the desired
pane from the list. To select all panes, select All Panels. By default, the
program searches all panes.
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Search pane
Figure 40
Search Pane selection list
(Show Pane List button, available only if the Pane list is not visible)
Makes the Pane list visible.
(Hide Pane List button, available only if the Pane list is visible) Hides the
Pane list.
(Search button) Searches the pane(s) selected in the Pane list for all
occurrences of the term you typed in the Search term box. If the program
finds a matching item, it expands the folder structure to make the
matching item(s) visible, makes the lettering of each item red and
highlights the item in yellow. Note: The search term is not case- sensitive,
but it must contain the entire name of the desired items.
Scroll buttons
(Available only after a search) Lets you scroll up and down the lists of
highlighted search items after a search.
(Clear button, available only after a search) Clears the search term from
the Search term box, and resets the color of any matching item to its
original color.
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Design Data pane – icons, special text, and buttons
Item
Comments
An unexpanded folder (domain) that contains subfolders or other items.
An expanded folder. The items that it contains are visible in the Navigator.
Expands a folder to show its contents.
Collapses a folder to hide its contents.
A folder that can contain microarray design(s) or bait librar(ies).
A folder that can contain probe group(s) or bait group(s).
An item that contains Agilent content that you must update from the eArray Web site
before you can use it.
An available microarray design with a status of Draft, or an available probe or bait
group with a status of Incomplete.
An available library or microarray design with a status of Review.
An available library or microarray design with a status of Completed.
An available library or microarray design with a status of Submitted.
An available probe or bait group with a status of Locked.
A methylation array design. This folder contains array data for the design, organized by
genome build.
A CGH array design. This folder contains array data for the design, organized by genome
build.
A gene expression array design. This folder contains array data for the design, organized
by genome build.
A ChIP array design. This folder contains array data for the design, organized by genome
build.
A genome build folder within a specific design folder. This folder contains arrays for the
specific genome build and design.
A folder that contains sample data for a design. The data are read-only to the user who is
currently logged in. The data apply to the indicated genome build, and are not used by
eArrayXD.
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Design Data pane – actions and shortcut menus
Item
Comments
A folder that contains sample data for a design. The data can be edited the user who is
currently logged in. The data apply to the indicated genome build, and are not used by
eArrayXD.
A single array data file.
Data from an individual microarray sample. The design from which the data were derived
is read-only to the user who is currently logged in. These data are not used by eArrayXD.
Data from an individual microarray sample. The design from which the data were derived
can be edited by the user who is currently logged in. These data are not used by
eArrayXD.
Data created from a multi-pack array.
red text An item that matches the search term in a search.
(Dock out button) Moves the Design Data pane from the Navigator, and opens it in a,
separate window.
(Collapse button, available only if the Design Data pane is not collapsed) Collapses the
Design Data pane, and shows its title bar at the bottom of the Navigator.
(Expand button, available only if the Design Data pane is collapsed) Expands the Design
Data pane.
Design Data pane – actions and shortcut menus
The Design Data pane of the Navigator shows available content items that
are stored on your server for the selected application type, and any
external content that you imported. It also shows the names of items in
the Agilent Catalog. For more information on the contents of the Design
Data pane, and how to use eArrayXD, see the eArrayXD Users Guide.
• Double- click any folder to expand or collapse it.
Data Folder
• Double- click a data type folder (CGH, ChIP, Expression, or CH3) to
expand it and display its contents.
• Double- click a design folder to display the genome builds for the design.
• Double- click a build folder to display the imported arrays for that build.
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Design Data pane – actions and shortcut menus
5
Main Folders
The Designs folder in the Design Data pane contains the following main
folders:
Folder
Contents
AgilentCatalog
Content from the Agilent Catalog that is available for the
selected application type.
<Workgroup Name>
This folder, which bears the name of your workgroup, contains
custom workgroup content that is available for the selected
application type.
Imported External Designs
Design information and array data for downstream processing
by the Agilent Genomic Workbench data analysis applications.
You cannot use eArrayXD to work with any of the items in
these folders. For more information, see the applicable user
guide for each data analysis application.
Custom Designs
Custom designs created using eArray.
AgilentCatalog Folder
• Right- click the name of a microarray design or probe group to open a
shortcut menu with available options. (Note – The availability of the
options varies by design status and ownership.) See the eArrayXD User
Guide for information on how to use these options.
<Workgroup Name> Folder
• Double- click the name of a folder to expand or collapse it.
• Right- click on an Array Design or Probe Group and select one of the
available actions (described in the eArrayXD User Guide).
• Right- click the name of a domain folder to open a shortcut menu. See
the eArrayXD User Guide for information on how to use these options.
Imported External Designs Folder
Imported external designs appear in this folder under folders for the type
of design they represent: ChIP, Expression, CGH, or Only Stats and
Params (for imported statistics and parameters from Feature Extraction).
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Design Data pane – actions and shortcut menus
• Right- click the Imported External Designs folder to open a shortcut
menu with an Import option. When you select this option, a menu
appears with the following options for file import:
Option
Description
Design File
Opens the Import Design Files dialog box, where you can select an
Agilent GEML-based (*,xml) file for import. See “Import” on page 222
and “To import Agilent GEML design files” on page 46.
Note: You cannot import Catalog designs. They must be downloaded
from eArray.
Axon Design File
Opens the Import Axon Files dialog box, where you can select
GenePix/Axon design (*.gal) files for import. See “Import” on
page 222 and “To import Axon design files” on page 47.
FE File
Opens the Import FE Files dialog box, where you can select an Agilent
Feature Extraction array data file to import. See “Import” on page 222
and “To import Agilent FE or Axon data files” on page 47.
Axon File
Opens the Import Axon Files dialog box, where you can select Axon
(*.gpr) files for import. See “Import” on page 222 and “To import
Agilent FE or Axon data files” on page 47.
UDF Files
Opens the UDF Files dialog box, where you can select a Universal Data
File (UDF) to import. See “Import” on page 222 and “To import a UDF
file” on page 49.
• Double- click an imported designs folder (CH3, ChIP, Expression, CGH,
Only Stats and Params) to display the imported designs for that data
type.
• Double- click the name of an imported design folder, and then
double- click the Design Samples folder to display a list of genome
builds.
• Double- click the name of a genome build folder to display imported
arrays for that build.
Design Folder
• Right- click the name of a design folder to display these options:
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Design Data pane – actions and shortcut menus
Option
5
Description
Download from eArray (Available only for Agilent microarrays) Updates the annotations for
your array design from the eArray Web site. Agilent regularly updates
annotations in eArray as new ones become available. See “To
download a design from eArray” on page 71.
Update from eArray
Updates the annotations for your array design from the eArray Web
site. Agilent regularly updates annotations in eArray as new ones
become available. See “To update probe annotation in design files” on
page 71.
Note: In order to use this function, you must enter your eArray
Username and Password in the Miscellaneous tab of the User
Preferences dialog box. See “User Preferences” on page 262.
Delete
Opens a Confirm dialog box. If you click Yes, the program permanently
deletes the design and all arrays associated with it.
Genome Build Folder
• Right- click the name of a genome build folder to display these options:
Option
Description
Show Properties
Opens the Design Properties dialog box. See “Design Properties” on
page 204.
Delete
Opens a Confirm dialog box. If you click Yes, the program permanently
deletes all of the arrays in this genome build folder.
Individual Arrays
• Right- click the name of an array to display these options:
Option
Description
Show Properties
Opens the Microarray Properties dialog box. See“Microarray
Properties” on page 229 and “To display or edit the attribute values of
a specific array” on page 66.
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Design Data pane – actions and shortcut menus
Option
Description
Rename
Opens an Input dialog box, where you can type a new name for the
array. Click OK to rename the array.
Delete
Opens a Confirm dialog box. If you click Yes, the program permanently
deletes the array.
• Drag an array from the Design Data pane to an experiment folder in
the Experiment pane to associate it with an experiment. You can drag
multiple arrays at once from one genome build in a design. Hold down
the ctrl key and click the additional arrays to select them. You can also
select a contiguous block of arrays; click the first array in the block,
then hold down the Shift key and click the last one.
Custom Designs Folder
This folder displays custom designs available from eArrayXD.
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Experiment pane – icons, special text, and buttons
5
Experiment pane – icons, special text, and buttons
Item
Comments
Click to expand a folder and display its contents.
Click to collapse a folder and hide its contents.
A folder that contains files or other folders.
A methylation array design. This folder contains array data for the design, organized by
genome build.
A CGH array design. This folder contains array data for the design, organized by genome
build.
A gene expression array design. This folder contains array data for the design, organized
by genome build.
A ChIP array design. This folder contains array data for the design, organized by genome
build.
A genome build folder within a specific design folder. This folder contains arrays for the
specific genome build and design.
A read-only genome build folder within a specific design folder.
A genome build folder, within a specific design folder, that you can modify.
An array that is not selected for view and analysis.
An array that is selected for view and analysis. The specific color of this icon can vary.
A calibration array.
An empty folder.
Data created from a multi-pack array.
blue
text
The active experiment. All data and results that appear in Chromosome, Gene, and Tab
Views are from this experiment.
red text An item that matches the search term in a search.
(Dock out button) Moves the Experiment pane from the main window, and opens it in a,
separate window.
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Experiment pane — actions and shortcut menus
Item
Comments
(Collapse button, available only if the Experiment pane is not collapsed) Collapses the
Experiment pane, and shows its title bar at the bottom of the Navigator.
(Expand button, available only if the Experiment pane is collapsed) Expands the
Experiment pane.
Experiment pane — actions and shortcut menus
Figure 41
Expanded Experiment Pane
You can use many items in the Experiment Pane of the Navigator to open
shortcut menus or take other actions.
• In general, double- click the Experiments folder within the Experiment
Pane, and the folders within it, to expand and collapse them. Exception:
Double- click the name of an unselected experiment to select it for
analysis. This action opens the Experiment Selection dialog box. To
select the experiment for analysis, click Yes.
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Experiments Folder
• Right- click the Experiments folder to display these options:
Option
Description
New Experiment
Opens the Create Experiment dialog box (see “Create Experiment” on
page 197), where you can name the new experiment, and open
another dialog box that lets you add microarray data to the
experiment. See “To create a new experiment” on page 58.
Export
Opens the Export Experiments dialog box, where you can export one
or more experiments as a single ZIP file. See “Export Experiments” on
page 216 and “To export experiments” on page 81.
Specific Experiment Folder
• In the Experiments folder, right- click the name of a specific experiment
folder to display these options:
Option
Description
Select Experiment
(Appears only if the experiment is not selected.) Opens the
Experiment Selection dialog box, which asks if you want to select the
experiment. Click Yes to select the experiment for display and
analysis.
Or, in the Experiments folder, double-click the name of an experiment
that is not selected to open the Experiment Selection dialog box. To
select the experiment for analysis, click Yes.
If you switch experiments, a Confirm dialog box asks if you want to
save the current result. Select one of these options:
• Overwrite Current Result – Replaces the selected experiment
result in the Data Navigator with the result that appears on your
screen.
• Create New Result – Opens the Save experiment result dialog box,
where you can save the result that appears in the main window as
a new experiment result. See “To save a result” on page 124.
• Continue Without Saving – The program does not save the result
that appears on your screen.
In some cases, the Confirm dialog box offers only Yes and No choices.
If you click Yes, the Save experiment result dialog box appears, where
you can save the result that appears on your screen with the name of
your choice.
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Experiment pane — actions and shortcut menus
Option
Description
Deselect Experiment
(Appears only if the experiment is selected.) If the results are unsaved,
a Confirm dialog box opens with these options:
• Overwrite Current Result – Replaces the selected experiment
result in the Data Navigator with the result that appears on your
screen.
• Create New Result – Opens the Save Experiment result dialog box,
where you can save the result that appears on your screen as a
new experiment result.
• Continue Without Saving – The program does not save the result
that appears on your screen.
In all three cases, the program then removes the experiment data and
results from all views.
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Share
Opens the Sharing for enterprise dialog box where you can add users
to share the experiment. See “Sharing for enterprise” on page 245.
Show Properties
Opens the Experiment Properties dialog box. Use this dialog box to
see the names of the arrays in the experiment, and also to add or
remove arrays from the experiment. See “Experiment Properties” on
page 209.
Edit Attributes
Opens the Sample Attributes dialog box, where you can change the
values for the attributes assigned to the arrays in the experiment. See
“Sample Attributes” on page 235. To add new attributes you must use
the Sample Manager. See Sample Manager User Guide.
Export
Opens the Export Experiments dialog box, where you can export this
and other experiments as a single ZIP file. See “Export
Experiments” on page 216, and “To export experiments” on page 81.
Export Attributes
Opens the Export Array Attributes dialog box, where you can save a
file that contains selected attributes of the arrays in your experiment.
See “Export Array Attributes” on page 212.
Edit Array Color
Opens the Edit Array Color dialog box, where you can select a display
color for each of the arrays in the experiment. See “Edit Array
Color” on page 207.
Edit Array Order
Opens the Edit Array Order dialog box, where you can change the
order of the arrays in the experiment pane of the Navigator, and in
Chromosome, Gene, and Tab Views. See “Edit Array Order” on
page 208.
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Experiment pane — actions and shortcut menus
5
Option
Description
Create Query
Opens the Create Query dialog box, where you select or create a Query
to find samples in the selected experiment, based on conditions set in
the query. See “Create Query” on page 201.
Rename
Opens an Input dialog box, where you can type a new name for the
experiment. Click OK to rename the experiment.
Delete
Opens a Confirm dialog box that asks if you want to delete the
Experiment. Click Yes to delete it.
Note: You can delete any experiment except the selected one.
Design Folder
• In the folder of a specific experiment, right- click the name of a design
to open a shortcut menu with a Delete command. If you click Delete, a
Confirm dialog box opens. Click Yes to disassociate all of the arrays
under the design from the experiment.
Genome Build Folder
• In the folder of a specific experiment, in a specific design folder,
right- click the name of a genome build to display these options:
Option
Description
Set for Calibration
Use of calibration arrays is not recommended by Agilent. This feature
has been deprecated.
Save As Text File
Opens the Save Design dialog box, where you can save all of the data
for the genome build and design within the experiment as a
tab-delimited text file.
Delete
Opens a Confirm dialog box that asks if you want to disassociate all
arrays under the design from the experiment. Click Yes to remove the
links between the arrays and the experiment.
Note:
• If you delete a design from an experiment, the program removes the
links between the experiment and the design and its arrays. The
actual design and array data stay in the Data folder.
• Saved results become unavailable if they involve arrays you delete
with this command.
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Experiment pane — actions and shortcut menus
Individual Arrays
• In a specific experiment, right- click the name of an individual array to
display these options:
Option
Description
Select
(Available only if the array is not already selected) Selects the array for
display and analysis.
Deselect
(Available only if the array is selected) Removes the array data from
Genome, Chromosome, and Gene views, and excludes it from the
analysis. Also removes the array data from the Selected Arrays tab in
Tab View.
Select for Calibration
Use of calibration arrays is not recommended by Agilent. This feature
has been deprecated.
Deselect from
Calibration
Use of calibration arrays is not recommended by Agilent. This feature
has been deprecated.
Rename
Opens an Input dialog box, where you can type a new name for the
array in this experiment. Click OK to accept the new name for the
array. The array name is changed only for the selected experiment.
Delete
Opens a Confirm dialog box that asks if you want to disassociate the
array from the experiment. Click Yes to remove the link between the
array and the experiment.
Note:
• If you delete an array from an experiment, the program removes the
link between the experiment and the array. The actual array data
remains in the Data folder.
• You cannot restore an experiment result that includes a deleted
array.
Show Properties
Opens the Microarray Properties dialog box, where you can view and
edit microarray attributes. See “Microarray Properties” on page 229.
For array files from the Agilent Feature Extraction program, you can
also view the headers and feature data from the file.
See “To display or edit the attribute values of a specific array” on
page 66.
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Option
Description
Edit Array Color
Opens the Select Color dialog box, where you can select a display
color for the array. See “Select Color” on page 237.
Edit Array Order
Opens the Array Order dialog box, where you can change the order of
the arrays in the Experiment pane of the Navigator, and in
Chromosome, Gene, and Tab Views. See “Edit Array Order” on
page 208.
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My Entity List pane – Icons, buttons, and special text
My Entity List pane – Icons, buttons, and special text
Item
Comments
Click to expand a folder and display its contents.
Click to collapse a folder and hide its contents.
A folder that contains files or other folders.
An individual gene list or track.
red regular
text
An item that is an exact match with the search term in a search, or a gene list that
has not been applied or that has red chosen as its custom color.
colored
italics
A gene list that has been applied.
red bold
italics
A track that is selected for display in Gene View.
black bold
italics
A “combined” track that is selected for display in Gene View. A combined track
contains information from two or more individual tracks associated by logical
criteria.
(Dock out button) Moves the My Entity List pane from the main window, and opens
it in a, separate window.
(Collapse button, available only if the My Entity List pane is not collapsed) Collapses
the My Entity List pane, and shows its title bar at the bottom of the Navigator.
(Expand button, available only if the My Entity List pane is collapsed) Expands the
My Entity List pane.
My Entity List pane – Actions and shortcut menus
Gene List folder
• Right- click the Gene List folder to open a shortcut menu with an
Import Gene List option. This command opens an Import dialog box
that you can use to import a gene list into the program. See
“Import” on page 222.
• Double- click the Gene List folder to show or hide its gene lists.
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My Entity List pane – Actions and shortcut menus
5
• In the Gene List folder, right- click the name of a gene list to display
these options:
Option
Description
View in Table
Opens the Gene List dialog box, where you can view the list of genes.
You can also edit the description of the gene list, and change the
display color of the genes. See “Gene List” on page 220 and “To
display the genes in a gene list” on page 75.
Rename
Opens an Input dialog box, where you can type a new name for the
gene list. Click OK to accept the new name.
Delete
Opens a confirm dialog box that asks if you are sure you want to
delete the gene list. Click Yes to confirm.
Save As
Opens a Save As dialog box, where you can save the gene list as a text
(*.txt) file. See “To export a gene list” on page 82.
Add to gene list
Opens the Add gene list dialog box, where you can add the gene list to
any other one in the Gene List folder. See “Add Gene List <name>
to” on page 183 and “To add one gene list to another” on page 76.
Highlight
(Available if the gene list is not selected.) Displays all genes in Gene
View, and highlights the genes from the gene list in their display color.
See “To show gene lists in Gene View” on page 98.
Show only
(Available only if all genes appear in Gene View, or if the gene list is
not selected) Restricts the genes in Gene View to those on the gene
list. No other genes appear. The program displays the genes in their
display color. See “To show gene lists in Gene View” on page 98.
Show All
(Available only for the selected gene list.) In Gene View, displays all
genes, without highlighting. See “To show gene lists in Gene
View” on page 98.
Tracks folder
• Right- click the Tracks folder to display these options:
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My Entity List pane – Actions and shortcut menus
Option
Comments
Import Tracks
Opens the Import Track dialog box, where you can import a
BED format track file into the program. See “Import Track” on
page 227 and “To import tracks” on page 53.
Export Tracks
Opens the Export Tracks dialog box, where you can select
tracks for export as a single BED format track file. See “Export
Tracks” on page 217 and “To export tracks” on page 82.
Combine Tracks
Opens the Combine Tracks dialog box, where you can
associate two or more individual tracks by logical criteria to
create a new combined track. See “Combine Tracks” on
page 191 and “To combine tracks” on page 77.
Track name
• Right- click the name of a track to display these options:
166
Option
Comments
Show in UI
Mark this option to display the track in Gene View next to the
data and results of the selected experiment. See “To show
tracks in Gene View” on page 100.
Show in Report
Mark this option to display the track in the reports.
Genomic Boundaries
Click to analyze data invoking the genomic boundaries for only
that track. You can choose to do this for only one track.
Show in UCSC
Opens the UCSC Genome Browser in your Web browser and
uploads the track. You can then view the track.
View Details
Opens a dialog box that displays information about the track.
See “Track” on page 254.
Rename
Opens an Input dialog box, where you can type a new name for
the track. Click OK to rename the track.
Delete
Opens a Delete Track dialog box that asks if you are sure you
want to delete the track. Click Yes to delete the track.
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My Networks pane
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My Networks pane
Contains the biological networks/pathways that you found using Network
Search or that you create using a literature search in eArrayXD. For more
information, see the eArrayXD Users Guide.
Tasks pane
Figure 42
Navigator Tasks pane
Many tasks that you do in Agilent Genomic Workbench generate jobs that
are completed in the background on your Agilent Genomic Workbench
server or on the eArray Web site. You use the Tasks pane to keep track of
the status of these jobs, and to take action on them when their results
become available.
Tasks pane – Icons, buttons, and special text
These icons, buttons, and special text items can appear in the Tasks pane
of the Navigator:
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Tasks pane – Actions and shortcut menus
Item
Details
An unexpanded folder (domain) that contains subfolders or other items.
An expanded folder. The items that it contains are visible in the Navigator.
Expands a folder to show its contents.
Collapses a folder to hide its contents.
A pending task with a status of Submitted. The task has been submitted to the job
queue, but no action has been taken on it yet.
A pending task with a status of Processing. The task has been submitted to the job
queue, and the program (or the eArray Web site) is processing the job.
A pending task with a status of Complete. The results of the job are now available for
your use.
A pending task with a status of Error. An error has occurred, and you must re-submit
the job. For probe or bait uploads, an error file is available that lists the errors in your
input file.
(Available when the Tasks pane is not collapsed.) Collapses the Tasks pane. When you
collapse the pane, its title bar appears at the bottom of the Navigator.
(Available when the Tasks pane is collapsed.) Expands the Tasks pane.
If the Tasks pane appears within the Navigator, this button detaches the pane and
opens it in a new window. If the Tasks pane appears in a window, this button
re-attaches the pane to the Navigator.
Tasks pane – Actions and shortcut menus
• Double- click the name of a folder to expand or collapse it.
• Right- click the name of a pending task to open a shortcut menu. The
shortcut menu contains commands that are appropriate to the type of
job, and its status.
For details on actions in the task menu, see the eArrayXD User Guide.
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Genomic Viewer
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Genomic Viewer
Genomic Viewer is the display for the DNA Analytics applications (CGH,
ChIP and CH3). It includes the three Views – Genome, Chromosome and
Gene Views – the Tab View and the View Cursor.
Genome View
Detach button
Resize
buttons
Selected
chromosome
The View Cursor
Figure 43
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Genome View (vertical orientation), with human chromosomes. The X chromosome is selected.
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Genome View
Genome View shows pictures of each of the distinct types of chromosomes
in the selected genome. A blue box is drawn around the selected
chromosome, and the View cursor appears as a blue line across the
chromosome.
Genome View actions and shortcut menus
• Click a chromosome to select it. When you select a chromosome,
Chromosome, Gene, and Tab Views show only genomic regions, genes,
and data for it. The specific location in which you click the
chromosome sets the position of the cursor. See “The View Cursor” on
page 177.
• On the selected chromosome, click anywhere to move the cursor. See
“The View Cursor” on page 177. This also moves the cursor in
Chromosome, Gene, and Tab Views.
• Right- click anywhere within Genome View to display a menu. If you
click View Preferences, the View Preferences dialog box opens, where
you can set preferences for the display. See “View Preferences” on
page 268.
• Click the Detach button
(located at the top center of the pane)
to remove Genome View from the main window and open it in a
separate window. To reattach the view, click its Close button
.
• Drag the side or bottom borders of the pane to resize it.
• On a border of the pane, click a resize button (for example, or ) that
points away from the pane to move that border all the way to the edge
of the main window. To move the border back to its previous location,
click the other resize button.
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Chromosome View
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Chromosome View
Detach button
Resize buttons
Log ratio axis
Chromosome name
Cytobands
Plot
area
Selected region
Chromosome
The View Cursor
Figure 44
Chromosome View, human X chromosome shown
Chromosome View shows a more detailed diagram of the chromosome you
select in Genome View.
• Cytobands and a plot area appear next to the chromosome.
• When you select arrays for display, their data appear in the plot
area.
• The cursor appears as a solid blue line across the chromosome and
the plot area.
• The selected region of the chromosome (if any) appears as a dotted
blue box in the plot area.
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Chromosome View
Chromosome View actions and shortcut menus
• Click a cytoband, any part of the chromosome, or anywhere in the plot
area to move the View cursor to that location. See “The View
Cursor” on page 177.
• Drag the pointer over any part of the plot area to select a chromosomal
region for display in Gene View. Drag parallel to the chromosome. This
also moves the cursor to the center of the selected region. See “The
View Cursor” on page 177.
• Right- click anywhere within Chromosome View to display a menu. If
you click View Preferences, the View Preferences dialog box opens,
where you can set preferences for the display. See “View
Preferences” on page 268.
• Click the Detach button
(located at the top center of the pane)
to remove Chromosome View from the main window and open it in a
separate window. To reattach the view, click its Close button
.
• Drag the side or bottom borders of the pane to resize it.
• On a border of the pane, click a resize button (for example, or ) that
points away from the pane to move that border all the way to the edge
of the main window. To move the border back to its previous location,
click the other resize button.
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Gene View
5
Gene View
Chromosomal
location axis
Displayed region
Detach button
Plot area
Log ratio
axis
CNV
Tracks
Genes
The View
Cursor
Figure 45
Gene View, with log ratio data from an experiment and CNV tracks.
Gene View shows a more detailed view of the chromosomal region you
select in Chromosome View. See “Chromosome View” on page 171.
• Regions that contain genes appear as small blue boxes. Gene names
appear nearby. You can customize the appearance of gene names.
Also, you can use a gene list to highlight genes of interest, or to
display only the genes in the list. See “To change the appearance of
genes in Gene View” on page 100, and “To show gene lists in Gene
View” on page 98.
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Gene View
• Log ratio data from selected arrays in the active experiment appear
as a scatter plot. You can also customize the scatter plot. See “To
change scatter plot appearance” on page 93.
• The location of the cursor matches the location of the cursors in
other views. See “The View Cursor” on page 177.
• The name of the chromosome, and the coordinates and size of the
displayed chromosomal region appear at the top of the pane.
• Imported tracks can also appear in Gene View. See “To show tracks
in Gene View” on page 100.
Scatter Plot
Figure 46
Scatter Plot command group in CH3 Gene View
You access the scatter plot command group in Gene View or through View
Preferences from the View tab. The commands are different for CGH, ChIP
and CH3 applications. Scatter plots appear in the Chromosome and Gene
Views but only if they have been selected in the View Preferences dialog
box.
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Methylation
Results
Mark the box to enable the Methylation Results scatter plot. Selection for
the CH3 Methylation Results scatter plot lets you color the methylation
results based on Z- score Values.
Log Ratios
Mark the box to enable the Log Ratios scatter plot. Selections for the CH3
Log Ratios scatter plot let you color the Log Ratios by Log Ratio Values or
Probe Score Values.
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Gene View
Configure Colors
and Ranges
5
Opens the Configure Coloring Ranges and Shades dialog box, where you
can set up the colors and ranges for the Methylation Results and Log
Ratios scatter plots. For more information, see “Configure Coloring Ranges
and Shades” on page 193.
Gene View buttons
Zooms in to see a smaller region in more detail.
Zooms out to see a larger region in less detail.
When in vertical orientation, scrolls up through the genes and data to
lower- numbered chromosomal coordinates.
When in vertical orientation, scrolls down through the genes and data to
higher- numbered chromosomal coordinates.
When in vertical horizontal orientation, scrolls left through the genes and
data to lower- numbered chromosomal coordinates.
When in vertical horizontal orientation, scrolls right through the genes and
data to higher- numbered chromosomal coordinates.
(Resize buttons) The button that points away from Gene View expands the
view. The other button restores the view to its original size. (These
buttons will appear horizontal if the view orientation is horizontal.)
(Detach button) Removes Gene View from the main window, and opens it
in a separate window.
Gene View shortcut menu and other actions
• Click anywhere in the plot area of Gene View to move the cursor to
that location. See “The View Cursor” on page 177.
• Drag an inside border of Gene View to resize the view.
• Right- click anywhere in the plot area of Gene View to display these
options:
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Gene View
Option
Description
Create Gene List
Opens the Create Gene List dialog box, where you can create a
new gene list based on the selected (or another) chromosomal
region. See “Create Gene List” on page 199 and “To create a
gene list” on page 74.
Upload Track to UCSC
Opens the Upload Track to UCSC dialog box, where you can set
parameters to upload the track to the UCSC (University of
California at Santa Cruz) Genome Browser in your Web
browser. You can then display the track and use the tools
available in the UCSC Web site to examine the data. See
“Upload Track to UCSC” on page 260.
Search Probes in eArray
Opens the Search Probes in eArray dialog box, where you can
set the region to search for probes in eArray.
Simple HD Search
Opens SimpleHD Probe Search in the Search tab, where you
can start a search of the Agilent eArray web site for probes in
the selected (or another) chromosomal region. See “Simple
HD Probe Search” on page 247. See the eArrayXD User Guide
for more information.
Chromosomal Location
Search
Opens Probe Search in the Search tab, where you can search
for probes based on their chromosomal locations. See the
eArrayXD User Guide for more information.
User Preferences
Opens the User Preferences dialog box, where you can set
user preferences on three separate tabs. See “User
Preferences” on page 262 for more information.
View Preferences
Opens the View Preferences dialog box, where you set
preferences for the Genomic Viewer. See “View
Preferences” on page 268.
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The View Cursor
5
The View Cursor
The View cursor reflects the center of the current chromosomal location of
interest. It appears in several Views:
• In Genome View, it appears as a blue bar across the selected
chromosome.
• In Chromosome View, it is a blue bar that appears across the
chromosome and across the plot area of the view.
• In Gene View, it is a blue bar that appears across the plot area and
tracks of the view.
Figure 47
Genomic viewer showing view cursors
The position of the cursor in one view is also the position of the cursor in
all views. The exact chromosomal location of the cursor appears in the
first cell of the Status bar. Several actions change the position of the
cursor:
• In Genome View, click anywhere on a chromosome to move the
cursor to that location.
• In Chromosome View, click a cytoband name, part of the
chromosome, or anywhere in the plotting area to move the cursor to
that location.
• In Gene View, click anywhere in the plotting area to move the cursor
to that location.
The cursor used in Gene View is the same cursor used for the tracks.
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Tab View
• In Tab View, click a row of a data table to move the cursor to the
chromosomal location for that row.
Tab View
Resize buttons
Detach button
Design tabs
Figure 48
Selected
array
Unselected
array
Selected row
Tab View
Tab View displays design annotation and log ratio data related to the
chromosome you select in Chromosome View.
• The exact column content of the tables depends on the specific tab
and design, but it always includes chromosomal locations of probes.
• The selected row of data appears highlighted in blue. This row
represents data that corresponds approximately with the location of
the cursor.
• Columns of log ratio data appear below the names of the specific
arrays to which they correspond. If an array is selected for display
in Chromosome and Gene views, a colored square appears next to its
name.
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Tab View
5
• Signal intensity (raw signals) and/or annotations appear if selected
from the View command ribbon.
Tab View tabs and buttons
You can see the following tabs and buttons in Tab View. See Figure 48 for
a diagram that identifies some of these elements.
Design tabs
A separate tab appears for each microarray design included in the active
experiment. The name of the design appears on each tab, along with an
icon:
– A methylation array design
– An aCGH array design.
– A gene expression array design.
– A ChIP- on- Chip array design.
When you click a design tab, the data and annotation for the arrays in the
design appear in Tab View. The program separates the arrays of the
design into the Arrays tab and the Calibration Arrays tab (see below).
Arrays tab
Selected Arrays
tab
(Available when you click a specific design tab.) Contains a table of data
and annotation for all arrays in a design that contain biological data.
Contains a table of data and annotation for the selected arrays from all
designs in the active experiment.
(Resize buttons) The button that points away from Gene View expands the
view. The other button restores the view to its original size
(Detach button) Removes Gene View from the main window, and opens it
in a separate window.
Tab View actions and shortcut menus
• Click the name of an array in a column heading to select the array for
display.
• Right- click the name of an array in a column heading to display these
options:
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Tab View
Option
Description
Rename Array
Opens an Input dialog box, where you can type a new name for
the array. This only changes the name of the array within the
active experiment.
Remove Array From
Experiment
Opens a confirmation dialog box. Click Yes to remove the link
between the array and the active experiment. This command
does not delete the data file from the program. To do this, see
“To remove data or design files from the program” on page 72.
Select Array
(Available if the array is not selected.) Selects the array for
display. A colored square appears next to the name of the
array.
Deselect Array
(Available if the array is selected.) Removes the array data
from scatter plots, and removes the column of the array from
the Selected Arrays tab.
Edit Array Color
Opens the Select Color dialog box, where you can change the
display color of the array. See “Edit Array Color” on page 207
and “To change the display color of an array” on page 89.
Edit Array Order
Opens the Edit Array Order dialog box, where you can change
the order in which the names of the arrays in a given design of
the active experiment appear in Tab View and in the Data
Navigator. In Gene View, when you view separate scatter plots
for each array, the plots also appear in this order. See “Edit
Array Order” on page 208 and “To change the order of arrays in
an experiment” on page 61.
Select All Arrays
Selects all arrays in all designs in the active experiment for
display. All arrays appear in the Selected Arrays tab.
Deselect All Arrays
Removes all arrays from display, and from the Selected Arrays
tab.
Scroll to Column
Opens the Scroll to Column dialog box, where you can select a
column in the current tab. The program then scrolls the data
table in the tab so you can see the selected column. See
“Scroll to Column” on page 236.
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Tab View
5
• Right- click a heading of a column other than an array data column
to open a shortcut menu with a Scroll To Column option. If you click
this option, the Scroll To Column dialog box appears, where you can
select a column in the current tab. The program then scrolls the data
table in the tab so you can see the column.
• Click an entry in a data table to select the row in which it appears.
This also moves the cursor to the location of the data point
corresponding to the selected row.
• Right- click a data table entry to display these options:
Option
Description
Find in column
Opens the Find in column dialog box, where you can search for
a specific text string within the column you clicked. See “Find
in column” on page 218.
Google
LocusLink
Opens your Web browser, and sends the column entry you
clicked as a search string to the selected site. The UCSC links
search the indicated University of California, Santa Cruz
database related to the indicated genome build. See “To
search the Web for information on probes in Tab View” on
page 110.
PubMed
UCSC HG15(April ‘03)
UCSC HG16(July‘03)
UCSC HG17(May‘04)
UCSC HG18(March‘06)
UCSC mm8(Feb‘06)
UCSC mm9(July‘07)
DGV(hg18)
GO
KEGG(HUMAN)
Customize Link
Opens the Customize Search link dialog box, where you can
create or edit a custom Web link that appears in this shortcut
menu. When you click a custom link, the program opens your
Web browser, and sends the column entry you clicked as a
search string to the site. See “Customize Search Link” on
page 203 and “To create a custom Web search link” on
page 110.
(other options)
If other options appear in this shortcut menu, they are custom
Web search links. Click them to open your Web browser, and
send the column entry you clicked as a search string to the
site.
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Status Bar
Status Bar
Cursor position
Genome build
Ratio type
Table size
Figure 49
Status bar
The Status Bar displays information related to the displayed data.
Cursor position
Genome build
Ratio type
The chromosomal location of the cursor. See “The View Cursor” on
page 177.
The genome build for the displayed data.
The mathematical type of the array data. The possible types are:
• ratio
• log2 ratio
• log10 ratio
• ln (natural log) ratio
182
Selected Row
The row in the displayed data table that is selected. The location of the
cursor is approximately the chromosomal location for this row.
Table size
The number of rows and columns in the displayed tab. The size appears
as
<# of rows> x <# of columns>.
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Dialog Boxes
5
Dialog Boxes
This section describes the dialog boxes specific to the interactive analysis
of the CH3 Application. They are listed in alphabetical order by title.
Add Gene List <name> to
Figure 50
Add Gene List <name> to
Purpose: Adds genes from one gene list (the source gene list) to another
(the target gene list).
To open: In the Design Data pane, right- click the name of a gene list,
then click Add to Gene List.
Select target
gene list
Build
The gene list to which genes are added. Select one from the list.
(Read- only) The genome build for the genes in the list. The builds of the
two gene lists must match.
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Agilent Feature Extraction Importer
Description
List of genes
Gene List Color
OK
Cancel
(Optional) Description of the combined gene list.
A list of the genes in the target gene list.
(Read- only) The display color of the target gene list.
Adds the genes from the source gene list to the target gene list.
Closes the dialog box without adding any genes to the target gene list.
Agilent Feature Extraction Importer
Figure 51
Agilent Feature Extraction Importer
Purpose: Lets you edit the name of the FE data file you will import and
to indicate whether you want to flip the red/green ratio for the data.
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Agilent Feature Extraction Importer
5
To open: In the Home tab, click Import > Array Files > FE File, select
the desired FE data file(s), then click Open.
Name
Dye Flip
Lets you edit the names of the FE files. You can change the names of the
files to names that are easier to recognize or remember.
For each array:
Select Normal if:
• The test samples were labeled with cyanine- 5 (red).
• The control samples were labeled with cyanine- 3 (green).
• The imported ratio (test/control) should be reported as is.
Select Flipped if:
• The test samples were labeled with cyanine- 3 (green).
• The control samples were labeled with cyanine- 5 (red).
• The imported ratio (control/test) should be reported with the ratio
inverted (test/control).
The program does not combine dye- flip pairs.
Overwrite arrays
with duplicate
names
Run in
Background
OK
Cancel
Mark this option to replace existing file(s) in the program with the
imported one(s), if they have the same name(s).
Imports the files, and lets you use your computer for other purposes while
the import occurs. This is especially useful if you have many files to
import.
Imports the files in the foreground. You cannot use your computer for
other purposes while the import occurs.
Cancels the entire import process without importing anything.
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Array Set
Array Set
Figure 52
Array Set dialog box.
Purpose: To select replicate arrays to combine in the analysis.
To open: Click Fuse in the Preprocessing ribbon.
The array set dialog box opens when you combine designs. See “To
combine (fuse) arrays” on page 115.
Table
Displays the arrays that are combined, in order of their values for the
ArraySet attribute. The program creates a separate combined array for
each group of arrays with a given value for ArraySet. The table also shows
the design ID for each array, and the design type.
Select
Normalization
Select Centralization normalization or none. See “Methylation Detection
and Measurement Algorithms” on page 278.
Remove arrays
from experiment
after fuse
Deletes the original individual arrays after creating combined arrays so
that the experiment won't contain duplicate data.
Continue
Cancel
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Click to create combined designs using the selected options.
Cancels any selections, and closes the dialog box.
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Batman Parameter Setup
5
Batman Parameter Setup
Figure 53
Batman Parameter Setup dialog box
Purpose: This dialog box is used to set the parameters for the Batman
tool of methylation analysis. For details on how this algorithm works, see
“Bayesian tool for methylation analysis (BATMAN)” on page 288.
To open: In the Analysis tab, under Batman, mark the Apply box.
Z-score
Select CPG
Window
Mark Use Z- score to use the Z- score in place of the log ratio values.
Select the local CPG window to use.
Select CPG File
Click Browse and select a FASTA file containing sequences with 400 bases
on either side of the center of the probe. The file must be in standard
FASTA format.
Select TM
mapping file
If the Tm information is present in the design, the TM map file will
display Available. If the Tm information is not in the design, you must
select a TM file to use. The Tm file must be a tab delimited text file with
probe name and Tm as two columns.
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Batman Report Dialog
Continue
Cancel
Click to apply the Batman parameters and display them in the views.
Click to close the dialog box without applying the Batman algorithm.
Batman Report Dialog
Figure 54
Batman Report Dialog
Purpose: This dialog box is used to select the output format and location
of the Batman report.
To open: In Reports, click Generate Batman Report.
Report Type
Output Format
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All methylation reports are probe- based.
Complete Genome – Creates report as .xls file for entire genome
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Batman Report Dialog
5
Per- Chromosome – Creates report for each chromosome as .txt file. Open
in spreadsheet program to see headers properly aligned.
Select File
Location
Click Browse to select a location for the file, and type a file name.
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Catalog and Workgroup Data
Catalog and Workgroup Data
Figure 55
Catalog and Workgroup Data dialog box
Purpose: Used to download Agilent Catalog and workgroup data sets from
the eArray website to the Agilent Genomic Workbench database.
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Combine Tracks
5
To open: In the Home tab, click Data.
After you install the Agilent Genomic Workbench client program, you can
transfer probe and bait sequences and annotation, as well as the genomic
coordinates of exon boundaries, from the eArray Web site. Although you
can transfer these data at any time after you install the client program,
you must transfer it before you can do certain tasks in eArrayXD that
require it. For example, to search for expression probes from the Agilent
Catalog, you must first transfer the Catalog expression probe data from
the eArray Web site to your server. See the eArrayXD User Guide for more
information.
Learn More
Download
NOTE
Opens an information box that describes the type of data that is
downloaded for the data type.
Submits a data download task that appears in the Tasks pane of the
Navigator. To view the status of the download, click eArrayXD > Job
queue > Tasks.
If your workgroup contains a large amount of data, the download may take a long time.
Combine Tracks
Figure 56
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Combine Tracks dialog box
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Combine Tracks
Purpose: Lets you create a combined track that contains elements of two
or more source tracks, associated by logical criteria. See “To combine
tracks” on page 77.
To open: In the My Entity List pane, right- click the Tracks folder, then
click Combine Tracks.
Name
New Condition
Delete Condition
Track
Operator
Reset
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The name of the combined track
Adds a new, empty row to the Track/Operator table in the dialog box.
Removes the bottom row from the Track/Operator table in the dialog box.
In each row, select a track to include in the combined track.
In each row, select the desired logical operator. This operator controls the
manner in which the program combines the track in this row with the
others. Select one of these options:
Operator
Comments
AND
Places an element in the combined track if it appears in both this track and any of
the others.
OR
Places an element in the combined track if it appears in either this track or any of
the others. If you set this operator for all tracks in the list, the result is a
nonredundant set of elements from all tracks.
MINUS
Removes the elements that appear in this track from the combined track, if they
otherwise appear there.
Removes all Track/Operator pairs from the table in the dialog box, and
clears the Name of the combined track.
Save
Creates the combined track, but does not close the dialog box.
Close
Closes the dialog box. Opens the Confirm track save dialog box if you
created a combined track, but did not save it.
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Configure Coloring Ranges and Shades
5
Configure Coloring Ranges and Shades
Figure 57
Configure Coloring Ranges and Shades dialog box
Purpose: This dialog box is used to enter ranges and select colors for
scatter plot options. Tabs show selections for Methylation Results and Log
Ratios options.
To open: In Gene View, move the mouse pointer over Scatter Plot to
display the scatter plot options and then click Configure. Or, click the
View tab and click View Preferences. Under Configure Coloring schemes,
click Configure Colors and Ranges.
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Configure Coloring Ranges and Shades
Table 4
Methylation Results Parameters
Parameter
Description
Z-score Values
Min
Type a minimum value for the range.
Max
Type a maximum value for the range.
Color
Click to open the Select Color dialog box, where you can select the
color you want to display for this range. See “Select Color” on
page 237 for more information.
Add Range
Click to add a row to the range table using the values displayed in Min
and Max, and the selected Color.
Remove Range
Click to remove the ranges with Edit/Delete box marked.
Edit Range
Click to edit range(s) with Edit/Delete box marked.
Range table
This table displays the defined ranges, including minimum and
maximum values, color for each range, and Edit/Delete selection.
Table 5
Log Ratios Parameters
Parameter
Description
Log Ratio Values
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Min
Type a minimum value for the range.
Max
Type a maximum value for the range.
Color
Click to open the Select Color dialog box, where you can select the
color you want to display for this range. See “Select Color” on
page 237 for more information.
Add Range
Click to add a row to the range table using the values displayed in Min
and Max, and the selected Color.
Remove Range
Click to remove the ranges with Edit/Delete box marked.
Edit Range
Click to edit range(s) with Edit/Delete box marked.
Range table
This table displays the defined ranges, including minimum and
maximum values, color for each range, and Edit/Delete selection.
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Configure Coloring Ranges and Shades
Table 5
5
Log Ratios Parameters (continued)
Parameter
Description
Probe Score Values
Min
Type a minimum value for the range.
Max
Type a maximum value for the range.
Color
Click to open the Select Color dialog box, where you can select the
color you want to display for this range. See “Select Color” on
page 237 for more information.
Add Range
Click to add a row to the range table using the values displayed in Min
and Max, and the selected Color.
Remove Range
Click to remove the ranges with Edit/Delete box marked.
Edit Range
Click to edit range(s) with Edit/Delete box marked.
Range table
This table displays the defined ranges, including minimum and
maximum values, color for each range, and Edit/Delete selection.
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Confirm Overwrite
Confirm Overwrite
Figure 58
Confirm overwrite dialog box
Purpose: When you import an experiment, it can contain designs and/or
arrays that have the same names as those already available in Agilent
Genomic Workbench. This dialog box lets you select which designs and/or
arrays to overwrite.
To open: This dialog box appears when you import a ZIP format
experiment file, and it contains designs and/or arrays that are already
available in Agilent Genomic Workbench. See “To import an experiment
file” on page 55.
Select the designs to overwrite
Design
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The names of the designs in the imported file that have the same names
as designs that are already available in Agilent Genomic Workbench.
Overwrite
Mark the check box for each design that you want to overwrite.
Select All
Marks all of the check boxes under Overwrite.
Deselect All
Clears all of the check boxes under Overwrite.
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Create Experiment
5
Select the microarrays to overwrite
Array
The microarray identification, usually a barcode.
Name
The names of the arrays in the imported file that have the same names as
arrays that are already available in Agilent Genomic Workbench.
Overwrite
Mark the check box next to each existing array that you want to
overwrite.
Select All
Marks all of the check boxes under Overwrite.
Deselect All
Clears all of the check boxes under Overwrite.
OK
Cancel
Overwrites the selected files (both designs and arrays) and closes the
dialog box.
Closes the dialog box, and returns you to the Import (experiments) dialog
box. See “Import (experiments)” on page 224.
Create Experiment
Figure 59
Create Experiment dialog box
Purpose: To create an organizational unit in the Experiment pane of the
Navigator to link to array data for display and analysis and to create the
links to the data for the experiment (see “Experiment Properties” on
page 209).
To open: In the Experiment pane of the Navigator, right- click the
Experiments folder, and click New Experiment, or click File > New
Experiment.
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Create Experiment
Name
Description
Briefly, describe your experiment with information that will help you
identify it.
Properties
Click to display the Experiment Properties dialog box where you can
select microarrays to add to your new experiment. See “Experiment
Properties” on page 209.
NOTE
198
Type a name for your new experiment.
Do not click OK until you have added arrays to your experiment in the Experiment
Properties dialog box or you will have an empty experiment. You can also add arrays to the
experiment later, by dragging and dropping the arrays from the Design Data pane of the
navigator. See “To add arrays to an experiment” on page 60.
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Create Gene List
5
Create Gene List
Figure 60
Create Gene List dialog box
Purpose: To limit the genes presented in Gene View to a preselected
number valuable for interpreting data
To open: Right- click Gene View, and click Create Gene List.
Name
Type in name of gene list.
Build
Select the genome build for the genes.
Description
Describe the type or nature of the genes in the list.
Set Chromosome Start-Stop
Select a chromosome and a region in Chromosome View for selecting the
genes in the list before you open the Create Gene List dialog box.
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Create Gene List
User Defined
Select to choose region from which the genes in Gene View are selected.
The chromosome selection list and the Start and Stop positions on the Y
axis are enabled when this option is selected. With this option, you can
override the selections you made before opening Create Gene List.
For complete
gene view
Select all the genes in Gene View.
For aberrant
region below
cursor
Select those genes that appear in the aberrant region just below the
cursor position in Gene View.
Chromosome
If you select User Defined, you can select a different chromosome than
had been selected before opening the Create Gene List dialog box.
Start
If you select User Defined, you type in a Start position to define the
region that contains the genes for the list.
Stop
If you select User Defined, you type in a Stop position to define the
region that contains the genes for the list.
Color
Change
200
Click to change the color of the gene list name in Data Navigator.
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Create Query
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Create Query
Figure 61
Create Query dialog box
Purpose: Used to find samples in an experiment, based on conditions that
you select. Also used to create and save an new query in the program.
Queries are used to find samples with attributes that match the conditions
of the query.
To open: In the Experiment pane, right- click the experiment folder, and
select Create Query.
Create Query
Query Name
Click the arrow and select a query from the list.
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Create Query
Execute
Select sample
from the
experiment
where
Executes the selected query. Results of the query appear in the Results
box.
Select conditions that are applied for the query. For each condition (row),
select a logical operator to apply. To add a condition (row) to the table,
click New Condition. Each condition has these elements:
• Conditions – The condition(s) against which the query searches.
• Logical Operation – (Available only if you configure more than one
condition.) The relationship between the condition and the next one in
the list. For example, if you select AND in Logical Operation for the
first condition, the query includes or excludes a sample if it passes
both the first condition and the next condition.
New Condition
Edit Condition
Delete Condition
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Opens the New Condition dialog box, where you create the parameters for
the condition. Adds a new, blank condition (row) to the table. See “New
Condition” on page 232.
Opens the New Condition dialog box, where you can change the
parameters for the selected condition.
Removes a condition from the list. To remove a specific condition, click
anywhere within the condition, then click Delete Condition.
New
Opens an Input dialog box, where you can type a name for the new query.
To accept the name, click OK. The program creates the query, and adds
the new name to the Query Name list.
Update
Saves any changes you make to the query conditions, without closing the
dialog box.
Delete
Opens a Confirm dialog box that asks you if you want to delete the
selected query. To delete the query from the program, click Yes.
Rename
Opens an Input dialog box where you can type a new name for the query.
To accept the name, click OK.
Results
Shows the sample results when you select a query and click Execute.
Clear
Clears the results of a query execution from the Results box.
Close
Closes the dialog box. If you created or changed a filter, but did not
update it, a Confirm dialog box opens. Click Yes to accept the changes, No
to reject the changes, or Cancel to return to the dialog box.
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Customize Search Link
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Customize Search Link
Figure 62
Customize Search Link dialog box
Purpose: This dialog box lets you create a custom Web search link in the
shortcut menu that appears when you right- click a table entry. The link
opens the URL of your choice, and sends the table entry to it as a search
string. See “To create a custom Web search link” on page 110.
To open: Right- click any entry in a tab in Tab View, other than a column
heading, then click Customize Link.
URL Name
URL
The name of the custom Web search link that appears in the shortcut
menu (see above). To edit an existing custom Web search link, select it
from the list.
The full uniform resource locator (URL) of the desired search page. For
the query string value, type <target>
For example, this URL sends the selected tab view entry to google.com:
http://www.google.com/search?hl=eng&q=<target>
New
Opens an Input dialog box, where you can type a name for a new custom
Web search link. Click OK to add the name to the URL name list.
Update
Saves the settings in the dialog box.
Delete
Deletes the selected custom Web search link.
Close
Closes the dialog box.
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Design Properties
Design Properties
Purpose: Gives general and detailed information about a given microarray
design. See “To display the properties of a specific design” on page 70.
To open: In the Design Data pane of the Navigator, right- click the name of
a genome build within a design folder, then click Show Properties.
Several tabs are available.
Attribute tab
Displays general identifying attributes of the array design, and statistics
such as the total number of features in the design. For Catalog designs,
the “Date” field is the date of the downloaded catalog design file.
Figure 63
Design Properties dialog box – Attribute tab
Non Unique Probes tab
Displays the nonunique probes in the design. Nonunique probes have more
than one mapping in the genome that is a perfect match.
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Design Properties
Figure 64
Design Properties dialog box – Non Unique Probes tab
S. No
The sequence order of the probes within the tab.
Probe
The name of the each nonunique probe.
Value
The chromosomal locations to which each of the probes binds. Because
these are nonunique probes, two locations appear for each probe.
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Design Properties
Data tab
Displays the names of the probes in the design and their target genomic
locations. The tab displays the probes for one chromosome at a time.
Figure 65
Select
Chromosome
Probe
Chromosome
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Design Properties dialog box – Data tab
The chromosome whose probes appear in the list. To display the probes
for another target chromosome, select one from this list.
The name (Probe ID) of each probe.
The name of the chromosome to which the probe is designed.
Start
The location on the selected chromosome of the first base pair for the
probe.
Stop
The location on the selected chromosome of the last base pair for the
probe.
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Edit Array Color
5
Edit Array Color
Figure 66
Edit Array Color dialog box
Purpose: To change the color of the arrays in an experiment
To open: Right- click the experiment name, and click Edit Array Color.
Select Array
Color
Mark the check box for the array(s) whose color you want to change.
Click to change the color for the selected array(s). If you selected more
than one, all the selected arrays will change to the same color.
Select All
Click to mark all the check boxes.
Deselect All
Click to clear all the check boxes.
Edit Color
Restore default
Click to change the color for the selected array(s). Same as Color button.
Click to restore the original color(s) to the selected array(s).
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Edit Array Order
Edit Array Order
Figure 67
Edit Array Order dialog box
Purpose: This dialog box lets you change the order of arrays in an
experiment, which defines the order in which the program displays arrays
and array data in the Experiment pane of the Navigator and in Tab View.
If you select the Stacked rendering style for scatter plots, the array order
also determines the order in which the scatter plots for the arrays appear.
To open: Right- click the name of an experiment, then click Edit Array
Order in the shortcut menu.
Array Name
Design
Order by
The arrays in the selected design, listed in their current order.
Select the name of a design. In Array Name, the program displays the
arrays associated with the selected design.
Select an attribute to use as a basis for ordering the list. For example, if
you select Barcode, the program reordered that Array Name list based on
Barcode.
Moves a selected array up in the Array Name list. To select an array in
this list, click the name of the array.
Moves a selected array down in the Array Name list. To select an array in
this list, click the name of the array.
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Experiment Properties
OK
Cancel
5
Applies the new array order.
Closes the dialog box without making any changes to the array order.
Experiment Properties
Figure 68
Experiment Properties dialog box
Purpose: To select the arrays to link to the experiment
To open: In the Create Experiment dialog box, click Properties, or in the
Experiment pane of the Navigator, right- click the experiment name, and
click Show Properties.
Experiment
Name:
Displays the name of the selected experiment.
Description
Text Box:
Displays the description of the experiment that was entered when the
experiment was created.
Select Design
Designs
Select the design whose arrays you want to add to the experiment.
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Experiment Properties
Genome Builds
Select the genome build for the design you selected, if the design has more
than one genome build.
Arrays
Array List
Displays the arrays in the selected design that are available for the
experiment.
• To select an array to move to the Selected Array List, click its name.
• To select additional arrays, hold down the ctrl key and click their
names.
• To select a contiguous block of arrays, click the name of the first array,
then hold down the Shift key and click the name of the last one.
Selected Array
List
Displays the arrays that you have selected for the experiment.
Moves the selected arrays in Array List to the Selected Array List. You can
move arrays from as many designs as you want, if they are all for the
same genome build.
Moves all of the arrays in Array List to the Selected Array List.
Removes an array from the Selected Array List. To select an array for
removal, click its name. If desired, you can re- add an array.
Clears the Selected Array List.
Display name by
OK
Cancel
210
Click to select how the array names are displayed in the experiment. The
Global Display name is the name assigned in Sample Manager for the
array. See the Sample Manager User Guide for more information.
Adds the selected arrays to the experiment and closes the dialog box.
Closes the dialog box without making any changes.
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Export
5
Export
Figure 69
Export dialog box – Several types of file exports use this dialog box. This specific example exports selected experiment(s) as a ZIP format file.
Purpose: Lets you select a location for an exported file.
To open: This dialog box appears after you select specific experiment(s),
track(s), or array attribute(s) to export. See “To export experiments” on
page 81, “To export tracks” on page 82, or “To export array attributes” on
page 80.
Look in
Displays the folder or other location whose contents appear in the main
pane of the dialog box. To select another folder or other location, click
.
Moves to the next higher folder level.
Displays the Desktop.
Creates a new folder in the selected location in Look in.
Displays the names, only, of folders, files, and other locations in the main
pane of the dialog box.
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Export Array Attributes
Displays both the names and information about folders, files, and other
locations in the main pane of the dialog box.
Main pane
Displays the folders, files, and other locations in the selected location in
Look in. Only files of the selected type are displayed. To select file, click
its name. To open a folder or other location, double- click its name.
File name
Displays the name of the file to which the exported content is saved. To
change the name, you can either select a file in the main pane of the
dialog box, or type a new name.
Files of type
Sets the type of files that are displayed. To show all files, click
select All Files.
Export
Saves the selected content to the location given in the dialog box.
Cancel
Cancels your selections and closes the dialog box.
, then
Export Array Attributes
Purpose: This dialog box lets you select arrays whose attributes you want
to export. It contains two tabs: an Array tab where you select the arrays,
and an Attribute tab where you select the attributes of the selected arrays
to export. See “To export array attributes” on page 80.
To open: In the Home command ribbon, click Export > Array Attributes,
or in the Experiment pane of the Navigator, right- click the name of an
experiment, then click Export Attributes.
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Export Array Attributes
5
Array tab
Figure 70
Export Array Attributes dialog box – Array tab
Select Design
Designs
Genome Builds
Displays all of the designs available in the program. Select the design for
arrays that you want to export.
Displays the genome build(s) for the design. Select the desired genome
build to display the arrays for a single genome build.
Arrays
Array List
Displays the arrays in the selected design that are available for export.
• To select an array to move to the Selected Array List, click its name.
• To select additional arrays, hold down the ctrl key and click their
names.
• To select a contiguous block of arrays, click the name of the first array,
then hold down the Shift key and click the name of the last one.
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Export Array Attributes
Selected Array
List
Displays the arrays that you have selected for export.
Moves the selected arrays in Array List to the Selected Array List. You can
move arrays from as many designs as you want, as long as they are all for
the same genome build.
Moves all of the arrays in Array List to the Selected Array List.
Removes an array from the Selected Array List. To select an array for
removal, click its name. You can later add a removed array.
Clears the Selected Array List.
Next
Cancel
Moves to Attribute tab to select attributes for export.
Closes the dialog box without exporting any array attributes.
Attribute Tab
Figure 71
214
Export Array Attributes dialog box – Attribute tab
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Export Array Attributes
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Attributes
Attribute List
Displays the attributes that are not exported for the selected arrays.
Selected
Attribute List
Displays the exported attributes for the selected arrays.
• To select an attribute to move to the Attribute List, click its name.
• To select additional attributes, hold down the ctrl key and click their
names.
• To select a contiguous block of attributes, click the name of the first
attribute, then hold down the Shift key and click the name of the last
one.
You must select the following mandatory attributes, or else you cannot import the attribute
file at a later time: Array ID, Global Display Name, Green Sample, Red Sample, Polarity.
NOTE
Removes an attribute from the Selected Attribute List. To select an
attribute for removal, click its name. You can add a removed attribute
later.
Clears the Selected Attribute List.
Moves the selected attributes in the Attribute List to the Selected Attribute
List.
Moves all of the attributes in the Attribute List to the Selected Attribute
List.
Back
OK
Cancel
Moves back to the Array tab for array selection or removal.
Opens the Export dialog box. See “Export” on page 211.
Closes the dialog box without exporting any array attributes.
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Export Experiments
Export Experiments
Figure 72
Export Experiments dialog box
Purpose: Lets you select experiments for export. The program exports all
array designs and data for the experiments as a single ZIP file. This file
does not include any parameter settings, array selections, or results. See
“To export experiments” on page 81.
To open: In the Home tab, click Export > Experiments.
Select experiments to export
Select All
Deselect All
OK
Cancel
216
Displays all experiments available for export. Mark each experiment you
want to export.
Selects all experiments for export.
Clears all check boxes under Select experiments to export.
Opens an Export dialog box. See “Export” on page 211.
Cancels the export and closes the dialog box.
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Export Tracks
5
Export Tracks
Figure 73
Export Tracks dialog box
Purpose: Lets you select tracks to export as a single BED format file. See
“To export tracks” on page 82.
To open: In the Home tab, click Export > Tracks.
Select tracks
Select All
Deselect All
OK
Displays all of the filters available in the program. Mark the check box
next to each track that you want to export.
Selects all available tracks for export.
Clears all of the check boxes under Select Tracks.
Opens the Export dialog box, where you can select a location for the
exported BED format file. See “Export” on page 211.
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Find in column
Cancel
Cancels the export and closes the dialog box.
Find in column
Figure 74
Find in column dialog box
Purpose: This dialog box lets you set search parameters for a specific
column entry for the selected chromosome. Based on these parameters,
the program can highlight the row of the first entry that matches. The
cursor then moves to the location defined in the row.
To open: Right- click any entry in a tab in Tab View other than a column
heading, then click Find in column in the shortcut menu.
Find in column
Direction
Type all or part of the entry you want to find.
Select a search direction:
• Up – Sets the search to move up the column from the selected location.
• Down – Sets the search move down the column from the selected
location.
Conditions
Mark any of these search options:
• Match Case – Mark this option to take case into account. For example,
if you mark Match Case, and you type aa351 in Find in column, the
search finds the next entry in the column that contains aa351. It does
not find entries that contain AA351 or Aa351.
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Find in column
5
• Match whole word – Mark this option to only find entries in which the
complete entry matches what you type in Find in column. For example,
if you type AA351 in Find in column, and mark Match whole word, the
program finds the next AA351 entry. It does not find entries such as
AA3512 or AA351992.
Find Next
Cancel
Finds the next matching entry in the selected column, and moves the
cursor to the location defined in the row that contains the entry. The
search is performed only for the chromosome selected in the Genome
View.
Closes the dialog box.
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Gene List
Gene List
Figure 75
GeneList dialog box
Purpose: Lets you view the names of the genes in a specific gene list and
to change the display color of the gene list. See “To display the genes in a
gene list” on page 75.
To open: In the My Entity List pane of the Navigator, right- click the
name of a gene list, then click View in Table.
Name
Description
220
(Read- only) The name of the gene list.
(Optional) Brief descriptive comments about the gene list, such as how it
was created or the nature of the genes in the list. You can edit the
description.
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Go To Gene/Genomic Location
S. No
Gene Names
5
The sort order number. This is the index number of each gene within the
gene list.
The names of the genes in the gene list.
Color
Opens the Choose Gene List Color dialog box, where you can change the
display color for the gene list. See “Combine Tracks” on page 191.
OK
Saves the gene list with any new description or display color, and closes
the dialog box.
Cancel
Closes the dialog box without making any changes to the gene list.
Go To Gene/Genomic Location
Figure 76
Go To Gene/Genomic location dialog box
Purpose: To find a specific gene location in Gene View by either selecting
the RefSeq by Symbol or by selecting the Genomic Location.
To open: Click Home > Go to Gene/Genomic location.
RefSeq by Symbol
Select the Reference Sequence accession symbol from NCBI, and click Go.
Genomic Location
• Chromosome – The chromosome number.
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Import
• Base Position – The position on the chromosome.
Click Go after selecting the chromosome number and the position of the
gene on the chromosome.
Cancel
Closes the dialog box.
Import
Figure 77
Import dialog box
Purpose: Lets you select files and import them into Agilent Genomic
Workbench.
To open: In the Home tab, click Import, then select any kind of import
except Genome Build or Track. The type of file for import appears in the
title of the dialog box. To import a gene list, right- click the Gene List
folder in the My Entities List pane of the Navigator, then click Import
Gene List.
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Import
5
Use the standard Windows Explorer commands in the dialog box to select
a file for import.
For some imports, you can select multiple files. Click the name of the first
file, then hold down the ctrl key and click the names of additional files. To
select a contiguous block of files, click the name of the first file in the
block, then hold down the Shift key and click the name of the last one.
File name
Files of type
Displays the name of a file you select for import.
Lets you select the types of files to display from the types shown in the
table below. To display all files, click
,then select All Files.
.
Import or Open
Cancel
File type
Extension
FE array File
*.txt
Axon array file
*.gpr
UDF file
*.txt
Design file (GEML)
*.xml
Axon design file
*.gal
Array attributes
*.txt
Experiments
*.zip
Filters
*.xml
Gene list
*.txt
Imports the file into the program. In some cases, the name of this button
is Open, rather than Import. Also, when you click Import, in many cases
one or a series of additional dialog box(es) lets you further define the
content for import. See the instructions for each type of import in
Chapter 2.
Cancels the import and closes the dialog box.
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Import (experiments)
Import (experiments)
Figure 78
Import dialog box (for experiments)
Purpose: Lets you select the specific experiments within a ZIP format
experiment file to import into the program. See “To import an experiment
file” on page 55.
To open: In the Home tab, click Import > Experiments. In the dialog box
that appears, select the desired ZIP format experiment file, then click
Import.
Select
experiments to
import
Select All
Deselect All
OK
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These columns appear:
• Import – Mark the check box for the experiment(s) to import.
• Experiment – The names of the experiments available for import in the
ZIP format experiment file.
Selects all of the experiments in the ZIP file for import.
Clears all of the check boxes under Import.
Imports the selected experiments into the program. If the name of an
imported array design or data file matches one that is already available in
the program, the Confirm overwrite dialog box appears, where you can
select the data and/or design files that you want to overwrite. See
“Confirm Overwrite” on page 196.
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Import GEML design files
Cancel
5
Cancels the upload and closes the dialog box.
Import GEML design files
Figure 79
Import GEML design files dialog box
Purpose: To display information in the design file and to remove any files
that you don’t want to import.
To open: In the Home tab, click Import > Design Files > GEML File.
Select the desired *.xml design files, then click Open.
File Name
ID
Type
The name(s) of the design file(s) for import.
The Agilent ID number for the design file
The application type; CGH, ChIP, miRNA, or gene expression.
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Import Genome Build
Species
Genome Build
Status
The species for the genome build. This appears automatically when the
Genome Build is selected.
The genome build for the design. If the genome build is not read
automatically, a “?” appears. Click Genome Build and select the correct
value from the list.
• Not Set – Appears if Genome Build and Species information is not
shown.
• Not Allowed – Appears if a Genome Build is selected that does not
match the design.
• Overwrite – Appears when the design file has been updated and will
overwrite any existing one of the same name.
• Valid – Appears when the file is new.
Remove
Start Import
Cancel
Click
to remove a specific design file from the list.
Starts the import of the design files in the list.
Cancels the upload and closes the dialog box.
Import Genome Build
Figure 80
Import Genome Build dialog box
Purpose: To import a new set of genome build files into Agilent Genomic
Workbench. See “To import a genome build” on page 52.
To open: In the Home tab, click Import > Genome Build.
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Import Track
Species
Build Name
5
The genome’s species of origin.
The name of the build to import.
Refseq File
The location of the RefSeq database file. This file contains chromosomal
locations of genes. To select a Refseq file, click Browse.
CytoBand File
The location of the applicable cytoband file. This file contains graphical
cytoband information for Gene View and Chromosome View. To select a
cytoband file, click Browse.
OK
Cancel
CAUTION
Imports the genome build and closes the dialog box.
Cancels the import and closes the dialog box.
Import only Agilent-supplied genome build files.
Import Track
Figure 81
Import Track dialog box
Purpose: Lets you import a BED format track file. See “To import
tracks” on page 53. Track information can appear in Gene view. See “Gene
View” on page 173.
To open: In the Home tab, click Import > Track.
Species
Select the species to which the track relates.
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Import Track
Build Name
Color
Track Name
Track File
Browse
OK
Cancel
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This list contains the available genome builds for the selected species.
Select the desired genome build.
The assigned display color for the track. To change this color, click
Change.
Type a name to identify the imported track.
Type the location of the BED track file that you want to import, or click
Browse to select a file.
Opens an Open dialog box, where you can select the BED track file to
import.
Imports the track into the program.
Cancels the import and closes the dialog box.
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Microarray Properties
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Microarray Properties
Purpose: Displays the properties associated with an array. You can also
edit the values of specific attributes. To add attributes to the list, see the
Sample Manager User Guide.
To open: For any array in the Design Data folder or Experiments folder,
right- click the array name, then click Show Properties. For non- Agilent
arrays, only the Attribute tab appears.
Attribute tab
Figure 82
Microarray Properties dialog box with list of Attributes and their values
• Attribute — Displays the attributes in an array by name. You can load
these from an Excel spreadsheet.
• Value – Indicates the values, if any, for each array.
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Microarray Properties
NOTE
Close
You cannot edit values for read-only arrays.
Closes the dialog box.
FE Headers Tab
Figure 83
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Microarray Properties dialog box with list of FE Headers and their values
Index
Displays a sequential index to help identify FE properties.
Name
Displays feature parameters, statistics and constants for the whole array.
Value
Displays the value for each parameter, statistic and constant.
Close
Closes the dialog box.
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FE Features Tab
Figure 84
Selection List
List Box
Microarray Properties dialog box with list of FE Features and associated data
Select the chromosome whose feature information you want to display.
Displays FE features and the associated data. The fields are:
Index
FeatureNum
ProbeName
gIsPosAndSignif
LogRatioError
PValueLogRatio
gProcessedSignal
rProcessedSignal
gMedianSignal
rMedianSignal
gBGSubSignal
rBGSubSignal
gIsSaturated
rIsSaturated
gIsFeatNonUnifOL
rIsFeatNonUnifOL
gIsBGNonUnifOL
rIsBGNonUnifOL
rIsPosAndSignif
gIsWellAboveBG
rIsWellAboveBG
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New Condition
New Condition
Figure 85
New Condition dialog box
Purpose: To create a condition for a sample query.
To open: In the Create Query dialog box, click New Condition or Edit
Condition. See “Create Query” on page 201.
Attributes
Attributes is the only type of condition you can set for the CH3
application type.
For an attribute conditions, do the following:
• In the list at the left- hand side of the dialog, select the sample attribute
for the condition.
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5
• In the second list, select an operator to apply to the range.
• Range – Type a start and stop value that defines a range for the
attribute.
OR
• Attribute Value – Type an attribute value for the attribute.
OK
Cancel.
Saves the new or changed condition and returns to the Create Query
dialog box.
Cancels the changes and closes the dialog box.
Probe Methylation Status Setup
Figure 86
Probe Methylation Status Setup dialog box
Purpose: To associate Tm (melting temperature) map files with the design
files for the arrays in the experiment or to make sure the Tm information
is already in the design file.
To open: Click Analysis, then mark Apply for the Probe Methylation
command
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Probe Methylation Report Dialog
Design Name
Tm Map File
Shows the names of the design files contained in the experiment
Click Browse to find the Tm map file for the design file. This is necessary
only if the design is a custom design or if Tm is still not available after an
attempted update.
If the design is an Agilent Catalog array, the Tm information is available
within the file as long as it has been updated. If the design file has not
been updated, the Browse button is active. If it has been updated, the Tm
Map File option says Available. If not, update it before continuing.
Continue
Cancel
The methylation algorithm calculates and displays the Z- score results.
Closes the dialog box without generating a report.
Probe Methylation Report Dialog
Figure 87
Probe Methylation Report dialog box
Purpose: To select whether the results are reported for the complete
genome or for individual chromosomes. See “Report Format” on page 272.
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Sample Attributes
5
To open: Click Reports, then click Generate Probe Report.
Report Type
Output Format
All methylation reports are probe- based.
Complete Genome – Creates report as .xls file for entire genome
Per- Chromosome – Creates report for each chromosome as .txt file. Open
in spreadsheet program to see headers properly aligned.
Select File
Location
Click Browse to select a location for the file, that you must name.
Sample Attributes
Figure 88
Sample Attributes dialog box
Purpose: To show, hide, or edit array attributes
To open: In the Experiment Pane of the Navigator, right- click the
experiment, and click Edit Attributes.
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Scroll to Column
This dialog box lets you enter or change the existing values for the
attributes listed for the arrays in the experiment. You can also show or
hide attribute columns. The columns that appear initially are the default
columns (Array ID, Global Display Name, Green Sample, Red Sample,
Polarity and Extraction Status) plus any that you selected to show.
Changes you make are applied globally. See the Sample Manager User
Guide.
Changes to array attributes you make in this table appear also in the Sample Manager
table.
NOTE
Scroll to Column
Figure 89
Scroll to Column dialog box
Purpose: This dialog box lets you select a column. The program then
scrolls the tab so that you can see the selected column.
To open: Right- click a column heading in Tab View, then click Scroll To
Column. in the shortcut menu.
Select column
OK
Cancel
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Displays the columns available in the selected tab. Select the one you
want to display.
Scrolls the current tab to show the selected column.
Closes the dialog box.
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Select Color
5
Select Color
Purpose: To select a color. Three tabs are available for selecting colors:
• Swatches tab - select colors based on samples (swatches)
• HSB tab - select colors based on an HSB schema (Hue, Saturation, and
Brightness)
• RGB tab - select colors based on an RGB schema (Red- Green- Blue)
To open: This dialog box opens when a function allows you to change a
color. For example, right- click on an array in an experiment, click Edit
Array Color and click the Swatches, HSB, or RGB tab.
Swatches tab
Figure 90
Select Color - Swatches tab
This tab is used to select a color based on color samples (swatches).
Preview
The Preview area shows how the selected color appears. When you change
the color, the original color appears at the top of the color box on the
right.
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Select Color
Recent:
OK
Cancel
Reset
Choose a recent color selection.
Click to select the color and close the dialog box.
Click to close the dialog box without changing the color.
Click to change swatches, HSB, and RGB colors back to the default colors.
HSB Tab
Figure 91
Select Color - HSB Tab
In this tab, you can select a color based on an HSB schema (Hue,
Saturation, and Brightness).
238
Hue
Click the H button, and move the slider up and down, or go up and down
the list of numbers, to select the hue or color of the array.
Saturation
Click the S button, and move the slider up and down, or go up and down
the list of numbers, to select the saturation level for the color.
Brightness
Click the B button and move the slider up and down, or go up and down
the list of numbers, to select the brightness level for the color.
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Select Color
RGB Numbers
Preview
OK
Cancel
Reset
5
Reflect the amount of red, green and blue in the resulting color.
The Preview area shows how the selected color appears. When you change
the color, the original color appears at the top of the color box on the
right.
Click to select the color and close the dialog box.
Click to close the dialog box without changing the color.
Click to change the swatches, HSB, and RGB colors back to default values.
RGB Tab
Figure 92
Select Color - RGB Tab
This tab is used to select a color based on an RGB schema.
Red
Move the slider to change the amount of red in the color. Or, click the up
or down arrow to select a number.
Green
Move the slider to change the amount of green in the color. Or, click the
up or down arrow to select a number.
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Select data type for experiments
Blue
Move the slider to change the amount of blue in the color. Or, click the up
or down arrow to select a number.
Preview
The Preview area shows how the selected color appears. When you change
the color, the original color appears at the top of the color box on the
right.
OK
Cancel
Reset
Click to select the color and close the dialog box.
Click to close the dialog box without changing the color.
Click to return the swatches, HSB, and RGB colors back to default values.
Select data type for experiments
Figure 93
Select data type for experiments dialog box
Purpose: Lets you select the mathematical form of the data in an
imported UDF file, and its associated application type. See “To import a
UDF file” on page 49.
To open: In the Home tab, click Import > Array Files > UDF File. In the
dialog box that appears, select the desired UDF file, then click Open.
Experiment Name
Data Type
By default, the experiment name is the name of the imported UDF file. To
change the name, double- click it, then edit it as desired.
Select the mathematical form of the array data in the UDF file. The
options are:
• ratio
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Select Experiment for Creating Query
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• log2 ratio
• log10 ratio
• ln ratio (base e)
Design type
Continue
Cancel
Select the application type (CH3, CGH, or expression, for example) for the
array data in the UDF file.
Accepts your selections, and goes to the next step in the UDF import
process.
Cancels the UDF import.
Select Experiment for Creating Query
Figure 94
Select experiment for creating query dialog box
Purpose: Used to select experiment(s) that will be used for a query.
To open: In the Experiment pane of the Navigator, right- click the
Experiment folder, and select Create Query.
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Select Users
Select
experiment for
creating query
Mark the check box next to each experiment you want to use for the
query.
Select All
Click to mark all check boxes.
Deselect All
Click to clear all check boxes.
OK
Cancel
Opens the Create Query dialog box where you can create a query for the
selected experiments. See “Create Query” on page 201.
Cancels the query and closes the dialog box.
Select Users
Figure 95
Select Users dialog box
Purpose: Select users who will share an experiment.
To open: From the Sharing for enterprise dialog box, click Add.
Click to select a user from the list. To select additional users, hold down
the Ctrl key and click the additional users to select them. You can also
select a contiguous block of users; click the first user in the block, then
hold down the Shift key and click the last one.
OK
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Adds the selected user(s) and closes the dialog box.
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Set genome build and species for Axon design files
Cancel
5
Closes the dialog box without selecting any users.
Set genome build and species for Axon design files
Figure 96
Set genome build and species for Axon design files dialog box
Purpose: Lets you set the species and genome builds for imported Axon
design file(s), and to remove specific designs files from the import, if
necessary. See “To import Axon design files” on page 47.
To open: In the Home tab, click Import > Design Files > Axon File. In
the dialog box that appears, select at least one Axon design file, then click
Import.
No.
File Name
An index number within the dialog box for each Axon file.
The names of each Axon design file selected for import.
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Set genome build and species for Axon design files
Species
Genome Build
Status
The species for each design file. If a species is incorrect, select the correct
one from the appropriate list.
The genome build for each of the design files. If a genome build is
incorrect, select the correct one from the appropriate list.
The status of the file is one of the following:
• Valid – The file is a new file that can be imported.
• Healthy – The file passes validation and can be imported.
• Not Set – Appears if Genome Build and Species information is not
shown.
• Not Allowed – Appears if a Genome Build is selected that does not
match the design, or if the design is an Agilent Catalog design.
• Overwrite – The file is a valid design file, but when you import it, it
will replace an existing design that has the same name.
• Corrupt – The file failed validation. When you start the import process,
the program ignores the file.
Remove
Start Import
Cancel
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Click
to remove a specific design file from the list. This is
useful if you select a design file in error, or if you do not want to
overwrite an existing one.
Imports the file(s) and closes the dialog box.
Cancels the import and closes the dialog box.
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Sharing for enterprise
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Sharing for enterprise
Figure 97
Sharing for enterprise dialog box
Purpose: Select users to share an experiment. Users you have shared the
experiment with can add or change the experiment.
To open: In the Experiment pane of the Navigator, right- click the name of
an experiment, and select Share.
Add
Remove
Opens the Select Users dialog box, where you select users you want to
share the experiment.
Removes the selected user(s) from the shared User names list.
OK
Accepts the changes and closes the dialog box.
Cancel
Cancels any changes and closes the dialog box.
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Show/Hide Columns
Show/Hide Columns
Figure 98
Show/Hide Columns dialog box
Purpose: Used to select the attributes for display in the Sample Attributes
dialog box and the Sample Utility tab. The Sample Utility tab is available
when you go to Sample Manager. See the Sample Manager User Guide for
information about Sample Manager.
To open: This dialog box appears when you click Show/Hide Attributes
at the bottom of the Sample Attributes dialog box.
All available attributes are shown in the Attributes column. Attributes
with a check- mark next to them are displayed in the Sample Attributes
and Sample Utilities tab for each sample. To select an attribute for display,
mark the Show in Table box next to it. To hide an attribute, clear the
Show in Table box.
Save
Select All
Deselect All
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Saves the current list of selected attributes and updates the Sample
Utilities table based on the selections.
Selects all the attributes in the list.
Clears all check marks from attributes in the list.
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Simple HD Probe Search
Close
5
Closes the dialog box. If changes have been made, the program asks if you
want to save your changes before closing.
Simple HD Probe Search
Figure 99
Simple HD Probe Search pane in the Search tab
Purpose: Lets you set up and submit a Simple HD Probe Search job to the
eArray Web site. Two main options are available for this type of search:
• Simple Genomic Intervals HD Search – (Available for CGH,
ChIP- on- chip, and methylation applications) Retrieves probes from the
Agilent HD probe database on the eArray Web site based on genomic
coordinates that you enter.
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Simple HD Probe Search
• Simple Gene Annotations HD Search – (Available for the CGH
application type) Retrieves probes from the Agilent HD probe database
on the eArray Web site based on gene annotations that you enter, such
as gene symbols or GenBank accession numbers.
NOTE
For more information on search functions for the eArray Web site, see the eArrayXD User
Guide.
To open: Right- click in the Gene View, and select Simple HD Search.
Search
Parameters
The table below describes the search parameters that can appear. You
must set the Search Name, the Species, the Select HD Search by setting,
and one or more Genomic Intervals or Gene Annotations. All other
parameters are optional, or can be left as is.
Parameter
Instructions/Details
Job Information
248
Search Name
Type a name that will help you to identify this search job and its
results.
Species
Select the desired species. The species that appear in the list
reflect the species available in the HD probe database for your
application type.
Build Number
(Read-only) The name of the applicable genome build for the
selected species. The HD probe database only contains probes
designed to the most current genome build for each species.
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Simple HD Probe Search
Parameter
5
Instructions/Details
Probe Options
Filters
Select one of the options below. If relevant, type a value for the
filter in FIlter Value. Filters restrict the list of returned probes,
based on specific criteria.
• None – The search does not apply any of the filters in the list.
• Average Spacing – In Filter Value, type the desired number of
base pairs. This defines the average number of base pairs
between the centers of the retrieved probes in each genomic
interval.
• Probes Per Interval – In Filter Value, type the desired number
of probes. This defines the maximum number of probes that
HD Search retrieves for each genomic interval.
• Total Probes – In Filter Value, type the desired number of
probes. This defines the total number of probes HD Search
collectively retrieves for all specified genomic intervals.
Filter Value
(Available if you select a filter) Type the desired value for your
selected filter.
Prefer Catalog Probes
(Available for HD-CGH probe searches, if you select a filter) To
give preference to Agilent catalog probes in the probe selection
process, mark this check box. If two probes are close to each
other for a given probe interval, HD search selects the Agilent
catalog probe.
Use TM Filter
Removes probes with TMs that produce unsatisfactory results on
the Agilent platform. The search always applies this filter.
Similarity Filter
The options below can appear. The availability of specific options
depends on species and application type.
• No Filter – The program does not apply a similarity filter. If you
select this option, the Use Non-Unique Probe Filter check box
becomes available (see below). Non-unique probes map to
more than one location in the target genome.
• Perfect Match Filter – Removes a probe from the search
results if it has more than one perfect match in the genome of
the selected species.
• Similarity Score Filter – Removes a probe from the search
results if it is designed to a given genomic region, but it also
has significant similarity to other parts of the target genome.
Probes such as this can cause cross-hybridization problems.
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Simple HD Probe Search
Parameter
Instructions/Details
Probe Options
Filters
Select one of the options below. If relevant, type a value for the
filter in FIlter Value. Filters restrict the list of returned probes,
based on specific criteria.
• None – The search does not apply any of the filters in the list.
• Average Spacing – In Filter Value, type the desired number of
base pairs. This defines the average number of base pairs
between the centers of the retrieved probes in each genomic
interval.
• Probes Per Interval – In Filter Value, type the desired number
of probes. This defines the maximum number of probes that
HD Search retrieves for each genomic interval.
• Total Probes – In Filter Value, type the desired number of
probes. This defines the total number of probes HD Search
collectively retrieves for all specified genomic intervals.
Filter Value
(Available if you select a filter) Type the desired value for your
selected filter.
Prefer Catalog Probes
(Available for HD-CGH probe searches, if you select a filter) To
give preference to Agilent catalog probes in the probe selection
process, mark this check box. If two probes are close to each
other for a given probe interval, HD search selects the Agilent
catalog probe.
Use TM Filter
Removes probes with TMs that produce unsatisfactory results on
the Agilent platform. The search always applies this filter.
Similarity Filter
The options below can appear. The availability of specific options
depends on species and application type.
• No Filter – The program does not apply a similarity filter. If you
select this option, the Use Non-Unique Probe Filter check box
becomes available (see below). Non-unique probes map to
more than one location in the target genome.
• Perfect Match Filter – Removes a probe from the search
results if it has more than one perfect match in the genome of
the selected species.
• Similarity Score Filter – Removes a probe from the search
results if it is designed to a given genomic region, but it also
has significant similarity to other parts of the target genome.
Probes such as this can cause cross-hybridization problems.
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Parameter
Instructions/Details
Use Non-Unique Probe Filter
(Available if you select no similarity filter) Mark this check box to
remove a probe if it maps to multiple locations in the target
genome.
You can set the stringency of this filter. See below,
See the eArrayXD User Guide for more information.
Max Perfect Genomic Hits
(Available if you select no similarity filter, and mark Use
Non-Unique Probe Filter) Sets the maximum number of
locations to which a probe can map in the target genome, and
still pass the non-unique probes filter. Type the desired number of
locations.
Example: Probe A maps to two locations in the target genome,
and Probe B maps to three locations. You select No Filter in
Similarity Filter, mark Use Non-Unique Probe Filter, and type 2 in
Max Perfect Genomic Hits. The filter removes Probe B, but does
not remove Probe A.
Interval Options
Select HD Search by
Genomic Intervals – Sets up the search to retrieve probes based
on genomic intervals that you enter.
Gene Annotations – Sets up the search to retrieve probes based
on gene annotations that you enter, such as gene symbols or
GenBank accession numbers.
Extend Interval Boundary
Type the number of 5' base pairs and 3' base pairs by which to
move out the start and end points of all of your genomic intervals.
This can help retrieve additional probes that lie outside the
initially defined regions of your genomic intervals.
Example: You type 500 in 5' base pairs and 300 in 3' base pairs.
eArray extends an original interval of 9000–10000 to 8500–10300.
Genomic Intervals
(Available if you select Genomic Intervals in Select HD Search
By) Type either a chromosomal location or a cytoband in the box.
Separate multiple intervals with pipe “|” characters. All of the
intervals must be of the same type. For information about how to
enter genomic intervals, see the eArrayXD User Guide.
Upload – Opens a File Upload dialog box, where you can upload a
file of chromosomal locations or cytobands.
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Parameter
Instructions/Details
Gene Annotations
(Available if you select Gene Annotations in Select HD Search
By) Type a gene annotation such as a GenBank accession
number (for example, NM_016660 or AY884282) or a gene symbol
(for example, H3N2 or CTSB) in the box. Use pipe “|” characters
to separate multiple annotations. eArray resolves annotations to
genomic intervals before it starts your search.
Upload – Opens a File Upload dialog box, where you can upload a
*.txt file of gene annotations. In the file, list one accession or
gene symbol per line. In a given search, the annotations must all
be of the same type.
Include Regions
(Available only for HD-CGH searches) Select one of the options
below. You can use this parameter to limit your HD-CGH probe
search to only exonic, or only intragenic regions of the genome.
• All – Retrieves probes in all of the specified genomic region,
both within and outside of gene boundaries.
• Exonic – Retrieves probes in exonic sequences within genes
in the specified genomic region. When you select this option,
the Gene Confidence list becomes available. Select the
appropriate Gene Confidence. See below.
• Intragenic – Retrieves probes found within gene boundaries,
whether or not they are found in exonic sequences. When you
select this option, the Gene Confidence list becomes available.
Select the appropriate Gene Confidence.
Gene Confidence
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(Available if you select Exonic or Intragenic in Include Regions.
See above.) Select the desired gene confidence level. This level
reflects the relative confidence in the source defining the gene
boundaries. For example, High confidence genes have known
proteins defined for them. If you set Gene Confidence to Low, the
search returns all High and Medium confidence genes as well as
ESTs and predicted genes.
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Simple HD Probe Search
Parameter
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Instructions/Details
Exclude Options
Standard Exclusion Interval(s)
eArray lets you select from among many sets of standard
exclusion intervals, based on annotation tracks. To ignore
genomic regions defined in one or more of these sets, mark this
option, then select the desired set(s) from the list that appears.
Control-click the names of additional sets to select them.
Custom Exclusion Interval(s)
To ignore the genomic intervals defined in a file of genomic
intervals, mark this option.
Upload – Opens a File Upload dialog box, where you can upload a
file of the desired genomic intervals.
You can set both standard and custom exclusion intervals in the
same search.
Search
Reset
Submits the search job to the eArray Web site.
Clears all parameters, or restores them to their default values.
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Track
Track
Figure 100 Track details dialog box
Purpose: This dialog box lets you view the chromosome locations in the
track.
To open: Click the Details link for the desired track in the Tracks tab of
the Preferences dialog box. See “Tracks tab” on page 262.
Track Parameters
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These parameters appear:
Parameter
Description
Name
The name of the track.
Species
The species to which the track applies.
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Track
Data
Parameter
Description
Format
The format of the track data. Agilent Genomic Workbench supports
the BED format.
Genome Build
The specific genome build of the species to which the track applies.
Description
Descriptive text saved with the track.
5
Tracks must contain entries for at least these four columns in the table:
Column
Description
Chromosome
The name of the chromosome
Start
The first base pair of the particular feature in the chromosome.
Stop
The last base pair of the particular feature in the chromosome.
Name
The name of the feature. This name appears next to the defined region
for the feature.
The other columns are additional BED track file columns that can appear
for some tracks. Agilent Genomic Workbench does not display these.
Close
Closes the Track dialog box.
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UDF Import Summary
UDF Import Summary
Figure 101 UDF Import Summary dialog box
Purpose: Reports how many lines of data were successfully imported from
a UDF file, and how many lines were skipped. Skipped lines are caused by
missing chromosome mapping information, or improper formatting of the
UDF file.
To open: Import a UDF file (see “To import a UDF file” on page 49). This
dialog box appears after you map the columns of the UDF file.
Table
OK
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Displays the file name of the imported UDF file, the number of lines that
were successfully imported, and the number of lines, if any, that were
skipped during import. If many lines were skipped, review the data for
improper formatting or missing chromosome mapping information.
Closes the dialog box.
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Universal Data Importer - Map Column Headers
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Universal Data Importer - Map Column Headers
.
Figure 102 Universal Data Importer - Map Column Headers dialog box
Purpose: Lets you set up a universal data file (UDF) for import. You select
several properties for the UDF, and identify the contents of each column
of data in the file. You can also save column mappings for re- use.
To open: As you go through the UDF import process (see “To import a
UDF file” on page 49), in the Select data type for experiments dialog box,
click Continue. See “Select data type for experiments” on page 240.
Species Info
Select Species
Select the species for the array data in the UDF.
Select Genome
Build
Sets the species- specific build to use.
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Universal Data Importer - Map Column Headers
Mapping Info
Select Mapping
Save Mapping As
Applies an existing column map to the current UDF. A column map
identifies the contents of each column of data. To create a new column
map for the current UDF, select CUSTOM.
Saves the column map under a new name. Opens an Input dialog box,
where you can type a name for the new map.
Array ID Info
Virtual Array ID
A number that uniquely identifies the data in the UDF. Typically, an
Agilent microarray slide has a physical barcode that Agilent Genomic
Workbench uses to generate an Array ID. The Array ID is used to track
the data from the slide as it goes through the steps of an analysis
workflow. A “virtual” Array ID is, by default, a system- generated ID that
serves the same purpose for data from UDFs. You can also create your
own virtual Array ID.
Use System
Generated Array
ID
By default, the virtual barcode assigned to the array data in a UDF is a
number that is created by the program. To create your own barcode, clear
Use System Generated Array ID, then type a new number in Virtual
Array ID.
Table
This table lets you identify the contents of the columns of data in the
UDF. The first row of the table displays the column heading information
from the UDF. The second row contains labels that you apply to each
column, and the rest of the table displays lines of data from the UDF. If
the UDF contains data from Agilent arrays, the column headings will
exactly match the labels in the lists.
In the list below each column heading, select the applicable label. You
must use each of the labels exactly once, except LogRatio, which you can
use more than once. These labels are available:
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Label
Description
ProbeName
Names of probes.
ChrName
Names of chromosomes.
Start
First chromosomal location for each probe.
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NOTE
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Label
Description
Stop
Last chromosomal location for each probe.
Description
Text annotation for the probe.
LogRatio
Array data values that correspond to each probe. You can use this
label more than once.
If you select an existing column mapping, then change or reset the column labels in the
table, the program changes or resets the saved column map as well.
Reset
Clears all the column labels in the second row of the table. If you have
selected an existing column mapping, this command also clears the labels
in the saved map.
Import
Imports the UDF file with the specified parameters, and opens the UDF
Import Summary dialog box (see “UDF Import Summary” on page 256).
Cancel
Cancels the import and closes the dialog box.
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Upload Track to UCSC
Upload Track to UCSC
Figure 103 Upload Track to UCSC dialog box
Purpose: Lets you select a track to upload to the UCSC Web site, where
you can view it in the UCSC genomic browser.
To open: Right- click in Gene View, and select Upload Track to UCSC.
Name
Type a name for the track. This name identifies the track when it appears
in lists and displays.
Build
(Available if you select User Defined in Set Chromosome Start- Stop.)
Select the genome build with which to associate the track.
Description
Set Chromosome
Start-Stop
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Type descriptive text to attach to the track for reference.
This parameter defines the region of the chromosome used for the track.
Select one of these options:
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Upload Track to UCSC
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• User Defined – Lets you define an arbitrary region of any chromosome.
If you select this option, select the desired chromosome in
Chromosome, then type the beginning (Start) and end (Stop) locations
of the desired interval.
• For complete gene view – The chromosomal region that appears in
Gene View.
• For aberrant region below cursor – All of the intervals that begin
before the cursor position and end after the cursor position.
Select Track
Source
The type of analysis result the program uses to construct the regions
defined in the track. Select one or both of these options:
• Methylation Score – Uses the current methylation score as the source
for the track.
Save as Track in
Genomic
Workbench
Saves the selected track in the Tracks folder in the My Entity List pane of
the Navigator.
OK
Creates the track. To display the track in Gene View, use the Tracks tab
of the User Preferences dialog box to enable it. See “Tracks tab” on
page 262. To export the track, see “To export tracks” on page 82.
Cancel
Closes the dialog box without creating a track.
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User Preferences
User Preferences
Purpose: This dialog box is used to set up preferences for display of
tracks, data storage locations, and licenses.
To open: From the Home tab, click User Preferences. Or, right- click in
the Gene View, Chromosome View, or Genome View, and click User
Preferences.
Tracks tab
Figure 104 User Preferences dialog box - Tracks tab
Purpose: To import and set up the appearance of tracks next to the Gene
View. Tracks are additional graphic displays of genomic information
loaded from an external file. They align with genomic coordinates in Gene
View.
To open: In the User Preferences dialog box, click the Tracks tab.
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User Preferences
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Font Options
Select the font type, style and size for the gene annotations that appear in
the selected tracks.
Tracks List
Track Name
Show in UI
Show in Report
Name of the track already loaded or imported
Mark the check box to display the track next to Gene View.
Mark the check box to display the track information in all the reports.
Genomic
Boundaries
Click to use the track to define only the regions that aberration detection
algorithms will run. You can choose to do this for only one track.
Delete
Mark the check box to delete the track from the list. Then, click Delete to
delete the track from the list.
Details
Click to display all the chromosome locations defined in the track.
Import
Click to import new tracks.
Delete
Click to delete the tracks selected in the Delete column.
Up
Down
Click to move a track up the list.
Click to move a track down the list.
Visualization Parameters
Genes
These options affect the appearance of the Track and Gene View.
• Orientation – Type a number to set the angle at which the Gene
Symbols will appear in Gene View and the Track Annotations appear in
the tracks.
• Show Gene Symbols – Mark to show gene symbols in Gene View, and
clear the check box to hide them.
Genomic
Boundaries
Tracks
These options let you include or exclude the Genomic Boundaries from the
analysis.
These options affect the appearance of the Track Views.
• Show Annotations – Mark to show the names of the gene regions for
the tracks, and clear to hide them.
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User Preferences
• Show Overlaid – Mark to overlay all the tracks that appear next to
Gene View, and clear the check box to display the information in
separate tracks.
Miscellaneous tab
Figure 105 User Preferences dialog box – Miscellaneous tab
Purpose: For data/content set- up, this dialog box allows you to set up
eArray access and to change the location for data.
To open: In the User Preferences dialog box, click the Miscellaneous tab.
eArray User
Details
Sets login details for the Agilent eArray Web site.
• URL – At present, https://earray.chem.agilent.com
• Username – The name registered on the eArray site.
• Password – The password registered on the eArray site.
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User Preferences
Error Model
5
(Not available for CH3) The DLRErrorModel (Derivative Log Ratio)
measures noise in the data for CGH analyses.
Data Location
The folder where the program stores array data and design files. To select
a location, click Browse.
Configuration
Parameters
Lets you set or change location for the database server. Do not change
these unless directed to do so by your network or database administrator.
• Database host – The fully qualified name for the computer that
contains the Agilent Genomic Workbench database.
• Database port – The port number on which MySQL Server listens for
its clients.
• Common Storage Location – (for display only) The location of the
common file storage area used to store and access files created during
various operations of Agilent Genomic Workbench. The location is set
during installation of Agilent Genomic Workbench server and cannot be
changed here.
Change
Click to enable configuration parameters.
Restore
Click to restore configuration parameters to the original settings.
Change Writer
Preferences
Opens the File Writer Preferences dialog box, where you select file types
that eArray creates for new microarray designs (in addition to the
defaults). See the eArrayXD User Guide for more information.
Edit Proxy
Settings
Opens the Edit Proxy Settings dialog box, where you select to use a proxy
and type the proxy settings to use for an Agilent Genomic Workbench
client. For more information, see the Product Overview Guide.
Edit Server Proxy
Settings
Opens the Edit Server Proxy Settings dialog box, where you select to use a
proxy server and type the proxy server settings to use for an Agilent
Genomic Workbench server. For more information, see the Product
Overview Guide.
Apply
OK
Cancel
Applies any changes to the preferences.
Accepts any changes and closes the dialog box.
Cancels all changes and closes the dialog box.
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User Preferences
License tab
Figure 106 User Preferences dialog box – License tab
Purpose: The License tab allows you to display and update your CH3
application license. This license enables the CH3 application, and allows
you to use it to analyze array data.
To open: the In the User Preferences dialog box, click the License tab.
Host Name
Select Analysis
Application
266
Displays the host computer name automatically.
Select the Agilent Genomic Workbench application for which you have a
license.
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Server Location
Select this option if you have a concurrent user license. To edit this name,
select Server Location, then type name of the computer used as your
license server. If you select this option, the Text License option is
unavailable.
Text License
Select this option if you have an application license (CGH, ChIP, CH3). To
change the license, delete the old license text, and paste the new license
text in the box.
OK
Accepts any changes you have made, and closes the dialog box.
Cancel
Closes the dialog box without changing any license information.
Apply
Accepts any changes you have made, but does not close the dialog box.
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View Preferences
View Preferences
Figure 107 View Preferences dialog box
Purpose: This dialog box allows you to configure how data and results
appear in Genome, Chromosome, and Gene views.
To open: In the View tab, click View Preferences.
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View Alignment
5
Selects the orientation and rendering style (described below).
Option
Description
Orientation
Horizontal
Stacks Genome, Chromosome, and Gene views horizontally in the
main program window. Genomic locations appear across the bottom
of each view.
Vertical
Displays Genome, Chromosome, and Gene views from left to right as
side-by-side panes in the main program window.
Rendering Style
Data Visibility
Overlaid
In Chromosome View and in Gene View, displays data and results as a
single, combined pane for all arrays. (Default)
Stacked
In Chromosome View and in Gene View, displays a separate pane for
each array.
For each view, or all views, selects the kind(s) of data and results to
display.
In View, select the view you want to configure. To set availability of
display items for all views, select All views. Some display items are only
available for certain views. When you select a display item, it enables the
item for display — for some items, you must take additional steps to
display them. For example, you may need to configure a specific algorithm
in the toolbar.
Mark any of the following options, as available:
Display item
Description/Comments
Scatter Plot
The plot(s) of individual log ratio data points.
Scatter Tool Tip
The ToolTips that appear when you place the pointer over specific data
points on the scatter plot(s) in Gene View. The tool tip shows the array
of origin and the numerical log ratio value for the data point.
Moving Average
The result of the Moving Average algorithm. See “To set up a moving
average (Log Ratio) calculation to smooth the data” on page 117.
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View Preferences
Rendering
Patterns
Display item
Description/Comments
ZScore
Marking this check box displays the results from the methylation
algorithm to produce the statistical likelihood of methylation or no
methylation in CpG Island regions of the genome.
Log Ratio Error
Envelope
Mark this check box to display the error envelope for log ratio values.
These options control the specific appearance of data and results in
Genome, Chromosome, and Gene views. You configure these options
separately for each type of array design.
• Design Type – Select the type of design to which the patterns are
applied: CH3, Expression, or Other.
• Styles – Select the display style for each of these elements:
Display element
Details
Log Ratios
Select how to display individual data points as: Color filled circles
(ellipses), circles, rectangles, filled rectangles, + signs, or x signs.
The latter two choices increase performance.
Moving Average
Select the line style for the moving average display. Lines appear in
the display color defined for each array. See “To set up a moving
average (Log Ratio) calculation to smooth the data” on page 117.
•
•
•
•
Scatter Plot (Chr
View) Point Size
NOTE
270
Continuous – A solid line.
Dashed – A dashed line.
Dotted – A dotted line.
Do not show area – No line.
Select a point size to use for display of scatter plot data points.
Rendering scatter plots for more than 10 high density arrays in the Chromosome View may
take significant time. Selecting filled circles as the rendering style for CH3 scatter plots can
also decrease performance. For faster performance, change the rendering style for CH3
data from the filled circle to the plus (+) or cross hair sign.
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View Preferences
Configure Scales
5
For Methylation Results or Log Ratios plots, mark Apply to enable the
custom scale. In Range, type the value to use as the range for the scatter
plot.
Configure
Coloring schemes
Use these options to change the display of the scatter plot in the Gene
View. These options are the same as those displayed in the Scatter Plot
ToolTip in the Gene View.
Show Memory
Monitor in Status
Bar
Displays a memory usage monitor in the eighth cell of the status bar. For
information about the Status Bar, see “Status Bar” on page 182.
OK
Applies the changes you made to all preferences and closes the dialog box.
Cancel
Closes the dialog box without applying changes.
Apply
Applies changes without closing the dialog box.
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Report Format
Report Format
The Methylation Report contains all the statistical results to help you
determine if there are methylated or unmethylated regions in the genome.
The report is created in .xls format for the complete genome or in .txt
format for individual chromosomes. Use a spreadsheet program to open
the report(s).
To learn the meaning of the values in the ZScore columns see “Overview
of Methylation Detection and Visualization Algorithms” on page 276.
Methylation Reports contain the following columns, in this order:
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Column
Description
CytoBand
Accepted name of the cytoband for each section of chromosome
ChrName
Name of the chromosome (for example, chr17)
ProbeName
Name of the probe (for example, P)
Start
The first base pair of the chromosomal location to which the probe
binds
Stop
The last base pair of the chromosomal location to which the probe
binds
Description
Name or phrase for the type of probe (for example, promoter)
GeneNames
Names of each gene in whose region probes are located
“Name of CpG Island
Track”
Name of each CpG Island in whose region probes are located
Combined ZScore
Combination of both the methylated and unmethylated ZScores. The
higher the positive combined ZScore, the more likely the probe is
methylated and vice versa.
ZScore_Methylated
Probe ZScore that are contained in right Gaussian. All of these values
are positive or small negative values.
ZScore_Unmethylated
Probe ZScore that are contained in left Gaussian. All of these values
are negative or small positive values.
logOdds
This number reflects how likely it is that a probe is methylated rather
than unmethylated. The higher the positive value, the more likely it is
methylated.
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Report Format
Column
Description
logRatio
Log ratio from the extracted FE image file for the probe
Tm
Melting temperature for the probe region
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User Guide
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Overview of Methylation Detection and Visualization Algorithms 276
Methylation Detection and Measurement Algorithms 278
Methylation status detection algorithm 278
Bayesian tool for methylation analysis (BATMAN) 288
Visualization Algorithms 293
Moving average (linear smoothing) 293
Triangular smoothing 294
Appendix 297
References 298
This chapter provides implementation details for the algorithms used in
the Methylation (CH3) application of Agilent Genomic Workbench 6.5. The
methylation algorithms facilitate the statistical analysis of methylated
genomic regions. The first section presents the methylation detection and
measurement algorithms. This is followed by a section that describes the
visualization options available for methylation analysis.
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Overview of Methylation Detection and Visualization Algorithms
Overview of Methylation Detection and Visualization Algorithms
The Methylation module of Agilent Genomic Workbench provides
algorithms for genomic methylation detection and visualization. The main
methylation detection algorithms are described below. Information on
algorithms that visualize the data follows.
Methylation detection and measurement algorithms
Methylation of cytosines in DNA is an epigenetic modification that can
play a role in the regulation of gene expression. Generally, methylation is
associated with repression of gene expression. Much of the aberrant DNA
methylation is found in CG- rich regions, termed CpG islands.
Agilent catalog methylation (CH3) arrays are specifically designed to
assess the methylation pattern within CpG islands and gene promoter
regions. The methylation detection algorithm is designed to be compatible
only with the Agilent protocol for DNA methylation analysis and the
Agilent methods for affinity- based methylated DNA enrichment.1
Methylation status detection algorithm
The methylation status detection algorithm is designed for two- color
assays, where the green (Cy3) channel is comprised of input DNA, and the
red (Cy5) channel is comprised of the affinity- enriched DNA. As a result
of constraints imposed on the array design, the probes that target genomic
regions with varying CG content do not share a uniform melting
temperature (Tm), which can result in compression of the log- ratios.
The methylation detection algorithm allows you to normalize the log- ratios
for each probe, based on its Tm, and returns the methylation status of a
probe. This sharpens the bimodal distribution observed with log- ratios
alone. For more information, see “Methylation status detection
algorithm” on page 278.
BATMAN algorithm
The BATMAN (BAyesian Tool for Methylation ANalysis) algorithm2 has
been adapted here for analysis of Agilent methylation microarrays. This
algorithm takes into account the local density of CpG dinucleotides in the
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genomic vicinity of the interrogating probes, and estimates methylation
events using this information. The algorithm is used with log ratio
observations and can additionally incorporate the effects of probe melting
temperature corrections based on the output Z- scores from the
methylation status detection algorithm. For more information, see
“Bayesian tool for methylation analysis (BATMAN)” on page 288.
Visualization algorithms
Moving average – linear
The moving average algorithm sets a fixed window size around every point
of interest. The points are either the probe log- ratio scores determined
prior to the methylation detection algorithm, or the combined scores that
result from application of the methylation detection algorithm. The
program reports the value for each point as the average of that point and
neighboring points within the window boundaries. For more information,
see “Moving average (linear smoothing)” on page 293.
Moving average – triangular smoothing
The triangular smoothing algorithm is a shaped algorithm based on a
moving average. As the moving average centers on a point of interest, the
program applies a maximum weight, and that weight falls off with
increasing distance. The program adjusts the point of interest as the
weighted mean of itself and neighboring points. For more information, see
“Triangular smoothing” on page 294.
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Methylation Detection and Measurement Algorithms
Methylation Detection and Measurement Algorithms
Agilent Genomic Workbench provides two algorithms for methylation
detection and measurement of the methylation levels detected by
Methylated DNA Immunoprecipitation (MeDIP). The methylation status
detection algorithm uses a melting temperature differential to discriminate
between methylated CpG regions and unmethylated regions. The Bayesian
tool for methylation analysis (BATMAN) algorithm uses conditional
probabilities from a calibration step to estimate methylation levels in CpG
regions. The BATMAN algorithm may be used directly on the log- ratios, or
it may be used on the resultant Z- scores from running the methylation
status detection algorithm.
Methylation status detection algorithm
You find epigenetic methylation predominantly in genomic areas of
increased GC content, known as CpG islands. Agilent human methylation
arrays specifically query approximately 25,000 CpG islands in the human
genome, in addition to gene promoter regions. The Agilent methylation
protocol uses antibody enrichment for 5- methylcytosine, which results in a
log- ratio of the relative abundance of genomic fragments with methylation
to those without.
Although the log- ratio scores give information about the relative
abundance of the genomic fragments, individual specificities of the probes
in each log- ratio score vary by melting temperature. Differences in melting
temperature can therefore decrease probe specificity. Normally, the probes
on Agilent arrays are chosen in such a way as to minimize probe- to- probe
differences in melting temperature.
The algorithm first bins the probes by their melting temperature. Within
these bins, you can make direct probe comparisons. When you compare
probes by log- ratio scores within each bin, you observe a bimodal
distribution — a mode comprised of those probes that were not
methylated, and a mode comprised of methylated probes. You also observe
a bimodal distribution in per- Tm Z- normalized scores.
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The algorithm determines the methylation status of a probe in a
Tm- dependent manner. To determine which probes are methylated, the
binning process compares log- ratios among probes of similar melting
temperatures.
To apply parametric statistical techniques, the algorithm fits the bimodal
distribution to a set of Gaussian curves, each of which requires only a
limited number of parameters to accurately fit the data. The algorithm
uses normalized Z- scores from the Gaussian distributions to effectively
judge the methylation status of a given probe on the array.
Purpose
Detection of methylation status is a visualization and report analysis that
determines the confidence score for probes that query known CpG islands
and gene promoter regions. To determine the probe methylation status, the
analysis uses a combination of log- ratio scores from competitive
hybridization methods, and the range in melting temperatures among
probes located in CpG islands.
Use
To establish a link between genomic methylation and gene expression,
detection of methylation status is used to identify probes likely to query
methylated genomic regions. CpG islands, measured on the Agilent catalog
methylation (CH3) design array, generally are either fully methylated or
fully unmethylated. You use the algorithm to identify methylation status
for probes. You can then visualize the probes in a genomic context — for
example, CpG islands within the genome.
Algorithm
The algorithm first bins the probes by their melting temperature. For each
bin, it applies Gaussian fits using one of three models. It fits the probe
log- ratios to Gaussians, using a local searching algorithm called random
hill climbing.3 Z- scores and p- values derived from the Gaussian data give
probabilities and confidence values for methylated and unmethylated
probe populations. The algorithm then calculates a methylation logOdds,
which gives the relative probability that a probe is more likely methylated
than unmethylated.
NOTE
The logOdds score indicates how likely a probe is to be methylated. It compares p-values
from the methylated to unmethylated populations. If they are the same, this value is zero.
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Step 1: Create a binned distribution of probes
To compare log- ratio scores between genomic fragments enriched by
methylation and those without methylation, the algorithm orders probes
into discrete melting temperatures.
7 Probes are binned according to their melting temperature. Each bin is
1ºC wide.
8 The algorithm fits the Gaussian distributions to the probe log- ratios
within each Tm bin. It does this by further dividing the probe log- ratios
into sub- bins (with default size of 0.1 ºC), to generate the observed
distribution of signals, as shown in Figure 108.
Figure 108 Overview of procedures in the methylation status algorithm
Step 2: Fit Gaussian curves to the binned data
For each bin of 1ºC, the algorithm applies Gaussian fits in one of three
models, depending on whether the data points are unimodal or
multimodal:
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• If the data are unimodal, then it fits one Gaussian distribution to the
data.
• If the data are bimodal (the most common fit for methylation data),
then it fits two overlapping Gaussian distributions to the data.
• If the data are trimodal, then it fits three overlapping Gaussian
distributions to the data.
NOTE
One Gaussian goodness of fit can exceed two Gaussians if, for example, no methylated
probes were pulled down during the antibody enrichment process, or if the number of
methylation events is exceedingly small. If this occurs, Agilent Genomic Workbench
automatically fits one Gaussian to the data, and the message “SINGLE GAUSS” appears in
the report. The Z-score in the visualization and report is then calculated from the single
Gaussian (that is, how likely the probe is to be methylated), instead of a combined Z-score
(which is reported from models using either two or three Gaussians).
The process to fit a Gaussian curve to the data has two steps:
1 Initial parameter approximation
2 Improvement of the model parameters using a local search procedure
called random hill climbing.3
Initial parameter
approximation
For each Gaussian curve, the parameters to be fit are the mean (μ),
standard deviation (σ), and mixture coefficient (α, the total Gauss
integral). For each of the three possible models, the algorithm
approximates the initial parameters as follows:
• First Gaussian
For any of the three models, the parameter estimations are done in
the following ways:
1 The mean is estimated by the maximum bin.
2 The standard deviation is estimated using the IQR (Inter- Quartile
Range) of the distribution, as inferred from the data left of the mean.
3 The mixture coefficient is estimated from the maximum bin height.
• Second Gaussian
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If the data points are bimodal or trimodal, the first Gaussian is
subtracted from the distribution. The mean, standard deviation, and
mixture coefficient are then estimated in the same way as the first
Gaussian.
• Third Gaussian
If the data are not well explained by either a unimodal or bimodal
distribution, then a third Gaussian curve can be fit to allow a better
estimate of the first and second Gaussian parameters. In this case,
the parameter estimates are:
1 The mean is estimated from the mean from the entire bin.
2 The standard deviation is estimated from the entire bin.
3 The mixture coefficient is estimated from the height of the bin used
to estimate the third Gaussian mean.
Parameter
improvement
procedure
Because many probes are generally present in each Tm bin (on the order
of thousands), the algorithm uses a straightforward local search procedure
called random hill climbing3 for parameter optimization. This procedure is
highly accurate and extremely fast when fitting the Gaussian models to
the data.
In each step, the algorithm alters a random parameter. It accepts this
alteration if it improves the target fitness function. To converge to a
(locally) optimal result, each random step gets shorter with each iteration.
This reduction in step is made logarithmically by n, the number of
iterations, and a default starting value of 100 (termed LB):
log ( LB + n )
----------------------------log ( LB )
(1)
The algorithm achieves convergence when a new step does not yield an
improvement in the target fitness function. The procedure converges if no
change occurs to the fitness value for 1000 iterations, or after the
algorithm has performed 50000 iterations.
Target fitness
function
282
The default target function is the Chi- squared goodness of fit. This
function is described using an observed distribution ODi=1…N, and an
estimated distribution function EF (for example, the convex combination
of two Gaussians), as follows:
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N
2
χ =
6
2
OD i – EF i
∑ --------------------------EF i
(2)
i=1
Step 3: Calculate Z-normalization scores
After the algorithm fits the Gaussian curve(s) to the data, it performs
parametric statistical analysis. The log- ratios from the Tm- binned probes
may differ in value. Agilent Genomic Workbench therefore uses a measure
from the Gaussian distributions themselves to normalize the scores. This
measure is called a Z- score:
log – ratio probe – μbin
Z = -------------------------------------------------------σbin
(3)
where μbin is the mean and σbin is the standard deviation of the Gaussian
distribution. The Z- score is a measure of the distance of a given probe
log- ratio score from the mean of any of the Gaussian curves, given the
standard deviation of that curve. The Z- score normalizes comparisons of
probes from different bins, by taking into account the Gaussian- fit curves
of the different bins.
Because the methylation model typically contains two Gaussians, the
algorithm calculates Z- scores for each one. The left Gaussian represents
those probes with a lower log- ratio score, and consists predominantly of
unmethylated probes. The left Gaussian is also the major mode, because
the majority of probes are generally unmethylated. The right Gaussian
represents probes with a high log- ratio score, and consists predominantly
of probes enriched for methylation. The algorithm calculates the following
Z- scores as statistical measures of whether or not a probe is methylated:
1 The Z- score derived from the left Gaussian. A negative or small positive
value means that the probe is likely to be unmethylated.
2 The Z- score derived from the right Gaussian. A positive or a small
negative value means that the probe is likely to be methylated.
3 The combined Z- score. This is the summation of the left and right
Gaussian Z- scores. It reflects the location of a probe log- ratio value in
relation to the Gaussian distribution(s) of probes with similar Tm. A
strong positive value of the combined score means it is methylated,
while a strong negative value indicates the probe is unmethylated. See
Figure 109 for more information.
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Figure 109 Effect of probe log-ratio score placement on a mixture of Gaussians – result
ranges for Z-normalization score
In addition to the Z- score calculation, Agilent Genomic Workbench
calculates a p- value that considers how much of a tail continues past the
log- ratio point on the Gaussian curve. Such a calculation is important
because the tails of the Gaussian distributions overlap; hence it is often
difficult to clearly assign a given probe log- ratio score to the left or the
right Gaussian if a pronounced valley exists between the two distributions.
See Figure 110 for more information.
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Figure 110 Calculating Z-scores and p-values from two overlapping Gaussian distributions
The following p- values are calculated for the usual case of two Gaussians:
1 The p- value derived from the left Gaussian. This is denoted pM in the
report, and is the confidence at which the non- methylation null
hypothesis is rejected (that is, the confidence at which you can call the
probe methylated).
2 The p- value derived from the right Gaussian. This is denoted pU in the
report, and is the confidence at which the methylation null hypothesis
is rejected (that is, the confidence at which you can call the probe
unmethylated).
3 Finally, the algorithm uses the bimodal distribution of log- ratio scores
among probes of similar Tm to calculate a logOdds score of the probe.
This final logOdds score reflects the likelihood that a probe is
methylated, and is calculated as ω:
pM
ω = – log ⎛ -------⎞
⎝ pU⎠
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Interpreting the
results
Visualization
As shown in Figure 110 above, the boundary to decide whether a probe is
methylated often comes from log- ratio scores that fall in a valley between
two Gaussians. Although the program can establish a ratio of the
contribution of each Gaussian by calculation of the logOdds score ω, a
region exists where ω approaches a value of 1, and a methylation status
decision cannot be made. In other words, a threshold on the logOdds
score decides the methylation status call. This threshold is not fixed, and
can be determined after you inspect the output in the context of a project.
Agilent Genomic Workbench can display a moving average for both the
probe log- ratio scores and the Z- normalization scores from the
methylation status algorithm. The Z- scores used in the moving average
visualization are the combined Z- scores, and reflect the position of the
probe log- ratio score on both Gaussian curves, as shown in Figure 109.
Figure 111 displays the Gene View results of the methylation status
algorithm for Chromosome 21. The left pane displays the Z- score moving
average. There is no defined cut- off value to determine whether a probe
or probe region is methylated. The middle pane shows the moving average
for the probe logOdds ratios, with genomic tracks annotated. The right
pane displays the CpG island track. Note that the probes fall specifically
within CpG islands, and upstream of genomic tracks such as coding
regions.
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Figure 111 Output of methylation status detection algorithm. On the right side of Agilent
Genomic Workbench, Gene View displays the moving average for both the
Z-score (left pane of Gene View) and the probe log-ratios (middle pane of Gene
View). Human CpG islands are displayed as a track along the right side of the
moving averages. Note that the probe log-ratios, which are only slightly positive in this region (colored red), may take on negative combined Z-scores (colored by sample).
Output report
The program generates an output report for each chromosome. The report
contains the following statistical measures:
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Table 6
Contents of methylation output report
Reported parameter
Meaning
Z-score unmethylated
Z-score derived from the left Gaussian. This is the probability
that the observed value for a probe comes from the
unmethylated population.
Z-score methylated
Z-score derived from the right Gaussian. This is the probability
that the observed value for a probe comes from the methylated
population.
Combined Z-score
Summation of the methylated and unmethylated Z-scores
pM
The p-value derived from the left Gaussian. This is the
confidence at which the non-methylated null hypothesis is
rejected (that is, the confidence at which you can call the probe
methylated).
pU
The p-value derived from the right Gaussian. This is the
confidence at which the methylated null hypothesis is rejected
(that is, the confidence at which you can call the probe
unmethylated).
logOdds
The logOdds score ω; the likelihood that a probe is methylated
rather than unmethylated.
Bayesian tool for methylation analysis (BATMAN)
Epigenetic methylation is found predominantly in genomic areas of
increased GC content, known as CpG islands. Agilent human methylation
arrays specifically query approximately 25,000 CpG islands in the human
genome, in addition to gene promoter regions. Like many MeDIP protocols,
the Agilent methylation protocol uses antibody enrichment for
5- methylcytosine, which results in a log- ratio of the relative abundance of
genomic fragments with methylation to those without.
Although the log- ratio scores give information about the relative
abundance of the genomic fragments, the scores do not reflect the
underlying distribution of CpG dinucleotides, the abundance of which can
increase fragment enrichment. Differences in CpG dinucleotide frequencies
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can therefore confound methylation state quantitation because the signal
log- ratios themselves depend upon the density of the methylated CpG
sites.
To obtain an absolute measure of methylation, the BATMAN algorithm
first calibrates a set of conditional probabilities based upon the distance
of a probe from any CpG sites. Based on expected fragment sizes during
DNA shearing, the total number of CpG sites per probe can be estimated,
yielding a Gaussian distribution of methylated sites to give the observed
probe signal. This conditional probability is then used as a likelihood to
generate a Bayesian posterior probability of the methylation status of any
CpG site given the probe signal. The signal used in BATMAN can be either
the probe log ratio or the Z- score from the methylation detection
algorithm.
In general, probes chosen for MeDIP- microarray experiments are limited
by their genomic location and therefore have a wider range of melting
temperatures. The methylation detection algorithm accounts for this by
binning the probes according to their melting temperature and
standardizing the probe scores. Agilent Genomic Workbench therefore
allows you to combine the output from the methylation detection
algorithm with the BATMAN algorithm, to account for the differences in
probe melting temperatures and the effects of inhomogeneous CpG
densities.
To increase the speed of the overall calculation, BATMAN uses the
assumption that the methylation state of neighboring CpG dinucleotides is
not independent, but rather homogeneous due to the effect of methylation
spreading and maintenance. A Monte- Carlo simulation is then used to
sample windows of fixed genomic size to establish methylation levels for
all CpG sites.
Purpose
BATMAN is a visualization and report analysis that determines the
absolute methylation level for CpG dinucleotides that are contained within
DNA fragments queried by probes across the microarray. These probes
typically capture known CpG islands and gene promoter regions. To
determine the absolute methylation level of individual CpG sites, the
analysis uses a calibration method to arrive at a distribution that
describes the conditional probability of the probe signal given a set of
methylation states. Bayesian inference is then used to obtain the
methylation states given the probe signal.
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Bayesian tool for methylation analysis (BATMAN)
Use
To establish a link between genomic methylation and gene expression, or
to observe epigenetic changes, Agilent Genomic Workbench uses the
BATMAN algorithm to quantify the methylation level of CpG dinucleotides
across probes that query methylated genomic regions. You can then
visualize the probes in a genomic context — for example, CpG islands
within the genome, or individual CpG dinucleotides within CG- sparse
regions.
Algorithm
The algorithm begins with a calibration step, which uses a regression to
fit a trend between local CpG density and either probe log- ratios, or
Z- scores if the methylation status detection algorithm is used. This is
followed by a modeling step, which uses the calibration results to
construct the conditional probability of the log- ratios (or Z- scores) given
the methylation state. A sampling step occurs to generate a large number
of possible methylation states. Bayesian methodology is used to invert the
conditional probability and summarize the methylation state per probe.
Finally, a methylation call step fits a distribution to the scores on a
per- chromosome basis. Additionally, for each CpG island, genomic
intervals are found with the same methylation call.
Step 1: Calibration step
As described in Down et al.,2 a coupling factor Ccp is defined as:
C cp = m
---n
where c is any given CpG dinucleotide, n is the number of fragments
containing c, and m is the total number of DNA fragments hybridizing to
any probe p. A linear regression is then fit to the plot of the log- ratios (or
Z- scores) versus Ctot, where:
C tot =
∑C cp
p
Since most CpG- rich areas are hypomethylated while most CpG- poor
regions are hypermethylated, the regression fit is increased by limiting the
least- squares method to the low- CpG regions. The least square estimates
are given by:
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∑( x – x ) ( yi – y )
β̂ 1 = ---------------------------------------2
∑( xi – x )
and:
β̂ 0 = y – β̂ 1 x
The variance of the predicted response (that is, the expected range of
values of y at 95% confidence) is given by:
⎛
2
( x d – x ) ⎞⎟
1
Var ( y d – [ α̂ + β̂x d ] ) = σ 1 + ---- + ------------------------⎜
2⎟
m
⎝
∑( xd – x ) ⎠
2⎜
This value is then used in the modeling step along with the regression line
slope and intercept.
Step 2: Modeling and sampling
This step uses the data from the calibration results to create a likelihood
model. The MeDIP model is given by:
f(A m) =
⎛
∏ζ ⎜⎝ Ap Abase + r ∑Ccp Mc,
p
c
– 1⎞
v ⎟
⎠
Where Ap is the log ratio (or Z- score) for the probe, Abase is the baseline
value given by the linear regression, and Mc, the methylation state at c, is
regarded as a continuous variable with a Gaussian distribution.
Possible combinations of methylation steps are sampled from the MeDIP
model using nested sampling.4
Step 3: Summarization (Bayesian inference)
The sampling step results are then summarized to create a score for each
probe. Each of the sampling states are weighted by the likelihood estimate
from the model. The IQR and median values are calculated across samples
and for every probe in every array.
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The responses are then trimmed to the regions of interested (for example,
track boundaries).
Step 4: Methylation calling
A beta distribution is fit to the scores per chromosome obtained in the
summarization step. (See “Methylation calling – the beta distribution” on
page 297 for more information). Agilent Genomic Workbench then
calculates the modes of the distribution and compares the score to the
mode, assigning methylated / status calls (+1 for methylated, - 1 for
unmethylated). If the modes are not unique, then zero is returned as a
call.
The beta distribution is fit using a local searching algorithm called
random hill climbing.3
Interpreting the
results
Visualization
292
For each CpG island, Agilent Genomic Workbench displays consecutive
regions that share the same call across each chromosome.
The consecutive regions appear as intervals for inspection.
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Visualization Algorithms
6
Visualization Algorithms
After you apply the methylation algorithm, it is useful to visualize the
general trend of the pre- algorithm probe log- ratios and post- algorithm
combined Z- scores, using a moving average line plot. To facilitate the
visualization of large datasets, Agilent Genomic Workbench employs a
sliding window across the genome, to smooth the data points for detailed
examination. The smoothing functions are available independently from
the main methylation algorithm. A change in window size in the
visualization algorithms does not affect the methylation calls.
To reduce the complexity of the data from probe- to- probe noise, you can
apply one of two smoothing algorithms. One algorithm applies linear
smoothing (the moving average algorithm), and the other applies a
triangular smoothing function. These algorithms use a sliding window of
fixed size, set in the user interface (UI). You can apply them to the log
ratios, the Z- score output from the methylation algorithm, or both.
The moving average visualization algorithm sets a fixed window size
around every point of interest. The algorithm reports the value for that
point as the average of that point and neighboring points within the
window boundaries.
Moving average (linear smoothing)
To compute a moving average, the program averages probe log- ratios or
combined Z- scores over a small subset of points in the genome. This
moving average window, w, may be simply a number of adjacent
measurements, or it may be over a positional window (such as every
megabase).
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Triangular smoothing
Triangular smoothing
The triangular smoothing algorithm is a shaped smoothing algorithm based
on a moving average. As the moving average centers on a point of interest,
the program applies a maximum weight, and that weight falls off with
increasing distance. Then the program adjusts the point of interest as the
weighted mean of itself and neighboring points.
Purpose
When you visualize or analyze array data, it is common to smooth the
data using a moving average. However, the moving average approach is not
the optimal means to reduce noise associated with each independent
point, because it can minimize log- ratio changes or methylation Z- scores
and obscure individual points. Triangular smoothing is a good compromise
that reduces noise of individual points, while remaining sensitive to true
localized or small- scale variations in the data.
Use
The smoothing functions are used for visualization purposes only, and do
not affect the methylation algorithm. However, the program applies the
moving average (Z- score) after the methylation algorithm, to smooth the
methylation Z- scores that you visualize in the user interface.
Algorithm
The number of neighboring points used for smoothing depends on the type
of moving average. If you select point input (pt input), the program keeps
constant the number of points that are averaged. It may use, for example,
3, 5, 7, 9, or 11 points, and it gives each point equal weight. Alternatively,
you can select a window of constant width (in Mb or Kb). The window
moves across the data and centers on the point of interest. All points
within its range are averaged to yield the moving average value for the
point.
NOTE
In linear smoothing, the user-defined point input (pt input) uses the same number of probes
as in triangular smoothing. However, an equal number of probes is taken in each direction
(to the left and right of the center probe) to compute the moving average. This might not be
ideal if the probes are placed at varying distances.
Two potential problems exist in linear smoothing:
1 Use of a fixed window width (by choice of a base pair from the UI)
causes a variable number of probes to be averaged at each smoothed
point. Therefore, the degree of averaging varies from probe to probe,
depending on how many probes are in the fixed- width window. Because
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6
varying numbers of measurements contribute to each smoothed point,
the degree of statistical noise reduction also varies for each point. This
can complicate the error analysis.
2 For so- called “zoom- in” arrays, where some genomic regions of interest
are covered more densely than neighboring regions, the appropriate
window size can vary greatly between the different genomic regions.
Smoothing windows that are appropriate for sparsely tiled regions
obliterate all structure in densely tiled regions. Windows appropriate for
densely tiled regions perform practically no averaging at all in sparsely
tiled regions.
Triangular smoothing avoids these problems, by use of smoothing windows
that contain a fixed number of probes, regardless of the total range of
sequences those probes span. This respects the fact that nearby probes are
more relevant than distant probes to the average at any point.
The concept is illustrated in Figure 112. Fixed- count smoothing includes
the same number of points in each average, but weights probes far from
the averaged point as much as points near it. In triangular smoothing with
pt input, the program enlarges a symmetric window around the averaged
point until it contains the number of points chosen for the fixed- size
window. These points may be on one or both sides of the averaged point,
depending on the probe density around the averaged point. These points
are then weighted appropriately for the triangular smoothing function,
depending on their distance from the averaged point.
When you enter a number of points from the user interface, the program
uses a variable window width, chosen to be the smallest window that is
symmetrical about the averaged point and includes the specified number
of points. Figure 112 illustrates the application of this method for pt
input.
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Triangular smoothing
Figure 112 Triangular smoothing with pt input
The program applies smoothing to a region of varying probe density. The
effective width of the smoothing window, W, depends on the length of the
smallest symmetrical region (Δ) that includes the specified number of
points. The weight given to each point is proportional to the height of the
triangle at that point.
Weights assigned to the log- ratio or combined Z- score values of these
probes are given by the equation:
w(x) = (W – x ) ⁄ W
2
(1)
where the effective window width, W, is determined by the length, Δ, of
the symmetrical region that includes the specified number of points:
W = Δ ⁄ (2 – 2)
Interpretation
Visualization
296
(2)
The smoothing algorithms affect the scatter plot in the Chromosome and
Gene Views. See Figure 111 for more information.
Agilent Genomic Workbench can display scores from the methylation
algorithms in the UCSC genome browser for visual interpretation.
Methylation (CH3) Analysis Guide
Statistical Algorithms
Appendix
6
Appendix
This section contains additional information about statistical algorithms
and is useful to understand algorithm steps in detail.
Methylation calling – the beta distribution
The beta distribution is given by:
α– 1
β–1
x
(1 – x)
--------------------------------------B ( α, β )
Where B in the denominator is an incomplete beta function given by:
Γ ( x )Γ ( y )
B ( x, y ) = -----------------------Γ (x + y)
And the gamma function is:
∞
Γ (z) =
∫
(t
z – 1 –1
e ) dt
0
The beta distribution is shown in Figure 113.
Figure 113 The beta distribution, illustrated with various parameter values
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References
References
1 “Agilent Microarray Analysis of Methylated DNA Immunoprecipitation Protocol, version 1.0” Agilent Life Sciences and Chemical Analysis
Protocols. © 2008.
<http://www.chem.agilent.com/Library/usermanuals/Public/G4170- 90012_
Methylation_Protocol.pdf>.
2 Down, T. A., Rakyan, V. K., et al. (2008). “A Bayesian deconvolution
strategy for immunoprecipitation- based DNA methylome analysis.” Nat
Biotechnol 26(7):779- 785.
3 Russell, Stuart J., Norvig, Peter (2003). Artificial Intelligence: A Modern
Approach (2nd ed.), Upper Saddle River, NJ: Prentice Hall, pp. 111- 114,
ISBN 0- 13- 790395- 2.
4 http://www.inference.phy.cam.ac.uk/bayesys/nest.pdf
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299
www.agilent.com
In this book
This guide describes how to
use the Methylation (CH3)
application of Agilent
Genomic Workbench 6.5 to
apply algorithms that help
identify methylated regions.
©
Agilent Technologies, Inc. 2010, 2015
Revision A1, October 2015
*G3800-90030*
G3800-90030
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