DeCyder 2D Software, Version 7.0

DeCyder 2D Software, Version 7.0
GE Healthcare
DeCyder 2D Software, Version 7.0
User Manual
Contents
1
Introduction
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
2
Start and the Organizer
2.1
2.2
2.3
3
Overview ........................................................................................ 35
Select a project for gel images import ..................................... 37
Add gel files to the import list .................................................... 39
Add, edit and remove items in the import list ........................ 40
Edit gels .......................................................................................... 42
Check settings for added images .............................................. 46
Import files .................................................................................... 48
DIA (Differential In-gel Analysis) Module
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
5
Start the software ........................................................................ 25
The Organizer ............................................................................... 26
Item properties ............................................................................. 32
Image Loader
3.1
3.2
3.3
3.4
3.5
3.6
3.7
4
Introduction .................................................................................... 7
DeCyder 2D Software Documentation ....................................... 7
Measuring differential protein abundance using
Ettan DIGE system ......................................................................... 8
Experimental Design using an Internal Standard .................. 10
Integration of DeCyder 2D Software with Ettan DIGE system
experimental design .................................................................... 13
Steps involved in Image Analysis using DeCyder 2D Software
15
Structure of DeCyder 2D Software ............................................ 16
What’s new in DeCyder 2D Software version 7.0 ................... 22
Overview ........................................................................................ 49
DIA Graphical User Interface ..................................................... 50
Viewing spot data ........................................................................ 51
Creating and opening workspaces ........................................... 66
Spot detection and quantitation ............................................... 69
Data analysis (optional) .............................................................. 73
Saving, printing and exporting .................................................. 83
Exporting data .............................................................................. 84
BVA (Biological Variation Analysis) Module
5.1
5.2
5.3
5.4
Overview ........................................................................................ 85
Graphical user interface ............................................................. 86
Viewing spot data ........................................................................ 88
Viewing protein data ................................................................. 102
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5.5
5.6
5.7
5.8
5.9
5.10
5.11
5.12
5.13
5.14
5.15
5.16
6
Spot picking in DIA
6.1
6.2
6.3
6.4
6.5
6.6
7
Overview spot picking in DIA ................................................... 175
Identification of reference markers ........................................ 176
Identifying Proteins of Interest using the DIA module ........ 178
Assigning Spots for Picking ...................................................... 180
Editing Pick Locations ............................................................... 181
Exporting Pick Lists .................................................................... 182
Spot picking in BVA
7.1
7.2
7.3
7.4
7.5
7.6
7.7
8
Protein quantitation .................................................................. 109
Creating and opening workspaces ......................................... 110
Defining spot map attributes ................................................... 113
Gel matching .............................................................................. 117
Match editing .............................................................................. 128
Protein statistics ........................................................................ 140
Protein Filter ................................................................................ 160
Molecular weight (Mw) and isoelectric point (pI),
(optional) ...................................................................................... 164
User-defined protein labelling (optional) ............................... 168
Protein database linking (optional) ......................................... 169
Saving and printing .................................................................... 172
Exporting data ............................................................................ 174
Overview spot picking in BVA .................................................. 183
Identifying Proteins of Interest using the BVA module ....... 184
Spot detection on the pick gel ................................................. 187
Identification of reference markers ........................................ 187
Assigning Spots for Picking in BVA .......................................... 190
Editing Pick Locations ............................................................... 193
Exporting Pick Lists .................................................................... 195
Batch processor
8.1
8.2
8.3
8.4
8.5
8.6
8.7
Overview ...................................................................................... 197
Setting up the DIA Batch List ................................................... 198
Setting up the BVA Batch List .................................................. 203
Editing items and workspaces (optional) ............................... 206
Saving the batch ......................................................................... 212
Running the batch ...................................................................... 214
Printing the batch ...................................................................... 216
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9
User Administration Tool
9.1
9.2
9.3
9.4
9.5
9.6
9.7
9.8
Overview ...................................................................................... 217
Open User Administration Tool ............................................... 217
Create an Administrator user account .................................. 218
Create a new user account ...................................................... 219
Edit a user account .................................................................... 220
Profile settings ............................................................................ 220
Change password ...................................................................... 221
Delete a user account ............................................................... 221
10 Database Administration Tool
10.1 Introduction ................................................................................ 223
10.2 Open Database Administration Tool ...................................... 224
10.3 Backup of database ................................................................... 224
10.4 Restore data from backup ........................................................ 226
10.5 Export data .................................................................................. 229
10.6 Import data ................................................................................. 232
10.7 Archiving from the database ................................................... 236
10.8 Retrieve archived data .............................................................. 239
10.9 Release item (Workspace, Projects or Gels) .......................... 240
10.10Other administration tools ....................................................... 241
11 XML Toolbox
11.1
11.2
11.3
11.4
Overview ...................................................................................... 243
Opening the XML Toolbox module .......................................... 244
Extracting data ........................................................................... 244
Tag definitions ............................................................................ 247
12 Tutorials Introduction
12.1 Scope of tutorials ....................................................................... 255
12.2 Tutorial files ................................................................................ 256
13 Tutorial I - Using DIA module for preliminary investigation
of protein changes
13.1
13.2
13.3
13.4
13.5
13.6
13.7
Objective ...................................................................................... 257
Overview ...................................................................................... 257
Experimental design .................................................................. 258
Analysis of control-control gel ................................................ 259
Analysis of control-treated gel ................................................ 268
Assigning protein of interest .................................................... 271
Spot confirmation ...................................................................... 273
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13.8 Save DIA workspace (optional) ................................................ 274
13.9 Exporting a Pick Lists and physically excising spot from the gel
274
14 Tutorial II - Employing an internal standard to Analyze
Protein Changes
14.1
14.2
14.3
14.4
14.5
14.6
14.7
14.8
Objectives .................................................................................... 275
Overview ...................................................................................... 275
Experimental design .................................................................. 276
Spot detection and quantitation ............................................. 277
Creating the BVA workspace ................................................... 281
Matching ...................................................................................... 286
Statistical analysis ..................................................................... 294
Assigning spots as proteins of interest .................................. 299
15 Tutorial III - Processing the Preparative Gel and
Generating a Pick List
15.1
15.2
15.3
15.4
Objective ...................................................................................... 303
Overview ...................................................................................... 304
Experimental design .................................................................. 304
Matching to analytical gels ...................................................... 313
16 Tutorial IV - Automated identification of differentially
expressed proteins
16.1
16.2
16.3
16.4
16.5
Objective ...................................................................................... 325
Overview ...................................................................................... 325
Experimental design .................................................................. 327
Protein spot filtering (optional) ................................................ 328
Processing multiple images ..................................................... 329
Appendix A Understanding the digital image
A.1
A.2
A.3
A.4
A.5
A.6
Image acquisition ....................................................................... 339
The digital image ........................................................................ 339
Formatting graphic files ........................................................... 340
Elements of the digital image .................................................. 342
Image dimensions ...................................................................... 343
Image quality .............................................................................. 345
Appendix B Spot processing algorithms
B.1
B.2
Summary of spot normalization procedure .......................... 347
Detailed description of spot normalization procedure ....... 348
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Appendix C Experimental examples
C.1
C.2
Example of a paired experiment ............................................. 353
Example of a two condition experiment ................................ 357
Appendix D Importing protein data
D.1 Import procedure in BVA .......................................................... 364
Appendix E Keyboard shortcuts
E.1
E.2
DIA module keyboard shortcuts .............................................. 365
BVA module keyboard shortcuts ............................................ 366
Appendix F Glossary
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Introduction 1
1
Introduction
1.1
Introduction
Two-dimensional electrophoresis (2-D electrophoresis) is a leading tool in
proteomics research today, capable of visualizing many components of
complex proteomes in a single gel. Ettan™ DIGE (Difference Gel Electrophoresis)
system is a method for pre-labelling protein samples prior to 2-D
electrophoresis. The system is based upon the specific properties of CyDye™
DIGE Fluor dyes which enable multiplexing of separate protein mixtures on the
same 2-D gel.
DeCyder 2D™ 2D software is an automated image analysis software suite which
enables detection, quantitation, matching and analysis of Ettan DIGE system
gels. Whenever “DeCyder 2D Software” is used in this manual, it implies DeCyder
2D version 7.0 software. The software was developed as part of Ettan DIGE
system, to exploit the multiplexing capabilities of the CyDye DIGE Fluor dyes.
Multiplexing, the co-migration of more than one sample per gel, enables the
inclusion of an internal standard. The internal standard is used to derive
statistical data within and between gels. This experimental design using the
internal standard, effectively eliminates gel-to-gel variation, allowing detection
of small differences in protein levels to be achieved. Using DeCyder 2D
Differential Analysis Software, system variability is minimized enabling
expression differences identified by 2-D DIGE to be confidently assigned to
induced biological change.
1.2
DeCyder 2D Software Documentation
The DeCyder 2D Software User Manual, this manual, is distributed as a PDF file
together with the DeCyder 2D Software. It can also be ordered separately as
printed matter.
The user manual is broadly divided into two main parts, the reference manual
(Chapters 1-11) and the tutorials (Chapters 12-16). It is recommended that new
users first work through the tutorials, in order to gain a rapid understanding of
the software’s capabilities. The tutorials are step by step guides that take the
user through the main applications of the software using real examples. The
tutorials are designed to be worked through without prior knowledge of the
reference component of the manual. The reference manual provides a detailed
technical account of the built-in functionality of DeCyder 2D Software. This can
be used as an information source for experienced users.
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1 Introduction
1.3 Measuring differential protein abundance using Ettan DIGE system
The DeCyder 2D Software Installation Guide and DeCyder 2D Software Getting
Started Guide are distributed as printed matter together with the DeCyder 2D
Software.
The DeCyder Extended Data Analysis module User Manual is a separate user
manual for the extensive Extended Data Analysis module. It is distributed as a
PDF file together with the DeCyder 2D Software. It can also be ordered
separately as printed matter.
Detailed information can also easily be found in the built-in online help included
in the software. Select Help:Help Contents and Index in each of the modules to
access the online help for the specific module.
1.3
Measuring differential protein abundance using
Ettan DIGE system
To compare protein abundance in different samples, conventional 2-D methods
require the separation of each sample on an individual gel. This “one-sampleper-gel” approach exposes the data to a high level of system variation, i.e. the
variation that arises from differences in protein uptake into the first dimension
strip, second dimension gel running, etc. This high level of system variation can
outweigh the often subtle, induced biological changes that the experiment is
intended to detect, for example, differences that are caused by a disease state,
drug treatment or life-cycle stage.
To compound this problem, it is also necessary to separate the induced
biological changes within an experiment from the inherent biological variation,
i.e. the differences between two individual animals, cultures, plants or flies, that
are present, irrespective of the applied experimental test conditions. To achieve
this, multiple sample replicates must be incorporated within each experimental
design. This requires the separation and analysis of a large number of samples
and can be a slow process if each sample has to be separated on a different gel.
Ettan DIGE system has been developed to address these problems. The system
includes CyDye DIGE Fluor Cy™2, Cy3 and Cy5 minimal dyes, which are mass
and charge-matched, spectrally resolvable fluorescent dyes. Additional CyDye
DIGE Fluor Cy3 and Cy5 saturation dyes have been developed specifically to be
used where only small amounts of sample are available. Please refer to Section
F.1 for ordering details of the Scarce Sample Labelling kit. A protein sample
labelled with any of the CyDye DIGE Fluor dyes, will migrate to the same position
on a 2-D gel. This permits the multiplexing of two or three samples within the
same 2-D gel, allowing the inclusion of an internal standard (see Section 1.4).
Gels are scanned using the Typhoon™ Variable Mode Imager, generating
overlaid, multi-channel images for each gel. Images can then be analyzed using
the DeCyder 2D Software, which contains novel algorithms for co-detection of
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Introduction 1
multiplexed gel images and has been specifically developed for use with Ettan
DIGE system (see Section 1.4 ).
Figure 1-1. Scheme showing the workflow for Ettan DIGE system
The benefits offered by Ettan DIGE system are:
•
Accurate quantitation and statistical analysis of protein abundance
changes
•
High sensitivity and wide dynamic range (5 orders of magnitude)
•
Minimization of system (gel-to-gel) variation
•
Easier matching between gels, with increased confidence
•
Fewer gels required per experiment
•
Faster analysis due to fully automated gel-processing workflow
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1 Introduction
1.4 Experimental Design using an Internal Standard
1.4
Experimental Design using an Internal Standard
Ettan DIGE system provides the ability to multiplex samples, enabling the use of
an internal standard within each 2-D gel. Ideally, the internal standard should
consist of a pool taken from all of the samples within the experiment. The
internal standard is labelled with one of the CyDye DIGE Fluor minimal dyes
(usually Cy2 if using CyDye DIGE Fluor minimal dyes or Cy3 if using CyDye DIGE
Fluor saturation dyes) and run on each gel in the experiment. This creates an
image that is the average of all experimental samples, with all proteins in the
experiment represented. The presence of the internal standard in every gel
provides an intrinsic link between samples. Ettan DIGE system is currently the
only 2-D gel electrophoresis protein difference analysis technique to utilize the
internal standard approach.
There are several benefits of using an internal standard in 2-D experiments.
Firstly, each protein spot in a sample can be compared to its representative
within the internal standard on the same gel, to generate a ratio of relative
protein levels. Quantitative comparisons of samples between gels are made
based on the relative change of sample to its in-gel internal standard. This
process effectively removes the system (gel-to-gel) variation enabling accurate
quantitation of induced biological change between samples (Figure 1-2). The
need to run gel replicates is also overcome, reducing the number of gels
required per experiment.
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Figure 1-2. Analysis of samples 1-4 in the absence of an internal standard would suggest
that the protein spot circled in red on gel 1 is absent in samples 3 and 4. However,
reference to the internal standard, which is an identical pool of all samples run on both
gels, shows that this protein has not entered gel 2. This indicates that the observed
absence of this protein spot in samples 3 and 4 is due to system variation (e.g. gel
distortions, differences in first dimension focusing etc.) and not to sample differences.
Similarly, analysis without the internal
standard of the protein spot circled in blue suggests that this protein is present in a
greater abundance in sample 4. Reference to the internal standard indicates that system
variation has resulted in this protein having an increased volume in gel 2 relative to gel 1.
Hence the abundance of this protein is unchanged in samples 1,2 and 4 and decreased in
sample 3.
A further benefit of using an internal standard is that matching between gels is
more straightforward. The internal standard image is common between all gels
in an experiment, therefore matching can be performed between internal
standard images which have the similar spot patterns. Conventional 2-D
electrophoresis requires matching between different samples on different gels,
which introduces differences in spot patterns from sample-to-sample and gelto-gel variation. Matching between internal standards allows matching
between identical samples, so variations in spot patterns are due only to
electrophoretic differences.
The internal standard approach can be applied to two-color or three-color
experiments by including the internal standard plus one sample or two samples
respectively on each gel.
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1 Introduction
1.4 Experimental Design using an Internal Standard
To maximize the quality of the data obtained when working with Ettan DIGE
system, the correct experimental design should be implemented.
Table 1-1shows an example of the experimental design used for a simple threecolor experiment using control and treated groups, each containing four
individuals
Gel
Cy2
Cy3
Cy5
1
Pooled internal standard
Control A
Treated B
2
Pooled internal standard
Control B
Treated C
3
Pooled internal standard
Treated D
Control C
4
Pooled internal standard
Treated A
Control D
Table 1-1. Using an internal standard for accurate measurement of protein abundance
changes between samples in an experiment (for an experiment using >2 samples).
Each gel contains a pooled internal standard labelled with CyDye DIGE Fluor Cy2
minimal dye. Four biological replicates (A-D) have been included for control and
treated samples that have each been labelled with CyDye DIGE Fluor Cy3 or Cy5
minimal dyes. A greater degree of statistical confidence can be assigned to the
experimental results by increasing the number of biological replicates
employed. Half of the control group are labelled using the CyDye DIGE Fluor Cy3
minimal dye, half using the CyDye DIGE Fluor Cy5 minimal dye and similarly for
the labelling of the treated group, thereby conforming to best experimental
practices.
For more detailed information about Ettan DIGE system and using an internal
standard, please refer to Ettan DIGE system User Manual (code no. 18-1173-17).
For more examples of experiments, see Appendix C , Experimental examples.
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1.5
Integration of DeCyder 2D Software with Ettan DIGE system experimental design
DeCyder 2D Software was developed specifically for the 2-D DIGE methodology
and therefore all the advantages of this approach are utilized in the software.
•
DeCyder 2D Software used in conjunction with Ettan DIGE system allows
the analysis of experimental designs with various degrees of complexity.
Simple control – treated experiments as well as complex multi-condition
experiments addressing factors such as dose and time can be performed
in a single analysis.
•
The relationship between any number of samples can be accurately
quantified and statistically analyzed in DeCyder 2D Software by employing
the internal standard (see Figure 1-2). This approach results in unparalleled
accuracy, allowing experimental conclusions to be drawn with high
confidence. No other 2-D electrophoresis technique available is capable of
resolving multiple samples in this manner and hence Ettan DIGE system is
unique in utilizing an internal standard on every gel.
•
The novel co-detection algorithm exploits the identical spot patterns
generated when multiple samples are resolved on the same gel. The
algorithm generates identical spot detection patterns on all images
derived from the same gel. Hence all spots on the same gel are effectively
matched with the identical spot boundaries.
•
Spot quantitation is performed automatically by normalizing spot volumes
against the internal standard (Figure 1-3). The co-detection algorithm
ensures that the internal standard and the quantified analytical spot have
an identical spot boundary. This results in a highly accurate and robust
protein quantitation.
•
DeCyder 2D Software utilizes experimental design incorporating an
internal standard and performs gel to gel matching on the standard
samples.
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1 Introduction
1.5 Integration of DeCyder 2D Software with Ettan DIGE system experimental design
Figure 1-3.
A. DeCyder 2D Differential Analysis Software utilizes an internal standard to
1) aid spot matching between samples within the same gel and
2) generate a ratio of protein abundance between the proteins of the internal standard
and each sample within the same gel.
Because the internal standard is the same sample run within each gel, this effectively
normalizes all the data. These functions are performed within the Differential In-gel
analysis (DIA) module of the software.
B. The Biological Variation (BVA) module is used to provide a quantitative comparison of
protein abundance between all samples within the experiment.
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1.6
Steps involved in Image Analysis using DeCyder 2D Software
Image analysis performed using DeCyder 2D Differential Analysis software uses
a number of complex algorithms, some of which are patent pending. They have
been designed specifically for use with multiplexed 2–D images. The image
analysis can be broken into the following processes:
•
Spot detection
•
Background subtraction
•
In–gel normalization
•
Gel artifact removal
•
Gel to gel matching
•
Statistical analysis
The complex algorithms associated with these steps form part of the in-built
functionality of the DeCyder 2D Software. For details, see Appendix B , Spot
processing algorithms. The various stages of gel processing are performed by
different modules within the software suite.
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1 Introduction
1.7 Structure of DeCyder 2D Software
1.7
Structure of DeCyder 2D Software
1.7.1
DeCyder 2D Software modules
The software consists of five modules. Four of them create and use files within
the DeCyder 2D database (see Figure 1-4)
.
Figure 1-4. Structure of DeCyder 2D Software
Image Loader - Imports image files into DeCyder 2D database making them
accessible for the other modules.
DIA (Differential In-gel Analysis) – Protein spot detection and quantitation on a
set of images, from the same gel.
BVA (Biological Variation Analysis) – Matches multiple images from different gels
to provide statistical data on differential protein expression levels between
multiple groups.
Batch Processor – Automated spot detection, matching of multiple gels,
interpretation and export to BVA without user interaction.
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XML Toolbox – Extracts user specific data facilitating automatic report
generation.
Image Loader module
The Image Loader module is used to import sets of gel images (each saved as
either 16-bit TIFF or customized .GEL files) into a specified project within the
DeCyder 2D database. Each image in the set is generated from samples labelled
with different fluors. The gel images are thereby made accessible to the other
modules within the DeCyder 2D Software. During the import, the Image Loader
module automatically groups the added images into gels, containing up to 3
images with different CyDye labels. The Dye chemistry setting is also automatic
if the gels are named as recommended.
2-D
DIGE gel
DeCyder
Database
.gel or .tif
Image Loader
.gel
.gel
Figure 1-5. Schematic representation of Image Loader module workflow
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1.7 Structure of DeCyder 2D Software
DIA module
The DIA module processes a set of up to 3 gel images from a single gel. Each
image in the set is generated from samples labelled with different fluors. The
images must be imported into the DeCyder 2D database via the Image Loader
module. Images must be processed in the DIA interface prior to data analysis in
BVA. The DIA algorithms detect spots on a combined image derived from
merging individual images from an in-gel set of images. This co-detection
ensures that all spots are represented in all images. DIA then quantitates spot
protein abundance for each image and expresses these values as a ratio,
thereby indicating changes in expression levels by direct comparison of
corresponding spots.
The data can be saved as a DIA workspaces in the DeCyder 2D database which
also can be used in the BVA module for multi-gel analyses. A spot pick lists can
be exported as a text file. This pick list is created from data generated in a single
DIA module analysis (i.e. an experiment based on a single gel). In addition, data
can be exported in an XML format which can be queried using the XML toolbox
or copied directly from the DIA module and pasted into applications such as
Microsoft Word and Excel.
DeCyder
Database
Pick list
.txt
.xml
DIA
.gel
.gel
.xml
.dia
.dia
.dia
XML Toolbox
.txt
.htm
Figure 1-6. Schematic representation of DIA module workflow.
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BVA module
The BVA module utilizes the DIA workspaces from the DIA module together with
the original gel images to match protein spots on different gels. Statistical
analysis is then performed to accurately assess protein expression changes
occurring in biological replicates, comparing different conditions/treatments.
This multi-gel approach, which allows analysis of replicates, provides greater
statistical validity to findings. The results can be saved as BVA workspaces in the
DeCyder 2D database, which can be re-opened in the BVA module. Furthermore,
data can be exported in XML format for data extraction using the XML Toolbox.
In addition, data can be copied directly from BVA and pasted into applications
such as Microsoft Word and Excel.
DeCyder
Database
.gel
.gel
Pick list
.dia
.dia
.txt
BV
.xml
.dia
.xml
.bva
.bva
.bva
XML Toolbox
.txt
.htm
Figure 1-7. Schematic representation of BVA module workflow.
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1.7 Structure of DeCyder 2D Software
Batch Processor
The Batch Processor integrates both the DIA and BVA modules enabling fully
automated processing of multiple gels without user intervention. The Batch
Processor can be configured to analyze several gels in the DIA module
exclusively. Alternatively, multiple gels can be processed through both modules
to produce DIA and BVA workspaces, a Batch workspace and a subsequent pick
list. The different workspaces are saved in the DeCyder 2D database while the
pick list is saved in a user defined area on the hard drive or the network.
Figure 1-8. Schematic representation of Batch workflow.
XML Toolbox
The XML Toolbox enables the extraction of user specific data from XML files
generated in either the DIA, BVA, or Batch modules. This data can be saved in
either text or html format enabling users to access data from DeCyder 2D
Differential Analysis Software workspaces in other applications.
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1.7.2
DeCyder 2D Software extra tools
The software has four extra tools (see Figure 1-9.):
Database Administration Tool - Tool for backup, archiving, import/export of
files in the DeCyder 2D database. Only for DeCyder 2D administrator users.
User Administration Tool - Tool for creating/deleting users, changing
passwords etc. Only for DeCyder 2D administrator users.
Main - Main window in DeCyder 2D Software from which all modules and the
Organizer can be opened.
Organizer - Tool for simple database functions such as create project, move
files and change access rights for project.
User
Administration
Tool
DeCyder
Administrator
users
DeCyder
Scientist
users
DeCyder
Database
Database
Administration
Tool
Import
Export
Backup
Restore
Archive
Retrieve
Release WS
Project
Gel images
DIA workspaces
Create project
Move/Copy/Delete
Change access
Edit information
Change password
BVA workspaces
Batch workspaces
Figure 1-9. Overview of the DeCyder 2D database and extra tools.
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1.8 What’s new in DeCyder 2D Software version 7.0
1.8
What’s new in DeCyder 2D Software version 7.0
The DeCyder™ 2D version 7.0 includes the following updates:
•
Warping (BVA)
•
Image Editor (Image Loader)
•
Improved Gel Image View (BVA)
•
Floating Master (BVA)
•
Gel Overlay (BVA)
•
Extended Spot Editing functions (BVA)
•
Extended Match Editing functions (BVA)
•
Simplified installation
•
Save as in DIA, BVA, and EDA
•
Improved EDA linking of BVA workspaces
•
Export of Log Standardized Abundance values from BVA
For detailed information, see below.
Warping (BVA)
Warping is now available to improve matching accuracy and to simplify the
process of evaluation of matching results.
The warping function reshapes the standard image of a gel to fit the master
standard gel image. The original gel images are always intact and are not
affected by warping.
Image Editor (Image Loader)
The Image Editor is a new DeCyder 2D function that is used to prepare the raw
images for further analysis, e.g. to crop and rotate the gel images. During
editing, the gels are represented as a three color overlay image.
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Introduction 1
Improved Gel Image View (BVA)
A number of changes are made in order to improve the usability of gel
visualization.
•
The area used for visualizing gel images is enlarged.
•
When switching view modes the gel image layout is unchanged.
•
Color coded headers indicate the current image sorting order.
•
New navigation tools for zooming and scrolling in the mosaic view.
•
The 3D views display all spot contours within the displayed area when
selecting a spot.
Floating Master (BVA)
In BVA Match mode, a separate ‘‘floating’’ window always displays the master
gel. The master gel image is always available in order to evaluate and edit the
match results.
Gel Overlay (BVA)
It is now possible to visualize differences between gel images by using the gel
overlay. The overlay function displays two gel images in the same view, the
master or standard image in yellow, and the other image in blue.
Extended Spot Editing functions (BVA)
In BVA Match mode it is now possible to manually edit spot detection using
these functions;
•
Merge (Improved)
•
Split merged
•
Split any (New)
•
Move (New)
•
Delete
•
Copy between gels
•
Add new (New)
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1 Introduction
1.8 What’s new in DeCyder 2D Software version 7.0
Extended Match Editing functions (BVA)
In BVA Match mode it is now possible to manually edit matches using these
functions:
•
Multi select spots in different gels for landmarking (Improved)
•
Confirm match sets (New)
•
Confirm single match
•
Add match
•
Break match
•
Comment on match set instead of single match (New)
Simplified installation
The complete installation package, including tutorial data, is now distributed on
a single DVD. The install and uninstall procedure has been simplified, and the
tutorial data is included in the database installation.
Save as in DIA, BVA, and EDA
It is now possible to save existing workspaces using ‘‘Save as’’.
Improved EDA linking of BVA workspaces
To increase the flexibility in linking of BVA workspaces, the linking is now
performed on common spot coordinates.
Export of Log Standardized Abundance values from BVA
The ratio values included in exported XML files from BVA are now the Log
Standardized Abundance values which are used in all statistics calculations. The
new format simplifies post processing of BVA data.
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Start and the Organizer 2
2
2.1
Start and the Organizer
Start the software
1
Select Start: All Programs: GE Healthcare: DeCyder 2D Software: DeCyder
2D. Alternatively, double-click the DeCyder 2D icon on the desktop.
The DeCyder 2D Main window appears with a Login dialog and a Licence
agreement dialog.
2
Read the licence agreement. Check the Do not show this during startup
box, if desired, and click Yes.
3
In the Login dialog, enter User name and Password. The DeCyder 2D
database is selected by default.
4
Optional: Click More>> to select another database or License option. Click
OK when desired options have been selected.
5
In the Login dialog, click OK to start the DeCyder 2D Software.
The license file has to be found before work is allowed in the software. See
also DeCyder 2D Software Installation Guide.
The Main window includes icons for all the modules of DeCyder 2D
Software. See also Chapters 3-8.
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2 Start and the Organizer
2.2 The Organizer
The Main window also includes icons for the Organizer (which can be used
for moving, copying, renaming and deleting files within the DeCyder 2D
database, see also 2.2) and the XML Toolbox (which can be used to export
spot data from DIA or BVA for downstream analysis, see also Chapter 11).
When moving the mouse over an icon a short explanation for the icon is
displayed in the field at the bottom of the window.
6
2.2
Start a module or tool by clicking the appropriate icon.
The Organizer
The Organizer is a tool that can be used to:
•
Create new projects, see 2.2.2.
•
Organize work into different projects, see 2.2.3.
•
Move, copy and paste files/projects, see 2.2.3.
•
Delete files/projects (can be performed by all users that have read/write
access to the file/project). see 2.2.3.
•
Change
- user passwords, see 2.2.4.
- access to projects, see 2.2.5.
•
Edit
- project information, see 2.2.6.
- workspace information, see 2.2.7.
- gel information, see 2.2.8.
•
Export images, see 2.2.9.
The user type Administrator, can change owner of all projects.
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Start and the Organizer 2
2.2.1
Open Organizer
1
Click the Organizer button in the DeCyder 2D Main window to open the
Organizer dialog.
2
All projects accessible to the current user are displayed in the left panel.
Each project can contain six sub-folders: Batch, BVA, BVAtemplate, DIA,
GEL, and EDA. When selecting a sub-folder the items therein are displayed
in the right panel. Sub-folders are created the first time that type of items
are saved to the project.
Note: When a project is selected in the organizer, the left panel shall show
the contents of that project, i.e. Batch, BVA, DIA, and Gel folder.
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2 Start and the Organizer
2.2 The Organizer
2.2.2
Create new project
1
Select File:New project to open the New Project dialog.
2
New Project upper list shows current Projects.
3
Enter Project name and a Description information (optional).
4
Check Public access if all users should have access to the project. If
unchecked, the project will only be visible for the owner.
5
Finish by clicking OK.
2.2.3
Move, copy, or delete a file in DeCyder 2D database
Note: Only the administrator and the project owner can delete projects. Only
users with Read/Write access can delete files.
1
In the left panel, select the project and a sub-folder.
2
In the right panel, select the file(s) to move, copy, or delete.
•
To move the selected file(s) from one folder to a folder of the same type,
use drag-and-drop.
•
To copy the selected file(s), select Edit:Copy and mark project/workspace
(in either the left or right panel) to which the file(s) is to be copied. Right
click and use Paste. Gels can not be copied between projects.
•
To delete the selected file(s), select File:Delete.
Note: It is possible to delete multiple files from the database, by selecting
one or several gels and then deleting them within one deletion
session.
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Start and the Organizer 2
2.2.4
Change user password
1
Select File:Change user password to open the Change password dialog.
2
Enter current password and then the new password.
3
Click OK to finish.
2.2.5
Change access to projects
Administrator users can change access to all projects. Scientist users can only
change access to projects they own.
1
Select a project in the left panel of the Organizer dialog.
2
Select File:Provide/Remove access... to open the Provide/Remove access
dialog. Only projects owned by the current user are displayed in the dropdown list in the dialog.
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2 Start and the Organizer
2.2 The Organizer
3
Select project in the drop-down list. The users and their access for the
selected project are displayed.
•
None = The user is not able to see the project at all
•
Read = The user can only see the project and its contents
•
Read/write = The user has full access to the project
Note: For projects with Public access checked, all users have Read/write
access and Provide access is not available (greyed out). Uncheck
Public access tick box to set individual user access.
4
Change access, then click Apply.
Note: Click Apply after each change!
5
Change access permission in a new project by repeating steps 3-4, or click
Close.
2.2.6
Edit project information
1
Select a project to edit in the left panel of the Organizer dialog.
2
Select File:Edit project information... to open the Edit project information
dialog.
3
Change Project name, Public access status and/or Description and click
OK.
Note: With Administrator rights it is also possible to change project
Owner.
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Start and the Organizer 2
2.2.7
Edit workspace information
1
Select a project and a folder in the left panel of the Organizer window.
2
In the right panel, right-click a workspace and select Edit workspace to
open the Edit workspace information dialog.
3
Change Workspace name and/or Description and click OK to finish.
2.2.8
Edit gel information
1
Select a project and a GEL folder in the left panel of the Organizer window.
2
In the right panel, right-click a gel and select Edit gel information to open
the Edit gel information dialog.
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2 Start and the Organizer
2.3 Item properties
3
Change Gel name and/or Description information and click OK to finish.
Note: When changing Gel name the images names in the Gel will not be
affected.
2.2.9
Export image
It is possible to export an Image file into it’s original file format (the format used
before it was imported via the Image Loader).
2.3
1
In the left panel of Organizer window, select a project, its’ GEL folder and
right-click the image to export.
2
Select Export image in the displayed menu.
3
Select where to save the image and enter a file name.
4
Click Save to finish.
Item properties
Properties can be displayed for all types of items in the DeCyder 2D Software
database.
2.3.1
32
Display properties
1
Right-click the object to display properties for an item to display the
Properties dialog.
2
Select desired tab. Different tabs (i.e. General, Description, Dependencies,
and Input items) are available for different types of items
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Start and the Organizer 2
3
General information of the items is shown in General tab. The current
status of the file or folder is shown in Attributes.
•
Read-only: The file cannot be modified. Create a copy to be able to modify
the information.
•
Locked: The file is in use on a different computer. The computer name is
displayed in the Description tab.
•
Archived: The file is archived. See Description tab for information of where
the file is archived.
4
In Description tab information about the file or folder is shown.
5
Item dependencies (Strong, Weak, or None) is shown in the Dependencies
tab, see 2.3.2.
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2 Start and the Organizer
2.3 Item properties
6
Item name and type, and project identity is shown in the Input items tab.
7
Click OK to close the dialog.
2.3.2
Dependencies
Strong dependency status of a Gel indicates that the corresponding
DIA Workspace made from data from that Gel can not exist if the Gel is deleted.
To protect data, it is not allowed to delete items with Strong dependency status.
Strong dependencies exist between the following relations:
•
Gels to DIA workspace
•
Gels to BVA workspace
Weak dependency is a concept for handling traceability. Weak dependency
status of a DIA Workspace indicates that a certain BVA Workspace has been
made from data from that DIA Workspace. The BVA Workspace can exist even
if the DIA Workspace is deleted but it is useful to keep track of which items that
have been used as input to a certain workspace. Therefor the term
Weak dependency has been introduced to define this state.
An item may be deleted if it has Weak dependency status. However, for tracking
reasons the system will inform you that traceability chains are broken before
deleting the item.
Weak dependencies exist between the following relations:
34
•
DIA workspaces to BVA workspace
•
BVA workspaces to EDA workspace
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Image Loader 3
3
3.1
Image Loader
Overview
Before starting the analysis of gel images in DeCyder 2D Software, the images
have to be imported into the DeCyder 2D database. The Image Loader module
is a simple tool to import gel images into a project within the DeCyder 2D
database.
1
To open the Image Loader module, click the Image Loader icon in the
DeCyder 2D Software main window. Image Loader window includes three
panels:
•
Left panel displays an import list of files selected for import with
Gel name and important gel information.
•
Center panel displays the database at the project level.
•
Right panel displays the gel files included in a selected project.
3.1.1
Images automatically grouped and named
Automatic grouping and naming
If gel images are named as recommended, Image Loader module automatically
groups the added images into gels, containing up to three images with different
labels. The gels are automatically named based on the image file names.
Recommended image file names
A standard name example is: Gel 01 Standard (Time 1) Cy3.gel
The name includes four parts
1
Part 1 is a general description, normally Gel followed by a number. However,
it is possible to set any name in part 1 except some key words used in the
second and third parts of the name (Do not use e.g. Standard, std, Control,
treated, Cy2, Cy3, Cy5).
Note: If you have more than 9 images and each image starts with a
sequence, use 01, 02, 03 etc., i.e. a leading zero to get a better sorting.
2
Part 2 contains a description of the function of the image (i.e. Standard,
Control, Treated). Other words, for example time and dose, can be used but
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3 Image Loader
3.1 Overview
must be within brackets.
Use short names with the most important parts of the name first for easy
sorting/finding.
3
Part 3 contains information of the labelling used to create the images (Cy2,
Cy3 or Cy5). By default the Cy2-gel will be placed as image 1, the Cy3-gel as
image 2 and the Cy5-gel as image 3.
4
The extension .gel or .tif is added at the end.
One example
•
Gel 01 Standard Cy2.gel,
•
Gel 01 Control (Time1_Dose2) Cy3.gel
•
Gel 01 Treated (Time2_Dose2) Cy5.gel.
The three images will be grouped into a Gel named Gel 01.
Note: If gel images are not named as recommended they will not be
automatically sorted. See 3.4.2 for renaming of gel images.
Note: If sample specific information is not within brackets, the images will not
be grouped but simply imported as single images into the DeCyder 2D
database.
Note: A gel with Standard included in the name will automatically be placed in
the experimental group Standard in the BVA module.
Note: Do not use the word ‘edited’ within an original file name. The Image
Editor will treat the file as an edited file and overwrite the file if edited.
3.1.2
Normal workflow
The normal workflow contains 5 main steps:
36
1
Select project, see 3.2.
2
Add gel files to the import list, see 3.3 and 3.4.
3
Edit gel images, see 3.5.
4
Check settings, see 3.6.
5
Import gel files into a project, see 3.7.
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Image Loader 3
3.2
Select a project for gel images import
1
In the Image Loader window, select a project in the Center panel into which
Gel images are to be imported. Current Gel files and Workspaces in the
selected project are shown in the Right panel. If required, create a new
project, see 3.2.1.
Note: If the automatically generated gelname (see 3.1.1) already exists in
the selected project, a suffix (_0 or _1 or _2 etc.) will be added to the
gel name automatically.
Note: If a project was not selected first, the renaming of the gel file must be
performed manually.
3.2.1
1
To create a new project
If there is no suitable Project present, click the New project button or right
click in the Center panel and select New project.
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3 Image Loader
3.2 Select a project for gel images import
38
2
Enter Project name and Description (optional project information). Check
Public access to allow other users to see and work with the project.
3
Click OK to finish.
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Image Loader 3
3.3
Add gel files to the import list
1
To add gel images to the import list, click Add... in the Image Loader main
dialog, or select File:Add file
2
Browse in Open file dialog to find the gel files to add to the list. Mark the
files and click Open. Alternatively, use Drag and Drop from Explorer.
Note: Only .tif and .gel files can be added. DeCyder 2D Software is
validated for file formats generated by GE Healthcare imager devices
recommended for Ettan DIGE system (see Appendix A).
3
The added files are displayed in the Left panel.
Note: Sometimes it is desired to add or delete gel files and images in the import
list. See 3.4.1 for instructions.
Note: If gel images have been named as recommended, see 3.1.1, Image
Loader module automatically groups the added images into gels,
containing up to three images with different labels. If the names of the
images do not follow the recommendations follow the instruction in 3.4.2.
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3 Image Loader
3.4 Add, edit and remove items in the import list
3.4
Add, edit and remove items in the import list
The Import list must sometimes be changed in order to get the desired gel files
and images imported. Some tools can be accessed by right clicking in the Left
panel. A dialog for Add new Gel row, Edit settings..., and Remove gel rows
and/or images is shown.
When moving images between different gels Drag and drop is also possible.
3.4.1
Add and remove gels and images to/from the import list
Add more image files:
1
Click Add... in the Image Loader main dialog, or select File:Add file
2
Browse in Open file dialog to find the gel files to add to the list. Mark the
files and click Open.
Remove gel files:
1
Select the desired gel and click Remove... in the Image Loader main dialog.
Remove separate image files from a gel file:
1
Click Edit to open the Edit settings dialog.
2
Select the desired image file tab and click Remove.
Remove all gel files:
1
Select Edit:Select all and the click Remove.
Add more images to a gel with less than three images:
1
To add gel images to the import list, select the desired gel and click Add...
in the Image Loader main dialog.
2
Browse in Open file dialog and select the image files to add to the gel. Click
Open.
3
Alternatively, use Drag and drop to move the image files to the gel.
Note: No automatic sorting of gels is performed when adding images this
way!
3.4.2
Manual grouping of gel images
If the gel images have not been named as recommended, see 3.1.1, the Image
Loader module can not automatically group the added images into gels.
Instead, the image files get one gel row each in the import list. However, the gel
images can be manually grouped by using Drag and Drop on the cells.
1
40
Use Drag and Drop to move the image file cells to the same row. The
following steps explains how to move one image file cell:
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Image Loader 3
a)
Click an image file cell to select it.
b)
Hold the mouse over the edge of the cell. The pointer changes symbol.
c)
Click and hold on edge of the cell, move the cell to desired row and
release the mouse button.
d)
Click OK. The cell is moved.
2
Delete the empty gel rows in the table by clicking Remove or selecting
Edit:Remove Empty Gels.
3
To change the name of the resulting gel:
a)
Click Edit to open the Edit settings dialog (Alternatively, right click the
gel and select Edit settings..., or select Edit:Edit settings).
b)
Select the desired Image Cy tab and change desired parameters. Click
OK.
c)
Now the changed Gel name is shown in Left panel (in this example
Gel2).
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3 Image Loader
3.5 Edit gels
3.5
Edit gels
The edges of the gel images could contain unwanted effects, artefacts. With
help of the Image Editor module these edges could be cut away during the
import process. The images originating from the same gel will be handled and
cropped simultaneously as one three-colored gel overlay image in the Image
Editor.
The Image Editor can be used within the Image Loader module or as a standalone module. See Section 3.5.3 for information on how to start the Image Editor
as a stand-alone module.
When cutting away edges the following points should be taken into
consideration:
•
Ensure that all relevant spots remain inside the image.
•
Areas that do not contain usable information should be removed.
•
It is more important to ensure consistent patterns than equal sizes.
•
Similar patterns facilitate correct matching.
The original image files are left intact. Edited versions of the image files are
stored in TIFF format in the same directory under the names
originalfilename(edited).tif. The gel will as default then consist of the new files,
i.e., the new image files will be in the gel rows in the left panel of the Image
Loader.
Note: Pick gels must still contain the internal reference marker. Define an “area
of interest” in the DIA module instead to facilitate proper matching, see
Section 4.6.1
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Image Loader 3
3.5.1
Cropping the gel images
1
Within the left panel of the Image Loader, select the gels to be edited. Click
e.g. on the numbers on the left hand side. Use shift and control to select
multiple gels.
2
Click the Edit Gel Images... button. The loading takes typically 5 sec. to 1
minute, depending on the images and the computer. The Image Editor
window opens, and every gel to be edited opens as a separate window
within the Image Editor window.
3
Click Tools: Crop. The cursor changes symbol. Clicking the crop icon gives
same result.
4
For every gel, i.e. within every window, perform step 5 - 7 below.
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3 Image Loader
3.5 Edit gels
5
Left click and hold to mark the first corner, drag the mouse to the opposite
corner and release the mouse button. The cropping rectangle is created.
6
Optimize the rectangle: Resize the rectangle by dragging in the corners.
Move the rectangle by picking it up at the sides. Consider the points stated
in Section 3.5 above.
Optional: use the zoom and brightness tools to help find optimum. If the
icons in the tool bar are not visible, select View:Toolbar, or use the Tools
menu instead.
7
Double-click in the rectangle to perform cropping according to the defined
cropping rectangle.
8
When all gels are edited satisfactorily, select File: Save All.
Note: If many gels are handled at the same time, it is best to save and close
each gel after it has been edited satisfactorily. This in order to
minimize the memory usage.
9
Close the Image Editor. (E.g. select File: Exit.)
Note: Perform the following steps to copy the size and position of an already
defined rectangle to another gel: Have the gels opened simultaneously
within the Image Editor. Perform step 7 above to the rectangle to copy.
The size and position will be suggested within the other gel windows.
Note:
44
If the Image Editor is used to edit a file with the word ‘edited’ in the
filename it will not make the change in a new file but overwrite the
existing file.
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Image Loader 3
3.5.2
Other functionalities of Image Editor
The Image Editor module also provides other functionalities:
•
To print a gel overlay image, select File: Print.
•
To preview before printing, select File: Print Preview.
•
To copy a gel overlay image to the clipboard, select Edit: Copy Image to
Clipboard.
•
To optimize contrast and brightness of the gel on your screen select
Tools:Contrast and Brightness Adjustment.
•
To get color information on which image file that corresponds to a certain
color, select File: Properties or right click and select Properties.
•
To get black background instead of white as in DeCyder 2D Software v 6.5,
select View: Inverted Images.
•
To be able to decide manually where the edited gel files are stored, use
File:Save As.
•
To rotate or flip a gel overlay image, select action within the Tools: menu.
•
To edit a separate image opened direct from the Image Editor, select File:
Open. This is not within recommended normal workflow.
3.5.3
Using the Image Editor as a stand-alone module
The Image Editor can be used within the Image Loader module or as a standalone module.
There are two ways to start the Image Editor as a stand-alone module:
•
Open via Start: All Programs: GE Healthcare: DeCyder 2D Software:
Image Editor.
•
Double-click a .gel file in the explorer. The .gel file will open in Image Editor
by default.
Once the Image Editor is open, handle the gels as described in Section 3.5.1 or
3.5.2.
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3 Image Loader
3.6 Check settings for added images
3.6
Check settings for added images
3.6.1
Default settings and automatic sorting
Image Loader module automatically groups added gel images with similarities
in file name into a gel (with at most three images). Gels with no name similarities
are added as a gel with a single image. The gel name and labels in the import
list, are set automatically if the information can be found in the added gel image
file names. See also 3.1.1.
In the group of three files included in a gel:
•
The Cy2 gel is by default set as image Cy2
•
The Cy3 gel is by default set as image Cy3
•
The Cy5 gel is by default set as image Cy5
If several gel images are included in a gel, the Dye chemistry is set, by default,
to DIGE min.
If only one image is included in a gel and Cy2, Cy3 or Cy5 are not found, the Dye
chemistry is set, by default, to PostStain and the label to Unassigned.
3.6.2
1
Checking settings
Before importing the gel files into a project, the settings must be correct for
all gels. Gels with invalid settings are shown in red text in the Left panel,
whereas gels with valid settings are displayed in black text.
Note: The settings can only be changed before importing the images.
2
46
If the settings are correct, continue with 3.7, otherwise continue below.
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Image Loader 3
3
To change settings, double click the Gel name to open the Edit settings
dialog. Alternatively, right click the gel and select Edit settings..., or select
Edit:Edit settings. Select desired Image Cy tab in order to edit setting for a
specific image.
•
The Gel ID is automatically generated at image import. It is a unique
number for the gel in the DeCyder 2D database. The Gel ID can be
changed manually in the dialog.
4
To remove a separate image, click Remove in the tab for that image.
5
Click OK when editing and deleting are completed.
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3 Image Loader
3.7 Import files
3.7
Import files
1
Make sure all information in the import list is displayed in black text, i.e. no
red text should be visible, see 3.6.2.
Note: A gel name is displayed in red in one project if the name already
exists in that project. This can be avoided by selecting the project
before adding the gels to the list, see 3.2.
48
2
To import files to the desired project, click Import.When the import is
finished, the imported files are displayed in the Right panel.
3
The files in the project are now ready to be analyzed in DIA or Batch module.
4
Exit the Image loader module by File: Exit.
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DIA (Differential In-gel Analysis) Module 4
4
4.1
DIA (Differential In-gel Analysis) Module
Overview
4.1.1
DIA Processes
DeCyder 2D Software DIA processes images from a single gel, performing spot
detection and quantitation.
The DIA module algorithms detect spots on a cumulative image derived from
merging up to three individual images from an in-gel linked image set. This codetection ensures that all spots are represented in all images processed. The
DIA module algorithms then quantitate spot protein abundance for each image.
The abundance values are expressed as ratios thereby indicating changes in
expression levels by direct comparison of corresponding spots. This ratio
parameter can be used, in small scale experiments, to directly evaluate changes
between two labelled protein samples. Alternatively, the ratio can be used for
protein spot quantitation of a sample against an internal standard to allow
accurate inter-gel protein spot comparisons (see Chapter 1 Introduction). Once
spot maps incorporating an internal standard have been analyzed in the DIA
module, the spot data can be used in BVA module for accurate quantitative
inter-gel studies. Generally, when multiple gel analyses are performed, only the
spot detection and quantitation algorithms are utilized in the DIA module. The
BVA module is then used for inter-gel analysis.
The DIA workspace can be saved and re-opened from within the DIA module.
The DIA workspace can also be used for multi-gel analysis in DeCyder 2D
Software BVA module. The DIA module can export pick list data in .txt format for
both Ettan Spot Picker or Ettan Spot Handling Workstation. Data can also be
exported in an XML format for either querying in DeCyder 2D Software XML
toolbox, or for copying and pasting into applications such as Microsoft™ Word™
and Excel™.
4.1.2
Normal workflow
The normal workflow includes the following 3 steps:
1
Create workspace, see 4.4.
2
Spot detection (Process Gel Images), see 4.5.
3
Save spot maps/workspace, see 4.7.
The DIA workspace can be used for further analysis in BVA module (see Chapter
6 Spot picking in DIA) or to produce a pick list (see Chapter 7 Spot picking in BVA).
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4.2 DIA Graphical User Interface
4.2
DIA Graphical User Interface
DeCyder 2D Software DIA graphical user interface is divided into four equally
sized inter-linked views.
•
Image View - primary and secondary gel images
•
3D View - a three dimensional representation of the gel localized on the
spot
•
Histogram View - graphical representation of data associated with the
spots displayed in the image view
•
Table View - tabulated data associated with spots displayed in the image
view
All four views are linked, therefore selecting a spot in, for example the Image
View, will display the spot in the Histogram View (in magenta), the 3D View and
the Table View. The role of the four views will be discussed in detail later in this
section.
Below the four views the Spot Control Panel is found. In this panel settings for
the selected spot can be changed.
Image View
Histogram
View
3D View
Table View
Spot Control Panel
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4.2.1
DIA main tool bar
The DIA main tool bar includes the following tools:
What's this?
Print
Properties
All Views
Table View
3D View
Histogram View
Image View
Contrast and
Brightness
Fit to Window
Create Workspace
Open Workspace
Save Workspace
Process Gels
Protein Filter
Exclude Filter
Area in 3D
Rotate in 3D
Zoom In
Zoom Out
Some of the icons are further described in the following sections.
4.3
Viewing spot data
This section describes various options for display of Spot data.
Spot data is displayed in four views:
•
Image View (for details, see 4.3.1)
•
3D View (for details, see 4.3.2)
•
Table View (for details, see 4.3.3)
•
Histogram View (for details, see 4.3.4)
4.3.1
Image View
The Image View simultaneously displays both primary and secondary gel
images. Which gel is selected as primary and secondary have impact on the
Histogram View and the Table View. The values in the Histogram View is the log
volume ratio of the primary gel/secondary gel. If the gel order is shifted, new
volume ratios are automatically viewed in the Histogram View and the Table
View.
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Functions
Five toolbar functions are associated with the Image View. All these functions
can also be accessed through menu options, indicated in parentheses.
(View:Image View). Expands the Image View to fit the workspace.
(View:Zoom:Zoom In). Zooms in to selected region of the image.
(View:Zoom:Zoom Out). Zooms out of selected region of the image.
(View:Zoom:Fit to window). Fits the entire gel image to Image View window.
(View:Contrast Brightness). Changes the brightness and contrast of the Image
View. Raising the positions of the slide bar increases either the contrast or
brightness. Selecting the Apply to all Images check box results in linking the
controls to all images. Altering the contrast and brightness does not change the
raw pixel data contained within the image, hence subsequent analyses are not
effected.
Zoom in/out with the mouse
Zooming in and out of the image can also be performed using the mouse by
dragging the mouse over a square area of the image to be zoomed. Dragging
the mouse from top left to bottom right results in zooming into the image, whilst
dragging the mouse from bottom right to top left result in resizing the image to
fit.
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Image View Properties
Properties associated with Image View are defined in the Image View properties
dialog and in the Spot Display Properties dialog.
Select View:Properties… or select the Properties icon then select the Image
View tab to display the Image View properties dialog.
•
Changing the Default radius of picking references allows the user to
define the reference marker for Ettan Spot Picker.
•
The Default picking head diameter is used to define the size of the
automated picking head.
For example, Ettan Spot Picker manual recommends a 2 mm picking head
diameter. Therefore with a
100 micron resolution image this translates to a 20 pixel picking head
diameter (the default value).
•
Select the Auto-center selected spots check box to automatically center
the selected spot in the image view.
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Click the Spot Display tab to display the Spot Display Properties dialog.
Select or deselect check boxes to display the preferred information in the Image
View.
54
•
Select Similar to display spot boundaries of spots with similar normalized
volume values when comparing the primary and secondary spot map (i.e.
both values lie between the upper and lower threshold values displayed in
the Histogram View (see also 4.3.4)).
Similar spots are displayed in green in both the Image View and the
Histogram View.
•
Select Decreased to display spot boundaries of spots where the
normalized volume values for the spot is higher on the primary spot map
than on the secondary (i.e. the log volume ratio is below the lower
threshold value displayed in the Histogram View (see also 4.3.4)).
Decreased spots are displayed in red in both the Image View and the
Histogram View.
•
Select Increased to display spot boundaries of spots where the normalized
volume values for the spot is lower on the primary spot map than on the
secondary (i.e. the log volume ratio is above the upper threshold value
displayed in the Histogram View (see also 4.3.4)).
Increased spots are displayed in blue in both the Image View and the
Histogram View.
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•
Select Excluded spots to display spots that have been excluded, either
manually or by applying an exclude filter.
Excluded spots are displayed in grey.
•
Select Picking references and pick locations to display all picking
references and pick locations in the Image View.
Picking references are displayed as yellow target circles.
Pick locations are displayed as yellow circles centred around the pick
location. By default the pick location is the same as the center of the picked
spot, but if the user selects to edit the pick location (see also 6.5.2), a yellow
line from the center of the picking circle extending to the center of the
corresponding picked spot is displayed. The pick locations are also
displayed in the 3D View as yellow cylinders when this option is selected.
•
Select Picked spots to display spots that are assigned to be picked. The
picked spots are displayed in black.
•
Select Protein of Interest spots to display spots that are set as POI, either
manually or by applying a filter. Protein of Interest spots do not have a
particular color assigned. The same color as for the abundance setting,
according to above, is used.
4.3.2
3D View
The 3D View function provides a three dimensional representation of the
primary and secondary images localized on the selected spot, representing the
raw image without filtering. The representation is plotted along the X-Y axes in
the plane of the gel. The Z axis scale is normalized between the two images
based on the volume ratios, facilitating direct visual comparison between the
two 3D spot images.
Functions
Three toolbar functions are associated with the 3D View. All these functions can
also be accessed through menu options, indicated in the parentheses.
(View:3D View). Expands the 3D View to fit the workspace.
(View:Area in 3D). Displays the area selected in the Image View in the
3D View.
(View:Rotate 3D). Rotates the 3D View, re-clicking the icon stops rotation.
Rotate manually
The 3D View can be rotated manually by dragging the mouse over the 3D
image.
Zoom in/out
Adjustment of the 3D View can be performed using the mouse. Holding down
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both mouse buttons and dragging the mouse upwards and downwards, results
in zooming in and out the 3D View, respectively.
Move spot image within 3D View
The position of the spot image within the 3D View can be adjusted by holding
down the right mouse button then dragging the mouse over the 3D View.
Reset orientation
The orientation of the scaling of the 3D View can be reset to the initial viewing
settings by selecting the Reset button at the top of the 3D View. Data associated
with each of the selected spots in the 3D View is displayed beneath the
corresponding spot. The table below describes the data displayed:
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Legend
Description
Spot number
Spot reference number (unique to a co-detected spot on
a set of images from the same gel).
Position
Gel X,Y co-ordinates of the co-detected spot
Volume
Spot pixel volume
Peak Height
Largest pixel value within the spot boundary (expressed
with background subtraction).
Area
Number of pixels within the spot boundary
Pick Position
Gel X,Y co-ordinates of the spot pick location
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3D View Properties
Properties associated with 3D View are defined in the 3D View properties dialog.
Select View:Properties… or select the Properties icon then select the
3D View tab, to display the 3D View properties dialog.
•
Spot Margin for displayed spots: The size of area displayed in the 3D View
can be altered by entering a positive integer between 3 and 80.
•
The Show caption colors check box allows the colored banner indicating
the upper edge of the 3D View (as seen in the Image View) to be removed.
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4.3 Viewing spot data
4.3.3
Table View
Displays data associated with selected co-detected spots, in a tabulated
format. The data within the table can be sorted into ascending or descending
order by clicking the column headers of the table. An arrow appears in the
selected column displaying the sorting order (see the example for the Area
column in the image below).
Clicking the Table View icon (or selecting View:Table View) expands the Table
View to fit the workspace.
The following table describes the information on co-detected spots that is
contained within the Table View.
Legend
Status
Spot No.
Abundance
Excluded
Volume Ratio
Picked
POI
Max Slope
Area
Max Peak Height
Max Volume
Protein ID
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Description
Indicates whether a spot has been confirmed by the
user (see Section 4.6.2).
Spot reference number (unique to a spot pair on a set of
images).
“Decreased”, “Similar” or “Increased” depending on
thresholds set in DIA Histogram View
Spot assigned by user or Exclude Filter for removal from
analysis set. An excluded spot is never completely
removed from the workspace and can be recovered by
the user. Excluded spots are not copied into BVA at
import of DIA workspaces.
Normalized Volume Ratio between co-detected spots in
the two visible gel views described in terms of fold
change. (A value of 2 indicates a 2-fold increase in
abundance. A value of -2 indicates a 2-fold decrease in
abundance, or a ratio of 0.5.)
The histogram is automatically recalculated if any of
the visible gel views are changed.
“Pick” designation indicates that a spot has been
selected for picking (see Section 7.4)
Indicates whether a spot has been selected as a protein
of interest, denoted by the letter “I” in the column.
Largest gradient associated with co-detected spots.
Number of pixels within the spot boundary.
Largest pixel value associated with co-detected spots.
Volume of the largest of the co-detected spots.
User defined protein identification (manually entered in
the Protein ID text box in the Spot Control Panel at the
bottom of the screen).
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Comment
PTM
User defined comment (manually entered in the
Comment text box in the Spot Control Panel at the
bottom of the screen).
Indicates whether a spot has been manually assigned
as a protein with a post translational modification,
denoted by the letters “PTM” in the column.
The information in the Table View for a specific spot can also be displayed in a
separate window:
•
To display the Spot Info dialog, right-click a spot in the Image View and
select Spot Info. Alternatively, select View:Spot Info...
The information in the dialog is automatically updated when clicking a new
spot.
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4.3 Viewing spot data
Table View properties
The spots displayed in the table can be selected by using the Table View
Properties Tab.
•
To display the Table View properties dialog, select View:Properties… or
select the Properties icon, then select the Table View tab.
By selecting and deselecting the check boxes the different categories of
spots can be selectively displayed. Deselecting all the check boxes results
in the Table View being blank. The Table View tab works on OR logic, i.e. a
spot only needs to conform to one criterion to be shown in the table.
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The following diagram illustrates some of the parameters in the Table View
associated with a spot.
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4.3.4
Histogram View
The histogram view is only visible when more than one image is being analyzed
(i.e. analyses employing either double or triple detection algorithms).
Clicking the Histogram View icon (or selecting View:Histogram View) expands
the Histogram View to fit the workspace.
Histogram
The histogram displays data associated with all detected spots in the primary
and secondary images. The detected spots are of three types:
Type of detected spot
Default Color in Histogram View
Increased
blue
Decreased
red
Unchanged
green
Spot data is plotted against log volume ratio on the X-axis, using two Y-axes.
62
•
Left Y-axis: displays the spot frequency. The red curve represents the
frequency distribution of the log volume ratios. The curve in blue
represents a normalized model frequency fitted to the spot ratios so that
the modal peak is zero (see Appendix B).
•
Right Y-axis: represents the scatter parameter selected in the histogram
selection box (right of the histogram). A plotted single data point on the
histogram represents an individual protein spot.
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The histogram is automatically recalculated when the primary and secondary
images are changed using the drop down menu in the Image View title bar.
Histogram selections area
The Histogram selections area contains two drop down menus and two
information boxes.
The following information is included in the area:
•
Scatter Parameter
The drop down menu can be used to plot either maximum peak height,
area, maximum slope, maximum volume (displayed on the right Y- axis of
the histogram) against log volume ratios.
•
Threshold mode
Thresholds are user-defined values that enable highlighting of spots that
differ between the primary and secondary image. Consequently, the
threshold functionality is predominantly used when performing smallscale experiments to determine expression differences in two samples run
on a single gel.
A variety of values can be selected in the threshold drop down menu.
•
X Model S.D. (standard deviation): number of standard deviations based
on the normalized model curve displayed in the histogram view.
•
X.X Fold: magnitude of volume ratios.
•
Manual: user-defined magnitude of volume ratios (entered in the
threshold text box).
The threshold values are represented by vertical black lines in the
histogram. Spot boundaries in the Image View and data points in the
histogram are automatically color coded to denote the spots that are
below, within or above the threshold values. The abundance column in
Table View is also updated automatically.
•
Threshold
Indicates the volume ratio selected in the threshold mode drop down
menu (e.g. the volume ratio represented by 2 model S.D.). See also 13.4.5.
•
2 S.D.
Indicates the volume ratio for 2 S.D. based on the raw data. In a normally
distributed data set 95% of data points fall within this value.
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4.3 Viewing spot data
Spot statistics area
The Spot statistics area displays information on the spot population illustrated
in the histogram view based on the user-defined threshold settings.
•
Decreased: the number of spots classified as decreased in their
abundance in the primary image, compared to the secondary image.
•
Similar: the number of spots classified as not differentially expressed in
the primary image, compared to the secondary image.
•
Increased: the number of spots classified as increased in their abundance
in the primary image, compared to the secondary image.
The Table View, Image View and Histogram View can be adjusted to selectively
display any of these subgroups:
Select the Spot Display and Table View tabs in the properties dialog box then
select/deselect the appropriate check boxes.
The number of spots designated as excluded and included (designated either
manually or by the Exclude Filter) are displayed, see next section for more
details.
Workspace information area
Displays frequency data contained within the entire workspace of:
64
•
Total number of detected spots
•
Number of excluded and included spots
•
Number of picked spots
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4.3.5
Customizing display colors
The colors used in the various views can be customized by selecting the Colors
tab in the Properties dialog box.
The dialog includes the following areas:
•
Spot colors: The colors of spot boundaries displayed in the Image Views
and the spot colors in the Histogram View can be altered by clicking the
colored circles and selecting a new color.
•
Histogram colors: The colors of the line graphs in the histogram view can
be altered by clicking the colored circles and selecting a new color.
Clicking Default restores the original settings.
Note: To use the new version 7.0 colors for a restored database, change the
color settings in DIA and BVA:
In both modules, click the Default button on the Colors tab and press OK.
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4.4 Creating and opening workspaces
4.4
Creating and opening workspaces
Before creating a DIA workspace, make sure the gel images have been imported
into the database with the Image Loader module.
Creating Workspaces
66
1
In the DeCyder 2D Main window, double-click the DeCyder 2D Differential
Analysis Software - DIA icon to open the software module.
2
Select File:Create Workspace to open the Create Workspace dialog.
3
Select project and select 1-3 images.
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4
When the desired files are added, click Create to create the DIA workspace.
Once the workspace has been created, spot detection and quantitation can be
performed, see 4.5.
Opening Workspaces
To open previously created and saved workspaces:
1
Select File:Open workspace and browse to locate the DIA workspace file.
2
When the workspace is located, select the file and click Open.
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4.4 Creating and opening workspaces
Workspace Properties
Properties associated with the workspace are displayed in the Workspace
Properties dialog.
To display the workspace properties dialog:
Select View:Properties… (or select the Properties icon), then select the
Workspace tab.
The dye tag and the dye chemistry settings selected at the image import in the
Image Loader module are displayed. The remaining information is derived from
the data processing performed.
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4.5
Spot detection and quantitation
4.5.1
Detection theory
The spot detection algorithm is designed to simultaneously process
1, 2 or 3 images derived from a single gel.
•
Single detection - one image
•
Double detection - two images
•
Triple detection - three images
Single detection is performed on images of fluorescently post-stained gels used
for picking, a case where there is a single image associated with the gel.
Double and triple detection takes advantage of the inherent co-migration
benefits of the CyDye DIGE Fluor dyes. A set of co-run images (2 images in
double detection and 3 images in triple detection) are merged together thereby
incorporating all spot features in a single image. Spot detection and spot
boundary definition is then performed using pixel data from all the individual
raw images and the merged image. The resultant spot map is overlaid back
onto the original image files. Since the spot boundaries and the detection areas
are identical for all images, the spots are effectively already matched. This
process results in highly accurate volume ratio calculations.
4.5.2
Spot detection algorithms
In this version of DeCyder it is possible to also use the spot detection algorithm
from DeCyder version 5.0 for detection of the spots in DIA and Batch processor.
Version 6.0 is default, recommended for normal gel quality, version 5.0 is
recommended for images with insufficient resolution. The two spot detection
algorithms differ in the splitting of the spots giving less spot splitting using the
6.0 algorithm. Due to an improved loading of the images minor differences
between the detection in DeCyder 5.0 and in DeCyder 7.0 with spot detection
algorithm 5.0 can be obtained at the right edges of the images.
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4.5 Spot detection and quantitation
Example of results obtained with the different spot detection algorithms is
shown in the figure below (spot detection algorithm 5.0 to the left and 6.0 to the
right).
In DIA the selection of spot detection algorithm is done in the Process Gel
Images dialog.
Information on the detection algorithm used in a saved workspace is also
displayed in this window.
When using the Batch Processor, the change of the spot detection algorithm
must still be done within DIA. The algorithm that was last used in a saved DIA
workspace will be the one displayed in the Batch Processor. In order to change
algorithm in the Batch Processor a DIA that has been processed with the desired
algorithm can be opened, reprocessed with the same algorithm and saved. This
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setting is Windows® user specific. In the Batch Processor the spot detection
algorithm that will be used for the present batch is displayed in Gel Properties.
4.5.3
1
Performing spot detection
Select Process:Process Gel Images to open the Process Gel Images dialog.
Select DeCyder detection algorithm 6.0.
Note: It is possible to use the DeCyder detection algorithm 5.0 instead. For
more information on the algorithms see section 4.5.2.
2
Enter an estimation (see also below) of the number of spots present on the
images, then click OK.
Estimation recommendation:
It is recommended that this value be overestimated to compensate for the
detection of non-proteinaceous spots on the image, e.g. dust particles
which are subsequently excluded from the analysis.
If all the spots have not been identified the spot detection process can be
repeated with a higher number of estimated spots.
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4.5 Spot detection and quantitation
For example, for a mammalian lysate run on an 24 cm pH 4-7 Immobiline™
DryStrip and a large format gel, such as the Ettan DALT Gel (20 cm x 26 cm),
a value of 2500 for Estimated Number of Spots should be satisfactory.
3
If spot detection is performed using estimated values in the high range (i.e.
up to 10,000 spots) the exclusion filter, see 4.6.1, can be used as a powerful
tool. An optimization of volume filtration generates a manageable number
of spots and an accurate detection.
4.5.4
Quantitation theory
Numerical data for individual spots are automatically calculated (e.g. volume,
area, peak height and slope).
•
Spot volumes (sum of pixel intensity within the spot boundary) are always
expressed with background subtracted. Background is subtracted on a
spot specific basis, by excluding the lowest 10th percentile pixel value on
the spot boundary, from all other pixel values within the spot boundary.
The spot volume is the summation of these corrected values.
•
Spot ratios are calculated (volume of secondary image spot/volume of
primary image spot). This ratio indicates the change in spot volume
between the two images.
These ratio values are normalized, so that the modal peak of volume ratios
is zero (since the majority of proteins are not up or down regulated). This
ratio parameter is referred to as the volume ratio. In all DeCyder 2D
Software DIA tables the volume ratio is expressed in the range of 1 to 1 000
000 for increases in spot volumes and –1 to – 1 000 000 for decreases in spot
volumes. Values between –1 and 1 are not represented, hence a two-fold
increase and decrease is represented by 2 and –2, respectively (and not 2
and 0.5 which is another way to express the same thing).
When two or three images are included in the DIA workspace, the images
displayed in the primary and secondary views can be selected using the drop
down menu in the Image View title bar. When using single detection the volume
ratio value is left blank, since there is no secondary image.
Note: The volume ratio is displayed in the Table View in this manner but all data
analyses performed in BVA module are based on the log of the normalized
ratio measurement (see Appendix B for further details).
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4.6
Data analysis (optional)
The DIA module does not require the user to perform any form of spot boundary
editing. After spot detection there are two optional post detection tasks that can
be performed before export of data:
•
Creation and execution of an exclusion filter to remove spots that do not
represent real proteins. See 4.6.1.
(Unnecessary if further analysis is to be performed in BVA module.)
•
Confirmation of detected spots. See 4.6.2.
This is used for small-scale experiments to confirm expression differences
in two samples run on a single gel.
To remove user-to-user variation from software analysis no spot editing is
allowed. Internal studies have shown that different users generate statistically
different results if spot editing is allowed. The spot detection process in DeCyder
2D Software has been demonstrated to be highly accurate (internal studies
have demonstrated 98% accuracy). The throughput is also higher without the
spot editing.
4.6.1
Spot exclusion (optional)
If further analysis of DIA gels is to be performed in BVA module, filtering, using
spot exclusion, is not necessary. Spots that do not represent real proteins will
not appear as significant in the BVA module as they will not appear in the same
place on different gels.
Original data is not changed by spot exclusion. Spot exclusion only provides help
to view selective data. However, when a BVA workspace is created, the data
from the DIA workspaces are copied into the BVA workspace. Excluded spots
and information on them are not included in the copying process and therefore
excluded spots are not included in the BVA workspace and are not possible to
analyze in the BVA module.
Defining an area of interest
The automated spot detection algorithm in DeCyder 2D Software DIA may
detect artifacts due to gel heterogeneity at the edges of the images. These spots
can be removed by setting an area of interest to define the gel without gel edge
artifacts. The spots outside of the defined area of interest are not removed until
an Exclude Filter is applied. If an area of interest was set before detection, all the
spots on the image will still be detected (even those outside the area of interest)
until an Exclude Filter is applied.
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4.6 Data analysis (optional)
To set an area of interest:
1
(Optional:) Click the Fit to window icon on the tool bar to fit the gel images
to the Image View.
2
(Optional:) Click the Image View icon on the tool bar to have a full screen
view of the gel images.
3
(Optional:) Remove the spot boundaries displayed in the Image View:
Click the Properties icon and select the Spot Display tab in the dialog that
now appears. Deselect Similar, Decreased and Increased and Click OK.
(Deselecting the spot boundaries is optional but helps the user to see the
gel more clearly).
4
Select Edit:Define Area of Interest. and using the rectangular target
pointer drag the mouse to draw a rectangle on either of the gel. Take care
to exclude edge artifacts. The area of interest is automatically defined on
the other image(s).
5
Click the Properties icon and select the Spot Display tab and reselect the
Increased, Decreased and Similar options and click OK.
6
Finish by creating an exclude filter as on page 75-77. All the spots on the
outside of the area of interest will automatically be removed when the
exclude filter have been performed.
Note: To remove the area of interest select Edit:Remove area of interest and
apply Exclude Filter to remove the Exclude status for the spots in Table
View, Image View and Graph View.
Defining an exclude area
It is possible to define a gel area to be excluded from the spot analysis. The spots
inside the excluded area will be automatically removed and the data will be renormalized automatically.
To set an exlude area:
74
1
Optional: Click the Fit to window icon on the tool bar to fit the gel images
to the Image View.
2
Optional: Click the Image View icon on the tool bar to have a full screen
view of the gel images.
3
Click the Properties icon and select the Spot Display tab in the dialog that
now appears. Deselect Similar, Decreased and Increased and click OK. This
removes the spot boundaries displayed in the Image View. (Deselecting the
spot boundaries on view is optional but helps the user to see the gel more
clearly).
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4
Select Edit:Define Exclude area and using the rectangular target pointer
drag the mouse to draw a rectangle on either of the gel. Take care to
exclude edge artifacts.
5
Confirm the exclusion in the Exclude spot dialog by clicking OK.
The spots are then excluded and the data is re-normalized. The exclude
area is automatically defined on the other image(s) in the DIA workspace.
Note: It is possible to define several Exclude areas by repeating steps 4-5.
6
Click the Properties icon, select the Spot Display tab, reselect the
Increased, Decreased and Similar options to view all spot boundaries
again, and click OK.
Note: Excluded spots can be returned to the experiment by running the
Exclude Filter on the whole image, see pages 75-77.
Creating an Exclude Filter
The Exclude Filter removes specific spots (based on defined characteristics) in
order to eliminate non-proteinaceous spots from further analyses.
The 3D View generally reveals if a detected spot is a gel artifact rather than a
protein spot. Dust particles normally have very steep sides and a pointed peak
when compared to the smoother curve of a protein spot. Therefore dust
particles have high slope values and low area values. The figure below
illustrates a protein spot and a dust particle spot.
Irregularities in the gel surface may also be detected as a spot, however such
artifacts have low peak height and volume values. Artifact spots can therefore
be excluded on the basis of slope, area, peak height and volume.
The appropriate filter parameter values can be efficiently obtained by ordering
the table according to each parameter in the filter.
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4.6 Data analysis (optional)
1
To order the table according to Max Slope, click once on the Max Slope
header on the table so that the largest maximum slope is at the top of the
column (if the smallest maximum slope is at the top, click again).
2
Click the first spot in the Table View.
3
Observe the spot in the 3D View. The 3D View clearly shows if the detected
spot is a dust particle rather than a protein spot.
a)
If the spot is a dust particle:
Jump to a spot with a value 0.5 to 1 lower than the Max Slope value for
the first spot and repeat step 3.
b)
If the spot is a protein spot:
Refine the search between the two values to find the last Max Slope
value before protein spots.
4
Note the value of the artifact immediately before a real protein spot in the
table. This is the value to enter into the slope parameter of the filter.
5
The same procedure can be applied with the Area, Max Peak Height and
Max Volume. The only difference with finding these parameters is that the
table has to be ordered so that the smallest value is present at the top of
the table before scrolling down. Within step 3a, jump to a value significantly
larger than the value for the first spot.
If there are three images in the workspace, toggle between the three
images, to ascertain more accurately the exclude filter parameters.
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6
After the filter parameters have been established, select Process:Exclude
Filter (or select the icon from the toolbar) to display the Exclude Filter dialog.
Enter the values as determined above and then click OK.
Note: Check Filter confirmed spots to allow the exclude filter to exclude
already confirmed spots that fall within the selected exclude filter
values.
The Exclude Filter will automatically exclude detected spots on the basis of
these values. If an area of interest has been defined, the spots outside of
the area of interest will also be removed. Excluded spots are by default
displayed with a gray spot boundary.
Note: Excluded spots are not removed from the DIA workspace, they are only
hidden. However, if using the DIA workspace in the BVA module, the
excluded spots in the DIA workspace cannot be analyzed.
Note: The excluded spots can be returned to the experiment by deselecting the
check boxes in the Exclude Filter dialog and then re-running the filter.
Alternatively, the values in the exclude filter can be edited, followed by rerunning the filter.
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4.6 Data analysis (optional)
Manual spot exclusion
Spots that do not represent real proteins, and that are not removed by the
exclusion filter, can be manually removed from the data set.
1
Highlight the spot, then select the Exclude check box at the bottom of the
screen.
2
Select Process:Re-Normalize to perform the normalization process again
with the manually excluded spots removed from the calculation.
Note: Always re-normalize abundance data if a large number of non-protein
spots have been removed.
Re-normalization is automatically performed when the Exclude Filter is
used.
4.6.2
Spot confirmation (optional)
Remaining, non-excluded spots can be manually confirmed, for the purposes of
visually verifying each spot. This is normally only used for small-scale
experiments.
Three options are available during spot confirmation:
Option 1:
Only confirm spots that are increased or decreased in their abundance. This
method is relatively rapid since the increased and decreased spots are
automatically identified by setting the threshold mode (see Section 4.3.4). There
is often little benefit in investigating spots that do not change in expression
levels.
1
Make sure the correct primary and secondary images are selected.
2
Select View:Properties and select the Table View tab. Ensure that the
Decreased, Increased and Picked Spots options are ticked, and that the
Excluded box is not ticked. It is useful to have the Confirmed option
checked. Click OK.
Note: If manual spot exclusion is to be performed, check the Excluded box
as well.
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3
Start the confirmation by sorting the Table View data based on a spot
characteristic (e.g. Max Volume):
Click the column header entitled Max Volume to sort the column with the
highest value at the top.
4
Scroll to the top of the table to make sure the spot with the largest volume
has been sorted to the top of the table. The spots with the largest Max
volume (and hence at the top of the sorted table) are almost exclusively
protein spots, which can be quickly confirmed.
5
Scrolling down the table to spots with lower Max volumes, spots that do not
represent real proteins start to appear. These can be manually excluded.
Spot confirmation relates to a Spot Number, therefore when using triple
detection a confirmed spot number will possess the confirm status irrespective
of which spot maps are displayed in the image view.
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4.6 Data analysis (optional)
Option 2:
Only confirm spots that have been assigned with a Protein of Interest status.
This is the recommended option if the protein filter has been applied prior to
spot confirmation. See also 4.6.3.
•
Make sure the correct primary and secondary images are selected.
•
Select only Protein of Interest spots in the Table View tab. The procedure
is then identical to option 1 from step 3.
Option 3:
Manually verify all spots (Decreased, Increased and Similar). This takes
approximately 1.5 hours for 1000 spots and is not recommended.
•
Make sure the correct primary and secondary images are selected.
•
Select the Similar, Decreased and Increased options on the Table View
tab. Click OK.
The procedure is then identical to option 1 from step 3.
4.6.3
Protein of Interest (optional)
Protein spots can be assigned as a Protein of Interest, thereby highlighting
these spots for further investigation. Protein of Interest status can be assigned
manually by selecting the Protein of Interest check box when highlighting the
desired protein spot.
Alternatively, the Protein filter can be used to automatically assign Proteins of
Interest using spot property as a selection criteria. See also 4.6.5.
4.6.4
PTM assignment (optional)
Protein spots possessing a post-translational modification (PTM), can be
denoted by checking the PTM check box.
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4.6.5
Protein Filter
The Protein Filter can be used to find spots based on user defined criteria. The
filtered spots can also be assigned with a Protein of Interest and/or Pick status.
1
Click the Protein Filter icon (or Process:Protein Filter...) to open the Protein
Filter dialog.
2
In the Protein Filter select the Assign Protein of Interest check box and/or
the Assign Pick Status check box. Proteins that meet the filter criteria,
specified in step 4, will be assigned with the selected status.
3
If the Select All check box is selected, all spots detected will be assigned
with the chosen status and it is not necessary to enter filtering setting.
Continue with step 5 immediately.
4
Alternatively, spots can be filtered on specific spot properties.
•
Selection due to change in expression:
Protein spots exhibiting a change in expression are commonly
analyzed. Therefore selecting proteins of interest on the basis of volume
ratio is useful for such spots. Spots can be selected for decreases or
increases in order to select spots that have diverged notably from each
other. Alternatively, spots can be selected for decreases and increases
allowing selection of spot volume ratios within a finite limit.
•
Selection on other characteristics:
Spots can also be selected on the basis of their physical characteristics
(i.e. area, max volume, max peak height) to ensure that spot selection
only occurs on spots that can potentially be successfully picked,
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4.6 Data analysis (optional)
digested then analyzed by mass spectroscopy.
The location of the spots on the X and Y axes can also be filtered so that
spots near or on the edge of the gel are not present in the pick list, or to
limit the eventual pick list to high or low molecular weight proteins. The
various filters can be used simultaneously to select spots on multiple
features.
•
Spot population to filter:
The filtering can take place on the whole spot population or only those
confirmed by selecting the Restrict to confirmed spots check box.
5
After entering the filter criteria, select the Filter button to ascertain the
number of spots that will be assigned with the selected status (POI or Pick).
6
If the number of resultant spots is unsuitable, the stringency of the filter can
be adjusted again (according to step 4) to produce an optimal number of
spots for selection.
7
Click OK to accept the spot filter parameters.
Spots assigned as Protein of Interest are denoted by the presence of the
letter I in the POI column of the Protein Table and have the POI check box
selected in the Spot Control panel.
Spots assigned as Pick are denoted by Pick in the Picked column of the
Protein Table and have the Pick check box selected in the Spot Control
panel.
Note: The protein of interest status of spots can be removed by selecting
Process:Unassign all Protein of Interest. The pick status of spots
can be removed by selecting Process:Unpick all.
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4.7
Saving, printing and exporting
Save workspace
DIA Workspaces are saved in the DeCyder 2D database, and contain all the data
associated with the loaded spot maps.
1
Select File:Save workspace to open the Save workspace dialog.
2
Select project where the workspace is to be saved.
Note: It is possible to create a new project by clicking the Create new
project icon.
3
Enter a file name, click Save.
Note: It is also possible to save an previously saved workspace without
overwriting the old version. Select File:Save workspace as and enter the
new name.
Printing
Select File:Print to open the Print dialog box.
Multiple check boxes can be selected for printing the required workspace views.
Note: To get optimal resolution, print images from Image View and not from All
Views.
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4.8 Exporting data
Using the clipboard
Any aspect of the different views within the workspace can be copied to the
clipboard for pasting into another application.
Click the part of the workspace to be copied, select Edit:Copy (the part of the
workspace selected is described in this drop down menu). The image is saved to
the clipboard and can then be pasted into other applications.
4.8
Exporting data
Each of the export functions in DIA is located under the File menu. The exported
files are placed in the file system, not in the DeCyder 2D database.
4.8.1
Export pick list
Select File:Export pick list to export the proteins, which have been assigned for
picking by the user, in the form of a .txt file that can be recognized by Ettan Spot
Picker or Ettan Spot Handling Workstation. See also Chapter 7 Spot picking in
BVA.
The pick list can also be exported in XML format if desired. For descriptions of
the xml-format, see EttanPickGel.pdf and EttanPickGel.xsd in the folder
Documents\Formats (in the folder where DeCyder 2D program is installed,
usually C:\Program Files\GE Healthcare\DeCyder 2D\Documents\Formats)
4.8.2
Export Workspace
Select File:Export Workspace to export all the data from the DIA workspace in
an XML format that contains all the data generated in the DIA workspace. This
can be opened by the DeCyder 2D Software XML toolbox.
For descriptions of the xml-format, see DeCyder XML.pdf and DeCyderML.xsd in
the folder Documents\Formats (in the folder where DeCyder 2D program is
installed, usually C:\Program Files\GE Healthcare\DeCyder
2D\Documents\Formats)
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5
5.1
BVA (Biological Variation Analysis) Module
Overview
5.1.1
Functionality
DeCyder 2D Software BVA processes multiple Ettan DIGE system gel images,
performing gel to gel matching of spots, allowing quantitative comparisons of
protein expression across multiple gels.
BVA processes gel images that have undergone spot detection in DIA. The BVA
module utilizes the files generated in DIA (Section 4.7) together with the original
scanned image files. The images are then matched to a single master image,
identifying common protein spots across the gels. Various experimental designs
can be assigned in BVA, allowing the statistical analysis tools to highlight
proteins that demonstrate significant protein changes under different
experimental conditions.
Additional built-in functionality allows post-matching activities to be performed:
•
Molecular weight calculation
•
Isoelectric point calculation
•
Database linkage
•
Statistical analyses
•
Spot pick list generation
Data can be saved then re-opened in a BVA workspace. Pick lists can be
exported as a text file for use in Ettan Spot Picker or Ettan Spot Handling
Workstation. Data can also be exported in an XML format for querying in
DeCyder 2D Software XML toolbox or copying and pasting into applications
such as Microsoft Word and Excel.
5.1.2
Normal workflow
The normal workflow includes the following steps:
1
Create workspace/Import DIA-workspace, see 5.6.
2
Assign experimental groups (in Spot Map mode), see 5.7.
3
Image level matching/warping (in Match mode), see 5.8.
4
Spot level matching (in Match mode), see 5.9. An optional step that could be
chosen later.
5
Statistical analysis (in Protein mode), see 5.10.
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5.2 Graphical user interface
6
Use Protein filter to assign protein of interest (in Protein mode), see 5.11.
7
Optional: Confirm the interesting spots (in Protein mode), see 5.13.2.
The workspace can be used to produce a pick list (see 5.15 and Chapter 7).
Note: If the available memory goes below 150 MB when creating or working
with a workspace, an alert is displayed. If this occurs, first secure the data
by saving and then try to free more available memory by terminating any
other running programs.
5.1.3
Large DIGE-experiments
It is recommended to use a maximum of 48 gels to keep the overview of the
workspace and to keep a good performance. Larger DIGE experiments should
be divided into several BVA workspaces and be analyzed in EDA. For
recommendation on how to set up BVA workspaces for subsequent statistical
analysis in EDA, see the first chapter of the DeCyder 2D v7.0 EDA User Manual
on how to prepare an EDA experiment.
5.2
Graphical user interface
The BVA graphical user interface is similar to DIA. It is divided into four interlinked views. Selecting a spot in, for example, the Image View will display the
same spot in the Graph View, 3D View and the Table View.
•
Image View: displays gel images. See also 5.3.1.
•
3D View: a three dimensional representation of the images localized on
the spot selected. See also 5.3.2.
•
Graph View/Experimental Design View: graphical representation of
data/options for experimental design. The window displayed in this area
depends on the mode selected. See also 5.3.4 and 5.7.2.
•
Table View: tabulated data. See also 5.3.3.
All views display different information depending on the mode selected. See
also the following page.
There is also a Data View control panel at the bottom of the workspace, which
incorporates specific functionality features. The contents of the Data View
control panel and the Table View are dependent on the mode selected.
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Create Workspace
Open Workspace
Save Workspace
Spot Map Mode
Match Mode
Protein Mode
Appearance Mode
Match Dialog
Protein Statistics
Calibrate pI and MW
Protein Filter
Area in 3D
Rotate in 3D
Sort and browse Images
Multiple Image Views
Zoom In
Zoom Out
Fit to Window
Contrast and brightness
Gel mosaic horizontal zoom in
Gel mosaic horizontal zoom out
Gel mosaic vertical zoom in
Gel mosaic vertical zoom out
Magnify Image View
Magnify Graph View
Magnify 3D View
Magnify Table View
Display All Views
View warped images
Display warp grid
Gel view color overlay
Properties
Print
What's this?
BVA (Biological Variation Analysis) Module 5
5.2.1
BVA main tool bar
The BVA main tool bar includes the following tools:
The main tool bar is very similar to that seen in the DIA module, but contain tools
for selecting modes (see also 5.2.2) and a few other tools usable in the BVA
module.
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5.3 Viewing spot data
5.2.2
BVA modes
The BVA module is composed of four different modes, which display data
manipulated through various tables and associated controls. Each mode
provides different functionality associated with specific processes in BVA
analysis.
Spot Map mode
The Spot Map mode is used to set up images for spot matching and statistical
analysis. The Table View in Spot Map mode lists data related to the Spot Maps
imported from DeCyder 2D Software DIA module.
Match mode
The Match mode is used for the processes associated with inter-gel matching.
The Table View lists all data associated with the matching algorithm.
Protein mode
The Protein mode is used to display and process data associated with the
protein spots identified across the gels. Each row in the Table View corresponds
to one protein spot, which may be present in several Spot Maps.
Appearance mode
The Appearance mode is used to display data associated with a selected
protein across the gels.
The table below briefly describes the function for each mode, and what
information is presented in the rows for each table type.
Mode
Spot Map mode (S)
Rows in table represent
Spot maps, corresponding to gel
images
Match mode (M)
Matched/unmatched spots for
Master Spot Map and Match Spot
Map
Protein mode (P)
Examine statistics of Proteins (spots in Master Spot
all proteins
Map)
Appearance mode (A) Examine statistics of Spot maps (all spot maps where
one protein
the protein is present)
5.3
Function
Set up experimental
design
Inter-gel matching
Viewing spot data
The general toolbar functions associated with the Image View, 3D View and
Table View are described in Sections 5.3.1, 5.3.2 and 5.3.3, respectively. There
are shortcuts for many menu choices, see Appendix E.
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5.3.1
Image View
The Image View allows 2-48 images to be viewed simultaneously. For a new
workspace, the Image View by default display and sort the standard images. Up
to 6 standard images are displayed by default. When a workspace is re-opened,
the images are displayed as they were shown when last saved.
Note: When working with many gels, deselect Contour and Annotations in the
Image View properties to optimize the display of the gels.
To select images displayed in multiple gel view:
The number of visible images can be changed by using the four gel view zoom
buttons: Gel mosaic horizontal zoom in, Gel mosaic horizontal zoom out, Gel
mosaic vertical zoom in and Gel mosaic vertical zoom out.
It is also possible to select the number of visible gels from a list displayed by
clicking the Multiple Image Views icon (or by selecting View:Display Multiple
Gel Views). The image below displays the selection 3 × 4.
Drop-down menu for image selection
The drop-down menu in the upper right corner of the title bar of each image is
used to select which image to display. The available gels in the drop down menu
are determined by the selected spot map sorting, see below.
Use the arrows of the scroll tool to move among the images included in the
workspace. Click the icon in the middle of the scroll tool to display the Image
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Layout window. The blue boxes indicate the gels currently displayed in the
Image View.
Spot map sorting
The spot maps included in a workspace can be sorted in various ways. It is
possible to change the sorting at any time.
New workspaces display all standard spot maps in S and M mode by default.
This simplifies the match editing procedure. When moving to P mode, the spot
maps are by default sorted after experimental groups. When returning to M
mode, the sorting from P mode is kept. In Match mode, a separate “floating”
window always displays the master gel. It is possible to move the window and
place it close to any spot map for comparison, or move it out of sight if it
temporarily is not needed.
Note: If you have a preparative gel for picking, place it in the group Unassigned
and set the function to Pick (P). The pick gel will then be sorted and
displayed together with the standards. The pick gel will also be easy
available if the image sorting Sort Freely is selected.
Any customized sorting (as described below) is kept when moving between
modes.
To change the sorting of the images displayed in the Image View, use the Sort
and Browse images icon or the menu View: Browse + Sort Gel Images.
When using the Sort and Browse images icon, click the icon and select what to
sort by (Standard, Experimental Group etc.). The Image Views are sorted
according to the selected criteria (e.g. Standard).
By selecting the option Sort Images Freely all images become available in all the
drop-down menus and thus you can sort the images any way you like.
The title bar can display a long or a short name, see Properties below.
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Zooming
If zooming a spot in one gel, all gels will be automatically zoomed (see Properties
on next page).
There are three spot zoom buttons: Zoom in, Zoom out, Fit to window. When
using a tool in one image, all images will be simultaneously zoomed
automatically. This feature is especially useful for landmarking.
The other functions in the Image View are the same as in DIA module, see 4.3.1.
Selecting spot maps in different modes
There are different ways to select spots and spot maps, how they are displayed
and what you can do with spots depending on which mode you are in. Warped
images (see also page 119) can be viewed in all modes.
•
Spot map mode
When selecting a spot map in Spot map mode the spot map header is
displayed in magenta. The spot map is selected by clicking the left mouse
button on the spot map header or by selecting the spot map in the spot
map table or in the Experimental Design View.
It is not possible to select spots in Spot map mode.
•
Match mode
In match mode a separate, floating window always displays the master
spot map with a yellow header. One more spot map can be selected by
clicking the left mouse button on the spot map header. The header of the
selected spot map, the match spot map, is displayed in magenta. Spot
maps can also be selected by clicking on a spot in a spot map.
In the Data View Controls area, different tools and options for manual
correction of spots and matches are available. See also Section 5.9.
•
Protein and Appearance mode
In Protein and Appearance mode you can select one or two spot maps to
determine which spot maps are presented in the 3D View. Click the left
mouse button on the spot map header to mark the header magenta, click
the right mouse button to mark it green. The colors are also displayed in the
3D View.
By left-clicking a spot, that protein and the spot map is selected (and thus
marked with magenta). In the Table View, 3D View and Graph View the new
selection is displayed. A selected spot is always displayed with a magenta
contour in all spot maps where it is matched.
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Properties
Properties associated with Image View are defined in the Image View Properties
dialog.
Select View:Properties… or select the Properties icon and then select the Image
View tab to display the Image View Properties dialog box.
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The dialog includes the following selections:
•
Long label in title bar: Select check box to display long image names
(example: Master: Gel 02 Cy2 Standard.gel (M)) in the Image View title bars.
If check box is unchecked, short image names (example: Gel 02 Cy2
Standard (M)) will be displayed in the title bars.
•
Spots present in table only: Select check box to display only the spots
displayed in the Table View.
•
Spot contour: Select to display the spot boundaries. (When working with
many gels in multiple view deselect Spot contour to optimize the display.)
•
Match vectors in Match Table View: Select to display the match vectors
(see Section 5.8.1).
•
Annotation: Select to display user determined annotation as text boxes on
the gel Image Views. The annotation displayed is selected from the list.
Only one category can be selected
Link Annotation: select to group spots derived from different isoforms of
the same protein (defined by the user by having the same Protein ID, AC,
name or comment) and labelled with a single label.
Filter Annotation: select to display only annotations for spots with pick
status or spots selected as proteins of interest.
Fixed Annotation Font: select and enter a value in the field to fix the font
size relative to the image resolution. This facilitates the generation of nice
copies of annotated gels for presentation purposes.
•
Link Image Views when scrolling: Select to link scrolling in all images.
•
Auto-center selected spots: Select to position selected spots in the center
of the spot map.
•
Picking Reference Data: The radius of the picking reference markers and
diameter of picker head are entered in the dialog boxes.
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5.3.2
3D View
A three dimensional representation of the two images selected in the Image
View, localized on the selected spot.
This view is only visible in the Match, Protein and Appearance modes. With the
exception of the data displayed below, each of the 3D images, the BVA and DIA
3D Views are identical (see Section 4.3.2).
Colored
banner
Colored
banner
A colored banner across one of the edges of the 3D View images corresponds
to the color of the title bar in the corresponding image view. The banner reveals
the orientation of the 3D View image, denoting the edge of the view closest to
the top of the gel.
Pick locations, when applicable, are displayed in the 3D View (see Section 7.6.2).
Moving the spot in the 3D View
To adjust the position of spot image within the 3D View, hold down the right
mouse button and move the mouse over the 3D View.
To rotate the 3D View, hold down the left mouse button and move the mouse
over the 3D View.
To zoom in and out in the 3D View, hold down both mouse buttons and move the
mouse over the 3D View.
To return to the original display of the spot, click the Reset button.
Properties
Properties associated with the 3D View are defined in the 3D View Properties
dialog.
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Select View:Properties… or select the Properties icon and then select the
3D View tab to display the Image View Properties dialog box.
•
Spot Margin for displayed spots: The size of area displayed in the 3D View
can be altered by entering a positive integer between 3 and 80.
•
The Show caption colors check box allows the colored banner, indicating
the upper edge of the 3D View (as seen in the Image View) to be removed.
5.3.3
Table View
The contents of the Table View are dependent on BVA mode selected. Similarly
the editable properties associated with the Table View are dependent upon the
mode selected (See also 5.3.5 and 5.3.7). The data within the table can be sorted
into ascending or descending order by clicking the column headers of the table.
The information for a specific spot in the table view can also be displayed in a
separate window:
•
Select a spot in a gel view using the left mouse button. Right-click a gel
view and select Spot Info (or select View:Spot Info) to display the Spot Info
dialog. The dialog displays the spot information for the selected spot in the
currently selected gel view.
If a spot is not selected in the selected gel view, the Spot Info dialog does
not contain any information. The information in the dialog is automatically
updated when selecting a new spot or gel view.
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5.3.4
Graph View and Experimental Design View
The Graph View is only displayed in Protein mode (P) and Appearance mode (A).
In Spot Map mode (S) the Graph View is substituted for Experimental Design
View, see also 5.7.2.
The Graph View in DeCyder 2D Software BVA allows the user to view scatter plot
representations of data points associated with individual proteins. For example
the manner in which the expression of a protein changes with time or drug
dosage can be graphically viewed. Examples of the graphical representations
are illustrated in the Section 5.10.
Properties associated with the Graph View are defined in the Graph View
Properties dialog box.
Select View:Properties… or select the Properties icon then select the Graph
View tab to display the Graph View Properties dialog box.
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The dialog includes the following areas:
Parameter visualization: Allows the user to define how data is displayed on the
graph by designating the parameters displayed on the X and Y-axis of the
graph.
•
X-axis: The options allows display of data according to group, conditions
or spot map label.
The sequence of the groups on the X-axis is determined by the order of
group folders in the Experimental Design view within the Spot Map mode.
This can be changed by dragging and dropping the group folder into a
new position in leftmost panel in the Experimental Design view.
•
Y-axis: Four options allow display of either: Abundance, Log Abundance,
Standardized Abundance and Log Standardized Abundance.
However, the statistical functions within DeCyder 2D Software only utilize
Log Standardized Abundance. Therefore the graphical representation of
this parametric value only reflect the data points used in the statistical
analyses.
•
Dashed line: The options allows display of dashed lines on the scatter plot
linking data points by the specified association. Samples either derived
from the same gel or possessing the same sample ID can therefore be
easily identified in the graph view.
Include in graph view: The options available determine the data points plotted
on the graph.
•
Spot: Select the check box to show all proteins spot data points associated
with the user defined X-axis parameter.
Colored circles represent these data points. The colors representing the
groups can be assigned in the Experimental Design view.
•
Mean Value Crosses: Select the check box to display the mean value of
the data points associated with the user defined X-axis parameter
(represented by a cross).
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•
Lines connecting Mean Values: Select the check box to display lines
connecting the mean values. Average marks are connected with a line
when the averaged subsets are related according to experimental group
or condition settings:
•
Groups are linked with a line when the X-axis displays groups.
•
Condition 1 assignments are linked with a line when X-axis display
Condition 2 values.
•
Condition 2 assignments are linked with a line when the X-axis display
Condition 1 values.
•
Standard: Select to display the Y-axis values corresponding to the internal
standard spots when Group or Spot Map Label is selected in the X-axis.
•
The DeCyder 2D Software standardization process defines all protein
abundance values associated with the internal standard to be 1 (or zero if
the log values are displayed). For further details see Appendix B.
•
Legend: Select to display legends defining the color coding of groups. The
legends are displayed when viewing the graph view in full screen mode.
Y axis settings: The Y-axis setting options enable the user to change the Y-axis
scaling:
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•
Automatic: Automatic optimization of the Y-axis scaling.
•
Manual: Manual definition of the maximum and minimum values on the Yaxis.
•
Manual with Automatic increase: Manual definition of the maximum and
minimum values on the Y-axis, but if the data points fall outside the
manual defined values the scale is increased automatically so that all
spots are shown.
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5.3.5
Spot Map Table
The Table View in the Spot Map mode displays data associated with the spot
maps. This data is derived from the DIA workspace generated in the DIA module.
The table below describes the data displayed in the Spot Map mode.
Legend
No.
Status
Image
Gel ID
Type
DIA Ws Name
Label
No. Of Spots
Matched
Function
Group
Group Description
Condition 1
Condition 2
Sample ID
Comment
Match Quality
Warp Status
Description
Spot map number in table
Indicates whether the image is Unmatched,
Matched, Landmarked, Master or Processing.
Gel image file name.
Gel ID is a unique number for gel in the DeCyder 2D
database.
Type of labelling chemistry used.
The name of the DIA workspace.
Dye used to label the protein (e.g., CyDye DIGE Fluor
Cy2, Cy3 and Cy5 minimal dyes).
Number of protein spots identified in the DIA.
Number of protein spots that have been matched to
the protein spots on the master.
Spot Map function (e.g. Master, Template, Analysis
and Pick).
Spot Map group (e.g. Control, Treated and Standard).
User defined description of group.
User defined condition assigned numerically
(example Time point 1,2,3…).
User defined condition assigned numerically
(example Dose point 1,2,3…).
User specified identification of sample.
User defined comment on Spot Map.
The Match Quality score displayed in the Spot Map
table is the average score for all spots in each Spot
Map.
The result of the warping if used in matching.
A failed warping may be caused by too big
differences in dimensions between the master and
match image.
It is possible to select one row in the Spot Map Table. The selected spot map is
also highlighted in the Experimental Design View.
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5.3 Viewing spot data
5.3.6
Spot Map Table properties
Properties associated with Spot Map Table are defined in the Spot Map Table
Properties dialog.
Select View:Properties… or select the Properties icon then select the Spot Map
Table tab to display the Spot Map Table Properties dialog box.
The dialog includes the following areas:
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•
Table Column Order and Visibility: The column titles selected will be
displayed in the Table View. Dragging the column titles in the list defines
the order of the columns in the Table View. Clicking Default restores the
original settings.
•
Conditions: Labels for condition 1 and condition 2 (e.g. time, dose) can be
selected from the drop-down lists (see also Section 5.10.8). New conditions
can be created by clicking Create new condition.
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5.3.7
Match Table
The Match Table displays spot specific information.
Legend
Pos.
Master No.
Status
Master
Match
Type
Description
Match Table row number.
Protein spot number on Master Spot Map.
Match confirmation status (confirmed or
unconfirmed).
Protein spot co-ordinates on Master Spot Map.
Protein spot co-ordinates on Match Spot Map.
Types of match:
Unmatched: Unmatched spots.
Auto Level1: Spots matched after first level of
matching.
Auto Level2: Spots matched after second level of
matching.
Comment
Match Quality
Match edit
Landmark: Spots matched by the user.
User defined comment regarding matched protein
spots.
A lower Match Quality score means a better match.
Added: Spots that have been added to the match spot
map.
Copied: Spots that have been copied to the match spot
map.
Merged: Spots that have been merged.
Master edit
Split: Spots that have been split.
Added: Spots that have been added to the master.
Copied: Spots that have been copied to the master.
Merged: Spots that have been merged.
Split: Spots that have been split.
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5.4 Viewing protein data
5.3.8
Match Table properties
Properties associated with Match Table are defined in the Match Table
Properties dialog box.
Select View:Properties… or select the Properties icon then select the Match
Table tab to display the Match Table Properties dialog.
•
5.4
Include in Match Table: Selects the categories of spots that are to be
displayed in the Match Table.
Viewing protein data
The information relating to proteins (such as those described in the Sections
5.11-5.14) can be viewed through the Protein Mode and the Appearance Mode
and is only possible to view once matching has been performed. The Protein
Mode displays several proteins simultaneously, allowing rapid comparison of
data from different proteins. The Appearance Mode displays a single protein
showing data from individual gels where the protein occurs.
Data can also be displayed by annotating protein spots in the Gel Images View,
which can be a useful tool for presentation of data.
5.4.1
Protein Table
The table below describes the information contained in the Protein Table:
Legend
Pos.
Master No
Status
Protein ID
Protein AC
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Description
Protein Table row number.
Protein spot number on Master Spot Map.
Confirmation status of protein.
Protein identification for database linking.
Protein accession number for database linking.
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Appearance
Indicates the number of Spot Maps in which the
selected spot appears, and the function these Spot
Maps have.
Example: “20(20) P,T” means that the spot is present in
20 out of 20 Spot Maps, P indicates that the protein is
present in at least one Pick Spot Map and T indicates
the protein is present in at least one Template Spot
Map.
T-test
p value calculated using the Student’s T-test.
Av. ratio
Average ratio between the groups selected in the
protein statistics dialog box.
1-ANOVA
p value calculated using One-way ANOVA statistical
test.
2-ANOVA –”Condition p value calculated using Two-way ANOVA statistical
1”
test with respect to Condition 1.
2-ANOVA –”Condition p value calculated using Two-way ANOVA statistical
2”
test with respect to Condition 2.
2-ANOVA-Interact
p value calculated using Two-way ANOVA statistical
test with respect to interactions between conditions 1
and 2.
Pick
Indicates whether the protein spot has been assigned
for picking in the selected pick list. If a different list is
selected, different spots are normally assigned for
picking.
Picked In
Indicates in which pick list/s the protein spot has been
assigned for picking, e.g. L1, L2, L3.
Pick spot vol.
Displays the volume of a spot, assigned for picking in
the selected pick gel (selected at the bottom of the
window), after spot detection and background
correction. This value can be used as an indication of
whether a spot can be identified by mass
spectrometry.
POI
pI
Mw
Name
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If the selected pick gel is changed, the volume will
change as well.
Proteins assigned Protein of Interest are marked with
an ‘I’ in this column.
Isoelectric point of protein (user-defined or calculated).
If only user-defined pI values are present in the table,
the table head is marked with pI* before calculation
and calculated values is shown in italics.
Molecular weight of the protein (user-defined or
calculated).
If only user-defined Mw values are present in the table,
the table head is marked with Mw* before calculation
and calculated values is shown in italics.
User defined protein names.
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Comment
Match Quality
User defined notes on proteins.
Morphological similarity metric describing deviation of
internal standard spot to an “average” internal
standard spot in protein set.
PTM
Indicates that the protein has a post translational
modification.
Paired T-test
Paired T-test significance level.
Paired Av Ratio
Paired Average Ratio between the groups selected for
protein statistics.
1-RMANOVA
Shows p-value calculated using one-way repeated
measures (i.e. for paired data) ANOVA statistical test.
2-RMANOVAShows the condition 1 p-value calculated using twoCondition 1
way repeated measures (i.e. for paired data) ANOVA
statistical test.
2-RMANOVAShows the condition 2 p-value calculated using twoCondition 2
way repeated measures (i.e. for paired data) ANOVA
statistical test.
2-RMANOVA-Interact Shows the interaction p-value calculated using twoway repeated measures (i.e. for paired data) ANOVA
statistical test.
Edit
If Merge/Split/Copy/Move or New spot has been made
in M mode, this column display “Yes”
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The columns displayed in the Protein Table can be changed using the Protein
Table Properties dialog box.
The dialog includes the following areas:
•
Protein Table Filter:
Select to display only proteins of interest or pick assigned proteins or both
in the Image View and the Table View.
•
Table Column Order and Visibility:
The selected column titles are displayed in the Table View. Clicking and
dragging the column titles in the list sets the order of the columns in the
Table View. Clicking Default restores the original settings.
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5.4.2
Appearance Table
The table below describes the information contained in the Appearance Table:
Legend
Image
Gel ID
DIA No.
DIA WS name
Type
Label
Function
Log Std Abund.
Std Abund.
Volume
Peak Height
Group
Group Description
Condition 1
Condition 2
Sample ID
Comment
Match Quality
Abund.
No.
Edit
Description
Gel image file name.
Gel ID is a unique number for gel in the DeCyder 2D
database.
Spot number in DIA.
Note: Different from the number in BVA!
The name of the DIA workspace.
Indicates the dye chemistry used.
Indicates the CyDye DIGE Fluor minimal dye label.
Spot map function (e.g. Master, Analysis, Pick and
Template). If the spot is assigned for picking in several
gels, this is represented by P1, P2, P3 etc. in this
column.
The logarithm (log10) of the standardized volume
ratio, or Volume of sample spot / normalized volume
of standard spot.
Protein volume ratio calculated relative to internal
standard described in terms of fold change. Same
value as in the Volume Ratio column in the DIA table.
(A volume ratio of 2 has the fold change value 2 and a
volume ratio of 0.5 has a fold change value of -2.)
Spot pixel volume (expressed background
subtraction).
Largest pixel value within the spot boundary
(expressed background subtraction).
Assigned spot map group.
Description of spot map group.
Condition 1 numerical value.
Condition 2 numerical value.
User defined sample identification.
User defined comment.
Morphological similarity metric describing deviation
of internal standard spot to an “average” internal
standard spot in protein set.
Relative volume of all spots representing a particular
protein in a BVA data set, used when no internal
standard is present.
Spot map number.
If Merge/Split/Copy/Move or New spot has been
made in M mode, this column display “Yes”
The Master No. for the protein is displayed above the table.
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The columns displayed in the appearance table can be changed using the
Appearance Table Properties dialog box.
Table Column Order and Visibility: The selected column titles are displayed in
the Table View. Clicking and dragging the column titles in the list sets the order
of the columns in the Table View. Clicking Default restores the original settings.
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5.4 Viewing protein data
5.4.3
Spot annotation
Displaying annotation
Various protein data elements can be displayed on the Image View. The
annotation can be defined using the Image View Properties dialog box. See
Section 5.3.1 for details.
Positioning the annotations
The position of the annotations in the Image View can be altered to ensure that
the different annotations are clearly visible. This feature is useful when one
annotation obscures another annotation.
•
To alter the position of an annotation:
Select Edit:Move Annotations so that a tick appears next to this option.
The annotation labels in the Image View can then be dragged to the new
positions.
5.4.4
Customizing display colors
The colors used in the various views can be customized by selecting the Colors
tab in the Properties dialog box.
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The dialog includes the following areas:
•
Match Colors: The spot boundary colors of the different categories of
spots displayed in the Match Mode’s Image View can be changed by
clicking the colored circles then selecting a new color.
•
Spot Colors: Spot boundary colors of the different categories of spots
displayed in the Protein and Appearance Mode Image Views can be
changed by clicking the colored circles then selecting a new color.
•
Annotation Colors: The colors of the gel image annotation text boxes and
linking lines can be changed by clicking the colored circles then selecting a
new color.
•
Spot Map Colors: The colors that designate different categories of spot
map in the Image Views can be changed by clicking in the colored circles
then selecting a new color.
The original settings can be restored by clicking Default.
Note: To use the new version 7.0 colors for a restored database, change the
color settings in DIA and BVA:
In both modules, click the Default button on the Colors tab and press OK.
5.5
Protein quantitation
When using DeCyder 2D Software BVA for inter-gel analysis it is recommended
that an internal standard is used in the experimental design (see Section 1.4). As
discussed in the previous section, the DIA quantifies the spots as a function of
the internal standard. This value is used in the BVA for analyses facilitating direct
comparison of spot maps, and is referred to as the Standardized Abundance.
Log10 of this value (referred to as the Log Standardized Abundance) is used to
aid scaling in graphical representations and is employed in all statistical
analyses.
If an internal standard is not used in the experimental approach DeCyder 2D
Software can express spot volumes of specific proteins across gels as a ratio
relative to the lowest volume spot of that protein. This parameter is referred to
as the Abundance in BVA. This latter approach is not recommended, since intergel quantitative comparisons are not as accurate as the quantitation using an
internal standard. It is not possible to perform the BVA statistical functionality
with this non-internal standardized data.
As with the Standardized Abundance, log10 of the Abundance value (referred to
as Log Abundance) is often used to aid scaling in graphical representations.
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5.6 Creating and opening workspaces
5.6
Creating and opening workspaces
5.6.1
Open the BVA module
In the DeCyder 2D Main window, double-click the DeCyder 2D Differential
Analysis Software - BVA icon to open the BVA module.
5.6.2
Creating workspaces
DeCyder 2D Software BVA requires DIA workspaces generated in the DIA
(Section 4.7) together with the original scanned image files, to create a BVA
workspace. It is recommended to include up to 48 gels in a BVA workspace.
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1
In the BVA module window, click the Create workspace icon to open the
Create Workspace dialog. Alternatively, select File:Create Workspace.
2
Select project in the left panel, select DIA workspaces to import in the
middle panel and click Add.
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The added files are displayed in the right panel.
3
More files can be added by repeating step 2.
Files can be removed by selecting the file and clicking Remove.
4
When the appropriate files have been added, click Create to create the BVA
workspace.
See 5.3 for information on how to view spots.
5.6.3
Opening workspaces
To open previously created and saved workspaces:
1
Select File:Open workspace, or click the Open workspace icon, and browse
to locate the BVA workspace file.
2
When the file is located, select the file and click Open.
See 5.3 for information on how to view spots.
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5.6 Creating and opening workspaces
5.6.4
Add template or DIA workspaces to an existing BVA workspace
To add a template or more DIA workspaces to an existing BVA workspace:
1
With the BVA workspace open, select File:Add Template/DIA workspace.
2
Select project and one or several DIA workspaces.
3
Click Add--> to add the DIA workspaces to the BVA workspace list in the
right panel.
Workspaces can be removed by selecting the file and clicking Remove
4
Click Add to add the DIA workspaces to the selected BVA workspace.
Note: If the added BVA workspace is a template, set the spot map as
function T, Template in the Data View Control panel of the Spot Map
mode (S).
5.6.5
Remove gel
Gels can be removed in any of the four modes.
1
Select a spot map in the gel that should be removed.
2
Select Edit:Remove gel.
The complete gel is removed, i.e. all spot maps are removed, not only the
one selected in step 1.
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5.7
Defining spot map attributes
All spot map attributes are assigned in the Spot Map mode. Select View:Spot
Map or select the toolbar icon to work in the Spot Map mode (if a new BVA
workspace has been created the interface will automatically open to the Spot
Map mode). This mode consists of three windows: Image View, Table View and
Experimental Design View.
5.7.1
Function assignment (optional)
Spot Maps can be assigned with up to four functions that indicate the role of the
Spot Map in the BVA processing. The Spot Map functions are:
•
Analysis (A) – Spot data will be included in quantitative analysis. Those that
should not be assigned as analysis images may include preparative gels
for spot picking and images to be used as a template.
•
Master (M) –One spot map can be assigned as the Master Spot Map in
each workspace. All other Spot Maps are matched to the Master. When
creating a new workspace, the Spot Map with the largest number of spots
is automatically set to be the Master Spot Map.
•
Template (T) - One spot map can be assigned as the Template spot map in
each workspace. Protein ID, Protein AC, Name, Comment, pI, Mw and
Protein of Interest assigned to the spots of the Template Spot Map will be
cross annotated to matching spots in all the other images. Templates can
also be used for linking in EDA, for further information see the first chapter
of the DeCyder 2D v7.0 EDA User Manual on how to prepare an EDA
experiment.
•
Pick (P) – Assign spot maps as Pick, if the gel will be used for generating a
pick list. Spot data from gels assigned as Pick are excluded from the
statistical calculations.
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5.7 Defining spot map attributes
To assign the spot map functions select View:Table View or click the Magnify
Table View icon to display the Table View only. The table shows the images that
have been loaded into the workspace and the number of spots that have been
detected on them.
Note:
A set of images from the same gel will have the same number of spots
since the DIA detection algorithm is designed to detect the same number
of spots on images from the same gel.
In the Function column all the images are, by default, assigned Analysis (A) and
one of the analysis images is assigned as Master (M). The analysis function
designation indicates that each of the images will be included in the statistical
analysis.
The Master function can be assigned to a different image:
Select the image to be assigned as master using the left mouse click, then select
the Master check box at the bottom of the workspace in the area entitled Spot
Map Functions.
All other function assignments are similarly performed.
5.7.2
Group assignment
A group is a collection of spot maps that for the purposes of the experiment
cannot be broken down into further sub-groups. Groups can include analysis
sets such as control groups, treated groups, time points and temperature points.
This spot map assignment is necessary to facilitate the inter-group statistical
analyses in DeCyder 2D Software.
The internal standard (non-experimental group) spot maps are assigned as
standard if the word standard, pooled or std is present in the image file name.
By default non-standard spot maps are assigned as “Unassigned”.
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The experimental group assignment is displayed in Experimental Design View
when within the Spot Map mode.
Spot Maps are located in the appropriate folder on the left side of the
Experimental Design View. The Spot Maps can be viewed by selecting the
desired group folder.
Add a new group
Before assigning groups new groups need to be added.
1
In the right panel, click Add.
2
Enter name, description (user defined information about, and linked to, the
experimental group) and condition values in the different fields.
Note: The conditions are selected according to page 156.
3
Click the color icon to select a color for the group.
4
Finish by clicking Confirm.
The new group folder is displayed in the left panel.
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5.7 Defining spot map attributes
Assign experimental groups
1
Click the Unassigned folder on the left to display the contents of the folder
both in the left and in the center panel of the Experimental Design view.
2
Drag and drop Spot Maps from the center panel to the appropriate group
folder in the left panel.
Several Spot Maps at a time can be moved.
The assigned groups are also displayed in the Spot Map Table in the Group
column.
To edit names of groups, see the following instructions.
Edit and remove groups
Any added experimental group except Standard and Unassigned can be edited
or removed.
Edit group names
All groups but Standard and Unassigned can be renamed.
1
In the left panel, mark the group folder to rename.
2
In the right panel, click the Edit Name button.
3
Change the name and click Confirm.
5.7.3
Comment assignment
The comment text box on the right of the data view control panel can be used
to enter a user defined comment that is specific to the selected Spot Map.
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5.8
Gel matching
All gel images must be matched to the master gel image, before data analysis.
The master gel is either defined by the automated matching to the gel with
highest number of detected spots or set by the user prior to automated
matching. The standard gel images of the gels are fitted to the standard gel
image of the master gel using automated matching with integrated warping or
using only the automated matching.
The result from using the automated matching with integrated warping is a
geometrically corrected gel image with very short match vectors. Match vector
indicate the positional difference for the same protein spot on different gels.
Using only the automated matching will generally result in much longer match
vectors.
To analyze the result, the color overlay function is useful. It is a gel image overlay
function which put two gel images, the master image in yellow color and other
image in blue color, on top of each other. Differences in spot position are thereby
easily found.
To further optimize the image matching, manual landmarks can be set in areas
where matching has been less successful and then rematching/re-warping can
be performed.
The workflow is described in next section. Additional information is found in
Sections 5.8.2-5.8.6 as described below.
•
Workflow, see section 5.8.1
•
Matching (theory), see section 5.8.2
•
Warping (theory), see section 5.8.3
•
Landmarking (theory), see section 5.8.4
•
Match result display, see section 5.8.5
•
Color overlay, see section 5.8.6
When the inter-gel matching has been checked, a more detailed checking of
matches, on spot level, can be performed before analyzing protein statistics. See
Section 5.9
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5.8.1
Workflow
Workflow overview
Phase 1 — Matching and warping, see page 118
Phase 2 — Alternative 1 — Checking the matching using color overlay images,
see page 119
Phase 2 — Alternative 2 — Checking the matching using match vectors, see
page 121 (This alternative is suitable for example when landmarking a pick gel.
Warping of pick gels may not always generate favorable results.)
Phase 3 — If required, set landmarks and re-match/re-warp, see page 122
Phase 1 — Matching and warping
1
Select Process:Match… or click the Match icon.
2
Select one of the options to match all the images, or just a specific set of
images.
•
Match Unmatched and Landmarked: matches spot maps that have
not been matched and those that have been landmarked after a
previous matching process. Recommended selection for any extra
matching procedures of the same spot maps.
•
Match All: matches all spot maps in the workspace (including those
already matched).
•
Match Selected: matches only the selected gel to the master.
The check box Optimize matching using warping is checked by default
and is recommended to use. Warping is then applied to all spot maps
selected by the radio buttons above. Uncheck to not execute the warping.
3
Click Match to commence matching. If the Optimize matching using
warping is checked, warped images are created. For every warped image
a warp symbol is presented in the upper right part of the image window.
For information on the Match Table, see 5.3.7.
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Phase 2 — Checking the matching
There are two alternatives for checking the matching results.
•
If warping has been performed the color overlay is a useful tool to find
mismatched areas, see Alternative 1 below.
•
If warping has not been used the match vectors is the main tool for
evaluating match results, see Alternative 2 on page 121.
Phase 2 — Alternative 1 — Checking the matching using color overlay images
If warping has been used it is recommended to first check gels and/or gel areas
where the warping has been less successful by examining the color overlay (se
more information on page 127).
1
Ensure that you are in Match mode.
2
Ensure that the warped images are displayed. Click the View Warped
Images button to alternate between warped gels and non-warped original
gels.
3
Sort the gel images by Standard to visualize all standard images in the
workspace, which will simplify the evaluation of the matching results.
4
It may be useful to click Show Warp Grid to display the grid pattern in the
warped version of the images. The Warp Grid illustrates the global
appearance of the warp transform.
5
Click the color overlay button and select Color Overlay Using Master
Image. The Master image within every gel is presented in yellow and the
Standard image is presented in blue.
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6
Right-click in a gel image and select, from the displayed list, not to show the
contours and to show the match vectors.
Note: Other options can be displayed, but showing too many options at the
same time can make it difficult to see patterns.
Note: It may be useful to turn different options on and off to find patterns.
For example, test on/off for grid, warp/no warp, color overlay, spot
contour and match vectors.
Figure 5-1. Example of warped images with displayed warp grid, match vectors and color
overlay using master image.
7
Examine all gels to single out any gels where the warping and/or matching
appears to generate less favorable results.
This can be determined by viewing areas where the yellow master color is
visible in parts of the image, or by viewing where the warping grid displays
an unusual level of deformation.
If the color overlay and the warp grid is not distinct, select Contrast and
Brightness and adjust the contrast and the brightness. It is possible to set
the contrast and brightness of the master (yellow) and match (blue) images
independently.
8
For images that have areas with unsatisfactory warping and/or matching
results, set landmarks in the areas where the warping and/or matching is
less successful. This can be done by confirming a few correct matches in
the area or by landmarking suitable spots (see also page 122).
Note: It may be necessary to turn on and off information to be able to
determine suitable landmark spots: warped images, warp grid, color
overlay, spot contours. Right-click in the gel image and choose action
to turn these on and off.
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Note: If there are large differences in size or large visual differences
between the master image and the match image the result of the
warping may be less successful. Try to landmark the particular
combination of images, or execute the matching without optimizing
using warping.
9
Re-match the gels with new landmarks (see also page 122):
•
Click the Match button.
The Match options dialog opens.
•
If you want to use warping, select Optimize Matching Using Warping.
The new warped images are created and matched. This can be done for
one gel at a time or for several gels at once.
10 Repeat steps 8 to 9 until the matching appears to be satisfactory in all gels.
11 Proceed to evaluating the matching result in more detail, see 5.9.
Phase 2 — Alternative 2 — Checking the matching using match vectors
If warping has not been used, check the overall gel matching by examining the
match vectors displayed in the Image View. The vectors should be orientated in
a similar direction across the gel. If an area of the gel does not follow this pattern
(e.g. the vectors are perpendicular or cross), it is highly likely that mismatches
are present in that area and should be looked at closely.
1
Ensure that you are in Match mode.
2
Right-click in a gel and select Show Match vectors.
3
Check the match vector patterns in all standard gels included in the
workspace.
4
For image areas with unsatisfactory matching (where match vectors that
are perpendicular or cross), set landmarks in the areas where the matching
is less accurate.
This can be done by confirming a few correct matches in the area or by
landmarking suitable spots (see also page 122).
Note: When matching has been optimized using warping, the match
vectors are normally very short. It may be necessary to turn on and
off information to be able to determine suitable landmark spots:
warped images, warp grid, color overlay, spot contours. Right-click in
the gel image and choose action to turn these on and off.
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5
Re-match the gels with new landmarks (see also page 122):
•
Click the Match button.
The Match options dialog opens.
•
Select options in the dialog and click Match.
If desired, warping can be used even though it was not used in the
previous attempt.
6
Repeat steps 4 to 5 until the matching appears to be satisfactory in all gels.
7
Proceed to evaluating the matching result in more detail, see 5.9.
Phase 3 — If required, set landmarks and re-match/re-warp
In some cases the automated warping may not succeed in completely aligning
all parts of an image to the master. In such cases, it is recommended to add one
or a few landmarks for spots in areas where the warping is less successful.
To use a spot as a landmark the spot must be present on both the match
image(s) and the master.
1
Use the Gel mosaic horizontal/vertical zoom buttons to select the number
of gels to be displayed, or click the Multiple Image Views icon (or select
View:Display Multiple Gel Views).
It is recommended to display only the standard images (spot maps) in the
analysis (since all other images were co-detected with its standard spot
map in the DIA module). For example, in an analysis with totally 12 images
including 4 standard images, display only 2×2 gels to simplify the
landmarking.
When reviewing the matching result, it is necessary to compare one gel
image to the master gel image. However, it is common that the master gel
view is not displayed among the currently visible set of gel views. To ensure
that the master is always available, the master image is displayed in a
separate floating (freely movable) window in M mode.
2
It is recommended to perform landmarking on the standard images. If, for
some reason, other images have been selected, click the Sort and Browse
images icon and select Sort images by standard to display the standard
images again (see page 90 for more information on sorting and selecting
images).
Unmatched spots have orange spot boundaries. See also Section 5.8.5.
If the spot boundaries are green in an unmatched set of spots then the
images are from the same DIA workspace as the master gel image (thus
landmarks are not necessary).
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3
To set a landmark, select a clearly defined spot in all spot maps. The spot
boundaries of the selected spots should become magenta (default).
To select a spot on more than one spot map, press the Ctrl-key and hold it
down, or check the Multi Select check box, and then click on the spots to
multi-select in the other gel views.
4
Click Add match (if all selected spots are unmatched) or Break+Add (if
some of the spots are already matched, but wrongly matched).
Note: Landmarking can be aided by viewing the pattern around the
selected spot in the 3D View to confirm that the two selected spots
do correspond to each other.
Note: The number of spots being displayed around the selected spot in the
3D View can be altered. Open the Properties dialog and select the 3D
View tab. Alter the numerical value in the Spot Margin For Displayed
Spot option, to adjust the parameter size and consequently change
the number of spots displayed around the selected spot. See also
page 95.
Note: Another way to set a landmark is to confirm a match, see “Confirm
single match” on page 131 or “Confirm match set” on page 131.
5
Repeat step 3-4 until all desired landmarks are set.
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6
When landmarking is completed, rematch the gels with new landmarks:
•
Click the Match button.
The Match options dialog opens.
•
If desired, select Optimize Matching Using Warping.
The new warped images are created. This can be done for one gel at a
time or for several gels at once.
During the rematching process, any confirmed matches (marked as type
Landmark in the Match Table) will be used as landmarks to improve the
matching process.
7
Repeat steps 5 to 6 until the matching appears to be satisfactory in all gels.
8
Proceed to Section 5.10.
5.8.2
Matching (theory)
All spot maps are matched to a master image sequentially. The spot-matching
algorithm is a pattern recognition algorithm that matches one single spot in one
gel to a single spot in another gel based on its neighboring spots.
Spot matching has to be performed before the BVA module can analyze the
abundance of protein spots in different gels. The matching is automatic and will
obtain the best and fastest matching results for gels without distorted pattern
caused e.g. by smiling or streaking and similar spot patterns.
The matching process is divided into two steps (Level 1 and Level 2).
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•
Level1: The algorithm first matches large spots spread evenly over the
whole gel. The algorithm matches by comparing positions and sizes of the
neighboring spots. The matched spots are then used as landmarks,
equivalent to manual matches. The algorithm then starts from these
landmarks and matches neighboring spots. In this way the matching
propagates out from the landmarks by matching a spot and then
matching its neighbors.
•
Level 2: Matching is performed by transforming the detected spot center
from one gel to the other gel. A spot center transformation is considered a
match if the transformed co-ordinates are close enough to a spot center
on the other gel. After each matching step a control is performed which
removes obvious mismatches.
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5.8.3
Warping (theory)
The warping function is used to correct distortions in the gel images, building
geometrically corrected gel images. The warping function reshapes the
standard image of the gel to fit the master standard gel image. The original gel
images are always intact and cannot be effected by warping. It is easy to switch
between viewing the warped versions of the images and viewing the original
versions of the images.
The center points of any manually landmarked spots in a combination of match
image and master image will also be used as landmark points in the warping
algorithm. The result from this is that manually landmarked spots should share
the same center point in both images after warping.
By warping the gel images, it is easy to reveal and review local mismatches on
an overview level as well as on a close level. Warping is a tool to improve and
optimize the final matching result.
First the optimum warp transformation, that will make a match image resemble
the master image as well as possible, is calculated. When this is done, the
transform is applied to the corresponding match image to generate the warped
version of the image. When the warped version of a gel image is being created
the text in the title bar of the corresponding gel view reads "Warping...”. This
happens after a matching operation is completed and when a BVA workspace
that contains warping information is opened. A BVA workspace only stores the
warp transform parameters rather than the complete warped images, which
saves a lot of space in the database. However, this implies that the warped
versions of the images need to be re-calculated each time the workspace is
opened.
5.8.4
Landmarking (theory)
Landmarks are manually pinned points used as start values in the matching and
warping. Landmarks may be set after matching if there are areas with many
mis-matched spots,
It is usually only necessary to set landmarks on those images that differ
significantly from the Master image. Often landmarks do not need to be set at
all.
To denote a spot as type Landmark in the Match table, you can confirm a match
or make a new landmark.
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Landmarked spots are also used to align the warping results. For details see
Section 5.8.3 and figure Figure 5-2. below.
Figure 5-2. Matching and warping results without landmarking (above) and with
landmarking (below). Note that for gel 3 in the top right corner, the landmarked spot has
resulted in a more correctly warped image in the area around the landmark, which can be
clearly seen in the color overlay.
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5.8.5
Match result display
Spot boundary colors
The automatic matches in the Table View are of two types: Auto Level 1 and Auto
Level 2. By default, the two types of matches are displayed differently in the
Image View:
•
Auto level 1 spots have by default green spot boundary highlights,
•
Auto level 2 spots have by default lilac spot boundary highlights.
•
Unmatched spot boundaries are colored orange.
In addition to these match types, there can also be matches of type Landmark,
see 5.8.4. Landmarked spot boundaries are purple.
Match vectors
The vector lines in the Image View indicate the positional difference for the
same protein spot on different gels.
5.8.6
Color overlay
The color overlay function in BVA is a gel image overlay function which put two
gel images, one yellow and one blue, on top of each other. Areas in the images
that are displayed in gray scale indicate areas where both of the images are
similar, while blue or yellow colored areas indicate differences between the
images. The color overlay function can be used to visualize the result of warping
and matching or to visualize protein variations in a gel.
•
To visualize the warping result select Color Overlay Using Master Image
and ensure that warped images are displayed. The fixed standard image
of the master gel is presented in yellow (as a yellow shadow) behind the
warped standard image presented in blue. Deviations can easily be found
by locating areas where the positions of blue spots and the corresponding
yellow spots are shifted, resulting in blue and yellow coloring of the image.
Note: It is not recommended to visualize the matching result of unwarped
images with the color overlay function. The deviations might be hard
to evaluate.
•
To visualize protein variations in a gel select Color Overlay Using Standard
Image. The Standard image of the gel is presented in yellow behind the
other (e.g. Treated) image presented in blue. For this option all spots should
generally be located in the same position. A blue spot indicates increased
protein abundance compared to the standard, and a yellow spot indicates
a decreased protein abundance compared to the standard. Any deviations
where the blue spot is not located precisely on top of the corresponding
yellow spot would indicate a difference in migration between the samples
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in the gel for that particular protein. This option is interesting to use in P
mode.
The color overlay feature is always available independent of the different view
modes: S, M, P, A, and independent of whether warped images are displayed or
not.
5.9
Match editing
After matching, manual checking of matches can be performed, but protein
statistics can be performed directly if desired. In the latter case, the protein
statistics can be used as an indication of which matches need manual checking.
The color overlay function together with the warping function are also powerful
tools to reveal local mismatches on a close level.
The workflow is described in next section. Additional information is found in
sections 5.9.2-5.9.3 as described below.
•
Workflow, see section 5.9.1
•
Available editing options, see section 5.9.2
•
Match Quality Metric, see section 5.9.3
Note: It may be useful to sort the spots in the match table in various ways.
Sorting on Master no is the natural way, but sorting on Status and Type
is useful to separate confirmed/unconfirmed spots plus
landmark/match level. If you want to find manually edited spots, sort on
Master Edit or Match Edit. If you use Match quality you can sort on that
column to sort out the spots with the highest match quality score (which
indicate high likelihood of mismatches).
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5.9.1
Workflow
For all match confirmation, the area at the bottom of the Match mode window
is used. Detailed descriptions of all available editing options are found in Section
5.9.2.
1
Ensure that you are in Match mode.
2
Click the spot to check in any of the images or in the Table View.
Note: When clicking a spot in an image which is not the master, that spot
map will be set as match spot map.
3
Check if the match is correct or not.
Deciding whether a match is accurate can be aided by viewing the selected
spot and the surrounding cluster in the 3D View as well as looking at the
matched spots in the Image View. Magnifying a View may also be helpful.
4
Confirm accurate matches, correct matching inaccuracies or correct
detection inaccuracies according to section 5.9.2.
5
Repeat from step 2 until all desired matches are confirmed.
Note: All spots that have been confirmed are denoted as type Landmark in the
Match Table.
5.9.2
Available editing options
The spots can be edited if the automatic detection is not satisfactory. Spot
editing can only be performed in Match mode and with the Warped Images
View deselected. The buttons and check boxes in the Data View Control panel
are used to select editing type.
Note: Always check the spot in the 3D View before editing. This is especially
important when performing split/merge.
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Table 5-1. Available editing options
Confirm accurate
matches as landmarks (A)
Correcting
matching
inaccuracies to
form landmarks (B)
Correcting
detection
inaccuracies (C)
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Editing option
Page
•
Confirm single match
131
•
Confirm match set
131
•
Commenting matches
131
•
Add Match
131
•
Break Match
131
•
Multiple select and Add+Break Match
131
•
Landmarking without prior matching
132
•
Merge spots
Note: Not available when viewing
warped gels.
133
•
Split merged spots
Note: Not available when viewing
warped gels.
134
•
Copy to master or selected
135
•
Remove from master or selected
135
•
New spot
Note: Not available when viewing
warped gels.
Note: Introduces user bias.
136
•
Split selected spot
Note: Not available when viewing
warped gels.
Note: Introduces user bias.
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•
Move selected spot
Note: Not available when viewing
warped gels.
Note: Introduces user bias.
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Confirm accurate matches as landmarks
Confirm single match
To confirm only the match in the selected spot map, click the Confirm Single
Match button.
Confirmed matches become landmarks and are denoted as type Landmark in
the match table.
Confirm match set
If the match is correct in all spot maps, confirm the match, by clicking the
Confirm Match Set button in the Data View Control panel.
Confirmed matches become landmarks and are denoted as type Landmark in
the match table.
Commenting matches
A comment can be associated with each matched protein by entering text in the
match comment text box. The comment is associated with all gels where the
corresponding master spot is matched.
Correcting matching inaccuracies to form landmarks
Add match
If the spot is unmatched, click the spot in the master, click the spot in the
selected gel and click Add Match.
Break match
Break Match is used for breaking an incorrect match in the selected spot map.
Select the spot in the selected spot map and click the Break Match button. The
button then changes into an Add Match button.
Note: If a match between a spot in the master gel and a match gel is broken,
that specific combination of spots will not be matched again by the
matching algorithm during a re-match.
Multiple select and Add+Break Match
For matching of a spot in several images to one master, or for breaking an
incorrect match in the selected spot map.
1
Select a spot. The matched spots in the other images will be displayed.
2
Press Ctrl and click the spots in the other images that should be matched
to the selected Master spot, or check Multi Select and click the spots.
3
Click the button Add Match (displayed if no matches need to be broken) or
Break + Add Match (displayed if one or several matches need to be broken)
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Landmarking without prior matching
Landmarking is a manual definition of matched protein spots in order to
improve the accuracy of the gel-to-gel matching process, when the samples are
particularly complex or the gels have not run well. This can be applicable for pick
gels for example as they often differ significantly from the Master image.
1
Use the Gel mosaic horizontal/vertical zoom buttons to select the number
of gels to be displayed, or click the Multiple Image Views icon (or select
View:Display Multiple Gel Views).
It is recommended to display only the standard images (spot maps) in the
analysis (since all other images were co-detected with its standard spot
map in the DIA module). For example, in an analysis with totally 12 images
including 4 standard images, display only 2×2 gels to simplify the
landmarking.
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2
Ensure that the gel to landmark is included in the display.
3
To set a landmark, select a clearly defined spot in all spot maps. The spot
boundaries of the selected spots should become magenta (default).
4
Select the spot which corresponds to the master image spot on the next
standard gel image, so that this spot becomes magenta.
A few seconds later, a vector line should appear showing that the spots
have been matched and the landmark has been set.
5
Repeat the landmarking for any remaining gels that may need
landmarking.
6
Repeat step 3-4 until approximately 10 landmarks have been set. It is
recommended that the landmarks are evenly distributed across the image,
as this aids the matching and warping process.
7
Click the Match icon,
8
When landmarking is completed, click the Match icon to open the Match
options dialog. Select Match all and if desired, Optimize Matching Using
Warping. Click OK.
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Correcting detection inaccuracies
Merge spots
A protein peak might be dissected into several spots despite appearing as a
single discrete spot. This is normally due to subtle gradient changes on the spot
surface that are not always apparent to the naked eye. These “split” spots can
be merged to form a single spot boundary.
1
In the Image View, select the matched spot that requires to be merged to
surrounding spot(s).
2
Select Edit:Merge Spots... or click the Merge check box, at the bottom of the
window, to display only the surrounding spots possible to merge with the
selected spot.
3
In the image view, click the spot you want to merge with the first spot.
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Both the image view and the 3D View display the new spot boundary
showing all the component spots. All statistics are recalculated
automatically.
Merged master spot
Match spot
When the merging is completed, the image is updated and display the new
spots that the spot now can be merged to.
4
Uncheck the Merge check box to finish the procedure.
The merging process can be reversed by selecting Edit:Split Merged or by
checking the Split Merged check box, see description below.
Split merged spots
To split merged spots, check the Split Merged check box or select Edit:Split
Merged. The merged spots are displayed in the Image View. Click the
appropriate spot to split. The spot is split and all statistics are recalculated
automatically. Splitting merged spots results in all the associated spots being
unmatched.
If you want to split another spot, check the Split Merged check box again and
repeat the procedure.
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Copy to Master
If a detected spot on the selected image has not been detected on the master
image for any reason, it can be copied from the selected gel to the master gel.
1
Select the spot on the selected image and then click the Copy to Master
button.
The spot boundaries are then transferred to the master.
2
Make sure that the new master spot is matched correctly to all gels by
performing a rematch. It is also possible to manually match the spots in the
area corresponding to the location of the new master spot in all gels.
Copy to Match
If a detected spot on the master gel has not been detected on the selected
image, it can be added to the selected image.
1
Select a gel view, which displays an image from the gel in which the spot
has not been detected, by clicking in the title bar of the corresponding gel
view.
2
Select the spot on the master image and then click Copy to Match. The spot
boundaries are then transferred to the selected gel.
Remove from Match/Master
Spots can be removed from any image. Select the spot on the selected image
and then click Remove from Match or Remove from Master. The spot
information is then removed from the selected/master image.
Note: This operation cannot be undone. When a spot is removed it is
permanently removed from the workspace.
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New Spot
Note: New spots can not be added when viewing warped images.
Note: If using this function on the master spot map, See “Master gel selection for
some editing functions” on page 138.
If you want to add a spot that has not been detected, check the New Spot box.
When moving the mouse over the selected gel, a pen will appear with which you
can draw spot boundaries manually.
Press the left mouse button where the new spot starts and move the mouse in
a suitable curve. When the left mouse button is released, the new spot contour
is closed.
A spot added in this way will receive a manually edited tag which is displayed in
the Edited column in the match table. A protein which contains a spot added in
this way will also receive a manually edited status which is visible in the protein
table.
Uncheck the New Spot check box to finish the procedure.
Note: If the BVA workspace shall be linked in EDA, avoid using the new spot
function in the master or template spot maps in the different BVA
workspaces as it is very difficult to manually perform the same
corrections on several BVA:s. Protein spots with different coordinates in
the master or template cannot be linked in EDA.
Note: It is important to know that by drawing new spots manually in this way,
user bias is introduced to the data set. It is thus recommended to avoid
using this type of editing.
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Split selected
Note: Spots can not be split when viewing warped images.
Note: If using this function on the master spot map, See “Master gel selection for
some editing functions” on page 138.
If you want to split a spot, check the Split Selected box. When moving the
mouse over the selected gel, a pen will appear with which you can draw a line
that splits the spot. Press the left mouse button when the tip of the pen rests on
a point on the contour of the spot to split. Hold the left mouse button down and
move the tip of the pen cursor to another suitable point on the contour of the
same spot and release the left mouse button.
Uncheck the Split Selected check box to finish the procedure. If the spot was
matched, both spots are now unmatched.
The action can be undone by merging the spots again.
A spot split in this way will receive a manually edited tag which is displayed in
the Edited column in the match table, and a protein which contains a spot split
in this way will also receive a manually edited status which is visible in the
protein table.
Note: If the BVA workspace shall be linked in EDA, avoid using the split function
in the master or template spot maps in the different BVA workspaces as it
is very difficult to manually perform the same corrections on several
BVA:s. Protein spots with different coordinates in the master or template
cannot be linked in EDA.
Note: It is important to know that by splitting spots manually in this way, user
bias is introduced to the data set. It is thus recommended to avoid using
this type of editing if possible.
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Move selected
Note: Spots can not be moved when viewing warped images.
Note: If using this function on the master spot map, See “Master gel selection for
some editing functions” on page 138.
1
If the spot has been mis-placed, check the Move Selected box to be able to
move the spot boundaries.
2
To move the spot position, move the displayed hand cursor to the center
point of the spot, press the left mouse button and drag the spot position to
a suitable new position, where the left mouse button is released.
However, be careful, the spot placement will be recalculated for the image!
3
Uncheck the Move Selected check box to finish the procedure.
A spot moved in this way will receive a manually edited tag which is displayed
in the Edited column in the match table, and a protein which contains a spot
moved in this way will also receive a manually edited status which is visible in
the protein table.
Note: If the BVA workspace shall be linked in EDA, avoid using the move function
in the master or template spot maps in the different BVA workspaces as it
is very difficult to manually perform the same corrections on several
BVA:s. Protein spots with different coordinates in the master or template
cannot be linked in EDA.
Note: It is important to know that by changing a spot location manually in this
way, user bias is introduced to the data set. It is thus recommended to
avoid using this type of editing if possible.
Master gel selection for some editing functions
To be able to use the functions New Spot, Split Selected and Move Selected on
the master spot map, the master spot map must be selected by clicking the
header. The border of the header will then be colored magenta.
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5.9.3
Match Quality Metric
Select Process:Calculate Match Quality to calculate and display the match
quality values. Match quality can be calculated for up to 30 gels.
Match quality values are typically between 1 and 15, expressed as real numbers
approximated to two decimal points. This value represents morphological, but
not positional similarities in matched spots. The match quality is calculated only
for the internal standard spots that are matched across a set of gel images, as
we know that these proteins should be the same.
The match quality value is calculated from the surface profile of each internal
standard on each gel matched to the master spot maps. The surface profiles of
all standard spots in a set of matched spots are combined to form an “average”
surface profile. Subsequently, all the individual internal standard spot surfaces
in the match are compared to this “normal” surface (including the spot derived
from the master spot map). The quality score is the calculated difference
between the individual matched spots and the “normal” spot.
The spot surfaces that deviate a great deal from the “normal” surface receive
the highest score, whereas the spot surfaces that have close resemblance to
the “normal” surface will be given the lowest score. Therefore, values
approaching zero indicate good spot similarity and hence accurate matching.
As the quality score increases the degree of similarity decreases revealing
incorrect matches. The match quality value in the Match Table can therefore be
used to rapidly identify incorrect matches when reviewing inter-gel matching.
In addition to quality score for each matched spot being displayed in the match
table, each set of matched spots (a protein) are also assigned a global match
quality score (displayed in the Protein table). This protein match quality score
represents the spot within the matched group that has the highest quality score.
This protein match quality value enables the identification of poorly matched
spots, thereby highlighting matched spots in the protein set that may contain
outliers. All the match quality values for all matched spots in a protein can be
viewed simultaneously in the Appearance Table facilitating the rapid
identification of the outlying data point.
There is also a quality score value designated for each spot map, which is
displayed in the Spot Map Table. This value refers to the average quality score
for all matched standard spots on each gel. Hence, gels that are poorly matched
possess high match quality values. High spot map match quality values are
often due to poorly run gels that cannot be matched efficiently without land
marking (see page 122).
Note: The match quality score is based exclusively on the surface profile of the
spots on the internal standard spot maps. Therefore, the quality score
cannot be calculated for experimental designs that do not contain an
internal standard. Furthermore, each co-detected spot for multiple
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images on the same gel all possess the same match quality value, which
is associated with the profile of the internal standard spot only.
Note: The match quality score is always calculated on the original images.
Warped images are not used in the match quality metric calculation.
5.10
Protein statistics
This section describes the various ways to perform statistical analyses and
includes the following sections:
5.10.1 Opening the protein statistics dialog box
5.10.2 Defining groups
5.10.3 Overview of Statistical tests
5.10.4 Independent and paired analyses
5.10.5 Student’s T-test and Average ratio
5.10.6 ANOVA
5.10.7 One-Way ANOVA
5.10.8 Two-Way ANOVA
5.10.9 Further statistical analyses
When the chosen analyses have been performed, continue with Section 5.11.
5.10.1
Opening the protein statistics dialog box
DeCyder 2D Software BVA possesses several statistical analysis methods that
can be employed to ascertain whether changes in expression of specific
proteins are significant between samples from different experimental groups.
To open the statistical analysis dialog select menu Process:Protein Statistics.
Alternatively, the statistical analysis dialog can be opened using the toolbar
icon.
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The dialog box contains various options allowing statistical analysis of protein
data from spot maps loaded in the DeCyder 2D Software BVA.
The Protein statistics dialog includes the following areas:
•
Type of statistical test: see 5.10.4 for more information.
•
Statistical tests: see Sections 5.10.5 to 5.10.8 for more information.
•
Apply false discovery rate (FDR) correction: see Section 5.10.3 for more
information
A text box with additional information in the bottom of the dialog gives guidance
on criteria that have to be fulfilled to be able to perform the selected statistical
analyses.
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5.10.2
Defining groups
A group in BVA is a collection of spot maps that for the purposes of the
experiment cannot be broken down into further sub groups.
For example, 3 replica gels from one sample are considered one group.
Alternatively, 3 gels of 3 different samples treated with exactly the same
experimental conditions can also be considered as a group.
There is no limit on the number of groups that can be assigned in DeCyder 2D
Software BVA.
The different experimental groups must first be identified in DeCyder 2D
Software BVA before statistical analysis of protein expression changes
(performed as in Section 5.7.2).
When performing the different statistical tests, populations should be selected.
See Sections 5.10.5 to 5.10.8 for more information.
5.10.3
Overview of Statistical tests
The BVA module has four “principle groups comparison” or “experimental
condition comparison” methods that can be applied to analyze protein spot
data.
•
Average ratio between two groups or two populations of groups. See also
5.10.5.
•
Student’s T-test: Statistical analysis between two groups or two
populations of groups. See also 5.10.5.
•
One-Way ANOVA (ANalysis Of VAriance): Statistical analysis between all
groups. See also 5.10.6 and 5.10.7.
•
Two-Way ANOVA: Statistical analysis between the two conditions in an
experimental design where there are two independent factors (e.g. timedose study). This analysis allows the internal and mutual effects of the two
factors to be quantified. See also 5.10.6 and 5.10.8.
•
Apply false discovery rate (FDR) correction: When testing thousand of
proteins for statistical significance with Student's T-test or ANOVA, many
of the proteins may appear to be statistically differentially expressed, but
several of the proteins may have achieved this significance by chance
alone.
Example: If 3,000 proteins are tested for differential expression with an
ANOVA value cut-off of 0.01, then the expected level of proteins to be
identified as significant by chance alone, even if there is no true differential
expression, is: 3 000 × 0.01 = 30 proteins. Multiple testing correction
methods, such as False Discovery Rate (FDR), adjusts the Student's T-test or
ANOVA values for each protein to keep the overall error rate as low as
possible. (See also the reference Benjamini,Y. and Hochberg,Y. (2000) On the
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adaptive control of the false discovery rate in multiple testing with
independent statistics. J. Educ. Behav. Stat., 25, 60-83.)
All tests can be performed as either independent or paired. See also 5.10.4.
The log standardized abundance is the only variable subjected to the above
statistical analyses within DeCyder 2D Software BVA. The standardized
abundance is derived from the normalized spot volume standardized against
the intra-gel standard. The logs of the standardized abundance values are used
in order that the data points approach a normal distribution around zero,
thereby fulfilling the requirements of the subsequent statistical tests.
Consequently, the statistical analysis functionality is not valid unless the
experimental design includes an internal standard on every gel.
General requirements
All spot maps included in the analysis need to be co-run with an internal
standard.
Only Spot Maps assigned as Analysis in the Spot Map Table are included in the
statistical analysis.
Automatic recalculation
Protein Statistics are recalculated automatically if any data that affects the
statistics are changed, e.g. if a match is broken or if a spot map is assigned to a
new group.
5.10.4
Independent and paired analyses
The Student’s T-test, One-Way ANOVA and Two-Way ANOVA can be either
independent (normal) or paired (individual). Both types are used to test the
hypothesis that a variable, in this case protein abundance, differs between
groups or experimental conditions.
The paired test is specifically used when each data point in one group
corresponds to a matching data point in the other group(s). A typical example
would be the same group of patients before and after a treatment. The unpaired
tests are more general techniques that can be used to test whether
standardized protein abundance differs between groups, and does not require
that the groups be paired in any way, or even of equal sizes.
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Examples:
The following graphs are examples illustrating independent and paired tests.
Independent test. Abundance of a specific protein in five diseased individuals
compared to a group of five non-diseased individuals. Data points are
independent since there are different individuals in each group.
Paired test. Abundance of a specific protein in the above five diseased
individuals before and after drug treatment. Data points are paired because the
same individuals are present in pre- and post-treated groups. Although there is
no significant difference between the means of standardized abundance in the
two groups, a paired T-test reveals that the two groups are significantly
different, since each individual exhibits an increase in protein abundance after
drug treatment. In this example, there is a single result (i.e. one image) for each
individual in each group. Experiments employing replicate images from the five
individuals can also be applied to a paired test. In this instance the means of the
replicas are calculated then subjected to pairing (ensuring the pairs are
independent).
Note: The above graphs are conceptual (not generated in DeCyder 2D
Software).
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Requirements for paired testing
In order to perform paired testing, the pairing of spot maps must be preassigned in the Spot Map Mode. This can be done by inserting a numerical
identifier in the text box labelled Sample ID at the bottom of the Spot Map Mode.
For example, spot maps of protein samples from individual 2 in each the of
groups can be assigned. In this way the dependency can be associated with
each spot map.
5.10.5
Student’s T-test and Average ratio
Average ratio
Log standardized protein abundance is the only variable analyzed in the
Student’s T-test within DeCyder 2D Software BVA module. The degree of
difference in the standardized abundance between 2 protein spot groups is
expressed as the average ratio. The average ratio value indicates the
standardized volume ratio between the two groups or populations. Values are
displayed in the range of -∞ to –1 for decreases in expression and +1 to +∞ for
increases in expression. Values between -1 and 1 are not represented, hence a
two-fold increase and decrease is represented by 2 and –2, respectively (not 2
and 0.5).
Note: The average ratio parameter is displayed in this manner but the statistical
analyses are based on the log of the true ratio measurement.
Student’s T-test
Student’s T-test, often known simply as the T-test, is one of the most commonly
used of all statistical tests. The Student’s T-test is used to test the hypothesis
that a variable differs between two groups. The Student’s T-test performed is an
equal variance two-tailed test, therefore, direction of change (i.e. increases and
decreases) in the standardized abundance parameter are considered.
The T-test requires a minimum of two data points in each of the two groups.
Greater statistical validity can be achieved using larger number of replicates. It
is recommended that the largest possible number of biological replicates are
performed for optimal validity. However, if biological replicates are not possible,
gel replicates can be used for the purposes of the statistical analysis. In these
instances it is recommended that gels be run, at least, in triplicate, hence each
group has a minimum of three data points.
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Null hypothesis
The Student’s T-test null hypothesis is that there is no change in the protein
abundance between experimental groups (i.e. the average ratio between two
groups is 1). Therefore the T-test p value (seen in the T-test column of the protein
table) represents the probability of obtaining the observed data if the two
groups have the same protein abundance. For example, if the T-test p value
between two groups is 0.01, then the probability of obtaining the observed
difference in protein abundance by stochastic variation alone is 1 in a 100.
Protein abundance differences are generally assumed to be statistically
significant when p≤0.05. However a critical value of 0.05 would mean that 5%
of the data points would be expected by stochastic events alone (e.g. 50 spots
out of 1000 tested). It is therefore advisable to review the critical value applied
to the data. Due to the small amount of experimental variation within Ettan DIGE
system and subsequent DeCyder 2D Software analysis, a critical value of 0.01 is
often applied.
Assumptions and limitations
The main assumptions of the Student's T-test are the following:
•
Log standardized abundance values within a group are normally or
approximately normally distributed. When the assumption of normality is
violated, the T-test tends not to perform well when the sample size is
small, or the significance level is small (e.g. p<0.01). However, two-tailed
tests are surprisingly robust even for skewed data.
•
The parametric variance of the two groups are equal. However, the test
tends to be robust when the sample sizes are approximately equal. If the
sample sizes are not equal, then the worst case occurs when the smaller
sample comes from the population with the larger variance.
Performing analysis (Average ratio and Student’s T-test)
The user can perform T-test calculations and average ratio calculations
between any two groups (or populations of groups).
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1
In the Protein Statistics dialog box, select the option button to indicate
whether the T-test is independent or paired. Pairing of spot maps has to be
pre-assigned in the Spot Map mode (see Section 5.10.4) for paired testing.
2
Select the two groups in the in the population 1 and population 2 lists
(population of groups can be selected by ctrl + left mouse clicking the
selected groups).
3
Select the Average ratio and the Student’s T-test check boxes.
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Click Calculate.
The T-test p values and average ratios will be displayed in the protein table.
5
The spots in the Protein Table can then be sorted by clicking the T-test
column header. This will order the table so that the protein spots exhibiting
the most significant changes are listed at the top of the table.
Since the T-test is two-tailed the statistical significance of both increases
and decreases in protein abundance quantified are ordered together.
A positive average ratio value indicates an increase from population 2 to
population 1 (the order is stipulated when defining groups in the protein
statistics dialog box). Conversely, a decrease in abundance is denoted by a
negative average ratio.
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Specific Requirements
If there are at least two members in each group, T-tests and average ratios will
be calculated. If there is only one member in each group only the “average” ratio
will be calculated.
Continue from 5.10.9 if no further calculations are to be performed.
5.10.6
ANOVA
Overview
Analysis of variance (ANOVA) is a family of methods that can be used to analyze
the results from both simple and complex experiments. It is one of the most
important statistical tests available for biologists and at its lowest level it is
essentially an extension of the logic of Student’s T-tests to those situations
where the concurrent comparison of the means of three or more samples is
required. Thus, when comparing two means, the ANOVA will give the same
results as the T-test for independent samples (if comparing two different groups
or observations). There is no restriction on the number of groups that can be
analyzed. It is equally valid for testing differences between 2 groups or among
20.
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ANOVA types
There are two types of ANOVA analyses in DeCyder 2D Software BVA module,
One-Way and Two-Way. The One-Way ANOVA evaluates differences in all
assigned groups, whereas the Two-Way ANOVA can also evaluate the statistical
significance of effects from independent factors (such as time, temperature and
dose). These are entered as conditions 1 and 2.
Replicate requirements
The ANOVA tests require a minimum of two replicate data points. Greater
statistical validity can be achieved using larger number of replicates. It is
recommended that the largest possible number of biological replicates are
performed for optimal validity. However, if biological replicates are not possible,
gel replicates can be used for the purposes of the statistical analysis. In these
instances it is recommended that gels be run, at least, in triplicate, hence each
group has at least three data points.
As with the Student's T-test the ANOVA tests can be either independent or
paired. Paired ANOVA tests require dependencies to be applied (see Section
5.10.4).
Null hypothesis
The ANOVA test null hypothesis is that there is no change in the protein
abundance between any of the experimental groups analyzed. The ANOVA test
compares the variance between groups with the variance within groups. The
ratio of the between-groups variance to the within-groups variance is known as
the F ratio, and can be used to generate a p value. If the F ratio is large, then it
shows that the variation between groups is large compared with the variation
within groups, and thus that the groups may be different. Therefore the p value
(displayed in the ANOVA column of the protein table) is a measure of the
probability of obtaining the observed data if the groups have the same protein
abundance.
For example, if the ANOVA p value between two groups is 0.01, then the
probability of obtaining the observed difference in protein abundance by
stochastic variation alone is 1 in a 100. Protein abundance differences are
generally assumed to be statistically significant when p≤0.05. However a critical
value of 0.05 would mean that 5% of the data points would be expected by
stochastic events alone (e.g. 50 spots out of 1000 tested will be false positives).
It is therefore advisable to review the critical value applied to the data. Due to
the small amount of experimental variation within Ettan DIGE system a critical
value of 0.01 is often applied.
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Assumptions and imitations
The main assumptions of the ANOVA tests are the following:
•
The populations from which the samples were obtained are normally or
approximately normally distributed.
•
The variance of the populations must be equal.
There is a certain amount of leeway in both these assumptions, particularly the
first: small deviations from a normal distribution are unlikely to be of any
consequence. However, if the variance among the groups differs greatly, the
ANOVA p value may have poor validity.
5.10.7
One-Way ANOVA
Overview
The simplest form of ANOVA is known as one-way ANOVA, and in DeCyder 2D
Software is used to test for differences in standardized abundance. For
example, a clinical trial might consist of three groups of patients according to
whether they were given drug A, drug B, or placebo. ANOVA could be used to
test the null hypothesis that there is no difference in standardized protein
abundance among the groups.
The test will not indicate which groups are different from which other groups,
just that there is an overall difference. In the above example, a significant
difference does not necessarily mean that drug A is different from drug B, or that
either drug is different from placebo. All a One-Way ANOVA test indicates is that,
somehow the patients are affected by one of the drugs.
Performing analysis (One-Way ANOVA)
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1
In the Protein Statistics dialog box, select Independent tests or Paired
tests.
A paired ANOVA test is called a repeated measures ANOVA (RMANOVA).
Pairing of spot maps have to be pre-assigned in the Spot Map Mode (see
Section 5.10.4) for paired testing.
2
Select the One-Way ANOVA check box (all assigned groups are included in
the One-Way ANOVA test).
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Click Calculate.
4
Select the Properties icon on the tool bar and ensure that the 1-ANOVA
check box is selected in the Protein Table tab.
The One-Way ANOVA p values will then be displayed in the protein table. As
with the T-test analysis, spots can be ordered by significance by clicking the
1-ANOVA column heading in the protein table.
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Specific requirements
All groups that have at least two members will be included in the calculation. To
analyze a subset of groups, those that are to be excluded have to be deselected
as analysis in the Spot Map function tab (see Section 5.7.2).
Continue from 5.10.9 if no further calculation are to be performed.
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5.10.8
Two-Way ANOVA
Overview
Two-Way ANOVA calculates the significance of the difference between groups
with the same condition 2 and different condition 1 value
(Two –Way ANOVA Condition 1) and the other way around
(Two –Way ANOVA Condition 2). The Two-Way ANOVA analysis also calculates a
significance value of the mutual effect of the two factors (Two –Way ANOVA
Interaction).
There are therefore three sets of hypothesis with the Two-Way ANOVA. The null
hypotheses for each of the sets are given below.
The population means of the first condition are equal. This is like the One-Way
ANOVA for condition 1 exclusively.
The population means of the second condition are equal. This is like the OneWay ANOVA for condition 2 exclusively.
There is no interaction between the two factors. A significant ANOVA Interaction
value indicates that the two factors affect each other due to synergy or
interference.
Examples of such effects are illustrated on next page:
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Graphical examples of Two-Way ANOVA analyses.
Graphs illustrate changes in protein abundance (y-axis) for a two-condition
experiment. Condition 1 (x-axis) represents two temperatures A and B; condition
2 (red and yellow circles) represents drug treated and control samples. Each
condition is in triplicate, hence there are four experimental groups with 3
samples in each group. Conditions 1 and 2 are used to link groups together
based on one common factor, i.e. group 1 and 2 may have the same condition
1 value (both temperature 1) but different condition 2 value (drug treated or
control). Groups 3 and 4 will have the other condition 2 value (both temperature
2) with different condition 2 values (drug treated or control).
•
Experiment 1, there is no variation in abundance from temperature A to B
or from treated and non-treated samples.
•
Experiment 2, temperature B results in increased protein expression
whereas drug treatment has no effect.
•
Experiment 3, drug treatment results in increased protein expression
whereas temperature changes have no effect.
•
Experiment 4, both drug treatment and increasing temperature result in
increased protein expression i.e. the two conditions are independent and
P12 is not significant.
•
Experiment 5, drug treatment only in conjunction with change in
temperature results in an increased protein abundance i.e. the two
conditions are synergistic hence P12<0.001.
P1, P2 and P12 are labelled as 2-ANOVA-“condition 1”,
2-ANOVA-“condition 2” and 2-ANOVA-interact in the Protein Table.
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Defining conditions
In order to perform Two-Way ANOVA analysis the experimental conditions for
each gel image must first be assigned.
1
Define condition labels:
Select the Properties icon and click the Spot Map Table tab.
The condition labels can then be selected from the drop-down lists.
Click OK to finish.
Condition label
selection from
drop-down lists
Note: To create new conditions, click Create new condition, enter name
and description and click OK.
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2
Enter numerical values for the experimental conditions of each group in the
Experimental Design View.
Up to two conditions can be designated for each spot map.
Note: It can, therefore, be seen that when assigning a two condition
experimental design the Condition labels must be defined for a TwoWay ANOVA test.
When assigning a single condition experiment the data is assigned
into Groups for One-Way ANOVA testing.
Performing analysis (Two-Way ANOVA)
1
In the Protein Statistics dialog box, select Independent tests or Paired
tests.
Pairing of spot maps has to be pre-assigned in the Spot Map Mode (see
Section 5.10.4) for paired testing.
2
Select the Two-Way ANOVA check box.
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3
Click Calculate.
4
Select the Properties icon on the tool bar and ensure that 2-ANOVA“condition 1”, 2-ANOVA-“condition 2” and 2-ANOVA-interact check boxes
are selected in the protein table tab.
The Two-Way ANOVA p values will then be displayed in the protein table. As
with the other analyses, spots can be ordered by significance by clicking
the appropriate 2-ANOVA column headings in the protein table.
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Specific requirements
The number of groups should be the number of different values in condition 1 ×
the number of different values in condition 2.
For example:
An experiment with 2 temperatures and 4 time points, require 8 groups for a
Two-Way ANOVA analysis. At least 2 spot maps are required in each group.
Otherwise there will be missing values and no result. The experimental design
should also be set up so that the following criteria are applied:
•
Condition 1: The experimental groups should have both condition values
filled in, so that there are at least 2 groups with different condition 1 values
and the same condition 2 value.
•
Condition 2: The experimental groups should have both condition values
filled in, so that there are at least 2 groups with different condition 2 values
and the same condition 1 value.
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Additional requirements for paired Two-Way ANOVA:
•
At least 2 different sample IDs in each group (spot maps with the same
condition 1 and condition 2 value).
•
A sample ID must be included in all groups.
For example: An experiment with 2 temperatures, 2 time points, and 3
individuals. If, for one individual (e.g. individual 3) for one protein, matches in all
groups does not exist, missing values will be the result. The individual will
therefore be removed and the remaining number of individuals has to be at
least 2 to perform the calculation. In this example there are 2 individuals left and
the calculation can be performed.
For further information on experimental design and set up examples, see
Appendix C Experimental examples.
5.10.9
Further statistical analyses
The data generated within DeCyder 2D Software can also be exported as an
XML file and then extracted using the XML toolbox, enabling post processing of
several parameters. The user can therefore apply their own statistical analysis
algorithms to normalized and pre-normalized data to suit their own specific
requirements. More statistical analysis options are also available in the EDA
application.
5.11
Protein Filter
The protein filter is a tool that allows the selection of proteins based on various
user-defined criteria. The original data is not changed by filtering. Filtering
allows selective data to be viewed. The selection is performed by entering
values (or not) for various information/criteria. The protein filter can also be used
to assign Protein of Interest and/or Pick status.
See also Section 5.4 for information on how protein information is displayed.
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5.11.1
Filtering proteins
1
Select Process: Protein Filter or click the Protein filter icon to open the
Protein Filter dialog.
2
Select settings and enter values (see 5.11.2 for detailed information).
3
If assigning Pick status, select pick list (see 5.11.2 for detailed information).
4
Click Filter to filter the proteins.
5
If the filtering is OK (a reasonable number of proteins are selected; not all,
not zero), click OK.
If the filtering is not OK, change settings as appropriate and repeat from
step 4.
6
Check that the filtered proteins are correctly matched (see also 5.9) and
check the graph.
If desired, continue with spot picking.
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5.11 Protein Filter
5.11.2
Protein filter selections
For examples of protein filter selections, see also Appendix C.
The Protein Filter dialog includes the following areas:
•
Filter action
The filter allows proteins to either be given a Protein of Interest or Pick
Status by selection of the appropriate check box.
If Assign Pick status in list is selected, click Change List to select a pick list.
The Select pick list dialog opens displaying the 10 available pick lists. The
number of proteins in the selected pick list is also displayed.
To change name on an existing list, click Change name... and change name
in the Change name dialog.
If the selected pick list should be emptied, click Clear list.... Alternatively,
select Process:Unpick All:in selected pick list.
Process:Unpick All:in all pick lists can be selected to clear all pick lists.
Click OK to return to the Protein Filter dialog.
Note: Proteins of interest from filters run before are not removed during
filtering. Select Process:Unassign all Proteins of Interest to remove
all proteins of interest before running the Protein filter again.
If proteins of interest are assigned first, they can all be assigned as Pick by
selecting Process:Assign all protein of interest as pick.
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•
General filter settings
The general filter settings allows the selection of all detected proteins by
selecting the Select all check box. This panel within the filter also permits
the restriction of the filtering to only a subset population of proteins either
those confirmed or those present on a specified number of spot maps.
•
Select proteins with
Note: It is only possible to select this filter in the Protein Filter dialog if
statistical tests have been performed prior to filtering the proteins
(see Section 5.10).
Proteins can be selected on the statistical parameters described in Section
5.10. Select statistical analyses check boxes (one or several) and enter
values in the appropriate text boxes.
See also next page.
For Two-way ANOVA:
•
If only one condition is to be used for selection:
check only one check box. (for example Time value)
•
If two conditions should be used for selection:
check both check boxes.
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5.12 Molecular weight (Mw) and isoelectric point (pI), (optional)
•
If one condition should be selected but NOT the other:
check both check boxes and
set one value to relevant, i.e. ≤ 0.05 and
set the other value to non-relevant, i.e. ≥ 0.05 and ≤ 1.
•
Use the Interaction value to see how the values interact or counteract
with each other.
Proteins can also be selected on the basis of their physical properties, such
as volume, X-co-ordinate and Y-co-ordinate. These physical properties
pertain to the spots on the Pick spot map selected from the drop down
menu.
Note: The use of the protein filter in selecting spots for picking is further
discussed in Section 6.4.2.
5.12
Molecular weight (Mw) and isoelectric point (pI),
(optional)
DeCyder 2D Software BVA provides the user with the functionality to calculate
and display the molecular weight (Mw) and isoelectric point (pI) of the proteins
on all the images being analyzed. The Mw and pI of known proteins (minimum
two) can be entered into the Protein Table, these values (known as the base list)
are then used to calculate the Mw and pI for all the other proteins on the images.
See also Section 5.4 for information on how protein information is displayed.
5.12.1
Entering Mw and pI of known proteins
To enter the Mw and pI of the known proteins in your experiment:
•
Select the protein in the Protein Table and enter the Mw and pI into the
Mw and pI text boxes, in the data view control panel.
The Mw and pI entered for a protein can be edited by clicking the protein
and changing the values in the Mw and pI boxes.
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Calculating Mw and pI
To calculate the Molecular weight (Mw) and isoelectric point (pI) of all the
proteins on the images:
1
Select Process:Calibrate pI and Mw or click the Calibrate pI and Mw icon
to open the Calibrate pI and Mw dialog.
The dialog displays the pI and Mw values that have been manually entered
into the protein table, using the data view control panel in the Protein mode.
These values are used as a base list to calculate the pI and Mw values for
all the proteins.
Calibration of the pI and Mw values can be user determined by selecting
Linear, Log Linear or Cubic Spline:
•
Linear: Creates a straight line between all values in the base list.
•
Log Linear: Similar to the linear function, except that the values in the
base list values are first logged (Log10). So the line is based on
logarithmic values.
•
Cubic Spline: Creates a line using the cubic spline function, which fits a
curved line through all points in the base list.
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5.12 Molecular weight (Mw) and isoelectric point (pI), (optional)
The type of first dimension Isoelectric Focusing (IEF) strip influences pI
calibration used. For example, a linear Isoelectric Focusing (IEF) strip makes
the Linear pI calibration method most suitable.
Mw calibration is influenced by the second dimension gel used. For
example, Log Linear Mw calibration method is most suitable for nongradient gels.
2
Select Linear, Log Linear or Cubic Spline for pI and Mw.
3
Click Calculate to perform the calculation.
The pI and Mw of the proteins should now appear in the Protein Table.
4
Click OK to confirm the calculation.
5
To clear all calculated pI and Mw values use Process:Remove calculated pI
and Mw values. Manually entered values are not affected by this operation.
Displaying the Mw and pI values on the images
To display the calculated pI and Mw values of the proteins on the gel images:
166
1
Click the Properties icon to display the Properties dialog. By default the
dialog should open on the Image View tab.
2
Select the Annotations check box then highlight the pI and Mw option from
the list of different annotations. Click OK to display these values on the
images.
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Use the Zoom in function to make the annotations more legible.
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5.13 User-defined protein labelling (optional)
5.13
User-defined protein labelling (optional)
In the Data View Control panel protein information is displayed and can be
entered.
See also Section 5.4 for information on how protein information is displayed.
Note: It is possible to enter data for more than one protein candidate in
Protein ID, Protein AC, and Name. Separate each entry by a comma or
semicolon.
5.13.1
Protein ID and Protein AC (optional)
Protein ID and Protein AC information found (see also 5.14) can be manually
entered. It can also be imported via File:Import Protein Data... See Appendix D
for more information. This type of information can also be included in a template
spot map which can be imported into the BVA workspace (see 5.6.4).
5.13.2
Confirmation (optional)
Protein confirmation allows the marking of proteins for user reference purposes
(e.g. for visually checked proteins). No part of the analysis in DeCyder 2D
Software BVA requires confirmation of proteins.
•
To confirm a protein, select the protein and then click Confirm in the data
view control panel.
•
If the selected protein is confirmed, it can be unconfirmed by selecting the
Unconfirm button.
5.13.3
Name (optional)
User-defined names can be entered in the text box in the Data View Control
panel. Protein names can also be imported via File:Import Protein Data..., see
Appendix D for more information.
5.13.4
Comment (optional)
User-defined comments can be entered in the text box in the Data View Control
panel.
5.13.5
Protein of interest (optional)
The Protein of Interest check box is selected for proteins that may warrant
further investigation by the user.
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5.13.6
PTM (optional)
Protein spots possessing a post-translational modification (PTM) can be
denoted by checking the PTM check box.
5.14
Protein database linking (optional)
DeCyder 2D Software provides the user with functions that enable rapid access
to both local and web based protein databases through Protein IDs and
Accession Numbers (AC). This functionality is designed to aid further study of
identified proteins using information from existing protein databases.
A list of web based protein databases are provided as default in DeCyder 2D
Software. The linking uses the default web browser to interact with the protein
databases. The Protein ID or AC is substituted into the web address of the
protein database. On condition that the protein ID entered in the protein table is
in the format that is recognized by the search algorithm of a protein database,
the user can directly access a selected protein from BVA.
Use the Protein database tab of the BVA module property pages to set up how
Protein IDs and ACs are inserted in the web address of different protein
databases.
See also Section 5.4 for information on how protein information is displayed.
5.14.1
Adding protein databases
To add a protein database to the default list of protein databases in BVA module:
1
Click the Properties icon (or select View:Properties) to open the Properties
dialog, then click the Protein database tab.
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5.14 Protein database linking (optional)
170
2
To add an additional database to the list, click the
Manage Online Databases... button.
3
The Web Links Settings dialog opens. Select desired accession number
type using the Show databases for accession number type scroll down
menu. Click Add....
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4
In the Add Database Properties dialog, give the database the desired
Name.
5
Enter the URL (web address) in the address text box. The address should
contain a place holder string for accession number (#AN#).
6
Test if the database connection is functional by adding accession number
for a known protein in Test Online Database, and click Test. The browser
should open and information on the test protein should be shown.
7
If the test is working, click OK to finish.
Example
•
In the Name field, enter: SwissProt: AC or ID search
•
In the URL field (with substitution string), enter:
http://www.expasy.ch/cgi-bin/get-sprot-entry?#AN#
•
In the Test field, enter the Protein AC number for Human myoglobin:
P02144
The web browser will open at the address:
http://www.expasy.ch/cgi-bin/get-sprot-entry?P02144
Accessing protein databases
1
Ensure that the required protein database is selected in the Properties
dialog by clicking the Properties icon and selecting the Protein database
tab.
2
Select the desired protein database(s) from the Database Selection list
then click OK.
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5.15 Saving and printing
3
Right mouse click the protein to be queried in the Protein Table, then select
the protein database.
If both Protein ID and Protein AC labels are available, a choice of the label
to be used is given. The web browser is then opened at the appropriate
address.
Note: The linking is dependent on the place holder string being compatible
with the selected protein database.
Removing and editing protein databases
Removing and editing are performed in the Protein database Properties dialog
box.
5.15
•
To remove a database:
Highlight the selected protein database and then click the Remove button.
•
To edit a database:
Highlight the selected protein database and then click the Edit button. The
protein database can then be edited.
Click OK to accept the changes.
Saving and printing
Save workspace
BVA workspaces are saved in the database, and contain all the data associated
with all the loaded spot maps. The BVA workspaces do not contain the image
files but do contain information on their file path.
1
Select File:Save workspace to open the Save workspace dialog.
2
Select project where the workspace is to be saved.
3
Enter a file name, click Save.
Note: It is possible to save an previously saved workspace without overwriting
the old version. Select File:Save workspace as and enter the new name.
Save as Template
A template is a spot map with protein information that may be transferred to a
different workspace where the same proteins are present.
1
Create a workspace with the appropriate protein specific data
(Protein ID/AC, Name, Comment, pI/Mw and Protein of Interest status).
Protein data can also be imported to the workspace by selecting
File:Import Protein Data.
Note: Pick status is never included when copying information from a spot
map.
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2
Make sure the spot map to save as template is the selected gel, with a
magenta title bar.
3
Select File:Save as Template to save the selected spot map.
When the template spot map is imported and matched with the spot maps of
another workspace (where the same proteins are present), the protein
information will be synchronized in all of the workspace if the spot map is set
function to T, Template. See 5.6.4 for more information on how to import a spot
map.
Printing
Select File:Print to open the Print dialog box.
Multiple check boxes can be selected for printing the required workspace views.
Using the clipboard
Any aspect of the different views within the workspace can be copied to the
clipboard for pasting into another application.
Depending on the BVA mode different views will be available for copying. Click
the part of the workspace to be copied and select Edit:Copy (the part of the
workspace selected is described in this drop down menu). The image is copied
to the clipboard and can then be pasted into other applications.
It is possible to copy all displayed gel views in a single operation by selecting
Edit:Copy All Visible Gel Images.
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5.16 Exporting data
5.16
Exporting data
5.16.1
Export pick list
Pick list data can be exported as a text file:
1
Select File:Export Pick List to open the Export pick list dialog.
2
Select pick list to export and click OK.
If the pick list to export includes unmatched spots, that is displayed in the
dialog. Either click Cancel and match those spots or continue the export.
Unmatched spots are not included in the export list.
3
Type in an appropriate file name for the pick list and select format and
location. The pick list can be saved as either a text file or an XML file by
appending the appropriate file extension to the file name (.txt or .xml). The
text file is used for Ettan Spot Picker and Ettan Spot Handling Work station.
See also Appendix D.
4
Click Save to finish.
5.16.2
Export workspace
Data associated with the entire workspace can be exported in an XML based
format (DeCyder 2D Software XML) by selecting File:Export Workspace. Note
where the XML file is saved to simplify the following work in DeCyder 2D
Software XML toolbox.
The DeCyder 2D Software XML file can then be opened in the XML Toolbox for
further analysis. See also Appendix D.
5.16.3
Other exports
The selected spot map can be exported by selecting Export selected spot map
(only in XML format).
Protein of interest can be exported by selecting Export protein of interest (only
in .txt format). Log standardized abundance and Volume are exported for all
proteins of interest.
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6
6.1
Spot picking in DIA
Overview spot picking in DIA
The DIA module of DeCyder 2D Software provides the user with the ability to
generate a spot pick list from a pick gel. The pick list assigns proteins for picking
by Ettan Spot picking robot or Ettan Spot Handling Workstation.
DIA module is only used for identification of proteins of interest and subsequent
picking when performing small-scale experiments utilizing 2 samples on a
single gel (e.g. control-treated samples). Spots can be accurately picked from
fluorescently post-stained gels and CyDye DIGE Fluor saturated gels.
Note: For all other cases, perform spot picking in BVA module as described in
Chapter 8.
6.1.1
Normal workflow
The generation of pick lists in the DIA module involves the following processes:
1
Process the gel to detect and quantify spots on the image (see Chapter 5).
Include identification of reference markers (see 6.2).
2
Identify proteins of interest (POI) (see 6.3).
3
Visually inspect the POIs and assign Pick status (see 6.4). Edit pick locations
if necessary (see 6.5). Confirm spots (see 6.4.1).
4
Save DIA workspace (see 5.7).
5
Export pick list (see 6.6).
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6.2 Identification of reference markers
6.2
Identification of reference markers
The reference markers placed on the preparative gels (seen as circles on the
images) act as reference points for Ettan Spot picking robot.
The reference markers can be detected automatically within the DIA module
during the spot detection process (see 6.2.1) but they can also be assigned
manually (see 6.2.2).
6.2.1
Automatic detection of reference markers
1
With the preparative gel image loaded into the DIA workspace, select
Process:Process Gel Images to open the Process Gel Images dialog.
2
Ensure the Autodetect Picking references check box is selected, then click
OK.
3
The reference markers will then be detected automatically during the spot
detection process.
6.2.2
Manual identification or editing of reference markers
The reference markers can be assigned manually.
176
1
Click the Image view icon to show the gel images only.
2
Select Edit:Define Picking Reference.
3
Use the circular mouse cursor that appears in the Image View and click the
center of the left reference marker of the selected (magenta) image.
4
Zoom into the area of this reference marker by holding down the left mouse
button to draw a rectangular area around the marker.
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5
To alter the size of the yellow circle:
- Click the Properties icon and select the Image View tab.
Set the value in the Default radius of picking references so that the circle
in the Image View is sufficiently large enough to accurately surround the
reference marker to make it simpler to center the cross over the reference.
6
To alter the position of the circle:
- Select Edit:Edit picking References.
- Place the hand shaped cursor that now appears on the circle, then drag
the circle so that the cross fits the center of the reference.
Note: Magnifying the area of the gel around the reference marker by
selecting the Zoom in icon may make accurate alignment easier.
7
Select Edit:Edit picking References to exit this mode.
8
When the left reference has been selected, use the same procedure to
select the reference on the right hand side of the preparative gel image.
6.2.3
Removing of all defined picking references in a workspace
It is possible to remove all defined picking references in the current workspace
by selecting Edit:Clear all picking references.
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6.3 Identifying Proteins of Interest using the DIA module
6.3
Identifying Proteins of Interest using the DIA module
Potential pick proteins on the gel are identified as protein of interest (POI). The
Protein filter with user defined criteria simplifies the procedure.
6.3.1
Manual identification
The simplest way to assign POI status is to manually choose spots then select
the Protein of Interest check box.
6.3.2
Identification via Protein Filter
Spots can also be selected using various criteria in the Protein Filter.
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1
Click the Protein Filter icon to open the Protein Filter dialog.
2
In the Protein Filter select the Assign Protein of Interest check box and
deselect the Assign Pick Status check box while testing the filtering. In this
way the proteins that meet the filter criteria, specified in step 4, will be given
a Protein of Interest status.
3
All spots detected can be assigned with protein of interest status by
selecting the Select All check box. If selected, filtering settings are not
necessary, continue with step 5.
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4
5
Alternatively, spots can be filtered on specific spot properties.
•
Selection due to change in expression:
Protein spots exhibiting a change in expression are commonly
analyzed. Therefore, selecting proteins of interest on the basis of
volume ratio is useful for such spots. Spots can be selected for
decreases or increases in order to select spots that have diverged
notably from each other. Alternatively, spots can be selected for
decreases and increases allowing selection of spot volume ratios within
a finite limit.
•
Selection on other characteristics:
Spots can also be selected on the basis of their physical characteristics
(i.e. area, max volume, max peak height) to ensure that spot selection
only occurs on spots that can potentially be successfully picked,
digested then analyzed by mass spectroscopy.
The location of the spots on the X and Y axes can also be filtered in order
that spots near or on the edge of the gel are not present in the pick list,
or to limit the eventual pick list to high or low molecular weight proteins.
The various filters can be used simultaneously to select spots on
multiple features.
•
Spot population to filter:
The filtering can take place on the whole spot population or only those
confirmed by selecting the Restrict to confirmed spots check box.
After entering the filter criteria, select the Filter button to ascertain the
number of spots that will be picked.
If the number of resultant spots is unsuitable, the stringency of the filter can
be adjusted (according to step 4) to produce an optimal number of spots for
selection and subsequent picking.
Note: It is possible to assign the filtered spots with a Pick status as well. See
6.4.2 for detailed information.
6
Click OK to accept the spot filter parameters.
Spots assigned as protein of interest are denoted by the presence of the
letter I in the Function column of the Protein Table and have the Protein of
Interest check box selected.
Note: The protein of interest status of spots can be removed by selecting
Process:Unassign all Protein of Interest.
7
The DIA workspace containing the protein of interest selection information
can then be saved (File:Save Workspace).
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6.4 Assigning Spots for Picking
6.4
Assigning Spots for Picking
6.4.1
Review proteins of interest and assign for picking
The spots assigned as POI should be visually inspected to remove those not
suitable for picking and subsequent analysis.
1
Initially, organize the Table View and Spot Display so that POI proteins are
exclusively displayed:
Select the Protein of Interest spots check boxes in both the Table View and
Spot Display tab of the Properties dialog.
The Table View then displays only the proteins with POI status (possessing
I status in the function column) and the Image View only displays spots with
POI status.
2
Review the POI assigned proteins in sequence to ensure that they are
suitable for picking.
Once the spot is deemed suitable for picking select the Pick check box in the
spot control panel.
Once the spot is assigned with a pick status it will have a black spot
boundary in the image view and Pick is displayed in the Picked column of
the Table View.
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3
Review, and if necessary edit, the pick location of the spot (according to 6.5).
4
Finish by clicking Confirm (see also 4.6.2) before reviewing the next spot in
the table. When clicking Confirm, next spot in the table is automatically
displayed.
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6.4.2
Assign pick status via filtering
Pick status can also be assigned using the protein filter function.
After step 6 on page 179:
6.5
1
In the Protein Filter dialog, select the Assign Pick Status check box.
2
Click Filter again to assign the filtered spots with a Pick status.
3
It is recommended to review and confirm the spots (according to 6.4.1 but
without assigning for pick) and, if necessary, edit the pick location of the
spots (according to 6.5) before generating a pick list.
Editing Pick Locations
6.5.1
Settings for editing pick locations
To simplify editing, display only picked spots in the image view:
1
In the Properties dialog, click the Spot Display tab.
2
Select only the Picking references and pick locations check box, then click
OK.
6.5.2
Review and edit pick locations
Each of the protein spots has a dot within the spot boundary denoting the
centre of mass of the spot and hence the centre at which a picking head will pick
from the gel. The centre of mass represents the optimal picking location for a
vast majority of spots.
However, it may be useful to edit the pick location when two spots are in very
close proximity in order to minimize the possibility of cross contamination. The
picking location can be edited in these cases.
1
Use the Zoom in icon to see the individual spots selected for picking.
The pick locations are displayed as a yellow (by default) transparent
cylinder and a yellow circle in the 3D View and Image View respectively,
when a picked spot on the pick gel is selected.
2
Edit the pick locations by zooming in on the selected spot in the Image View,
then selecting Edit:Edit Pick Locations.
3
Place the hand shaped cursor that appears over the centre of the pick
location that requires editing, in the Image View, then drag the pick circle to
the desired location. This can be repeated for all pick locations that require
editing.
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6.6 Exporting Pick Lists
Original pick location
4
6.6
Edited pick location
Select Edit:Edit Pick Locations to exit this mode. To return all pick locations
to the initial position of centre of mass select Edit:Restore Default Pick
Locations.
Exporting Pick Lists
Pick lists can be exported for Ettan Spot Picker or Ettan Spot Handling
Workstation.
To export a pick list:
1
Select File:Export Pick List.
2
Save the pick list as either a text file or an XML file by selecting the
appropriate file extension to the file name (.txt or .xml). The text file is used
for Ettan Spot Picker or Ettan Spot Handling Workstation.
For descriptions of the xml-format, see EttanPickGel.pdf and
EttanPickGel.xsd in the folder Documents\Formats (in the folder where
DeCyder 2D program is installed, usually C:\Program Files\GE
Healthcare\DeCyder 2D\Documents\Formats)
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7.1
Spot picking in BVA
Overview spot picking in BVA
The BVA module of DeCyder 2D Software provides the user with the ability to
generate one or several spot pick lists, from one or several pick gels. Multiple
pick lists can be useful, for example, when separating proteins with high
abundance from proteins with low abundance on the same gel. The pick lists
assign proteins for picking by Ettan Spot picking robot or Ettan Spot Handling
Workstation.
Identification of proteins of interest and subsequent picking is normally
performed in the BVA module. Only in small-scale experiments, utilizing two
samples on a single gel, pick lists are generated in the DIA module, see 6.1.
7.1.1
Normal workflow
The generation of pick lists in the BVA module normally involves the following
processes:
1
Analyze analytical images in DIA module and then in BVA module (see
Chapters 4 and 5).
2
Identify proteins of interest (POI) using the Protein filter in BVA module (see
6.3).
3
Process the pick gel in DIA module (see 7.3). Include automatic identification
of reference markers on the pick gel in DIA module (see 6.2).
4
Save DIA workspace (see 4.7).
5
Add the DIA workspace with the pick gel to the BVA workspace including the
analytical gels. (see 7.4.3)
6
Landmark and match the pick gel to the master gel of the analytical gels in
BVA module (see 15.4.3). POIs from the master gel are thereby transferred
to the pick gel.
7
Check matching and re-match if necessary in BVA module (see 15.4.3).
8
Visually inspect the POIs on the pick gel and set desired proteins as Pick for
a selected pick list. (see 6.4.1). This can also be performed via the Protein
Filter, see 5.11. Edit pick locations if necessary (see 6.5).
9
Export pick list (see 7.7).
Note: Spots can be accurately picked from fluorescently post-stained gels (such
as Deep Purple) and CyDye DIGE Fluor saturated gels.
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7.2 Identifying Proteins of Interest using the BVA module
7.2
Identifying Proteins of Interest using the BVA module
Potential pick proteins on the analytical gels are identified as protein of interest
(POI) in the BVA module. The Protein filter with user defined criteria simplifies the
procedure. Protein statistics can be used for filtering spots only within BVA
module.
The identified proteins of interest are transferred to the pick gel when matching
with the analytical gels (step 6 in 7.1.1).
7.2.1
Manual identification
The simplest means to assign POI status is to manually choose spots then select
the POI check box when in Protein mode.
7.2.2
Identification via Protein Filter
Alternatively, identification of proteins for picking can be based on the statistical
information generated from a BVA workspace containing several spot maps
that have been subjected to statistical analysis within the BVA module.
Proteins can be selected using various criteria via the protein filter.
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1
With an analyzed BVA workspace open, click the Protein Filter icon to open
the Protein Filter dialog box (see also Section 5.11 for further information).
2
In the Protein Filter select the Assign Protein of Interest check box and
deselect the Assign Pick Status in list check box while testing the filtering.
In this way the proteins that meet the filter criteria, specified in step 4
below, will be given a Protein of Interest (POI) status.
3
All protein spots detected can be assigned protein of interest by selecting
the Select All check box. If selected, filtering settings are not necessary,
continue from step 5.
4
Alternatively, proteins can be filtered for picking based on the statistical
analysis results (i.e. based on the statistical criteria described in Section
5.10).
•
General filter settings:
The filtering can take place on the whole spot population or a subset of
the proteins or spot maps. Select the Restrict to confirmed proteins
check box to ensure that only proteins with a confirm status are filtered.
Alternatively, select the Restrict to protein present in check box to
ensure that only spots that appear on the designated number of spot
maps are filtered.
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7.2 Identifying Proteins of Interest using the BVA module
•
Selection on protein statistics:
Filtering can be limited to certain statistical subsets. Select the desired
statistical method and enter values for the filtering.
Note: It is only possible to select this filter in the Protein Filter dialog if
statistical tests have been performed prior to filtering the proteins
(see Section 5.10).
•
5
Selection on physical characteristics:
Filtering can be limited to spots of a specified volume by selecting the
Volume check box then entering the volume values required.
The location of the spots on the X and Y axes can also be used for
filtering in order that spots near or on the edge of the gel are not
selected or to limit selecting too high or too low molecular weight
proteins.
The selection criteria for volume and gel region can be based on either
the Master gel or the Pick gel (if present) by selecting the appropriate
spot map in the drop down menu.
After entering the filter criteria, select the Filter button to ascertain the
number of spots that will be assigned as proteins of interest.
If the number of resultant spots is unsuitable, the stringency of the filter can
be adjusted (according to step 4) to produce an optimal number of spots for
selection and subsequent picking.
6
Click OK to accept the spot filter parameters.
Spots assigned as protein of interest are denoted by the presence of the
letter I in the POI column of the Protein Table and have the POI check box
selected.
Note: It is possible to assign the filtered spots or all POIs with a Pick status
as well. See 6.4.2 and 7.5.5 for detailed information.
Note: The protein of interest status of spots can be removed by selecting
Process:Unassign all Protein of Interest.
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7.3
Spot detection on the pick gel
The pick gel is normally prepared after the analytical gels have been
investigated. Therefore, it is often necessary to perform spot detection
independently on the pick gel.
7.4
1
Prepare and scan the pick gel(s) (the gel(s) to pick spots from) as described
in Ettan DIGE System User Manual.
2
Import the gel image(s) to the database using Image Loader, see Chapter 3.
3
Perform spot detection as described in Section 4.5.1 and, if preferred,
perform automatic detection of reference markers as described in Section
6.2.1.
Identification of reference markers
The reference markers placed on the pick gels (seen as circles on the images) act
as reference points for Ettan Spot picking robot.
The reference markers can be detected automatically within the DIA module
during the spot detection process (see 6.2.1) but they can also be assigned
manually (see 6.2.2).
7.4.1
Automatic detection of reference markers in DIA module
1
With the pick gel image loaded into the DIA workspace, select
Process:Process Gel Images to open the Process Gel Images dialog.
2
Ensure the Autodetect Picking references check box is selected, then click
OK.
The reference markers will then be detected automatically during the spot
detection process.
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7.4 Identification of reference markers
7.4.2
Manual identification or editing of reference markers
The reference markers can be assigned manually, in either DIA module or BVA
module.
Note: Picking references can not be defined nor edited when viewing warped
images.
1
Click the Image view icon to show the gel images only.
2
Select Edit:Define Picking Reference.
3
Use the circular mouse cursor that appears in the Image View and click the
center of the left reference marker of the selected (magenta) image.
4
Zoom into the area of the left reference marker by holding down the left
mouse button to draw a rectangular area around the marker.
5
To alter the size of the yellow circle:
- Click the Properties icon and select the Image View tab.
- Set the value in the Default radius of picking references so that the circle
in the Image View is sufficiently large enough to accurately surround the
reference marker to make it simpler to center the cross over the reference.
6
To alter the position of the circle:
- Select Edit:Edit picking References.
- Place the hand shaped cursor that now appears on the circle, then drag
the circle so that the cross fits the center of the reference.
Note: Magnifying the area of the gel around the reference marker by
selecting the Zoom in icon may make accurate alignment easier.
188
7
Select Edit:Edit picking References to exit this mode.
8
When the left reference marker has been selected, use the same procedure
to select the reference marker on the right hand side of the pick gel image.
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7.4.3
Add the DIA workspace with the pick gel into BVA
The DIA workspace(s) with the pick gel(s) (as generated in Section 7.3) need to be
added into the BVA module. The pick gel(s) then need to be matched against the
analytical gels.
1
With the BVA workspace open, select File:Add Template/DIA workspace.
2
Select project and one or several DIA workspaces.
3
Click Add--> to add the DIA workspaces to the BVA workspace list in the
right panel.
Workspaces can be removed from the list by selecting the file and then
clicking Remove
4
Click Add to add the DIA workspaces to the selected BVA workspace.
5
Match the pick gel(s) (see Section 5.8.1) to transfer the protein of interest to
the pick gels. Pick reference marker assignments are also included.
Landmarking may be required to accurately match pick gels (see
page 122).
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7.5 Assigning Spots for Picking in BVA
7.5
Assigning Spots for Picking in BVA
7.5.1
Assign function for pick gel(s)
The pick gel(s) should be assigned as Pick Spot Maps (using the function check
boxes in the Spot Map mode).
Pick status for a spot map denotes that the co-ordinates for generating the pick
list are based on the designated pick gel.
Note: When the pick gel has been assigned as pick it will be sorted and
displayed together with the standards. The pick gel will also be easy
available if the image sorting Sort Freely is selected.
7.5.2
Multiple pick lists
Multiple pick lists can be used to pick spots from a gel into several groups. For
example, high abundance proteins can be picked into one group and low
abundance into a second group. The multiple pick lists are thereby used as a
tool for grouping spots.
Multiple pick lists can also be used when several pick gels are included in the
experiment to make sure all interesting spots are picked. In this case one or
several pick lists per pick gel can be used and exported.
In the BVA module, there are 10 different pick lists into which spots for picking
can be assigned. The pick list to add spots for picking to, can be selected in the
dropdown list in the Data View Control panel at the bottom of the Protein Mode
window.
Alternatively, the pick list can be selected in the Protein Filter dialog.
In both cases, the pick list can be renamed by clicking Change list.
The final combination of pick list and pick gel is performed when exporting the
pick list(s). See also 6.6.
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7.5.3
Review proteins of interest and manually assign for picking
It is recommended that the spots assigned as POI are visually inspected to
remove those not suitable for picking and subsequent analysis. This can be
performed in the Protein mode of the BVA module.
1
First, organize the Protein Table so that POI proteins are exclusively
displayed in the Protein Table:
Select the Protein of Interest (I) only check box in the Protein Table tab of
the Properties dialog.
The POI column header in the Protein Table then displays the proteins with
protein of interest status (possessing I status in the function column).
2
The POI assigned proteins can then be reviewed on after each other to
ensure that they are correctly matched and suitable for picking. Once the
spot is deemed suitable for picking, select pick list and the Pick check box
in the Data View Control panel.
Spots assigned with a pick status have a red spot boundary in the image
view and the Protein Table column Picked In displays which pick list the
spot is added to.
The current pick list name is displayed in the Data View Control panel and
any spots assigned as Pick in that list have a Pick in the Pick column of the
Protein Table.The spot volume for the selected spot and gel is displayed in
the column Pick Spot Vol.
3
Review, and if necessary edit, the pick location of the spot (according to 7.6)
before reviewing the next spot in the table.
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7.5 Assigning Spots for Picking in BVA
7.5.4
Assign pick status via filtering
Pick status can be assigned using the protein filter function as well. After step 6
on page 186:
1
In the Protein Filter dialog, select the Assign Pick status in list check box.
2
Click the Change list... button to
•
select a pick list,
•
change pick list selection,
•
change a pick list name, or
•
remove proteins from the pick list.
The Change Pick List dialog opens displaying the available pick lists. The
number of proteins in the selected pick list is also displayed.
If the selected pick list should be emptied, click Clear list.... Alternatively,
select Process:Unpick All:in selected pick list. Process:Unpick All:in all
pick lists can be selected to clear all pick lists.
Click OK to return to the Protein Filter dialog.
3
Click Filter again to assign the filtered spots with a Pick status.
4
Review, and if necessary edit, the pick location of the spot (according to 6.5)
before reviewing the next spot in the table.
7.5.5
Assign pick status to all POIs without reviewing
It is possible to assign all Proteins of Interest with a Pick status by simply
selecting Process:Assign All Protein of Interest as Pick.
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7.6
Editing Pick Locations
7.6.1
Settings to simplify editing
Before reviewing and editing pick locations, the picking locations must first be
displayed in both the image view and 3D View when in Protein mode. The pick
location is only displayed on the spot map assigned as pick (provided it has the
picking references defined).
To achieve the above and to simplify the review of proteins with pick status, use
the following settings to only display proteins assigned with a pick status (in the
Image View of the BVA module when in Protein mode):
1
Open the Properties dialog.
2
On the Image View tab, ensure that the check box Spots present in table
only and that the Picking references and pick locations check box is
selected.
On the Protein Mode tab ensure that Pick is selected in the View list for
Proteins Table Filter.
The Protein Table and the image view will then display only the proteins that
have been assigned with a pick status.
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7.6 Editing Pick Locations
7.6.2
Review and edit pick locations
Each of the protein spots has a colored dot within the spot boundary denoting
the centre of mass of the spot and the centre at which a picking head will pick
from the gel. The centre of mass represents the optimal picking location for a
vast majority of spots.
However, it may be useful to edit the pick location when two spots are in very
close proximity in order to minimize the possibility of cross contamination. The
picking location can be edited in these cases.
Note: Pick locations can not be edited nor restored when viewing warped
images.
1
Use the Zoom in icon to see the individual spots selected for picking.
The pick locations are displayed as a yellow (by default) transparent
cylinder and a yellow circle in the 3D View and image view respectively,
when a picked spot on the pick gel is selected.
2
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Edit the pick locations by zooming in on the selected spot in the Image View,
then selecting Edit:Edit Pick Locations.
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3
Place the hand shaped cursor that appears over the centre of the pick
location that requires editing, in the Image View, then drag the pick circle to
the desired location. This can be repeated for all pick locations that require
editing.
Original pick location
Edited pick location
4
7.7
Select Edit:Edit Pick Locations to exit this mode.To return all pick locations
to the initial position of centre of mass select Edit:Restore Default Pick
Locations.
Exporting Pick Lists
The final combination of pick list and pick gel is performed when exporting the
pick list/s. Pick lists can be exported for either Ettan Spot Picker or Ettan Spot
Handling Workstation.
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7.7 Exporting Pick Lists
To generate a pick list for the pick gel:
1
Select File:Export Pick List to open the Export Pick List dialog.
2
Select pick list and pick gel to export then click OK.
3
Select a location for saving the pick list file.
The file name can be changed. Default file name is gel name - list name.
4
Select to save the pick list as either a text file or an XML file by selecting the
appropriate file extension to the file name (.txt or .xml). The text file is used
for Ettan Spot Picker or Ettan Spot Handling Workstation.
For descriptions of the xml-format, see EttanPickGel.pdf and
EttanPickGel.xsd in the folder Documents\Formats (in the folder where
DeCyder 2D program is installed, usually C:\Program Files\GE
Healthcare\DeCyder 2D\Documents\Formats)
5
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Click Save to export the pick list.
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8
8.1
Batch processor
Overview
The Batch Processor links both the DeCyder 2D Software DIA and BVA modules
to perform all stages of the 2-D DIGE analysis process. Once the Batch Processor
has been set up, the gels are processed after each other without user
intervention. The Batch Processor is, therefore, a means of automation and does
not introduce further processing and analysis to the spot map data. All the
concepts for processing the gel images have been discussed in the DIA and BVA
sections of the manual and are not further elaborated upon in this section.
References to the appropriate sections are made where necessary. It is
recommended to use a maximum of 48 gels to keep the overview of the
workspace and to keep a good performance. Larger workspaces should be
analyzed in EDA. For recommendation on how to set up BVA workspaces for
subsequent analysis in EDA, see the DeCyder 2D v7.0 EDA User Manual on how
to prepare an EDA experiment.
8.1.1
Normal workflow
The normal workflow contains 4 main steps:
1
Set up the DIA batch list, see 8.2
a) Select images to add
b) Enter settings
2
Set up the BVA batch list, see 8.3
a) Define spot map attributes
b) Define protein statistics settings
c) Define protein filter values
3
Save the Batch, see 8.5
4
Run the Batch, see 8.6
Recommendations
Review the BVA workspace prior to generating the pick list. A workflow of using
the Batch Processor to only assign proteins of interest is recommended.
This involves following the workflow but only selecting the Protein of Interest
check box (without selecting Pick status) in the Protein Filter dialog box, then
avoiding generating a pick list by cancelling the Set Pick List Filename dialog
box.
In this way the subsequent BVA workspace will have protein differences
highlighted as proteins of interest, which can be manually reviewed then
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8.2 Setting up the DIA Batch List
confirmed, before assigning the proteins of interest with a Pick status (see
Chapter 15 Tutorial III - Processing the Preparative Gel and Generating a Pick List).
8.2
Setting up the DIA Batch List
8.2.1
1
198
Opening the Batch Processor module
In the DeCyder 2D Main window, click the Batch Processor icon to open the
Batch Processor window.
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8.2.2
Select images to add to the DIA batch list:
1
In the Batch Processor window, right-click in the grey area and select Add
DIA item to open the Image Item selection dialog. Alternatively: select
File:Add DIA item.
2
Select the project from which images will be imported by clicking the
project name in the left panel.
Available images
Projects
The center panel displays available images in the selected project. The
available images are by default displayed as a list of file names. To display
details, right-click in the list and select Show details.
Note: If the required images have not yet been imported to the database,
click Import images... to open the Image Loader module and import
the images. (See Chapter 3 Image Loader for more information.)
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8.2 Setting up the DIA Batch List
3
In the center panel, select the images to add and click Add.
To add all available images in the selected project, click Add all.
Number of gels and
images added to DIA list
Added images are displayed in the list in the right panel. The settings for gel
names, dye chemistry and labels are as set up in Image Loader.
Remove items from the list
•
To remove entire gels or separate images from the list:
Select the entire gels (click in the gel column) or separate images (click in
the image column) to remove and click Remove.
Note: If separate images are removed, the remaining images are reordered
automatically.
•
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To remove all gels with their images:
Click Remove all.
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8.2.3
Set gel properties
The settings selected here are applied to all added gels. Changes and individual
settings for each gel can be made later in the Edit DIA item dialog (see 8.4.1).
1
Enter a value for the estimated number of spots. (See 4.5.3 step 2 for further
details.)
2
Check the Include in BVA batch list box if the images should inter-gel
matched and analyzed with the BVA module as well.
If checked, the BVA item settings dialog will be automatically opened when
clicking OK.
Set spot detection algorithm
In the Batch Processor the spot detection algorithm that will be used for the
present batch is displayed in Gel Properties. Version 6.0 is default. For more
information on the differences between the algorithms, see Section 4.5.2.
When using the Batch Processor, the change of the spot detection algorithm
must still be done within DIA. The algorithm that was last used in a saved DIA
workspace will be the one displayed in the Batch Processor. In order to change
algorithm in the Batch Processor a DIA that has been processed with the desired
algorithm can be opened, reprocessed with the same algorithm and saved. This
setting is Windows® user specific.
Pick gel properties
One gel per batch can be defined as pick. If several gels are pick gels this can be
defined in BVA module after the Batch processing.
If a pick gel is included in the DIA batch item list pick reference markers will be
automatically detected. If several pick gels are included, manually select the
Auto detect pick reference markers box in the Edit DIA item dialog (see 8.4.1)
for the required gels.
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8.2.4
Spot exclusion filter (Optional)
The settings selected here are applied to all added gels. Changes and individual
settings for each gel can be made later in the Edit DIA item dialog (see 8.4.1).
1
If spot exclusion is used, check appropriate boxes and fill in values for the
different properties. The filtering values are best pre-determined in the DIA
using one of spot map pairs to be batch processed. See Section 5.6.1 for
further details.
2
To define Area of interest and Exclusion area, check the boxes and enter
numerical values in the fields. (Origin=upper left corner of the rectangle; X=
width; Y=height). The values entered are graphically displayed in the box.
Alternatively, click the radio button Area of int. or Excl. area to draw the
selected area with the mouse in the box to the right.
Note: If the text box frame becomes red and the OK button is disabled, the
Area of interest or Exclusion area has invalid values. If that
happens, move the rectangle representing the area slightly to the left
or decrease the value of the coordinate by one. This will generate a
valid value and the image selection can be used.
3
Both area types can be used and they can even overlap.
The defined Spot exclusion filter values are used on all added gels. To define
values for separate gels, edit DIA item settings, see 8.4.1.
8.2.5
Finish DIA batch list settings
Click OK to finish the DIA batch list settings.
Note: The OK button is only available if selected values are valid. Invalid value
fields are marked with a red frame.
If the Include in BVA batch list box was checked, the BVA item settings dialog
will be automatically opened.
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8.3
Setting up the BVA Batch List
Note: It is possible to add existing DIA workspaces from the DeCyder 2D
database for use in the BVA analysis (see 8.4.5).
As with the Spot Map mode in the BVA module, attributes (i.e. spot map function,
group, conditions, sample ID and spot map comments) can be assigned to
individual spot maps in the BVA batch list. The assignment is performed in the
BVA item settings dialog. The dialog can be opened by selecting Edit:Edit BVA
item settings.
8.3.1
Define spot map attributes
All groups are assigned as in the Spot Map mode (Section 5.7.1 and 5.7.2). All
images set as Std (performed in the Image Loader module) are automatically
sorted into the Standard group.
1
Drag and drop the images from the center panel to the correct
experimental group in the left panel.
2
Make sure the function assignment is correct for each gel. All gels are by
default assigned as A, Analysis. It is possible, but not necessary, to assign
one each of M, Master; T, Template and P, Pick.
Note: If P, Pick, is selected for a BVA item, its DIA item will automatically be
set to Auto detect pick reference markers.
3
Optional: Enter Sample ID (only numbers, maximum number 65535) and
Comment.
Note: If no master image is assigned before running the batch, the BVA module
will select a master during the Batch processing.
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8.3 Setting up the BVA Batch List
8.3.2
Define protein statistics settings
Note: Statistical calculation can, if preferred, be performed in BVA module once
match quality has been checked, see 5.10.
1
Check the Setup protein statistics and filter check box to include protein
statistics calculations and protein filtering in the BVA analysis.
2
Click OK/next to save and continue to next dialog.
Note: OK saves and exits the dialog. Cancel exits the dialog without saving.
3
The Protein Statistics dialog box automatically appears. This allows the
user to perform statistical analysis.
4
Set up the protein statistical analysis as required. See also Section 5.10.
Note: When using paired t-test in batch, spot maps must have sample ID.
See Section 5.10.4.
204
5
Click OK/next to confirm and save the statistical analysis entered and
continue to next dialog.
6
The Protein Filter dialog box appears automatically.
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8.3.3
Define protein filter values
Protein filtering allows either the highlighting of interesting proteins for further
investigation or the direct identification of proteins for picking and subsequent
generation of a pick list, if a pick gel is present in the batch list (see Chapter 7
Spot picking in BVA). For more information on how to enter values for the protein
filter, see Section 5.11.
Click OK when desired values have been entered.
Note: It is recommended that the Protein Filter is used to highlight interesting
proteins for further investigation. These proteins can subsequently be
reviewed by the user in the BVA module prior to generating a pick list (see
Section 6.4).
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8.4 Editing items and workspaces (optional)
8.4
Editing items and workspaces (optional)
8.4.1
Edit DIA item settings
To edit settings for a DIA item:
1
2
Open the Edit DIA item for gel:x dialog. There are four ways to open the
dialog:
•
Double-click a DIA item in the DIA batch list
•
Right-click a selected DIA item and select Edit DIA item
•
Select a DIA item in the DIA batch list and then select
Edit:DIA item settings.
•
Select several DIA items, then right-click and select Edit:DIA item. This
option allows editing DIA settings for all selected items, one after the
other.
Edit settings.
Note: If Auto detect pick reference markers is removed for an item, the P,
Pick setting is automatically removed from its BVA item.
3
206
Select item for application of settings:
•
This item only: Settings will be applied to the selected item only. The
general settings will be overruled by the individual settings.
•
Subsequent items: Setting will be applied to the selected item and all
subsequent items in the DIA batch list. The general settings will be
overruled by the individual settings.
•
All items: Settings will be applied to all items in the DIA batch list.
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4
Click OK to apply the settings.
If several items were selected in step 1, the Edit dialog for the next item will
open. Repeat steps 2-4 until all dialogs are edited. To stop the editing, click
Cancel.
8.4.2
Edit Protein statistics settings
To edit settings for protein statistics:
1
Select Edit:BVA protein statistics settings.
2
In the Protein Statistics dialog that appears, edit the settings and click OK.
(See also Section 5.10 for guidance.)
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8.4.3
Edit Protein Filter settings
To edit settings for protein filtering:
208
1
Select Edit:BVA protein filter settings.
2
In the Protein filter dialog that appears, edit the settings and click OK. (See
also Section 5.11 for guidance.)
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8.4.4
Edit Batch and BVA workspace properties
To edit properties for Batch and BVA workspace:
1
Select File:Properties to open the Workspace Properties dialog.
2
Edit settings as appropriate.
Select conditions as required from the dropdown lists. There must be 2
different conditions selected to perform a batch run.
If one BVA batch item is set to Pick, a pick list will be generated. Therefore
a Pick list file name with an absolute path (for example: c:\DeCyder
2D\Picklist_folder\040802_picklist_high_score) must be entered. The file
must have the extension .txt or .xml.
To change file name, location or extension, click Set... and enter a file name
(.txt or .xml) and browse to find a suitable folder for the file.
Note: Only one BVA batch item can be set to Pick.
3
Click OK.
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8.4 Editing items and workspaces (optional)
8.4.5
Add items
To add DIA batch items:
Proceed as in 8.2.2.
Note: The settings of the last DIA item in the list will be used as default
values for the added DIA item.
To add DIA workspaces to BVA list:
1
Select File:Add DIA workspace item to BVA list.
Alternatively, right-click in the BVA list and select Add DIA workspace item
to BVA list.
The Add DIA workspace to BVA list dialog opens.
2
Select project in the left panel and then DIA items in the right panel.
Note: It is possible to add a template into the BVA list.
3
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Click Add to add the selected DIA workspaces to the BVA batch list.
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8.4.6
Remove items
To remove DIA or BVA batch items:
1
In the DIA or BVA batch list, select the item to remove.
2
Right-click the selected item and select Remove DIA item or Remove BVA
item. Alternatively, Select File:Remove DIA batch item or File:Remove BVA
batch item as appropriate.
3
If any other gels include the selected images, these gels are automatically
selected as well and a Confirm dialog is opened.
Note: If a DIA item is removed, the associated BVA items are removed as
well.
Note: If a BVA item is removed, the associated DIA item’s Include in BVA
check is removed in the DIA batch item list.
Click Yes to continue.
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8.5 Saving the batch
8.5
Saving the batch
Before the batch can be processed, it has to be saved to the database. The
batch is saved as a batch workspace and a BVA workspace.
1
Select File:Save batch to open the Save batch dialog.
2
Select a project into which the batch (and the BVA workspace, if at least one
BVA item exists) should be saved.
3
Enter a Batch name (and a BVA workspace name, if at least one BVA item
exists) in the fields.
4
If a BVA batch item is assigned as Pick, enter a Picklist file name with an
absolute path, for example: c:\DeCyder
2D\Picklist_folder\040802_picklist_high_score.
5
Click Save to finish. If the name conflict dialog appears, see 8.5.1.
When saving, the batch status will be changed to Workspace saved and
the color of the box will turn green.
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8.5.1
Name conflict
When saving the batch workspace:
If one or several DIA workspaces have file names that are already used within
the selected project, then the names must be edited. This is detected on saving
and a warning dialog is displayed:
Note: The dialog appears every time a name conflict is detected at saving.
•
In this case the DIA workspace name already exists in the selected project
and the DIA workspace has also been used in at least one BVA workspace.
It can therefore not be overwritten with the same name.
Change the DIA WS name in red. Valid names are displayed in black text.
•
In this case the DIA workspace name already exists in the selected project,
BUT it has no connection to an existing BVA workspace. It can therefore be
overwritten with the same name.
To change the name of the DIA workspace anyway, deselect the Overwrite
check box and then change the name.
•
Overwrite all: click to select all overwrite check boxes. This is useful if
testing batch run of only DIA workspaces several times with different
settings without saving the workspaces between the runs.
•
Rename all: click to deselect all Overwrite boxes. All DIA WS names
automatically appear in red text and need to be changed.
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8.6 Running the batch
8.6
Running the batch
The batch processing can be started when the Batch and BVA workspaces have
been saved. The batch state should be Workspace saved in a green box.
8.6.1
Saving Batch and BVA workspaces
1
Select Process:Save Workspace.
2
Select a Project in the left list or create a new project using the Create new
project... button.
3
Enter names in Batch WS name and BVA WS name boxes.
4
Click Save.
8.6.2
Starting the Batch Processor
1
Select Process:Run batch to open the Process start dialog. Click Start.
2
When the batch is processing, the status for the batch is displayed in the
box at the bottom of the Batch Processor window. See Table 8-2 for
detailed information.
3
The status for each item is displayed in the status column in the batch list.
See Table 8-1 for detailed information. It is always possible to double-click
in the status column to display the Status dialog for the specific item.
Note: Only items with state Pending will be processed.
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4
When processing is finished, the status displayed is Workspace saved in a
green box.
Table 8-1. Item status options.
Item status
Description
The item has not yet been processed or changed
after process.
The item is currently being processed.
Detection successfully completed.
only for DIA item
Processing failed.
If a DIA item fails, associated BVA items are set to
failed as well.
The item was excluded due to failure of the DIA item.
only for BVA item
Matching completed.
only for BVA item
The item selected as master (either manually by the
user or automatically by the BVA module).
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8.7 Printing the batch
Table 8-2. Batch status options.
Batch status
Description
New batch workspace can be created.
Data is entered or changed.
Current batch workspace is saved; an
existing batch workspace is loaded from
the database or batch processing has
successfully completed.
Batch processing is running.
Processing has completed and one or
several batch list items has entered failed
state.
A started process is being aborted. The
batch processing is halted once the
currently running detection or analysis is
completed.
8.7
Printing the batch
When printing the batch information, both the DIA batch list and the BVA batch
list are printed.
1
To print the batch, select File:Print.
2
In the dialog that appears, click OK to continue the printing.
To change the printer, click Cancel and then change the printer settings
before printing.
Note: The batch information is printed in a landscape format, 2 pages
wide. The pages are annotated Page 1 left, Page 1 right, Page 2 left
etc.
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9
9.1
User Administration Tool
Overview
The User Administration Tool is a program that enables administration of the
users of DeCyder 2D Software.
Note: Only users in the group User Administrator in the application User
Administration Tool can open and use the User Administration Tool.
The following actions can be performed in the User Administration Tool module:
•
Create a user account
•
Edit a user account
•
Change password
•
Remove a user account
9.1.1
Passwords
Passwords have to be changed at the first login when using a new account. It is
important to keep the password in a secure place for at least one user with
access to the User Administration Tool.
9.2
Open User Administration Tool
Note: Only users in the group User Administrator in the application User
Administration Tool can open and use the User Administration Tool.
1
In the Start menu of the DeCyder 2D Software database computer, select
All Programs: GE Healthcare: DeCyder 2D Software: User Administration
Tool to open the program.
2
Enter User name and Password in the login-dialog.
Note: At the first login to the User Administration Tool, use the default user:
User name: DEFAULT_ADMIN
Password: DECYDER_00
The predefined default user only has access to the User
Administration Tool.
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9.3 Create an Administrator user account
3
9.3
The User Administration Tool window is displayed.
Create an Administrator user account
At the first login, create a user called Administrator which will have access to
everything, see 9.4.
1
Select New Type required information. Red text indicates required fields.
Click OK. Enter further user information if desired.
2
Select application DeCyder.and select access groups Scientist and
Administrator.
3
Select access group User administrator in User Administration Tool and
click OK. Now, a user with full access in the system has been created.
4
Generate a temporary password.
TIP! Copy the password (normally including several special characters)
before exiting.
5
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Exit User Administration Tool and login as a user with Administrator
privilege before creating the other users.
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9.4
Create a new user account
To create a new user account Administrator user group level is required. See 9.3.
1
Click New... to open the Create User Account dialog.
2
Enter login name and full name in their respective fields (mandatory). If
desired, enter the position of the user, e-mail address and phone number
in their respective fields. Click OK
3
The new user account is added to the User Accounts list shown in the left
panel of the User Administration Tool window.
4
To get a temporary password click Generate pwd.
TIP! Copy the password (normally including several special characters)
before exiting.
Note: The generated password can only be used once. The user must
change password at the first login in order to log on.
5
Select the appropriate application (DeCyder 2D or User Administration
Tool) in the Select application drop-down list.
6
If DeCyder 2D was selected in step 5, check the box for the desired access
in the Access groups for DeCyder 2D list:
•
Scientist
The user will have access only to the DeCyder 2D application.
•
Administrator
The user will have access only to Database Administration Tool.
•
Scientist and Administrator
The user will have access to the DeCyder 2D application and Database
Administration Tool.
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9.5 Edit a user account
If User Administration Tool was selected in step 5, check the box in the
Access groups for User Administration list:
•
7
User Administrator
The user will have access to the User Administration Tool.
To save the settings and exit edit mode, click OK.
See 9.5 for information on how to edit other settings for a user account.
TIP! Add an extra user with unlimited access and without password expiry.
9.5
Edit a user account
1
In the User Accounts list, select the user account to be edited.
2
Click Edit. The fields in the right panel are activated.
Note: It is not possible to edit a user account if the user is currently logged
in. To see if a user is logged in or not, click the Refresh button and
check the status of the user account in the Status column in the User
Accounts list (Log In or Log Out).
3
Edit the settings.
Note: For more information on the available fields, see the User
Administration online help.
4
9.6
Click OK to save the new settings and exit edit mode.
Profile settings
See the Online help for information on profile settings.
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9.7
Change password
Administrator users can change password in the User Administration Tool.
Scientist users can also change password in Organizer, see 2.2.4.
9.8
1
Select Actions:Change password to open the Change password dialog.
2
Enter the new password in the New field and re-enter the new password in
the Confirm field.
3
Click OK to save and exit the dialog.
Delete a user account
1
In the User Accounts list, select the user account to be removed.
2
Click Delete.
Note: It is not possible to delete a user account if the user is currently
logged in. To see if a user is logged in or not, click the Refresh button
and check the status of the user account in the Status column in the
User Accounts list.
3
A dialog appears asking for confirmation of the removal of the user
account. Click Yes to remove the user account.
Note: The user account can not be reopened and a new user account can
not be given the same name as the removed user account.
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9.8 Delete a user account
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10 Database Administration Tool
10.1
Introduction
The DeCyder 2D Software has an integrated Oracle 10g database in which all
generated data is stored. To administrate the database a specific tool called
DeCyder Database Administration Tool is provided. The DeCyder Database
Administration Tool can only be run on the computer where the DeCyder 2D
Software database is installed.
The DeCyder Database Administration Tool comprises the following functions:
•
Manual backup of the DeCyder 2D Software database
•
Restore an earlier generated backup
•
Export of Workspace and Gel data
•
Import of Workspace and Gel data
•
Archive of Projects
•
Retrieval of archived data
•
Release item used to release items (such as workspaces, projects and gels)
that have been locked, due to for instance lost database connection
In addition, the following general function is provided:
•
Proxy settings configurations
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10.2 Open Database Administration Tool
10.2
Open Database Administration Tool
1
In the Start menu of the computer with the DeCyder 2D database installed,
select All Programs: GE Healthcare: DeCyder 2D 7.0 Software: DeCyder
Database Administration Tool to open the tool.
2
Enter User name and Password and click OK. The DeCyder 2D database is
set as default
Note: Only users with Administrator privileges, see 9.3, can open and use
the DeCyder Database Administration Tool.
3
10.3
The DeCyder Database Administration Tool opens.
Backup of database
Purpose:
Use backup to secure the data in the DeCyder 2D database. A manual backup
should be performed at least once a week. Backup should also be performed
before archiving data, as a safety precaution.
Note: Do not use backup to move small amounts of data. For that purpose, use
Export data. See Section 10.5.
Perform backup:
1
Click Manual backup button in the DeCyder Database Administration
Tool.
Note: If the installation of the Oracle database not was performed
according to DeCyder 2D Software Version 7.0 Installation Guide, it
might be required to enter the Oracle database SYS password.
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2
Information of users currently logged in is shown. If there are logged in
users contact them in order to log out, or do the backup later. To continue
click Next >>.
Note: It is not recommended to do a backup if users are logged in since
unsaved data will not be backed up and the backup process is slower.
3
Information of Location of backup file and Filename (autogenerated) is
shown. Click Finish to complete the backup process.
Note: Due to ORACLE restrictions it is not possible to choose backup
directory.
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10.4 Restore data from backup
4
The manual backup is now completed. Click Browse to review the backup
file and log file.
5
Copy the backup file and the log file to a safe place.
Note: Do not store the backup in the same hardware as the original data.
10.4
Restore data from backup
Purpose:
To restore the database to an earlier backup.
Note: All data entered into the database after the last backup will be erased.
Note: Users that are logged in during the Restore backup will lose their data
and will be logged out during the process.
Perform Restore:
226
1
Click Restore backup button in the DeCyder Database Administration
Tool.
2
If you have not performed a backup, see 10.3, click Yes in the dialog box.
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3
If you already have performed a Manual backup click No to continue the
Restore backup.
4
Ensure that the backup file is placed in the Export folder.
5
Click Browse to select the backup file to restore.
6
Browse for backup file. Select file and click Open.
7
In the DeCyder Database Administration Tool, check that the correct file
is selected and click Finish to complete the Restore backup.
8
If there are users logged on, the Restore backup - logged users window
shows user identity information. Get the logged in users to log out before
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10.4 Restore data from backup
continuing Restore backup. Click << Back to pause or Next >> to continue
the process.
Note: If you continue Restore backup without getting the logged in users
to log out they will be disconnected and all their current data will be
lost.
9
After Restore backup has been performed the application has to be closed.
Click OK in the dialog box.
Note: After restore all users are requested to change password at first login.
Note: To use the new version 7.0 colors for a restored database, change the
color settings in DIA and BVA:
In both modules, choose View:Properties and select Colors:Default and
press OK.
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10.5
Export data
Purpose:
Export data from the DeCyder 2D database to be able to share data with other
users of DeCyder 2D Software. When exporting a dump file (.dmp) is created.
Note: It is possible to export all types of information except Projects, Batches,
individual Images and BVA templates.
Perform export:
1
Click Export button in the DeCyder Database Administration Tool.
2
Browse and select desired workspaces and/or gels to export, then click the
right-directed arrow.
Note: If one wants to export both DIA and BVA workspaces for an experiment,
they will have common images with identical names. The database do
not allow two images with the same name in the same project. Below is
described how to deselect gel images.
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10.5 Export data
3
The selected workspaces are now shown in the Workspace name list and
their related gel images are shown in the Gel name list. Deselect any
workspace or gel image by marking the object in the list and click the leftdirected arrow corresponding to the Workspace name or Gel name lists.
Note: No in the Workspace name Gels list indicates that there is no Gel
related to the Workspace.
Note: If a DIA or BVA Workspace is exported without its gel images it is only
possible to import the Workspace into a database already
containing those gel images.
230
4
To get the related gel images which have been deselected back to the DIA
or BVA workspace, select the workspace again in the Workspace name list
and click the right-directed arrow. Then the selected gels will appear in the
Gel name list.
5
After finishing selecting workspaces and gel images click Next >> to
continue the export process.
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6
The software suggest a default Export file name. Change name if desired.
Click Next >> to continue the export process.
7
Click Finish to complete the export process.
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10.6 Import data
8
10.6
Click Browse to view export file and export log file.
Import data
Purpose:
The Import data function is used for importing data from one installation of the
DeCyder 2D database to another. Import in DeCyder Database Administration
Tool can only be performed with dump files (.dmp) created as described in
Section 10.5, using DeCyder 2D Software versions 6.0 or later.
Note: It is possible to import one or several DIA / BVA workspaces without any
or with only some associated images. However, the missing images must
already be present in the database to be able to import the workspaces.
Perform import:
1
Click Import button in the DeCyder Database Administration Tool. Browse
for desired .dmp file and click Next >> to continue the import process.
Note: The .dmp file to import must be placed in the Export folder. If not, the
.dmp file has to be moved to the Export folder before continuing the
Import.
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.
2
Select an existing Project into which the dump file is to be imported and
click Next >> to continue the import process.
Or create a new project into which the dump file can be imported by
clicking Create project and follow the instructions below.
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10.6 Import data
234
3
Enter Project name. Click Public access box to allow public access. It is
optional to enter Description text with relevant project information. Click
OK when all information is entered.
4
Check that the desired import file is selected. Click Finish to complete the
Import.
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5
Import searches for gel images already existing in the DeCyder 2D software
database. If a gel image is already present in the DeCyder 2D software
database a dialog box appears. Click Yes if you want to avoid import of
existing gel images (imported workspaces will be connected to existing
gels), or click No to import the gel images.
Note: Avoid importing gel images if possible since gel images take a lot of
space in the database.
6
In the DeCyder 2D database all item names within a Project must be
unique. If any item among the imported has a name already present in the
Project Import shows a dialog box. Enter a New name and click OK to
continue.
7
Import is now completed. See Import Log file for information.
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10.7 Archiving from the database
10.7
Archiving from the database
Purpose:
To archive project data and free up space in the DeCyder 2D database. Archived
project data is deleted from the DeCyder 2D database. If required, however, the
archived data can later be retrieved back into the database, see 10.8.
Note: An archived Project can only be retrieved to the same DeCyder 2D
database it was archived from.
Perform Archive:
1
Click Archive button in the DeCyder Database Administration Tool.
2
A dialog box is opened. Click Yes to perform a Manual backup before
archiving data as a safety precaution. See 10.3. If Manual backup has been
done, click No to continue Archive procedure.
3
Click Project data and click Next >> to continue Archive procedure.
Note: Archive Audit trail is currently not used. Do not select this option.
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4
Browse the DeCyder 2D database to select the project to Archive. Selected
item is shown in the Archive data - choose project list. When the selection
is completed click Next >> to continue Archive procedure.
5
If any workspace in the project to Archive is opened by another user, the
workspace is locked and Archive is not possible. A Lock items failed
message is shown. Contact the user to close the workspace. If the
workspace is incorrectly locked see Section 10.9 for how to release the
workspace.
6
If you try to Archive a project which contains a gel image linked to a
workspace in another project Archive project not possible - dependencies
window is shown.
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10.7 Archiving from the database
238
7
Check that the desired project is selected. Click Finish to complete Archive
procedure.
8
When Archive is completed, click Browse to check Log file information.
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10.8
Retrieve archived data
Purpose:
To access earlier archived data and retrieve it into the DeCyder 2D database.
Perform retrieval of archived data:
1
Select Retrieve in the DeCyder Database Administration Tool. Browse to
Select archive file. Click Next >> to continue Retrieve procedure.
2
Check that the correct archive file is selected. Click Finish to complete
Retrieve.
3
When Retrieve is completed open Retrieve log file for information.
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10.9 Release item (Workspace, Projects or Gels)
10.9
Release item (Workspace, Projects or Gels)
Purpose:
A workspace is locked each time it is opened via a DeCyder 2D application to
avoid that two users work on the same workspace simultaneously. However, if
any problem with the network or the client occurs while a workspace is open,
the workspace might be locked in the database even if the workspace is not
opened. Release item must then be used in order to release that workspace to
make it accessible for further work.
Note: If the database is restarted all locked workspaces will automatically be
released.
Perform release of workspace (or projects or gels)
Project and Gels are released the same way as Workspaces.
1
Select Release item in the DeCyder Database Administration Tool. Now,
all locked workspaces in the database are shown with information of
Workspace name, Username, Locked date, and Connected status (i.e. True
or False).
2
Search and select workspaces with Connected status False in the list. Click
Release selected item and the workspace is not locked anymore. A Release
item succeeded dialog box is shown
Note: It is not allowed to release workspaces with Connected status True. The
True status indicate that the workspace is actively used and releasing
that workspace means that the current work is lost. If one tries to release
such workspace a Release item failed dialog box is shown.
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10.10 Other administration tools
The proxy settings internet access can only be used together with the EDA
module. In this manual only a brief instruction of how to enter dialog
information is supplied. For detailed information of how to use these functions,
see instructions for applicable databases and servers and the DeCyder
Extended Data Analysis Software User Manual.
10.10.1 Proxy settings
Purpose:
To allow clients and server access the internet through a proxy server.
Perform proxy settings access:
1
Select Proxy Settings in the Other admin (other administration tools) in the
DeCyder Database Administration Tool to display the Proxy settings
dialog.
2
Select Enable to enable proxy access.
3
Specify Host and Port. For some internet destinations User name and
Password is required.
4
Test the connection to a proxy server by clicking the Test button.
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10.10 Other administration tools
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11 XML Toolbox
11.1
Overview
Data can be exported from the DeCyder 2D Software modules using a common
XML format called DeCyder 2D Software XML. XML is a structured universal
tagged language with tags that can be custom designed, making it easy to
access data from DeCyder 2D Software analysis workspaces for other
applications. The XML format can be used to make data available for post
processing by other software packages. For example, a database building
package can be used with XML formatted DeCyder 2D data to create a userdefined database (different to the DeCyder 2D database).
The DeCyder 2D Software XML Toolbox is a toolbox shell housing different tools
for extraction of the DeCyder 2D Software data from the different XML files that
can be exported within DeCyder 2D Software. This enables users to create tools
to convert their data into text files or html files (potentially other data formats
can be supported for conversion). Two basic tools are supplied to create Tabbed
text files and Web tables. For descriptions of the xml-format, look in the folder
Documents\Formats (in the folder where DeCyder 2D program is installed,
usually C:\Program Files\GE Healthcare\DeCyder 2D\Documents\Formats).
Prerequisites
To run the XML toolbox:
•
Fully disable any pop-up blocker software. Otherwise the result window
that displays the extracted data is blocked.
•
Set the security settings in Internet Explorer so that ActiveX controls can be
used.
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11.2 Opening the XML Toolbox module
11.2
Opening the XML Toolbox module
Click the Toolbox icon to open the XML Toolbox.
The XML Toolbox can extract data in two formats: Tabbed text files and Web
tables.
Tabbed text files
The Tab Separated Tables tool is used to export DeCyder 2D Software XML data
in a tabbed text format, for further data processing in other software that can
import this format of data (e.g. Microsoft Excel or SpotFire™). The data in the
resulting output table is configured such that the top row consists of an
identifying line of text, containing the name of the source XML file, and the type
of data extracted from the file. The second row in the output table is a tabseparated list of column headers, and the following rows consist of the actual
tab separated raw data.
To utilize the Tab Separated Tables tool click the Tab Separated Tables button.
Web tables
The Web Tables tool is an example of how the DeCyder 2D Software XML format
in combination with XSL and other web technology can be used to customize
reports in HTML format. The Web Tables tool can be used as a template for user
designed report tools.
To utilize the Web Tables tool click the Web Tables button.
The interface for both tools is similar.
11.3
Extracting data
The XML files must be exported from DIA module or BVA module, see Sections
4.8.2, 5.15 and 5.16, before they can be further handled in the XML Toolbox.
11.3.1
Loading the XML files
XML files can be loaded at the top of the tool page by typing the file pathway
directly into the list box and pressing enter on the keyboard or by browsing for
the required file. The tool verifies whether the selected file is a valid XML file
exported from DeCyder 2D Software-DIA or DeCyder 2D Software-BVA. The user
is alerted if the selected file is not valid.
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11.3.2
Type of proteins in the output table
Once the DeCyder 2D Software XML file has been opened, select the types of
proteins to be included in the output table. For XML files exported from BVA, four
selections are available, Picked proteins, Proteins of interest, All matched
proteins, and All proteins. For XML files exported from DIA, only the Picked
proteins, Proteins of interest and All proteins selections are available.
11.3.3
Category of data in table columns
The data is categorized into four groups:
1
Experiment data. The table contains general experimental set up
parameters valid for the current DIA or BVA experiment.
2
Image data. The data in this table contains the different parameters that
are unique to each spot in the individual gel images contained in the
selected DIA or BVA experiment.
3
Gel data. The data displayed in this table is unique for each detected spot
pair within each set of gel images that originate from the same gel.
4
Protein data. All protein data that is unique to a set of matched spots
across the different gels in the current BVA experiment is displayed in this
table.
The interfaces diverge when selecting categories of data:
•
Web tables. Each of the four lists of data items represents a type of result
table. So, if at least one item in each list is selected, the resulting output
contains four different result tables. Multiple selections are possible in all
lists. The content of the four data lists also changes, depending on
whether an XML file is exported from the DIA or BVA module (Protein data
are not available when exporting from DIA module).
•
Tabbed text files. Select a category of DeCyder 2D Software data to
include in the result output from the drop down menu. Depending on the
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11 XML Toolbox
11.3 Extracting data
selection of category and type of XML file selected, a number of items are
displayed in the list box below. Multiple selections are possible for all
categories.
11.3.4
Extracting Data
Once the data output has been specified, the data can be extracted by clicking
the Extract data button.
Note: Any pop-up blocker features in the Internet Explorer browser needs to be
completely turned off to be able to extract XML data. Otherwise the
output result window will be blocked and no data be available.
The data extraction process can take from a few seconds to several minutes
depending on the size of the selected file, the amount of data extracted and the
performance of the computer. Whilst the data is being extracted, a progress
window displays the size of the XML file. Once extracted, the Result Table is
displayed in a separate browser window. A floating Save As button is present in
the upper right hand corner of the output window. This button can be used to
save the extracted output directly to a designated file for use by other
applications.
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11.4
Tag definitions
11.4.1
BVA parameters
Experimental data
•
Image name. Gel image filename.
•
DIA experiment. File name of the corresponding DIA workspace.
•
Gel ID. A unique number for a gel in the DeCyder 2D database.
•
No. of included spots. Number of spots included in the data set.
•
Dye chemistry. Indicates the type of dye used. Minimal CyDye, saturation
CyDye or PostStain labelling.
•
Spot map function. Spot map function in DeCyder 2D Software BVA. M, A,
T and P represent master, analysis, template and pick respectively.
•
Dye label. Indicates the fluor used to label the protein.
•
No. of matched spots. Number of spots matched to the master image.
•
Group. Name of the experimental group to which the spot map has been
assigned.
•
Group Description. A description of the experimental group to which the
spot map has been assigned.
•
Condition 1. Numerical condition 1 assigned to the spot map group.
•
Condition 2. Numerical condition 2 assigned to the spot map group.
•
Sample ID. Numerical identifier assigned to spot maps for paired
statistical analysis.
•
Spot map comment. User defined comment on selected spot map.
Image data
•
Volume. Sum of the detected pixel values above background within a spot
boundary.
•
Peak height. Pixel value at the X,Y position of the spot. This represents the
highest pixel value within the spot boundary.
•
Normal volume. Normalized volume obtained by dividing the calculated
volume for each included spot by the center of volume of the
corresponding spot map.
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Gel data
•
Volume ratio. Ratio of the normalized volumes of a pair of spots from a
spot map pair. The extracted values are the Log Standardized Abundance
values visible in the Appearance table and graph view in BVA.
•
Spot comment. User defined comment on selected spot.
•
Picked status. Indicates whether the protein has been assigned for
picking.
•
Coordinates. Position of spot peak along the horizontal and vertical axes
of the spot map.
•
Pick coordinates. Gel X and Y coordinates of the spot pick location.
•
PTM. Indicates that the protein has a post translational modification.
Entered by the user in DIA module or BVA module.
•
POI. Indicates that the protein has been assigned as Protein of interest.
•
Protein ID. Unique protein identifier that can be used to search databases
providing it is in the correct format for the specific database.
•
Area. Area as expressed in pixels within a spot boundary.
•
Match confidence. Indicates the type of match achieved i.e. Unmatched,
Auto Level1, Auto Level2, Manual or Added (to Master Spot Map).
•
Match status. Indicates whether match has been confirmed.
•
Spot number. DIA spot number as given by the spot detection algorithm.
•
Match quality. Morphological similarity metric describing deviation of
internal standard spot to an “average” internal standard spot in protein
set.
Protein data
248
•
Picked status. Indicates whether the protein has been assigned for
picking.
•
Picked in. Contains the names of the pick lists that include the protein.
•
Protein ID. Unique protein identifier that can be used to search databases
providing it is in the correct format for the specific database.
•
Protein AC. The protein accession number as designated by the selected
database.
•
Protein Name. Protein name given by selected database.
•
Protein comment. Note typed in the protein comment box
•
POI. Indicates that the protein has been assigned as Protein of interest.
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•
PTM. Indicates that the protein has a post translational modification.
Entered by the user in DIA module or BVA module.
•
Match quality. Morphological similarity metric describing deviation of
internal standard spot to an “average” internal standard spot in protein
set.
•
pI. Isoelectric point of the protein.
•
pI Status. Indicates whether displayed isoelectric point was entered for
reference purpose of the algorithm, calculated from the selected
algorithm or manually edited (designated Template, Calculated and
Manual respectively).
•
Mw. Molecular weight of protein.
•
Mw Status. Indicates whether displayed molecular weight was entered for
reference purpose of the algorithm, calculated from the selected
algorithm or manually edited (designated Template, Calculated and
Manual respectively).
•
T-test value. Student’s T-test p value.
•
Average ratio. Average ratio between the groups selected for protein
analysis.
•
ANOVA1. One-way ANOVA (ANalysis Of VAriation) p value.
•
ANOVA2 condition1. Two-way ANOVA for condition 1 p value for condition
1.
•
ANOVA2 condition2. Two-way ANOVA for condition 2 p value for condition
2.
•
ANOVA2 interaction. Two-way ANOVA interaction p value for the
interaction between condition 1 and condition 2.
•
Paired T-test value. Paired Student's T-test p value.
•
Paired Average Ratio. Paired average ratio between the groups selected
for protein analysis.
•
RM ANOVA1 (Repeated Measures ANOVA). Paired One-Way ANOVA p
value.
•
RM ANOVA2-condition1. Paired Two-Way ANOVA p value for
condition 1.
•
RM ANOVA2-condition2. Paired Two-Way ANOVA p value for
condition 2.
•
RM ANOVA2-interaction. Paired Two-Way ANOVA p value for the
interaction between conditions 1 and condition 2.
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11.4 Tag definitions
11.4.2
DIA parameters
Experimental data
•
Primary Image. Refers to the left hand gel image present in the image
view.
•
Secondary Image. Refers to the right hand gel image present in the image
view.
•
Tertiary Image. Refers to the third image included in the DIA workspace.
•
DIA experiment. Gel name/designation.
•
Gel ID. A unique number for a gel in the DeCyder 2D database.
•
No. of included spots. Number of spots designated as included for
subsequent analysis.
•
Dye chemistry. Indicates the type of dye used. Minimal CyDye, saturation
CyDye or PostStain labelling.
•
Estimated no. of spots. User defined approximation of the number of
spots present in spot map image entered prior to spot detection.
•
No. of similar spots. Number of spots within the threshold mode set.
•
No. of increased spots. Number of spots showing an increase in
abundance, above the threshold mode set from the secondary image
compared to the primary image.
•
No. of decreased spots. Number of spots showing a decrease in
abundance, above the threshold mode set from the secondary image
compared to the primary image.
•
Threshold mode. Value above or below which spots are classed as being
differentially expressed.
•
Threshold value. User defined value that enable highlighting of spots that
differ between the primary and the secondary image.
•
2SD value. 2 standard deviation of the spot ratio distribution, 95% of the
spots lie within this ratio for normally distributed data.
Image data
250
•
Volume. Sum of the detected pixel values above background within a spot
boundary.
•
Peak height. Pixel value at the X,Y position of the spot. This represents the
highest pixel value within the spot boundary.
•
Spot slope. Gradient associated with the 3 dimensional attributes of a
spot map pair.
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Gel data
•
Volume ratio. Ratio of the normalized volumes of a pair of spots from a
spot map pair. A value of 2.0 represents a two-fold increase while -2.0
represents a two-fold decrease, whilst a value of 1.00 represents an
unchanged spot.
•
Spot comment. User defined comment on selected spot.
•
Picked. Indicates whether the protein has been assigned for picking.
•
Coordinates. Values giving the position of the center of mass of a spot on
the spot map.
•
Pick coordinates. Gel X and Y coordinates of the spot pick location.
•
PTM. Indicates that the protein has a post translational modification.
Entered by the user in DIA module or BVA module.
•
POI. Indicates that the protein has been assigned as Protein of interest.
•
Protein ID. Spot identifier, which after confirmation assignment will
appear with the relevant spot in the table view.
•
Area. Area as expressed in pixels within a spot boundary.
•
Peak height ratio. OBSOLETE! Ratio of the normalized peak heights of a
pair of spots from a spot map pair. A value of 2.0 represents a two-fold
increase while -2.0 represents a two-fold decrease, whilst a value of 1.00
represents an unchanged spot.
•
Spot radius. Refers to the radius that the spot area would have if it was
circular.
•
Excluded. Indicates whether the spot has been excluded.
•
Confirmed. Indicates whether a spot has been manually confirmed by the
user.
•
Auto Confirm Assignment. Refers to the spot status after application of
the exclude filter. Spots are either designated as excluded or included.
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11.4 Tag definitions
11.4.3
Template parameters
For information on templates see 5.15 and 5.16.
Experimental data
•
Image name. The name of the image.
•
DIA experiment. Gel name/designation.
•
Gel ID. A unique number for a gel in the DeCyder 2D database.
•
No. of included spots. Number of spots designated as included for
subsequent analysis.
•
Dye chemistry. Indicates the type of dye used. Minimal CyDye, saturation
CyDye or PostStain labelling.
•
Dye label. Indicates the type of CyDye used.
•
Spot map function. Indicates if the spot map has been labelled as a
analysis, pick, template or master spot map.
•
Spot map comment. A comment for the spot map.
Image data
•
Volume. Sum of the detected pixel values above background within a spot
boundary.
•
Peak height. Pixel value at the X,Y position of the spot. This represents the
highest pixel value within the spot boundary.
•
Normal volume. Normalized volume with respect to the total volume.
Gel data
252
•
Picked status. Indicates whether the protein has been assigned for
picking.
•
Coordinates. Values giving the position of the center of mass of a spot on
the spot map.
•
Pick coordinates. Gel X and Y coordinates of the spot pick location.
•
PTM. Indicates that the protein has a post translational modification.
Entered by the user in DIA module or BVA module.
•
POI. Indicates that the protein has been assigned as Protein of interest.
•
Area. Area as expressed in pixels within a spot boundary.
•
Protein ID. Spot identifier, which after confirmation assignment will
appear with the relevant spot in the table view.
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•
Protein AC. The protein accession number as designated by the selected
database.
•
Protein Name. Protein name given by selected database.
•
Comment. Note typed in the comment box
•
pI. Isoelectric point of the protein.
•
pI Status. Indicates whether displayed isoelectric point was entered for
reference purpose of the algorithm, calculated from the selected
algorithm or manually edited (designated Template, Calculated and
Manual respectively).
•
Mw. Molecular weight of protein.
•
Mw Status. Indicates whether displayed molecular weight was entered for
reference purpose of the algorithm, calculated from the selected
algorithm or manually edited (designated Template, Calculated and
Manual respectively).
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12 Tutorials Introduction
12.1
Scope of tutorials
The following tutorials provide an introduction to the functionality of Ettan
DeCyder 2D Software, within the context of an actual experiment. The tutorials
have been designed to be a step by step guide utilizing gel images and DeCyder
2D Software files which are co-installed with the tutorials. The four tutorials
cover different aspects of the software suite. They are all self contained and can
be undertaken independently.
To assist the user each tutorial includes a completed version of the DeCyder 2D
Software file, which the tutorial is designed to generate. These files all include
the word finished in their names. Small deviations in the generated files might
occur since an improved matching algorithm has been implemented in the
software.
Tutorial I
The DIA is used exclusively to demonstrate a small-scale experiment to assess
protein changes in samples that are limited. See Chapter 13.
Tutorial II
The second tutorial encompasses a similar experiment to that found in tutorial
I. However, this tutorial utilizes gels that have already undergone spot detection
in the DIA. The processes involved in configuring the BVA, without the Batch
Processor, are demonstrated. See Chapter 14.
Tutorial III
The processes required to analyze a “pick” gel for subsequent spot picking,
digestion and mass spectrometry analysis are demonstrated. See Chapter 15.
Tutorial IV
The tutorial demonstrates how to ascertain protein expression changes in an
experiment containing replicate gels in order to generate a pick list for Ettan
Spot Picker. The entire workflow is co-ordinated via the Batch Processor, thereby
providing a fully automated process. See Chapter 16.
The tutorials described above introduce the concepts and functionality of
DeCyder 2D Software. It is recommended that these tutorials are performed
before using the software for actual analyses to gain a preliminary
understanding of the software. Different elements of the tutorials can also be of
help when analyzing an actual experiment incorporating an internal standard.
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12.2 Tutorial files
12.2
Tutorial files
The tutorial files comes pre-installed in the database. They are also provided as
database export files ( .dmp) on the installation DVD for backup.
Perform the following actions to restore tutorial files:
1
Copy the export files ( .dmp) you would like to import from the DVD to the
export directory defined during installation.
2
Start the Database Administration Tool and select Import on the left side.
3
Select the file that was copied from the DVD and then click Next >>.
4
Select project where the imported files shall be placed. The
recommendation is to create a new project for tutorial data. Click Next.
5
Click Finish. The import procedure starts.
Note: In version 6.0 all tutorial workspaces were marked read only which meant
that they could not be changed or deleted. In version 6.5 and 7.0 all
imported tutorial data can be changed or deleted.
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13 Tutorial I - Using DIA module for preliminary
investigation of protein changes
13.1
Objective
This tutorial describes how to find protein abundance differences above
experimental variation between a control and a treated protein lysate of
bacterial cultures using DeCyder 2D Differential Analysis Software DIA
(Differential In-gel Analysis). The procedure in this tutorial can be applied to any
two protein mixtures/lysates.
This experimental design can be used to rapidly identify proteins that show a
substantial change in abundance from control to treated sample and is often
used in the preliminary stages of an experiment.
The method is used to gain early information for the samples in question, and to
check that the experimental procedures are appropriate for the new samples.
The major drawbacks of this approach are that only one control sample and one
treated sample are studied. There are no gel replicates so the statistical validity
of an altered protein is impaired. This experimental design only addresses
differences above system variation (see Section 1.3 for different sources of
variability). Since there are no biological replicates the inherent biological
variation within a population is not considered. This is also true if the biological
control and treated sample are pools of control and treated sets of population,
as this approach is looking at averages but not variation associated with the
different sets of population.
13.2
Overview
1
An experimental design using two gels is set up, “control-control” gel and
“control-treated” gel.
2
The control-control gel is first analyzed in the DIA module. From this, an
indication of overall system variation is determined by finding the threshold
value that 95% of the similar spots lie within, e.g. 95% of spots on controlcontrol gel are within 1.5 fold.
3
The control-treated gel is subsequently analyzed in the DIA module. As the
control-control gel gave a result that 95% are within X fold (e.g. 1.5 fold, with
the specified tutorial files the precise value is 1.41545) then any spots with
a fold change greater than X fold (e.g. 1.5 fold) has a 95% confidence that
it is a difference between the control and treated sample above
experimental variation.
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13.3 Experimental design
4
The spots greater than X fold (with the specified tutorial files the precise
value is 1.41545) on the control-treated gel are looked at in detail. These
can be assigned with a protein of interest status and can be confirmed in
preparation for spot picking.
Spot picking from a preparative gel is covered in tutorial III.
13.3
Experimental design
Two gels were loaded with bacterial lysates as indicated in the table below.
Gel
1
2
Cy3
Control
Control
Cy5
Control
Treated
The control-control gel (gel 1) is analyzed to determine the level of experimental
variation. The protein abundance variation calculated in DIA indicates the
degree of similarity between the protein spots from two images from the same
gel. Ideally there should be no difference between the proteins from the two
control-control gel images (because the same sample is being run on the same
gel). However, due to different experimental factors (for example, pipetting
variation) there is some intrinsic variability.
The data analysis is performed in 2 parts using the DeCyder 2D Differential
Analysis Software DIA module:
258
•
Analysis of control-control gel
•
Analysis of control-treated gel
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13.4
Analysis of control-control gel
13.4.1
Selecting gel images
1
Start the DeCyder 2D Software, see 2.1.
2
In the DeCyder 2D Main window, click the DeCyder 2D Differential Analysis
Software - DIA icon to open the software module.
3
Select File:Create Workspace.
4
Locate the project entitled Tutorial I. Select the images
Gel01 Control Cy3 and Gel01 Control Cy5.
5
Click Create to create the DIA workspace.
13.4.2
1
Spot detection
After the images have been loaded select Process:Process Gel Images.
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13.4 Analysis of control-control gel
2
In the Process Gel Images dialog box which now appears enter the value
2500 for the estimated number of spots (see 5.5 file for an explanation of
this value).
3
Click OK to begin spot detection on the images.
Before going any further, read the description of the main tool bar.
Create Workspace
Open Workspace
Save Workspace
Process Gels
Protein Filter
Exclude Filter
Area in 3D
Rotate in 3D
Zoom In
Zoom Out
What's this?
Print
Properties
All Views
Table View
3D View
Histogram View
Image View
Contrast and
Brightness
Fit to Window
The user interface is divided into four main windows, the Image View, the
Histogram View, the 3D View and the Table View. In addition the Spot Control
Panel at the bottom of the screen allows user defined attributes to the spot
selected in the four views
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.
Image View
Histogram
View
3D View
Table View
Spot Control Panel
The four views are all linked. Clicking a spot on the Image View highlights the
spot in magenta in the Histogram View. The spot is also represented three
dimensionally and is highlighted in gray in the Table View.
4
To display the entire gel image, click the Fit to window icon.
5
After detection it is important to save the workspace. Select File:Save
Workspace, and enter an appropriate name in the dialog box, which
appears.
The detected spots are of three types: increased, decreased and similar
(colored blue, red and green, respectively, in the Histogram View)
6
If, after detection, the Table View remains empty click the Properties icon
to bring up the properties dialog box. By default this will open on the
Workspace tab.
7
Change to the Table View options by clicking the Table View tab.
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8
By selecting and deselecting the check boxes the different categories of
spots can be selectively displayed. Deselecting all the check boxes results
in the Table View being blank. The Table View tab works on OR logic, i.e a
spot only needs to conform to one criterion to be shown in the table.
Similarly, spots can be selectively displayed in the Image View using the
Spot Display tab. See 13.4.3.
Selecting different protein spots on the images reveals that some of the
spots that have been detected are in fact either dust particles or artifacts
from the gel. To remove these artifacts use an Exclude filter on the images.
Some values must be set before using the Exclude filter.
13.4.3
Assigning an area of interest
Note: For DIA workspaces that will be further analyzed in the BVA module, this
step is not necessary.
It is not necessary to assign an area of interest on the tutorial gel Gel01.
Continue immediately with 13.4.4.
However, the procedure can be performed anyway for training purposes or if
other gels than the specified are used in the tutorial process. In that case,
continue below.
Due to gel heterogeneity at the edges of the images, there are artifacts that
need to be removed. These can be removed by setting an area of interest. The
spots outside of the defined area of interest are not removed until an Exclude
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Filter is applied. If an area of interest was set before detection, all the spots on
the image will still be detected (even those outside the area of interest) until an
Exclude Filter is applied.
1
Click the Image View icon on the tool bar to have a full screen view of the
gel images.
2
Click the Fit to window icon on the tool bar to fit the images to the Image
View.
3
Next, click the Properties icon to bring up the properties dialog.
4
Select the Spot Display tab and deselect Similar, Increased and
Decreased, so that the check boxes are identical to those shown in the
figure below. Click OK.
5
To set an area of interest, select Edit:Define Area of Interest.
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13.4 Analysis of control-control gel
6
Use the rectangular target pointer which now appears, drag the mouse to
draw a rectangle around the gel, ensuring that edge artifacts are outside
the Area of Interest.
If there are Reference Markers on the gel (see 7.2 for more information), it
does not matter if they are inside or outside the Area of Interest.
7
Click the Properties icon to bring up the properties dialog again.
8
Select the Spot Display tab and select Similar, Increased and Decreased.
Click OK.
9
Apply the defined Area of Interest, by performing the exclude filter as
described in 13.4.4.
Note: To get the same values as displayed in this Tutorial, make sure an
Area of Interest is not used. Remove Area of Interest as described in
4.6.1 on page 74.
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13.4.4
Gel artifact removal (optional)
Note: For DIA workspaces that will be further analyzed in the BVA module, this
step is not necessary.
A 3D View clearly shows if a detected spot is a gel artifact rather than a protein
spot due to the very steep sides and pointed top of an artifact compared to the
smooth curve of a protein spot.
In order to exclude these artifacts from subsequent analysis a set of Exclude
filter parameters must be generated.
1
With all 4 Views displayed, click the column header Max Slope, in the Table
View, until the spot with the highest max slope is present at the top of the
table.
2
Click the first spot in the Table View.
3
Observe the spot in the 3D View. The 3D View clearly shows if the detected
spot is a dust particle (continue with step 4 below) or a protein spot
(continue with step 5 below).
4
If the spot is a dust particle:
Jump to a spot with a Max Slope value 0.5 to 1 lower than the Max Slope
value for the first spot and repeat step 3.
5
If the spot is a protein spot:
Move backwards in the Table View list (to spots with higher Max Slope
values) to find the last artifact spot before a real protein spot.
6
Note the Max Slope value of the last artifact spot found above. This is the
value to enter into the Max Slope parameter of the Exclude filter.
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7
Repeat this procedure for Area, Max Peak Height and Max Volume, but in
each case ordering the table so that the smallest value is at the top of the
table. Within step 4, jump to a value significantly larger than the value for
the first spot.
If there are three images in the workspace, it may be necessary to toggle
between the three images, to ascertain more accurately the exclude filter
parameters.
8
When all the values have been found, select Process:Exclude Filter to
display the Exclude Filter dialog and enter the found values (for tutorial file
Gel01, use the values in the image below.) Click OK.
The Exclude Filter will automatically remove detected spots on the basis of
these values. If an area of interest has been defined, the spots outside of
the area of interest will also be removed.
The table below shows an example of a non-stringent set of filter parameters
that can be used to run a light filter:
Property
Slope
Area
Volume
Peak Height
266
Suggested value
1.1
100
10000
<80 and >65000
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13.4.5
Ascertaining the 2 S.D. (standard deviation) threshold value
(optional)
Note: For DIA workspaces that will be further analyzed in the BVA module, this
step is not necessary.
The 2 S.D. value calculated for the control–control gel is the fold ratio that
encompasses 95% of the spots and can be used as a guide to the level of
experimental variation. This value should be noted and can be set as the
threshold value when analyzing the control-treated gel (in 13.5.4).
(In this tutorial, when no Area of Interest is used, the 2 S.D. value is 1.41545 and
the amount of similar spots are then 94,3% for the control-control gel.)
The protein differences being observed on the control-treated gel will be above
the experimental variation.
In the Histogram View there is a Histogram Selections toolbar with two drop
down menus and two information boxes. Make a note of the value in the
information box entitled 2 S.D.
Once this is noted, the analysis of the control-control gel (step 2 on page 257) is
completed, and it is time to move on to next step.
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13.5 Analysis of control-treated gel
13.5
Analysis of control-treated gel
13.5.1
1
Select File:Create workspace (there will be an automatic prompt to save
the current workspace. This is unnecessary in this tutorial, therefore click
NO to continue).
2
Locate the project entitled Tutorial I. Select the GEL folder and then the
Gel02 folder.
All images within the Gel02 folder are automatically selected.
3
Click Create to create the DIA workspace.
13.5.2
268
Selecting gel images
Spot detection
1
After the images have been loaded, select Process:Process Gel Images.
2
In the Process Gel Images dialog box which appears, enter the value 2500
for the estimated number of spots (see help file for an explanation of this
value).
3
Click OK to begin spot detection on the images.
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13.5.3
Gel artifact removal (optional)
1
Optionally, assign an Area of Interest as described in 13.4.3.
2
Perform an Exclude Filter as described for the control-control gel (see
13.4.4), using the same filter parameters.
13.5.4
Setting a threshold
Thresholds can be set to highlight protein spots that differ in their abundance by
using the Threshold Mode drop-down menu in the Histogram Selections toolbar
of the Histogram View.
For example, to select spots whose volume ratios differ between samples by 2
fold or more, select 2.0 fold from the drop down menu.
A variety of values in the Threshold Mode box can be selected, such as 2 S.D.
which is based on the model histogram (blue line) in the Histogram View.
Alternatively the threshold value can be set manually by selecting Manual and
entering a figure in the Threshold information box.
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13.5 Analysis of control-treated gel
1
For the purpose of this tutorial, select Manual and enter the 2 S.D. value
ascertained in the control-control gel (1.41545, as in 13.4.5) into the
Threshold information box.
As previously discussed, the threshold value represents the inherent
experimental variation. It can therefore be hypothesized that spots with
ratios which fall outside this threshold in a control/treated experiment have
some level of significance. As a general rule, the higher the threshold set the
more confidence that differences above the value is significantly different
above experimental variation.
The amount of similar spots for the control-treated gel is 60.8%.
Comparing the similar spots for the control-control gel (94.3%) to the
control-treated gel (60.8%) shows that 33.5% spots are differentially
expressed between the two gels.
The analysis of the control-treated gel (step 3 on page 257) is now
completed, and next step is to assign proteins of interest.
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13.6
Assigning protein of interest
Note: For DIA workspaces that will be further analyzed in the BVA module, this
step is not necessary. It should be performed in the BVA module instead.
This process can be performed prior to, or after spot confirmation. The amount
of time spent on spot confirmation can be reduced by performing filtering first.
The Protein Filter selects protein spots based on user defined criteria such as
volume ratio, above a maximum volume or peak height. The protein filter is
therefore used as a filter to find proteins that are interesting which can then be
confirmed.
1
To select those proteins that have been found to be different on the
control–treated gel, select the Protein Filter Dialog icon.
2
Ensure that the Assign Protein of Interest check box is selected and that
the Assign Pick status is not selected.
This results in the proteins successfully filtered being given a Protein of
Interest (POI) status.
3
For the purpose of this tutorial, set the parameters identical to those in the
figure below.
The selections are: select to assign protein spots with a volume ratio
greater the 2 SD value from the control-control gel and with a max volume
between 105 and 108, for example.
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13.6 Assigning protein of interest
The volume ratio value 1.41545 is the threshold value gained from the
control-control gel. It represents the system/experimental variation, not the
biological variation.
4
Click Filter to calculate the number of spots that meet the specified criteria.
These values can be adjusted and the Filter button clicked again to see
how many spots now meet the criteria. The purpose of this filtering is to
narrow down the users search for interesting proteins that have
significantly changed.
5
Click OK when the number of spots that meet the specified criteria is
acceptable.
6
The subset of spots that met the criteria will now be assigned as protein of
interest in the protein table (denoted by the letter I in the POI column).
The protein of interest assignment (for step 4 on page 258) is now
completed. Confirmation and preparation for spot picking remain to be
performed.
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13.7
Spot confirmation
Note: For DIA workspaces that will be further analyzed in the BVA module, this
step is not necessary.
Three options are available when confirming spots:
•
Option 1: Only confirm spots that are Increased (blue) and/or
Decreased (red) in their abundance.
This is the recommended option if proteins of interest have not been
assigned.
Select View:Properties, and go to the Table View tab. Ensure that the
Decreased, Increased and Confirmed options are selected, and that the
Excluded and Unconfirmed box is not selected. Click OK.
•
Option 2: Only confirm spots that have been assigned with a protein of
interest status
Recommended option when the protein of interest assignment option has
been performed (as in this tutorial). This is the quickest option of the 3.
Select View:Properties, and go to the Table View tab. Ensure that only the
Protein of Interest spots checkbox is selected. Click OK.
•
Option 3: All spots, Decreased (red), Increased (blue) and Similar (green)
are manually verified.
This takes approximately 1.5 hours for 1000 spots - this is not
recommended as only those spots that are increasing or decreasing in
abundance are of interest.
Select View:Properties, and go to the Table View tab. Ensure that the
Similar, Decreased and Increased options are selected, and that the
Excluded box is not selected. Click OK.
For the purpose of this tutorial, option 2 is used, as Proteins of Interest have
already been assigned. (295 spots in the tutorial file are assigned as POI.)
Only spots that are interesting for further analysis are confirmed. When
confirming spots, a decision is made as to whether the spots selected are trueprotein spots or non-protein spots, such as a particle of dust that has not been
removed by the Exclude Filter function. (In this tutorial, no dust particles are left
after filtering with the exclude filter and the protein filter as described
previously.)
Other spots that are sorted as unconfirmed are spots that are too weak, mixed
or unresolved. Streaks in the gel also fall into the unconfirmed category.
The Image View and the 3D View are helpful tools in this decision process.
1
Adjust the Table View options according to option 2.
2
Select the first spot in the Table View.
3
Zoom in on the selected spot in the Image View.
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13.8 Save DIA workspace (optional)
4
Inspect the spot in the Image View and in the 3D View.
5
a) If the spot is a genuine protein: click Confirm in the Spot Control panel.
b) If the spot not a protein: select Exclude in the Spot Control panel and then
click Confirm.
6
13.8
The next spot in the list is automatically displayed.
Continue with steps 3-6 until you have confirmed all the relevant spots.
Save DIA workspace (optional)
1
The DIA workspace can be saved by selecting File:Save Workspace to open
the Save Workspace dialog.
2
Select a project in which to save the workspace.
Note: It is possible to create a new project by clicking the New project icon.
3
13.9
Enter a file name, click Save.
Exporting a Pick Lists and physically excising spot from the gel
To excise the spots from the gel a separate preparative gel should be used. This
is then matched to this analytical gel set in DeCyder 2D Software BVA module
and the protein of interest status is transferred to the relevant matching spots
on the preparative gel. The pick list is based upon these proteins. The pick list
with x, y co-ordinates of spot positions is then exported based on the
preparative gel. The pick list with the physical preparative gel is then taken to
Ettan Spot Picker or Ettan Spot Handling Workstation for spot excision and later
analysis by mass spectroscopy.
Generating a pick list is covered in more detail in Tutorial III.
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14 Tutorial II - Employing an internal standard to Analyze
Protein Changes
14.1
Objectives
Tutorial I describes the analysis of a simple experimental design using only the
DIA module to evaluate samples where an internal standard is not possible due
to sample scarcity. This tutorial extends to a more complex experimental design
incorporating an internal standard with several replicate samples.
This tutorial describes how to find proteins that exhibit statistically significant
changes between control and treated groups of bacterial cultures using
DeCyder 2D Differential Analysis Software. The procedure in this tutorial can be
applied to any two groups of protein mixtures with either replicate gels or
replicate biological samples. The tutorial outlines the various stages in
experimental design, sample organization, protein detection and quantitation,
gel matching and statistical analysis.
14.2
Overview
The different stages involved in identifying all proteins that are differentially
expressed in a given system, and which, therefore, warrant further investigation
are described below:
1
An experimental design is devised that will generate statistically significant
results; a design which minimizes or eliminates in-gel and gel-to-gel system
variation.
2
Eight sample lysates that form the basis of the experiment are prepared.
This consists of four lysates derived from four bacterial cultures treated
with benzoic acid and four control flasks that have not been treated. Since
the gel to gel variation in this system is low, gel replicates are not
compulsory if biological replicates are available.
3
Aliquots from each sample are taken and pooled to prepare a standard
sample. All three sample types (standard, control and treated) are labelled
with an appropriate CyDye DIGE Fluor minimal dye (Cy2, Cy3 or Cy5 dye) as
described in the table overleaf. Each sample is then applied to the gels.
4
The samples are separated within 4 gels by 2-D electrophoresis. Three
images are produced from each gel, by scanning at appropriate
wavelengths for each of the three fluors. Typhoon 9000 series Variable
Mode Imager is recommended for scanning.
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14.3 Experimental design
5
The gels are cropped and imported to the database using the Image Loader
module and the Image Editor.
6
The images from the first gel are loaded into the DIA module.
7
Spot detection and calculation of the spot properties are performed for all
images from the same gel.
8
Protein spots are normalized using the in-gel linked internal standard.
9
The DIA workspace for the first gel is saved for subsequent matching and
analysis in the BVA module.
10 The DIA workspace for the first gel and DIA workspaces from the remaining
gels are loaded into the BVA module.
11 Spot maps (images) are assigned to groups and the experimental design is
set up in the BVA module.
12 Matching, including warping, is then performed.
13 Spots that conform to certain criteria such as statistical significance and
above a certain ratio change are assigned as proteins of interest.
14 These spots are confirmed for eventual inclusion into a pick list.
The individual stages involved in the experiment are now described more fully.
14.3
Experimental design
Four replicate gels are loaded with bacterial lysates as indicated in the table
below.
Gel number
Cy2
Cy3
Cy5
Gel 1
Standard
Control 1
Treated 1
Gel 2
Standard
Treated 2
Control 2
Gel 3
Standard
Control 3
Treated 3
Gel 4
Standard
Treated 4
Control 4
Each gel contains a standard sample to normalize control and treated samples
against. The standard sample is derived from control and treated lysates, which
are pooled in equal concentration.
The set of three images generated from a single gel are processed in sequence
using the DIA module, in order to perform spot detection and spot quantitation
against the internal standard.
The data from DIA analysis is saved as a workspace for further analysis in the
BVA module to evaluate protein abundance differences.
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14.4
Spot detection and quantitation
Spot detection and quantitation by normalizing against the internal standard is
performed using the DeCyder 2D Software DIA (Differential In-gel Analysis)
module. Gels are processed in sequence in the DIA module.
14.4.1
Selecting gel images
1
Start the DeCyder 2D Software, see 3.1.
2
In the DeCyder 2D Main window, click the DeCyder 2D Differential Analysis
Software - DIA icon to open the software module.
3
Select File:Create Workspace.
4
Select the project Tutorial II and the folder Gel01. All images Gel 01 Cy2
Standard, Gel 01 Cy3 Control and Gel 01 Cy5 Treated will be selected.
Note: All the images are named in the style Gel O1 Cy X Standard, Control
or Treated.
5
Click Create to create the DIA workspace.
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14.4 Spot detection and quantitation
14.4.2
Spot detection and quantitation
1
Once the images have been opened select Process:Process Gel Images.
2
Enter the value 2500 for the estimated no. of spots (see Section 5.5 for an
explanation for this value).
3
Click OK to begin spot detection and quantitation on all three images.
The DIA analysis takes between 1 and 3 minutes depending on computer
specifications.
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14.4.3
1
Viewing the DIA workspace
Upon completion of the DIA processing, the spot map data is displayed in
the workspace (as shown below).
Image View
Histogram
View
3D View
Table View
Spot Control Panel
The user interface is divided into four main windows that show the Image
View, the Histogram View, the 3D View and the Table View.
The four views are linked. Clicking any spot on the Image View highlights
the spot in magenta in the Histogram View. The spot is also represented
three dimensionally and is highlighted in the Table View. Each of the four
views can be expanded to fill the whole screen.
2
Click the View icons on the main toolbar to expand any view into full view
or to return to display of all views.
Alternatively, in the View menu select the desired view.
3
If, after detection has been completed, the Table View remains empty, click
the Properties icon to bring up the properties dialog box. Alternatively click
View:Properties.
4
Click the Table View tab.
5
Select and deselect the check boxes to display the different categories of
spots selectively. Deselecting all the check boxes results in the Table View
being blank. The Table View tab works on OR logic, i.e. a spot only needs to
conform to one criterion to be shown in the table.
Similarly, spots can be selectively displayed in the Image View using the
Spot Display tab.
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14.4 Spot detection and quantitation
6
The gel images displayed in the image view can be changed using the drop
down menus in the image view title bar.
When using an experimental design that includes an internal standard, as
in this case, it is conventional that the primary image view stays as the
image of the internal standard, whereas the secondary view displays the
images of the analytical gels.
Spot quantitation, expressed as a volume normalized against the internal
standard, is calculated in the DIA module then displayed in the Table View
under the Volume Ratio column. This column is automatically updated
when selecting to view a different analytical gel in the secondary gel view.
14.4.4
Saving the DIA workspace
The data associated with the spot boundaries and spot quantitation are
employed by the BVA module to perform inter-gel matching and further
statistical analysis. The data is therefore saved as a DIA workspace in the
DeCyder 2D database.
280
1
Select File:Save Workspace.
2
Select the project Tutorial II.
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3
Enter the DIA workspace name Gel01, then click Save to finish.
The DIA module can now be closed.
14.5
Creating the BVA workspace
14.5.1
Selecting gels
1
In the DeCyder 2D Main window, click the DeCyder 2D Differential Analysis
Software - BVA icon to open the BVA module.
2
Select File:Create workspace.
3
Locate the DIA workspaces Gel01, Gel02, Gel03 and Gel04, in the project
Tutorial II.
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14.5 Creating the BVA workspace
4
Select the 4 gel files by holding down the Ctrl key and clicking each image
using the left mouse button, click Add.
5
Finish by clicking Create.
The DIA workspace and image files will be automatically loaded into the
BVA workspace.
The BVA interface consists of four different modes:
Spot Map mode (S), Match mode (M), Protein mode (P) and the Appearance mode
(A).
Each screen in the BVA interface possesses a similar layout, consisting of:
•
Image View: Displays the gel images making up the experimental set, with
the currently selected spot shown in magenta.
•
Table View: Depending on the mode selected, the Table View displays
either information on the images used in the experiment, different
properties for a single spot across the gels in the analysis set or protein
spot specific statistical values.
•
3D View: Representation of the selected spot in 3 dimensions (not
displayed in Spot Map mode).
•
Graph View: Graphical representation of the protein abundance data for a
single spot across the different images in the analysis set.
Note: The Graph View is only displayed in Protein mode (P) and Appearance
mode (A). In Spot Map mode (S) the graph view is substituted for
Experimental Design view (see Section 5.3.4).
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Create Workspace
Open Workspace
Save Workspace
Spot Map Mode
Match Mode
Protein Mode
Appearance Mode
Match Dialog
Protein Statistics
Calibrate pI and MW
Protein Filter
Area in 3D
Rotate in 3D
Sort and browse Images
Multiple Image Views
Zoom In
Zoom Out
Fit to Window
Contrast and brightness
Gel mosaic horizontal zoom in
Gel mosaic horizontal zoom out
Gel mosaic vertical zoom in
Gel mosaic vertical zoom out
Magnify Image View
Magnify Graph View
Magnify 3D View
Magnify Table View
Display All Views
View warped images
Display warp grid
Gel view color overlay
Properties
Print
What's this?
Tutorial II - Employing an internal standard to Analyze Protein Changes 14
All the tables in BVA can be ordered by clicking the headers at the top of each
column.
3DView
Image View
Table View
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Graph/
Experimental
Design View
Data View Control Panel
The main BVA tool bar is shown below.
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14.5 Creating the BVA workspace
14.5.2
Function assignment
1
Ensure that the Spot Map mode icon is selected.
2
Select View:Table View or click the Magnify Table View icon to display the
Table View only.
The Table View shows the images that have been loaded into the
workspace and the number of spots that have been detected on them.
Note: A set of images from the same gel will have the same number of
spots since the DIA detection algorithm is designed to detect the
same number of spots on images from the same gel.
Under the column Function all the images will be labelled with the function
Analysis (A) and one of the analysis images (the standard image with the
largest number of detected spots, e.g. the Cy2 standard image) will be
labelled with the function Master (M).
The analysis function (A) designation indicates that each of the images will
be included in post matching statistical analysis (with one exception: pick
gels labelled with the function (A) are not included in the statistical analysis
as they only contain one spotmap/gel and do not include a standard).
3
284
Reassign Spot Maps if necessary.
The Master function can be assigned to a different image, by selecting the
image to be assigned as master using the left mouse click. Tick the box
entitled Master at the bottom of the table in the area entitled Function for
Spot Map.
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14.5.3
Experimental group assignment
To generate statistical data for the proteins which differ between the control
and treated groups it is necessary to identify the different experimental groups
within the software. The three groups involved in this experiment are Control,
Treated and Standard.
Group assignment is performed via the Experimental Design view.
1
The standard images will automatically be assigned to the group Standard
by the software if the word standard or std appears in the image name. The
standard images will be located in the folder entitled standard.
By default non-standard spot maps are assigned as Unassigned. To assign
the control and treated images to their respective groups, these groups
must first be created.
2
Create a control group:
Click Add, then enter Control in the Group text box and click Confirm. The
new group folder Control is subsequently displayed. Add the treated group
in the same way.
3
Assign Spot maps to either the control or treated group folders by selecting
the Unassigned folder, then dragging and dropping spot maps from the
center panel of the Experimental Design view to the appropriate folders on
the left of the screen.
4
Select colors for the experimental groups by clicking the round Color
button and select colors, for example red and blue.
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14.6 Matching
14.6
Matching
1
Click the Match icon, ensure that Match all and Optimize Matching Using
Warping are selected and click Match to commence the matching process.
The view switches to automatically display warped images and the warped
images will be displayed when they become available.
2
286
Select Match mode (M button). This screen displays data on the gel to gel
matching of the different protein spots and allows match confirmation.
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The match mode consists of the Image View, 3D View and Table View. Each of
the three Views are linked, in such a way that when selecting a spot in the Image
View, that spot is displayed in 3 dimensions in the 3D View and the same spot is
highlighted in the Table View list. The vector lines in the Image View indicate the
positional difference for the same protein spot on different gels. When matching
has been optimized using warping the vectors are normally very short.
The matches in the Table View are of two types:
Match type
Match
Default spot boundary in Image View
Auto Level 1
High probability
Green
Auto Level 2
Low probability
Lilac
The boundaries of unmatched spots are colored orange (these colors can be
altered by clicking the properties icon and selecting the colors tab. The Colors
dialog allows the user to define colors to the different matches).
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14.6 Matching
Performing match assessing
The purpose of the match assessing is to get an overall impression of the
matching accuracy. This can be done in several ways. One way is to look at the
match vectors for each gel, if the match vectors are long in some areas, this may
indicate an area of mismatches. This area should be landmarked and rematched.
288
1
Click the Properties icon, and deselect Spot contours on the Image View
tab.
2
Click the icon Gel View Color Overlay and select Color overlay using
master image in the dropdown menu.
3
Select to view the warp grid by clicking the icon Warp grid.
4
If not already displayed, select to view all standard images by clicking the
Sort and browse images icon and select Standard to display the standard
images.
5
The master spot map (with a yellow header) is displayed in a separate
floating window which can be moved around. Move the floating master
window (with yellow header) and place it on top of the master image in the
Image View.
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6
To get an overall impression of the matching accuracy, examine all gels to
single out any gels where the warping algorithm has been less successful.
This can be determined by viewing areas where the warping grid displays
an unusual level of deformation and/or where the yellow master color is
visible in large parts of the image. This can also be done evaluating match
vectors. Zoom into the area where the warping grid displays high level of
deformation, or where the color overlay indicates large differences, by
pressing the left mouse button, drawing a square around the selected area
and then releasing the left mouse button.
7
It may be necessary to turn on and off information (warped images, warp
grid, color overlay, spot contours) to be able to determine suitable landmark
spots. Right-click in the gel image and choose action to turn these on and
off.
Now deselect the warp grid. This will give a clear overview of the warping
result in color.
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14.6 Matching
When matching has been optimized using warping the vectors are
normally very short. If the match vectors are long in some areas, this may
indicate an area of mismatches. In such cases these areas should be
landmarked and the corresponding gel re-matched.
The color overlay function in BVA is a gel image overlay function which put
two gel images, one yellow and one blue, on top of each other. The fixed
standard image of the master gel is presented in yellow (as a yellow
shadow) behind the warped standard image presented in blue. When the
intensity levels in the different color channels is the same, the image will be
displayed in gray scale for that area, in this way the colored areas in the
color overlay enhances parts of the images that are different. Deviations
where the blue spot is not located precisely on top of the yellow spot can
easily be found.
In some cases the automated warping may not succeed in completely
aligning all parts of an image to the master. In such cases, it is
recommended to add one or a few landmarks for spots in areas where the
warping is less successful.
8
Set a few landmarks in each found area.
To use a spot as a landmark the spot must be present on both the match
image(s) and the master. It is recommended to perform landmarking on the
standard images. To set a landmark, select a clearly defined spot in the
master gel image (click the Magnify Image View icon to simplify viewing).
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Decide whether the spot in the match image corresponds to the spot on the
Master image. Deciding whether a match is accurate or not can be aided
by viewing the selected spot and the surrounding cluster in the 3D View
(click the Display all views icon to see the 3D View) as well as looking at the
matched spots in the Image View.
The spot boundaries of the selected spots should become magenta
(default). To select a spot on more than one spot map, click while holding
down the Ctrl-button, or check Multi Select and then click the spots.
Click Add match (if all selected spots are unmatched) or Break+Add (if
some of the spots are already matched, but wrongly matched).
If the spot selected on the master image is incorrectly matched, click Break
Match.
Note: Break Match can only be performed on one image at a time.
Then select the spot on the match image that corresponds to the correct
match on the master image. If this spot is also incorrectly matched click
Break Match.
Finally, select the two corresponding spots so that both spots become
magenta and click the Add Match button.
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14.6 Matching
Setting landmarks in the areas with unsatisfactory warping and/or
matching results can also be done by confirming a few correct matches.
•
If the spot selected on the master image is correctly matched, click
Confirm Match Set, to set the spot as landmark on all the gels.
•
If the spot selected on the master image is correctly matched and the
spot will be landmarked on the selected match image, click Confirm
Single Match.
Note: Landmarking can be aided by viewing the pattern around the
selected spot in the 3D View to confirm that the two selected spots
do correspond to each other.
During the rematching process, any manually matched and confirmed
matches (marked as type Landmark in the Match Table) will be used as
landmarks to improve the matching and warping process.
9
292
When landmarking is completed, rematch the gels with new landmarks:
•
Click the Match button. The Match options dialog opens.
•
Select the appropriate match option and select Optimize Matching
Using Warping.
The new warped images are created. This can be done for one gel at a
time or for several gels at once.
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10 Repeat landmarking until the matching appears to be satisfactory in all
gels.
The effect of the landmark on the warped image is reviewed using color
overlay. Comparing the picture above with the picture on page 290 shows
that the landmarking should be focused to the images where the warping
is less successful. Generally if the warping is successful for a gel image, the
matching is successful as well, so less attention is needed to examine
match results for these images. As can be seen above, after landmarking
the match vectors are shorter and less of the yellow and blue colors are
seen. This means that the warping has been successful.
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14.7 Statistical analysis
14.7
Statistical analysis
1
Click the Protein icon or select View:Protein mode.
The Protein Table lists each of the matched spots, across all the images in
the experiment. The Protein mode consists of an Image View, a 3D View, a
Graph View and a Table View. Each of the four views are linked, in such a
way that when selecting a spot in the Image View, that spot is displayed in
3 dimensions in the 3D View, it is highlighted in the Table View and the
abundance values of that spot across all the images on which the spot
occurs are graphically displayed in the Graph View.
The Protein mode allows you to view those proteins that show a significant
difference in expression between the control and the treated group. The
statistical level of significance is calculated using the T-test (if more than
two groups, the ANOVA test is used).
2
294
To perform statistical calculations select Process:Protein statistics. For the
purpose of this tutorial, set the parameters to those identical to the figure
below. (FDR correction is used to minimize the risk of finding false positive
results, see also 5.10.3.)
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3
Click Calculate.
The spot specific statistics along with the average spot volume ratio now
appear in the table.
A low T-test value indicates a high level of significance due to the fact that
the statistical calculations are performed using the null hypothesis that the
differences in protein standardized abundance (i.e. the abundance relative
to intra-gel standard and/or biological variation within groups) of control
and treated samples is not significant.
4
To sort the spot in order with the statistically most interesting spots on top
of the list, first alter the Graph View:
Open the Properties dialog. Click the Graph View tab and ensure that the
view parameters are identical to those shown below. Click OK.
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296
5
Click the T-test column header to order the table so that the lowest value is
at the top of the table. Click a spot with a low value in the Table View. This
spot will now be displayed in all four views.
6
In Properties:Image view, check Spot contours again and uncheck the
Color overlay with master.
7
Click the headers of two spot maps to be displayed in the 3D view. Left-click
for magenta header and right-click for green header.
8
Click the color overlay icon and select to display standard images to
highlight differences in expression.
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The Standard image of the gel is presented in yellow behind the other (e.g.
Treated) image presented in blue. For this option, all spots should generally
be located in the same position. A blue spot indicates increased protein
abundance compared to the standard, and a yellow spot indicates a
decreased protein abundance compared to the standard.
Any deviations where the blue spot is not located precisely on top of the
corresponding yellow spot would indicate a difference in migration
between the samples in the gel for that particular protein.
9
In the Graph View, the dashed lines link data points from sample spots to
their respective standard data point. Therefore, in this experiment there are
four matched control spots and four matched treated spots (associated
with four standard data points).
To magnify the graph view click on the Magnify Graph View icon. To revert
click on Display all Views.
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14.7 Statistical analysis
The standards are the same on each gel, and hence the normalization
calculates how samples change with respect to their in-gel standard. A
ratio of sample to standard of, for example, 2.5 means that the sample
protein is 2.5 times greater than its standard protein, or a ratio of 2.5 to 1.
When this methodology is used, all standards are 1. Thus the standard is
displayed graphically as 1 and the samples displayed relative to their
standard. The log of 1 is zero and therefore when the log standardized
abundance is displayed, all standards are zero.
The protein table shows average ratio (i.e. Average abundance of
control/Average abundance of treated) as well as the statistical
significance of the differences.
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14.8
Assigning spots as proteins of interest
14.8.1
Selecting proteins of interest
This process can be performed prior to spot confirmation or after spot
confirmation. Doing this first can reduce the amount of time spent on spot
confirmation.
Select those proteins that have been found to be statistically different on the
analytical gels:
1
Select the Protein Filter Dialog icon.
The Protein Filter selects protein spots based on user defined criteria such
as Student's T-test value, average ratio or volume. The protein filter is
therefore used as a filter to find proteins that are interesting which can then
be confirmed.
2
Ensure that the Assign Proteins of Interest check box is selected and that
the Assign Pick status is not selected. This results in the successfully
filtered proteins being given a Protein of Interest status.
For the purpose of this tutorial, protein spots with a T-test score less than
0.001 and with an average ratio greater than or equal to 2 or less than or
equal to -2. Set the values identical to those in the figure on next page.
3
Click Filter. The number of spots that meet the criteria are displayed in the
dialog.
4
The values can be adjusted and the filter button clicked again to see how
many spots now meet the criteria. The purpose of this filtering is to narrow
down the users search for interesting proteins that change significantly.
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14.8 Assigning spots as proteins of interest
5
When the number of Proteins of Interest is acceptable, click OK.
Note: The volume filtering criteria is applied to spot volumes on the Master
gel. If a preparative gel is assigned the parameters can be applied to
this pick gel (see Tutorial III).
6
The subset of spots that meet the criteria will now be assigned with a
protein of interest status in the protein table.
Alternatively, individual spots can be assigned manually by clicking the spot
of interest and clicking in the POI check box at the bottom of the Protein
Mode window.
7
Select View:Properties or click the Properties icon. Click the Protein Table
tab and in the Protein Table Filter area, select Protein of Interest (I) only.
Click OK.
The Protein Table now displays only those spots that are of most interest
and which can now be manually confirmed.
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14.8.2
Spot confirmation in Protein Table
A spot should be confirmed when a significant difference has been identified,
and the user is certain that it is a real protein spot and it has been correctly
matched on all gels.
1
To simplify the confirmation procedure, click the Multiple Image Views icon
and select All to display one column with all control and one with all treated
images.
2
To confirm the first spot in the Protein Table, select the spot in the Table
View. The spot is automatically selected in all the other views.
3
View the spot in image and 3D view and determine whether the spots have
been matched correctly between the images. Zooming in the views might
be of help.
a) If the spots have been correctly matched, continue from step 4.
b) If the protein spots are incorrectly matched move to the Match Mode
View by clicking M and alter the match as described in Section 5.9.
4
When all the matches are correct the protein spot can be confirmed by
clicking the Confirm button. This records that the spot has been manually
checked. The next spot in the table is automatically displayed when clicking
Confirm.
5
This confirmation should be performed for all the significant differences
that have been assigned with a protein of interest status.
6
If, after manual scrutiny, the user decides that the spot should not be
assigned as POI, deselect the protein of interest status for the given spot by
unchecking the box marked POI at the bottom of the Protein Mode.
The row disappears from the Table View.
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14.8 Assigning spots as proteins of interest
14.8.3
Exporting the Pick List and physically excising spots from the
gel
To excise the spots from the gel a separate preparative gel should be used. This
is matched to the analytical gel set and the pick status is eventually transferred
to the relevant matching spots on the preparative gel. The pick list with x, y coordinates of spot positions is then exported based on the preparative gel. The
pick list along with the physical preparative gel is then taken to Ettan Spot Picker
or Ettan Spot Handling Workstation for spot excision and later analysis by mass
spectrometry. For a detailed description of the workflow associated with pick list
generation, see Chapter 5 BVA (Biological Variation Analysis) Module.
Generating a pick list is covered in more detail in Tutorial III.
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15 Tutorial III - Processing the Preparative Gel and
Generating a Pick List
15.1
Objective
As can be seen in tutorials I and II, proteins of interest, which warrant
identification and further investigation, can be obtained in both the DIA and BVA
modules. The Protein Filter tool is applied to the analytical gels to rapidly
ascertain which proteins are affected by the experimental conditions. When
using either CyDye DIGE Fluor minimal dyes or CyDye DIGE Fluor saturation
dyes, the highlighted proteins of interest have to be matched to a preparative
gel, from where the protein spots can be directly excised using Ettan Spot Picker
or Ettan Spot Handling Workstation (see Ettan DIGE system user manual). The
workflow for generating a pick list from a preparative gel in both CyDye DIGE
Fluor dye types is identical. Here in tutorial III a preparative gel for a CyDye DIGE
Fluor minimal dye experiment is used to demonstrate the various steps in
generating a pick list.
The aim of this experiment is to generate a pick list on a fluorescently poststained gel, relating to the most significant differentially expressed proteins in
control and treated bacterial lysates determined from Ettan DIGE system
analytical gels.
When using the CyDye DIGE Fluor minimal dyes, a fluorescently post-stained
pick gel is required rather than picking directly from an Ettan DIGE system gel
because the CyDye DIGE Fluor minimal dye label only <5% of the protein. This
results in the majority of the (unlabelled) protein migrating slightly different and
hence not ending at exactly the same place as the (labelled) protein seen on the
gel. Therefore a post stain (total stain, i.e. Deep Purple™) is used on a preparative
gel for spot excision. The image generated is then matched to the analytical set.
The positions of the significant differences are transferred to the fluorescently
post-stained gel and a pick list is created. This pick list, along with the
fluorescently post-stained gel, is transferred to the automated Ettan Spot Picker
or Ettan Spot Handling Workstation for spot excision and subsequent digestion,
spotting and mass spectrometry analysis.
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15.2 Overview
15.2
15.3
Overview
1
This Tutorial follows on from Tutorials II. Tutorial II concentrates on finding
differences between different biological groups. This tutorial describes how
to excise these spots for further protein identification analysis.
2
An experiment is designed as described in tutorial II, with the addition of a
separate preparative gel and a fluorescently post-stained gel containing
two reference markers.
3
The analytical gels are analyzed in DeCyder 2D Software to ascertain the
proteins of interest, as described in Tutorial II.
4
The preparative gel is analyzed in DeCyder 2D Software DIA. The reference
markers are assigned in DIA (alternatively, this can be performed in BVA).
5
The preparative gel spot map is saved as a DIA workspace and then added
to the BVA workspace that contains the analytical gels.
6
The preparative gel spot map is landmarked and matched to the master gel
of the analytical set.
7
The protein of interest assignments are transferred from the analytical set
to the preparative gel.
8
The proteins of interest are assigned a Pick status, then the pick locations
are visually inspected (and edited if necessary).
9
A pick list is exported from the preparative gel. The gel and the pick list are
then taken to Ettan Spot Picker or Ettan Spot Handling Workstation for spot
excision (See the relevant User Manual for instructions).
Experimental design
Ettan DIGE system and DeCyder 2D Software are used to investigate differential
protein expression in experimental groups. These identified proteins can then be
picked from a preparative gel for digestion and subsequent mass spectrometry
analysis.
Gels 1 to 4 were generated to determine which proteins were differentially
expressed in control and treated bacterial samples. These gels have been preanalyzed in DeCyder 2D Software and saved as a BVA workspace (named
Control Treated for picking in the project Tutorial III in the DeCyder 2D
database).
A fluorescently post-stained preparative gel (gel 5, named Pick), loaded with a
pool of control and treated samples was prepared for spot picking. The spots on
this pick gel are first detected within the DIA module, then matched to the
master gels (gels 1 to 4) in DeCyder 2D Software BVA.
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There is also a pre-analyzed pick gel, named Pick finished, in the project
Tutorial III.
Gel
number
Cy2
Cy3
Cy5
Fluorescent
post-stain
Gel 1
Standard
Control 1
Treated 1
-
Gel 2
Standard
Treated 2
Control 2
-
Gel 3
Standard
Control 3
Treated 3
-
Gel 4
Standard
Treated 4
Control 4
-
Gel 5 (Pick)
-
–
–
Control + Treated
15.3.1
Identifying protein spots on post-stained gel
1
In the DeCyder 2D Main window, click the Differential In-gel Analysis - DIA
icon to open the software module.
2
Select File:Create Workspace.
3
Locate the project entitled Tutorial III and the GEL folder. Select the pick gel
image, then click Create to create the workspace.
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15.3 Experimental design
4
As the image was fluorescently post-stained, the initial image contrast may
require adjusting (not necessary for the Tutorial III Pick gel). Select the
contrast/brightness icon and alter the bars to make the image clearer.
15.3.2
306
Spot detection
1
After the image has been loaded select Process:Process Gel Images.
2
In the Process Gel Images dialog which appears, enter the value 2500 for
the estimated number of spots (see Section 4.5). Select the Autodetect
Picking references check box.
Click OK to begin spot detection on the images.
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Before going any further, read the description of the main tool bar.
What's this?
Print
Properties
All Views
Table View
3D View
Histogram View
Image View
Contrast and
Brightness
Fit to Window
Create Workspace
Open Workspace
Save Workspace
Process Gels
Protein Filter
Exclude Filter
Area in 3D
Rotate in 3D
Zoom In
Zoom Out
The user interface is divided into four main windows, the Image View, the
Histogram View, the 3D View, and the Table View.
Image View
Histogram
View
3D View
Table View
Spot Control Panel
The four views are all linked. Clicking a spot on Image View highlights the spot
in gray in the Table View. (The Histogram View is disabled when analyzing single
images such as fluorescently post-stained gel images). The spot is also
represented three dimensionally.
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15.3 Experimental design
After detection
If, after detection, the Table View remains empty:
1
Click the Properties icon to bring up the Properties dialog box.
2
Click the Table View tab.
3
Select the check boxes to display the different categories of spots.
Deselecting all the check boxes results in the Table View being blank. The
Table View tab works on OR logic, i.e a spot only needs to conform to one
criterion to be shown in the table.
Similarly, spots can be selectively displayed in the Image View using the
Spot Display tab.
Selecting different protein spots on the images shows that some of the
spots that have been detected are in fact either dust particles or artifacts
from the gel. To remove these artifacts an Exclude Filter can be used on the
images.
If further analysis of DIA gels is to be performed in BVA module (as in this
tutorial), gel artifact removal is not necessary. Non-proteinaceous spots will
not appear as significant in the BVA module as they will not appear in the
same place on different gels. For training purposes Sections 15.3.3 and
15.3.4 can be performed, but it is also possible to proceed with 15.4
immediately.
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15.3.3
Gel artifact removal (optional) - Step 1:
Assigning an area of interest
This step is not necessary to perform on the Tutorial III Pick gel. It is only included
for training purposes.
The automated spot detection algorithm in DeCyder 2D Software DIA may
detect artifacts due to gel heterogeneity at the edges of the images. These spots
can be removed by setting an area of interest to define the gel only. Those spots
outside of the area of interest will be automatically removed when the Exclude
Filter is applied. The area of interest function is only operational when the
Exclude Filter is executed.
1
Optional: Click the Image View icon on the tool bar to have a full screen
view of the gel image.
2
Optional: Click the Fit to window icon on the tool bar to fit the image to the
Image View.
3
Next, click the Properties icon to bring up the properties dialog.
4
Select the Spot Display tab and deselect Similar, Increased and
Decreased, so that the check boxes are identical to those shown in the
figure below. Click OK.
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15.3 Experimental design
5
To set an area of interest:
- Select Edit:Define area of interest.
- Use the rectangular target pointer which now appears and drag the
mouse to draw a rectangle around the gel, ensuring that edge artifacts are
not included. It does not matter if the Reference Markers are inside or
outside the Area of Interest, as these have been set automatically (in
15.3.2.) and they are not regarded as spots.
6
Click the Properties icon to bring up the properties dialog.
Select the Spot Display tab, reselect the Similar, Increased and Decreased
options and click OK.
All the spots on the outside of the area of interest will automatically be
excluded when the exclude filter (see 15.3.4) has been performed. With
default color settings (in Properties dialog, Colors tab) excluded spots have
a gray outline in the Image View. Renormalization is automatically
performed when using the exclude filter, and volume ratios in the Table
View are updated as is the Graph View.
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15.3.4
Gel artifact removal (optional) - Step 2: Exclude filter
A 3D View clearly shows if a detected spot is a gel artifact rather than a protein
spot due to the very steep sides and pointed top of an artifact compared to the
smooth curve of a protein spot.
Artifacts can be excluded from subsequent analysis by generation of a set of
Exclude filter parameters.
1
With all 4 Views displayed, click the column header Area, in the Table View,
until the spot with the smallest area is present at the top of the table.
2
Observe the spot in the 3D View. The 3D View clearly shows if the detected
spot is a dust particle (continue with step 3 below) or a protein spot
(continue with step 4 below).
3
If the spot is a dust particle:
Jump to a spot with an Area value 10 to 20 higher than the Area value for
the first spot, and repeat step 2.
4
If the spot is a protein spot:
Move backwards in the Table View list (to spots with lower Area values) to
find the last artifact spot before a real protein spot.
5
Note the Area value of the last artifact spot found above. This is the value
to enter into the Area parameter of the Exclude filter.
6
Repeat this procedure for Max Volume. (Larger jumps than for the Area
values are possible in step 3.)
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7
When both values have been found, select Process:Exclude Filter to display
the Exclude Filter dialog box.
Select the Area and Volume check boxes and enter the noted values. For
the Tutorial III Pick gel, enter values as displayed in the dialog below.
Click OK.
An example of a non-stringent set of filter parameters that can be used to
run a light filter is as follows:
Parameter
Suggested value
Area
50
Volume
20000
8
It is possible to continue the work with either the DIA workspace analyzed
as described hitherto, or with a pre-analyzed DIA workspace named Pick
finished in the project Tutorial III.
If continuing with the DIA workspace analyzed in this tutorial, continue
below.
If continuing with the pre-analyzed DIA workspace, continue with 15.4.
9
Save the DIA workspace for subsequent gel to gel matching in BVA by
selecting File:Save workspace to open the Save Workspace dialog.
10 Select a project of your own, or create a new project in the Save Workspace
dialog.
11 Enter Pick as the DIA workspace name and click Save.
At this point the DIA workspace can be closed.
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15.4
Matching to analytical gels
The fluorescently post-stained picking gel has to be matched to analytical gels
in order to identify the proteins on the picking gel that show differential
expression and hence require picking.
15.4.1
Preparing the workspace
1
In the DeCyder 2D Main window, click the Biological Variation Analysis BVA icon to open the BVA module.
2
Select File:Open workspace.
3
In the Tutorial III project, select the BVA workspace Control treated for
picking. Click Open to open the selected workspace (this may take a few
minutes depending on the specifications of the computer).
This BVA workspace contains the experiment described in the experimental
design section of this tutorial. The workspace has been pre-analyzed in the
BVA module and several proteins, that demonstrate a statistically
significant change upon treatment, have been given a Protein of Interest
status and, therefore, require picking and subsequent identification.
The BVA interface consists of four different modes, Spot Map mode (S), Match
mode (M), Protein mode (P) and Appearance mode (A). Each screen in the BVA
interface possesses a similar layout, consisting of:
•
Image View: Displays the gel images making up the experimental set, with
the currently selected spot shown in magenta.
•
Table View: Depending on the screen selected, displays either information
on the images used in the experiment, experimental design, different
properties for a single spot across the gels in the analysis set or protein
spot specific statistical values.
•
3D View: Representation of the selected spot in 3 dimensions.
•
Graph View: Graphical representation of the protein abundance data for a
single spot across the different images in the analysis set.
All the tables in BVA can be ordered by clicking the headers at the top of each
column.
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15.4 Matching to analytical gels
Create Workspace
Open Workspace
Save Workspace
Spot Map Mode
Match Mode
Protein Mode
Appearance Mode
Match Dialog
Protein Statistics
Calibrate pI and MW
Protein Filter
Area in 3D
Rotate in 3D
Sort and browse Images
Multiple Image Views
Zoom In
Zoom Out
Fit to Window
Contrast and brightness
Gel mosaic horizontal zoom in
Gel mosaic horizontal zoom out
Gel mosaic vertical zoom in
Gel mosaic vertical zoom out
Magnify Image View
Magnify Graph View
Magnify 3D View
Magnify Table View
Display All Views
View warped images
Display warp grid
Gel view color overlay
Properties
Print
What's this?
The BVA module main tool bar is shown below.
4
Ensure that the S button is active (i.e., the Spot Map mode is selected). This
screen consists of an Image and a Table View.
5
Select View:Table View or click the Magnify Table View icon to display the
Table View only. The table shows the images that have been loaded into the
workspace and the number of spots that have been detected on them.
Note: 2 or 3 images from the same gel will have the same number of spots
since the DIA co-detection algorithm is designed to detect the same
number of spots on image pairs from the same gel.
Under the column Function all the images will be labelled with the function
Analysis (A) and one of the analysis images will be labelled with the function
Master (M). The analysis function designation indicates that each of the
images will be included in the post matching statistical analysis.
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6
The Master function can be assigned to a different image. However, the
Master should not be changed here or all the matching will have to be
repeated.
If Master function by any reason should be assigned to a different image,
select the image to be assigned as master using the left mouse click. Then
tick the box entitled Master at the bottom of the table in the area entitled
function for spot map.
7
Add the pick gel spot map (the post-stained preparative gel spot map) to
the BVA workspace:
a) Select File:Add Template/DIA workspace.
b) Select the project and DIA workspace file saved in Section 15.3.4 or the
preanalyzed file, Pick finished in the Tutorial III project.
c) Click Add-->.
d) Click Add.
8
Once loaded, the post-stained gel spot map can be assigned as a Pick gel,
ensuring that the eventual pick list will be based on the spot co-ordinates
of this post-stained gel image:
Select the post-stained spot map in the table view, then select the Pick
check box in the data view control panel. Deselect the default Analysis
check box.
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15.4 Matching to analytical gels
15.4.2
Editing picking reference markers
Two reference points are needed on the preparative gel to make sure the
software, controlling the picking robot, locates the spots correctly when picking.
The reference markers placed on the preparative gels (seen as perfect circles on
the images) act as these reference points.
The position of the reference markers can be automatically detected during the
spot detection process (as performed in Section 15.3.2). However, it is advisable
to review the position of the reference markers and edit them, if necessary.
316
1
With all 4 Views displayed, select the Pick.gel from the drop down list in the
name bar in the image view and get it selected by either clicking on the
name header or click on the name in the Spot Map Table (magenta header
and image name indicates that the image is selected).
2
Zoom into the area of this reference marker by holding down the left mouse
click to draw a rectangular area around the marker.
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3
The size of the target can be altered by clicking the properties icon and
selecting the Image View tab. A value of approximately 30 should be
entered in the Selected Image View, Default radius of picking references
box of the picking references data area of the dialog.
4
- Select Edit:Edit picking Reference to alter the position of the target. Place the hand shaped cursor that appears on the target and hold down
the left mouse click.
- Move the target so that it fits exactly around the reference. Magnifying the
area of the gel around the reference marker by selecting the Zoom in icon
may make accurate alignment easier.
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15.4 Matching to analytical gels
5
After reviewing the left reference marker, zoom into the right reference
marker, then check and edit its’ position in an identical manner. Uncheck
Edit:Edit picking References to exit this mode.When the picking references
have been reviewed it is necessary to match the preparative gel to the
master image, thereby matching the preparative image to all the analytical
images.
15.4.3
1
Matching and landmarking
To match the images, first select the M button in the tool bar (i.e. select the
Match mode).
From the images it is clear that the post-stained image is slightly different
from the analytical images. It is therefore necessary to set landmarks.
Landmarking allows the user to manually define matched protein spots in
order to improve the accuracy of the gel-to-gel matching process.
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2
Click the Magnify Image View icon to display the gel images only.
Click the Multiple Image View icon and select 1x2 to display the Pick gel and
the Master.
Click and drag the floating Master image to the right image window.
In the left image select the Pick.gel in the drop down menu in the image
header.
3
If the spots on the images are not clearly visible it may be necessary to alter
the contrast/brightness settings of the images.
Click the contrast/brightness icon, ensuring the Apply to all gel images
check box is not selected. Alter the position of the bars to alter the contrast
and brightness of the images until only the most intense spots are visible.
4
To set manual matches as landmarks:
a)
Click a clearly defined spot on the master image (the spot boundary
should become magenta).
b)
Now select the spot on the Pick gel image which corresponds to the
master image spot, so that this spot becomes magenta.
c)
Click the Add Match button in the manual corrections area. A vector
line should appear showing that the spots have been matched and the
landmark has been set. When next spot to landmark is selected the
newly manually matched spot will have a purple outline.
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5
Set approximately 10 landmarks on the images, spread evenly across the
images.
As the images are slightly different it will be necessary to scroll the two
images separately. This can be done by clicking the Properties icon and
selecting the Image View tab. Deselect the Link image views when
scrolling option.
6
Match the Pick gel by clicking the Match icon.
7
Select the Match Unmatched and Landmarked and uncheck the Optimize
Matching Using Warping option in the dialog which appears, click Match.
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15.4 Matching to analytical gels
8
When matching has been completed, it is recommended to look through
and check the matching result. If needed use the Add/Break functions and
redo the matching. The vector lines which appear will be very long due to
the difference in size of the analytical and preparative gel. These vector
lines can be hidden by clicking the properties icon and selecting the Image
View tab. Deselect the Match vectors in Match Table View option.
It is also possible to select the Link Image views when scrolling again, that
was unchecked during Landmarking.
In addition, Auto-center selected spot can be selected for later use in for
example 15.4.4.
9
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It is only necessary to determine whether the matches between the master
image and the preparative image are accurate. The only matches that need
to be confirmed are those that match to proteins assigned with a pick
status. Therefore, instead of confirming matches in the match mode, they
should be confirmed in the protein mode.
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15.4.4
Assign and inspect pick spots
1
Select the Protein mode and Display All Views by clicking the P button and
the Display All Views icon in the tool bar.
2
Open the properties dialog, Protein Table tab and select the Protein of
Interest in the Protein Table Filter area.
Click OK.
In the protein table, only proteins assigned as protein of interest will be
listed.
3
In the Image View, select the Master gel in the right image (from the drop
down menu in the image header) and the Pick.gel in the left image.
4
Select the master gel by clicking on it's header with a left mouse click (the
header will become magenta). Then click on the header of the pick gel with
a right mouse click to select this gel as secondary selected gel (the header
will become green). When a spot is now selected from the protein table, that
spot will become selected in the two images and it will also be displayed in
the 3D view (if the spot is present in both images).
5
Start at the top of the table and click the first protein in the list.
Check if this spot is matched to the pick gel, and if so, whether it is matched
correctly or not.
a) If this the match is correct, select the Pick check box in the data view
control panel. Then check the next spot in the list.
b) If the match is incorrect, switch to the Match Mode and correct the match.
Then move back to the Protein Mode, set the match as Pick and then check
the next spot in the list.
c) If the spot is not present on the preparative gel and hence cannot be
picked (this protein will not have a Pick status), move to check the next
protein in the list.
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6
Whilst reviewing the match accuracy of the preparative gel, the pick
locations should be simultaneously verified.
Spots assigned with a Pick status have a yellow transparent cylinder and a
yellow circle denoting the picking location in the 3D and image views,
respectively. The picking location of all spots to be picked is reviewed to
assess whether the pick location requires editing (see Section 6.5.2).
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322
To edit a pick location:
a)
Zoom in on the selected spot in the image view.
b)
Select Edit:Edit Pick Locations.
c)
Place the hand shaped cursor that appears in the image view over the
centre of the pick location (when in edit position, the hand will change
to a pointing shape), click and drag the pick circle to the new location.
d)
Select Edit:Edit Pick Locations to exit this mode.
To return pick locations to their original position select Edit:Restore
Default Pick Locations.
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15.4.5
Exporting the Pick List
Now that the post-stained gel has been matched and the pick proteins have
been reviewed, the Pick list has to be exported for use on the appropriate Ettan
picking system.
1
Select File:Export Pick List to open the Export Pick List dialog.
2
Select pick list and pick spot map to export then click OK.
Note: In this tutorial, unmatched spots should not be set as Pick (see 15.4.4,
step 5c). If unmatched spots have been set to Pick anyway, a warning
dialog will appear. Either click Cancel and match the unmatched
spots or continue the export. Unmatched spots are not included in
the export list.
3
Select where to save the pick list, type in an appropriate file name for the
pick list and select format.
The pick list can be saved as either a text file or an XML file by appending
the appropriate file extension to the file name (.txt or .xml). The text file is
used for Ettan Spot Picker and Ettan Spot Handling Work station.
4
Click Save to finish.
The actual post-stained gel is then taken to the appropriate picking system. The
pick list contains the relevant information to instruct the picker to excise the
spots. For more details refer to the relevant spot picker User Manual.
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16 Tutorial IV - Automated identification of differentially
expressed proteins
16.1
Objective
This tutorial describes how to automatically find statistically significant proteins
that are differentially expressed between control and treated groups of
bacterial cultures using DeCyder 2D Differential Analysis Software. The
methodology of this tutorial can be applied to any two groups of protein
mixtures with either replicate gels or replicate biological samples. The tutorial
outlines the different stages in experimental design, sample organization,
protein detection and quantitation in a gel, gel matching, statistical analysis and
generation of a pick list.
16.2
Overview
The following list describes the different stages involved in identifying all the
proteins that are differentially expressed in a given system, and so be worthy of
further investigation.
The stages are:
1
An experimental design is devised that will generate statistically significant
results; a design which minimizes or eliminates in-gel and gel-to-gel system
variations.
2
Eight sample lysates that form the basis of the experiment are prepared.
This consists of four lysates derived from four bacterial cultures treated
with benzoic acid and four control flasks that have not been treated. Since
the gel to gel variation in this system is low, gel replicates are not necessary
if biological replicates are available.
3
Aliquots from each sample are taken and pooled to prepare a standard
sample. All three sample types (standard, control and treated) are labelled
with an appropriate CyDye DIGE Fluor minimal dye (Cy2, Cy3 or Cy5 dye) as
described in the table overleaf.
4
The samples are applied to the four replicate Ettan DIGE system gels, and
the gels are then run. A preparative gel for picking is also run. Three images
are produced from each gel, by scanning at appropriate wavelengths for
the three fluors. The Typhoon Variable Mode 9000 series Imager or the
ETTAN DIGE Imager are recommended for scanning.
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16.2 Overview
5
Optionally, protein spot filtering can be performed to remove dust particles
from the gels. Analyses can be performed without protein spot filtering, and
this step is in that case omitted.
Protein spot filtering includes the following two steps:
a) Two representative images from a single gel are initially processed using
DeCyder 2D Differential Analysis Software DIA (Differential In-gel Analysis)
module. The DIA module performs spot detection and generates spot
information for all images simultaneously (in this case standard and control
images).
b) The data is examined to identify appropriate values for a number of
variables that are set in the Exclude filter (or artifact filter). These values are
automatically applied later in the experiment to remove all non-protein
spots from all the gel images.
6
326
The Batch Processor is run to perform a fully automated comparative
analysis of the control and treated samples. This analysis involves the
following steps:
•
Running a series of five sequential DIA analyses (for each of the four
gels and the pick gel).
•
Performing spot detection and calculation of the spot properties.
•
(Optional) Removing non-protein spots using the previously determined
exclusion filter parameters.
•
Normalization of the protein spots using the in-gel linked internal
standard.
•
Loading of the derived spot maps and associated data into the BVA
module.
•
Inter-gel matching of spot maps.
•
T-test analysis of proteins in control and treated samples.
•
Generation of a pick list for Ettan Spot Picker.
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16.3
Experimental design
In order to generate statistically valid data, a minimum of 2 replicates must be
used. These can be either biological replicates from each group or, if no
biological replicates are available then replicate gels must be employed. For
biologically relevant answers to a hypothesis, it is strongly advised that
biological replicates of each group are used. The greater the number of
biological replicates, the more biological variation is taken into consideration
and therefore, the more biologically relevant the results are to the system being
investigated. For the purpose of this tutorial, we describe the analysis of four
replicate gels loaded with bacterial lysates as indicated in the following table.
Each gel contains a standard sample to allow normalization of all control and
treated samples. The standard sample is obtained by mixing aliquots of control
and treated lysates, pooled in equal concentration.
Gel Number
Cy2
Cy3
Cy5
Gel 1
Gel 2
Gel 3
Gel 4
Gel 5 (Pick)
Standard
Standard
Standard
Standard
Control 1
Treated 2
Control 3
Treated 4
Treated 1
Control 2
Treated 3
Control 4
Fluorescently
post-stain
Control+Treated
The data analysis is performed in two parts, using the different DeCyder 2D
Software modules:
•
(Optional) Determining the protein spot filtering parameters.
•
Fully automated multi-gel processing using the Batch Processor module.
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16.4 Protein spot filtering (optional)
16.4
Protein spot filtering (optional)
This step is optional. Analyses can be performed without filtering workspaces to
remove dust particles. Continue at Section 16.5 if performing only the fully
automatic procedure.
In order to identify and exclude artifacts from subsequent analyses, the DIA
module is initially run on a representative pair of images from the same gel. This
is performed using the DeCyder 2D Differential Analysis Software DIA
(Differential In-gel Analysis) module, which identifies protein spots and allows
subsequent filtering to remove dust particles and other gel artifacts. The user
can examine the results and specify a series of parameters that will
automatically filter out all spots that fail to meet the set parameters.
It is possible to omit filtering because a dust particle on a specific gel is very
unlikely to be in the same position in other gels and therefore will not be
matched and will not be in the final analysis. However, filtering does “clean up”
the gel and may make the matching algorithm faster with possible greater
accuracy.
To perform protein spot filtering, follow the instructions in Tutorial I,
Sections13.4.1 -13.4.2, but select Gel 01 Cy2 Standard and Gel 01 Cy3 Control
in project Tutorial IV.
16.4.1
Gel artifact removal
It is not necessary in this tutorial to remove gel artifacts with the Exclude Filter
in the DIA module, as further analysis will be performed in the BVA module.
If the exclude filter is run anyway, information on this can be found in 13.4.4.
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16.5
Processing multiple images
Detection and subsequent matching of protein spots on the images derived
from the four gels must now be performed. Rather than analyzing a large
number of individual gels using the DIA module, an automated module, the
Batch Processor, can be used. This automatically runs a series of sequential DIA
analyses with no need for user intervention and can be set up to then
automatically match the individual spot maps, perform statistical analysis and
then generate a pick list in the BVA module.
16.5.1
Setting up the DIA Batch list
The first step is to create a DIA batch list by defining which gel images are to be
processed.
1
In the DeCyder 2D Main window, click the Batch Processor icon to open the
Batch Processor window.
2
In the Batch Processor window, right-click in the grey area and select Add
DIA item to open the Image Item selection dialog.
3
Locate the project entitled Tutorial IV in the left panel and select by clicking.
4
Click the folder GEL to display all gel images within the Tutorial IV project.
5
In the center panel, select all gel images, click Add all.
6
In the Gel properties area, enter the value 2500 for the estimated number
of spots. Ensure that the Include in BVA batch list check box is selected.
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7
The Spot exclusion filter area should be left empty as no filtering is
necessary.
8
Click OK to finish the DIA batch list settings.
16.5.2
Setting up the BVA batch list
To generate statistical data for the proteins that are expressed differentially
between the control and treated groups, it is necessary to identify the three
different spot map groups (control, treated and standard) within the software,
and to assign every image to one of the three groups.
The group names have to be created so that the appropriate images can be
assigned to them. The standard spot maps will be automatically assigned to the
standard group if the text Standard or Std is in the image name.
1
The BVA item settings dialog automatically appears after the previous
step.
2
Create the groups Control and Treated:
Click the Add button in the Experimental group area of the dialog.
Type the name Control and click OK
Click the Add button again, then type the name Treated and click OK.
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3
Once the two experimental groups have been created the spot maps can
be assigned to their respective group:
Select the Unassigned folder, then drag and drop the images from the
center panel to the correct experimental group in the left panel.
Note: It is possible to select more than one image at the time by using the
Ctrl or Shift key.
4
Click the gel in the center panel to enable the function boxes. Make sure the
function for all images is Analysis, A.
5
The last spot map is the pick gel. This gel does not belong to an
experimental group and is therefore left Unassigned.
Make sure the function for the pick gel is Pick, P, and deselect the Analysis
check box, A.
6
Check the Setup protein statistics and filter check box to include protein
statistics calculations and protein filtering in the BVA analysis.
7
Click OK/next to save and continue to next dialog.
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16.5 Processing multiple images
16.5.3
Protein statistics
Immediately after setting the spot map attributes the Protein Statistics dialog
box opens automatically. The level of statistical significance in protein
differences between the two experimental groups is calculated using the T-test.
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1
Select Independent tests.
2
In the box entitled Population 1, select Control, and select Treated in the
box entitled Population 2.
3
Check the check boxes for Average Ratio, Student’s T-test and the Apply
false discovery rate (FDR) correction.
4
Click OK/next.
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16.5.4
Protein Filter
Immediately after setting the protein statistics parameters the Protein Filter
dialog box automatically opens. The Protein Filter assigns a Protein of Interest
and/or Pick status (dependent on which Filter Action check box is selected) to
protein spots based on user defined criteria such as Student's T-test value,
average ratio or volume.
1
Select the Assign proteins of interest and Assign pick status in list check
boxes in the Filter action section of the dialog box.
For the purpose of this tutorial, set the parameters to those identical to the
figure below. Click OK.
The selections are: Select to assign protein of interest and pick status to
spots that are present (matched) in >=9 spot maps (75% of the 12 spot
maps in this experiment) with a T-test score less than 0.001 and with an
average ratio greater than or equal to 2 or less than or equal to -2.
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16.5 Processing multiple images
16.5.5
Saving the Batch workspace
1
Select File:Save batch to open the Save Workspace dialog.
2
Click Create new project... to create a project (see also 2.2) where the
workspace can be saved. Finish by clicking OK.
3
In the Save Workspace dialog, select the new project into which the batch
workspace should be saved.
4
Enter a Batch name (i.e. Tutorial IV Batch)
and a BVA workspace name (i.e. Tutorial IV BVA) in the fields.
5
Browse for a location where to save the pick list, and enter a Picklist file
name. Ensure that the extension is .tif or .xml.
6
Click Save to finish.
The Batch Processor workspace is now completed and ready to be run
Note: All items in the Batch Processor workspace can be edited by double
clicking the relevant cell in the DIA and BVA batch list. Protein statistics
and Protein filter settings can be edited by selecting Edit:Protein
statistics or Edit:Protein filter settings.
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16.5.6
Running the batch processor
The batch processing can be started when the Batch and BVA workspaces have
been saved. The batch state should be Workspace saved in a green box.
1
Select Process:Run batch.
2
A dialog opens up and prompt the user to make sure that no DIA or BVA
workspaces are open. If any other workspaces are open, close them and
click OK to start the batch run.
Note: The Master will be set automatically by the BVA module. The Master
can also be set when assigning the spot map attributes by selecting
the Master check box for the desired master spot map.
3
During the run:
•
The status for the batch is displayed in the box at the bottom of the
Batch Processor window. See Table 8-1 for detailed information.
•
The status for each item is displayed in the status column in the batch
list. See Table 8-1 for detailed information.
It is always possible to double-click in the status column to display the
Status dialog for the specific item.
Note: Only items with state Pending will be processed.
When processing is finished, the batch status displayed is Workspace saved in
a green box.
Upon completion of the batch run the DIA, BVA and pick list files are
automatically generated in the requested folders. The preparative gel and pick
list can then be taken to the appropriate Ettan spot picking instrument for
automated spot excision.
It is recommended that the BVA workspace is reviewed prior to generating the
pick list. A Batch Processor workflow that assigns proteins of interest only is
therefore recommended.
This involves following the above tutorial but only selecting the Protein of
Interest check box (without selecting Assign pick status) in the Protein Filter
dialog box.
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16.5 Processing multiple images
In this way the subsequent BVA workspace will have protein differences
highlighted as proteins of interest, which can be manually reviewed then
confirmed, before assigning the proteins of interest with a Pick status (see
Chapter 15 Tutorial III - Processing the Preparative Gel and Generating a Pick
List).
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Appendices
Scope of Appendices
The appendices cover various information not included in the chapters of the
User Manual nor in the tutorials.
Appendix A
Understanding the digital images
Appendix B
Spot processing algorithms
Appendix C
Experimental examples
Appendix D
Importing protein data
Appendix E
DeCyder 2D Software software keyboard short-cuts
Appendix F
Glossary
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Understanding the digital image Appendix A
Appendix A
Understanding the digital image
This Appendix deals with the acquisition and definition of the digital image. It
explains some of the more common terms used in association with digital
images and gives a brief insight into the GEL file format, which is the Typhoon
imaging systems default format.
A.1
Image acquisition
In order for any digital computer processing to be carried out on an image, it
must first be acquired and stored within the computer in a suitable form. Image
acquisition is a critical step and all primary data should ideally be stored exactly
as it is recorded by the imaging device without significant data compression, as
this may affect the accuracy of the recording. It should also be of the highest
quality required for image resolution in the particular application. Acquisition
can be achieved using a variety of scanners or digital imagers. These are usually
based on PhotoMultipier Tubes (PMTs) or Charge-Coupled Devices (CCD).
A PMT is an electro-optic device that converts light energy into electrical current
and amplifies the current, whilst a CCD is a silicon-based integrated circuit
consisting of a dense matrix of photodiodes that operate by converting light
energy in the form of photons into an electric charge.
A.2
The digital image
The most practical way of storing an image as digital data is to divide the image
into a grid of very small regions called “picture elements,” or “pixels”. In the
computer this digital grid or “bitmap” represents the image. Each pixel is
identified by its position in the grid, as referenced by its row (x) and column (y)
number. Each pixel has a different color or gray scale value and together they
form a representation of the image.
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Appendix A Understanding the digital image
A.3 Formatting graphic files
The images below show the digitalization procedure:
Original image.
A.3
Image is
partitioned into a
two-dimensional
array of square
sections. Each
division will be
used to form a
pixel.
The scanning
device then
collects a single
value to represent
the entire square,
which it then
transmits to the
computer.
After the computer
has received
values for each
section, the entire
image can be
reconstructed
using the pixel (x,y)
values.
Formatting graphic files
Once an image has been acquired, it is converted to a particular file format for
storage. There are a number of widely used image formats, such as TIFF, GIF etc.
and knowing which format to use is a key issue. Some formats are proprietary
and only useful in the program used to modify or acquire the image. Others are
useful for exchange between programs and computer platforms or for
presentation in web pages.
Image files include a certain amount of technical information, which is stored in
an area called the image header or tag. The image header may be of use in
displaying the image (e.g. length and width in pixels), identifying the image (e.g.
name or source), or identifying the owner.
Scientific images should be acquired and archived using an information
preserving or non-destructive format. The compression algorithms associated
with some file formats actually distort some of the pixel information when the
file is saved, while others do not. Loss of data may be unacceptable for use in
quantitative analysis and as a consequence, DeCyder 2D Differential Analysis
Software only supports specific compression formats.
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A.3.1
Tagged Image File Format (TIFF) images
TIFF, an extremely complex and flexible image format is used to exchange files
between platforms and software applications. The TIFF file consists of a number
of labels (tags) that describe certain properties of the file (such as gray levels,
color table, byte format, compression size etc.). After the initial tags, comes the
data, which may be interrupted by more descriptive tags.
Although the TIFF file is an industry standard it has many variants. DeCyder 2D
Differential Analysis Software has been validated for file formats generated by
GE Healthcare imaging devices recommended for Ettan DIGE system
applications. The 16-bit TIFF format has 216 = 65536 levels of signal resolution
and is the most commonly used file format for images. The image below shows
a schematic diagram of a TIFF file:
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A.4 Elements of the digital image
A.3.2
Gel Image File Format
Images scanned on the Typhoon are stored as GEL files, which are a variation of
the 16 bit TIFF and are purely gray scale files with one bit for each pixel. These
images have very wide dynamic ranges; typically images with 105 or 1-100,000
levels of signal resolution are possible. The GEL format uses a square root
algorithm to compress the possible 100,000 levels of an image into the 65536
levels available; this is located in the TIFF private tag domain. The square root
compression also provides higher signal resolution at the low end where small
changes in signal are more critical. This is important when discriminating
between two small values. The TIFF file will assign the same number to two
values that differ by a small amount, whereas the GEL file accurately represents
the data. The recommended instrument for scanning Ettan DIGE system gels for
DeCyder 2D Differential Analysis Software analysis is the Typhoon Variable
Mode Imager.
A.4
Elements of the digital image
A.4.1
Pixel intensity value
Each of the pixels that represent a stored image has a pixel intensity value,
which describes how bright that pixel is. This intensity value represents a
measured physical property i.e. emitted light. This value is the average for the
whole area covered by the pixel. A pixel's address is denoted by its row and
column co-ordinates in the two dimensional image.
A.4.2
Pixel or bit depth
Pixel or bit depth (referred to as bit depth from now on) refers to the amount of
information allocated to each pixel in a graphic image. Pixels have different bit
depths, which determine how many grayscales are available to the image. Most
image file formats store grayscale information in the header section of the file.
An 8-bit image, 28 or 256 grayscale values, a 16-bit image 216 or 65 536
grayscale values.
Due to variations in how graphics programs and conversion algorithms work, it
is best to start off with as much bit depth as possible. Large bit depth also
increases the dynamic range available when collecting images of, for example,
gels with protein or nucleic acid bands that are going to be quantified by pixel
intensity of the bands. When working with images that have high bit depths,
remember that to actually see this information on the computer, the display
must also be set to a high bit depth (thousands or millions of colors).
For black and white or binary images, pixels need only two bits of information
(black or white), and hence the bit depth is 4.
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Another type of image is the grayscale image, which is one in which the only
colors are shades of gray. The number of grayscales used can vary but 16-bit
systems have 65, 536 shades of gray, 0 being black and,
65 535 being white.
Table A-2. Bit depth and their corresponding number of different combinations
available.
Depth
1 bit
2 bit
4 bit
8 bit
12 bit
16 bit
A.4.3
Example
0
10
0110
01010011
011010011101
1011000100101101
Combinations
2
4
6
256
4096
65536
Pixel frequency histogram
The frequency histogram refers to the frequency representation of different
shades of gray or color in the image. A frequency histogram displays the
number of pixels representing each grayscale or color value. Frequency
histograms most commonly represent grayscale images and have many uses:
the histogram may reveal an under-or overexposed image (too many pixels with
values close to 0, or too many with values close to 255 respectively), and the
histogram can be manipulated to change the image: frequency values can be
deleted, and upper and lower thresholds can be set.
A.5
Image dimensions
A.5.1
Resolution
Image resolution is often confused with pixel dimension and refers to the
number of pixels displayed per unit length of an image. Pixel resolution is the
fineness of the divisions into which the scanner partitions the image. When the
divisions are extremely small, the scanner is said to have high resolution; when
the divisions are course, the scanner has low resolution. DeCyder 2D Differential
Analysis Software requires images which have a pixel resolution of 100 µm.
Anything less does not contain the required amount of information, whilst
anything more adds no further advantage to the image analysis procedure.
Resolution determines the area occupied by the images in conjunction with the
pixel dimension and is a measurement of clarity, or detail. It can also refer either
to an image file or the device, such as a monitor, used to display it. The
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Appendix A Understanding the digital image
A.5 Image dimensions
relationship between number of pixels and area is commonly expressed by
number of pixels per inch (ppi) the more pixels per inch the better the resolution.
Output (print or display) resolution is more commonly expressed in terms of dots
per inch (dpi). The resolution of a scanned image is also expressed in dpi where
the number of dpi is equal to the number of ppi i.e. an image scanned in at 300
dpi will give you an image resolution of 300 ppi. Image-file resolution and output
resolution combine to influence the apparent clarity of a digital image when it is
viewed. The display monitor used will also influence apparent image quality.
The amount of resolution is often ruled by practical considerations e.g. the
higher the dpi number the more information in the file, and the greater the
ability to enlarge a detail from that image. If the principal life of an image is on
screen e.g. an image for a web page, as opposed to being printed out, and if
details for enlargement are not required from it, a resolution of 100 dpi will be
sufficient. So, just as with image type, resolution needs to be matched to the
purpose of the scan. The Table below shows the size of an uncompressed 1" x
1" image in different types and resolutions:
Resolution (dpi)
400x400
300x300
200x200
100x100
A.5.2
2-bit Black and
white (Kb)
20
11
5
1
8-bit grayscale
(Kb)
158
89
39
9
24-bit color (Kb)
475
256
118
29
Dynamic range
Dynamic range is the ability of an imaging system to quantitatively detect very
dim and very bright features within a single image and is related to bit-depth. It
is a measurement of the number of bits used to represent each pixel in an image
and hence determines the number of colors or shades of gray (grayscale) that
can be represented in a digital image.
The dynamic range of a system is a function of the analogue-to-digital
converter, the purity of the illuminating light, colored filters, and any system
noise. It is measured on scale from 0.0 (perfect white) to 4.0 (perfect black), and
the single number given for a particular imaging system tells how much of that
range the unit can distinguish.
Variations in dynamic range of a system impact the quality of the digitized
image more than simple resolution does. High-end imaging systems are more
sensitive to the range of colors in the spectrum and can record minor
differences between two almost identical colors.
Several variables determine a system’s dynamic range: pixel depth (number of
bits per pixel per color), sensitivity of the image capture device i.e. CCD / PMT,
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accuracy of the focusing optics, and precision of the measurement of the black
and white points.
A.6
Image quality
Visual image quality is the cumulative result of the scanning resolution, the
dynamic range of the scanned image and the scanning device or technique
used. Image quality is often expressed in terms of resolution, but other factors
also affect the quality of an image file. Images are often stored at much higher
quality than they are displayed on a monitor because most printing devices are
capable of a much higher resolution than screen displays.
A key trade-off in defining an appropriate level of image quality is the balancing
of file size and resulting storage requirements with quality needs. Since pixel
dimensions and color depth of a graphic image are directly proportional to the
file size of the image, the higher the quality of an image, the more storage space
it will occupy. High quality images also require more system resources e.g.
higher bandwidth, networks, increased memory requirements and increased
time and cost of the scanning process. Effective image compression provides a
key to maintaining quality while using less storage space and system resources.
However, it is highly recommended that images be archived onto CD-ROM to
preserve storage space, particularly if using a network file server.
A.6.1
Background & noise
Background is defined as undesired signal often resulting from
autofluorescence or light scatter from a matrix or sample support. It can be
minimized by the selection of appropriate matrix or sample support e.g. low
fluorescence glass. Noise is defined as the statistical uncertainty inherent in a
measurement, such as the standard deviation associated with measured
background counts, e.g. with 2–D gels noise can be attributed to contaminants
with fluorescent properties similar to the specific fluor being used. By ensuring
that only high quality reagents are used and recommended procedures are
followed noise can be minimized. The sensitivity of the imaging system can be
adjusted by changing exposure times for CCD based systems or voltage settings
for PMT based systems. The specific signal can be optimized to give the highest
signal-to-noise ratio thus ensuring the maximum amount of information is
obtained from the image.
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A.6 Image quality
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Appendix B
B.1
Spot processing algorithms
Summary of spot normalization procedure
The normalization in DeCyder 2D Software Software version 7.0 is based on the
assumption that a majority of all proteins in a gel keep their expression level
between the different samples in an experiment. For this reason it is important
to detect a high number of protein spots in the gels. A minimum of 100 detected
spots is recommended, but it is preferable to include as many valid spots as
possible, e.g. 2000-3000 spots.
The spot background intensity levels are calculated in DeCyder 2D DIA based on
the 10th percentile pixel value on the spot border. The background
compensated spot volumes are then used to calculate the log volume ratio for
each spot in the gel. The volume ratio of a protein spot in DeCyder 2D DIA is
calculated by dividing the volume in the secondary gel view by the volume in the
primary gel view.
A histogram over all log volume ratios in a gel is created for each combination
of primary and secondary gel image. Since a majority of the protein spots
should have unchanged expression between the images, the main peak of the
log volume ratio histogram should be centered on the value zero. Usually the
main peak of the histogram is however not centered on zero when calculating
the raw log volume ratios, so in order to get the desired log volume ratio
histogram, the volume values in the primary spot map are multiplied by a
normalization factor, giving the normalized volume values. The normalization
factor is found by fitting a Gaussian curve to the main peak of the raw log
volume ratio histogram. The normalized volume ratios are given by dividing the
raw volume value for a spot in the secondary gel view by the normalized volume
value for the spot in the primary gel view.
Neither the normalized volume values nor the normalization factor can be
viewed in the DeCyder 2D DIA or DeCyder 2D BVA applications, only the raw
volume values and normalized volume ratios are visible. The normalization
factor can however be found in XML files exported from DeCyder 2D DIA.
The volume ratios in the DeCyder 2D DIA table view are values based on the
normalized log volume ratios that describe the expression level of proteins in
terms of fold increase or fold decrease. A volume ratio value of 1.5 would
indicate a 1.5 fold increase in protein expression, while a value of -1.5 would
indicate a 1.5 fold decrease in protein expression between samples. The Xvalues in the DeCyder 2D DIA histogram view are however the normalized log
volume ratios. When the standard image is viewed in the primary gel view in
DeCyder 2D DIA, the histogram X-values are the same as the standardized log
abundance values in the DeCyder 2D BVA graph view.
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Appendix B Spot processing algorithms
B.2 Detailed description of spot normalization procedure
B.2
Detailed description of spot normalization procedure
In DeCyder 2D DIA version 7.0 it is possible to change which gel images that are
displayed in the primary and secondary gel views. The histograms for all
possible combinations of gel images as primary and secondary image are
calculated during the spot detection procedure. When changing images the
histogram and table views are thus updated as well to reflect the current
combination of primary and secondary images.
For the DeCyder 2D DIA description below to conform to the standardized
experimental design used in DeCyder 2D BVA, the standard image in the gel
would be viewed in the primary gel view in DeCyder 2D DIA. The volumes V1i
below would thus be the volumes in the standard image. To switch between the
different sample images in the secondary gel view of DeCyder 2D DIA thus gives
the same corresponding expression ratios for the spots as when viewed in
DeCyder 2D BVA.
B.2.1
DIA spot normalization procedure
To clarify some of the procedures involved in the normalization of the ratio
histogram displayed in DeCyder 2D-DIA, this step by step algorithm description
is provided.
1
Calculate spot volumes
Based on the result of the spot detection algorithm, the volumes of the
detected spots are calculated and compensated for the appropriate
background level, based on the 10:th percentile pixel value on the border.
2
Calculate spot ratios
All the spot volume pairs are combined to create the set of ratio values for
the entire spot map. The ratios are calculated according to
Ri = log10(V2i/V1i), (i)
Where V1i is the volume of spot i in the left, or primary, gel image and V2i is
the volume of spot i in the right, or secondary gel image. The index i runs
over all spots that are included in the analysis. Spots that have status
excluded are thus not part of the normalization. The ratio Ri is also limited
to the range [-6, 6] to avoid infinite ratios for zero volumes.
3
Calculate data histogram
The ratio values Ri are then combined to a histogram. The current
resolution of the ratio histogram is 0.02.
4
Optimize a model histogram curve
A normal distribution is fitted to the main peak of the ratio histogram. The
tallest peak of the histogram C is used as the starting center position of the
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model curve, and all histogram data extending from the tallest peak in both
directions are included until the histogram value reaches below 10% of the
main peak height. At this point any data outside is excluded from the model
curve fitting procedure. This procedure is illustrated in the figure below. The
model curve parameters are then optimized to the selected histogram data
using a standard LMS gradient descent algorithm. When the optimization is
terminated, the center of the model curve is denoted C', also called
normalization factor.
5
Normalize the primary spot map
The spots volumes in the primary spot map are normalized using the
normalization factor C' in following way
V1i' = V1i * 10 C' (ii)
Where V1i' is the resulting normalized volume of spot i in the left, or primary,
gel image. The modified spot ratios are also calculated in this step
according to
Ri' = log10(V2i/V1i'), (iii)
This parameter Ri' is termed standardized log abundance in the DeCyder
2D-BVA module and is used for the statistical analysis.
6
Re-calculate data histogram
The modified ratio values Ri' are then combined to a new histogram of the
same resolution as the previous one described in 3 and 4 above.
7
Calculate data standard deviation
The standard deviation of all the spot volume ratios according to (iii) is
calculated. This gives a rough indication of the spread of the data set.
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Appendix B Spot processing algorithms
B.2 Detailed description of spot normalization procedure
B.2.2
Additional comments on spot normalization in DIA
The volume values displayed in the DeCyder 2D-DIA table view and 3D view are
always the raw background subtracted values, so the normalized volume
values can currently not be viewed in DeCyder 2D-DIA.
For the normalization procedure to be accurate, the number of spots needs to
be quite large. It is recommended that more than 100 spots are present in the
normalization. However, the more valid spots that are present the better. If the
number of spots to normalize is lower than 50, a model curve is not calculated.
In that case the position of the tallest histogram peak is used as C' for the
normalization calculations described in (ii).
When spots in the workspace have been manually excluded, the workspace
should be re-normalized based on the new set of included spots. This becomes
exceedingly important if a large number of spots have been manually excluded.
In the DeCyder 2D-DIA environment, the expression ratios are displayed
differently in the table than in the histogram. The histogram ratio representation
Ri' is illustrated above, while the expression ratio, E, presented in the table is
calculated according to
E = 10 Ri' for (Ri' >= 0) (iv)
E = -1/10 Ri' for (Ri' < 0)
This results in a value less than -1 for under expressed ratios and a value larger
than 1 for over expressed ratios. Equal ratios are expressed as a ratio of 1. The
values are used to be able to compare increased expression ratios and
decreased expression ratios in a similar way as fold changes. For example, a 1.5
fold increase in expression would represent a standardized log abundance
value of 0.1761, and a 1.5 fold decrease in expression would represent a
standardized log abundance value of -0.1761.
The normalization factor C' is not displayed anywhere in the DeCyder 2D-DIA
user interface, but it is available in exported XML files. The value can be found in
the <experimentalFeatures> section of the XML file in the dataAttr attribute of
<featureData> elements that have the name attribute set to NormalFactor. For
more information concerning this and other details in the XML format please
consult the XML format description.
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B.2.3
BVA spot normalization procedure
The standardized normalization procedure in DeCyder 2D BVA is based on the
concept of having one of the gel images in each gel as a standard reference
image. The sample that generates the standard reference image is a pool of all
samples in the experiment, and the same standard sample is used for all gels in
the experiment. It is generally recommended to use the Cy2 dye for the
standard image and to randomly assign the Cy3 and Cy5 dyes to the different
samples in the experiment. The standard image can then be used to normalize
the expression ratios between the different gels in an efficient way.
The standardized volume ratio for each standard image from the different gels
is set to the value 1.0. The expression ratio for each sample spot is then related
to its corresponding standard spot in the same gel, thus making it possible to
compare ratios between matched protein spots in the different gels.
The standardized results in DeCyder 2D BVA can be viewed in three different
ways:
1
In the Std. Abund column in the Appearance Mode view.
The value that is displayed in this column is the same value that is displayed
as the volume ratio in the DeCyder 2D DIA table view when the standard
image is visible in the primary gel view. It is thus the expression ratio termed
E using the notation in (iv) above. The value of each standard image is
always 1.0.
2
In the graph view as Standardized log abundance.
This is the preferred setting for the graph view that reflects the values that
are used for all statistics calculations. It thus displays the value Ri' using the
notation above.
3
In the graph view as Standardized abundance.
It is also possible to view the value of the plain normalized volume ratio
V2i/V1i' without applying the logarithm function in the graph view.
The volume values displayed in the appearance table of the DeCyder 2D BVA
application are the raw background subtracted volume values, just as they are
displayed in DeCyder 2D DIA.
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Appendix B Spot processing algorithms
B.2 Detailed description of spot normalization procedure
Calculation of Average Ratio in BVA
One way to measure difference in protein expression between two experimental
groups is to calculate the average ratio value for a protein. This value can be
found in the Av. Ratio column in the Protein Table of DeCyder 2D BVA. There are
two ways to calculate this value depending on the type of statistical analysis
that is selected for the experiment.
1
Unpaired statistics.
This is the default type of experimental design where the different samples
in the experimental groups are not considered to be related in any
particular way. In this case the average ratio, Rmean, for a protein is
calculated by taking the ratio of the means in standardized abundance
values for the protein spots in the corresponding groups, expressed in the
notation used in (iv) above.
R1 = mean standardized abundance for all matched sample spots in group
1.
R2 = mean standardized abundance for all matched sample spots in group
2.
2
Paired statistics.
This type of experimental design links samples together between the two
groups, indicating that the samples are related in some way. Usually the
link indicates that the two linked samples are taken from the same
biological individual, before and after a treatment for instance. In this case
the average ratio, Rmean, for a protein is calculated by taking the average
difference in standardized abundance between the linked sample pairs,
expressed in the notation used in (iv) above.
R1 = ratio between standardized abundance in group 2 and group 1 for
individual 1.
R2 = ratio between standardized abundance in group 2 and group 1 for
individual 2.
RN = ratio between standardized abundance in group 2 and group 1 for
individual N.
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Appendix C
Experimental examples
In this appendix there are examples of complex experimental designs and
statistical analyses. There are descriptions of both a paired testing and TwoWay ANOVA, which include details of the experimental design, setting up the
statistical analyses and interpretation of example proteins.
C.1
Example of a paired experiment
Experimental objective
An experiment is designed to investigate changes in protein expression caused
by a drug treatment after 24 hours in human subjects.
Experimental design
Blood samples are taken prior to drug treatment from five volunteers. Blood
protein was individually extracted and stored. The drug was then administered
to each individual, blood samples were taken 24 hours later and protein was
extracted. Ten aliquot samples derived from the five individuals were taken and
pooled to act as the internal standard. The pooled standard was labelled with
CyDye DIGE Fluor Cy2 minimal dye. Pre-treated and treated blood samples from
each volunteer were independently labelled with either CyDye DIGE Fluor Cy3 or
Cy5 minimal dye. Equivalent amounts of labelled standard, volunteer 1 pretreated, volunteer 1 post-treated were mixed and subjected to 2–D gel
electrophoresis. The samples from the other 4 volunteers were similarly
subjected to 2–D gel electrophoresis on separate gels (as described in the table
overleaf – standards on individual gel not shown).
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Appendix C Experimental examples
C.1 Example of a paired experiment
Assigning groups and individuals in DeCyder 2D Differential Analysis
Software
Spot data can be assigned into two experimental groups: pre- and post-drug
treatment represented by 1 and 2, respectively.
The same 5 volunteers are present in both groups, therefore the data can be
paired (i.e. assignment of individual volunteers within groups). The CyDye DIGE
Fluor Cy2 minimal dye labelled internal standard samples are omitted from the
table below.
Number
Gel
CyDye
Individual
Group
1
1
Cy3
1
1
2
1
Cy5
1
2
3
2
Cy3
2
2
4
2
Cy5
2
1
5
3
Cy3
3
1
6
3
Cy5
3
2
7
4
Cy3
4
2
8
4
Cy5
4
1
9
5
Cy3
5
1
10
5
Cy5
5
2
The above parameters can be designated either in the DeCyder 2D Software
Batch Processor or from DeCyder 2D Software BVA module. The resultant spot
map table is illustrated.
The experiment assesses the difference in protein expression between two
experimental groups of paired data, therefore a paired Student's
T-test is applicable.
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The Student's T-test and the Paired Tests check boxes must therefore be
selected in the statistics dialog box. The Average Ratio check box can also be
selected to calculate the magnitude of any protein expression change between
groups.
Example proteins
Fig C-1. Statistical data displayed for protein 580.
The independent T-test p value indicates that there is no significant difference
in the mean expression of protein 580 from pre- to post-drug treatment groups.
However, when data pairing is accounted for, the Paired T-test p value indicates
that there is a significant change.
Protein 580 expression levels appear to consistently increase regardless of the
initial protein abundance.
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Appendix C Experimental examples
C.1 Example of a paired experiment
Fig C-2. Statistical data displayed for protein 1142.
The independent and paired T-test p values indicate that there is no significant
difference in expression of protein 1142 from pre- to post-drug treatment
groups. The observed decrease in protein expression between experimental
groups is not due to a consistent abundance change of this protein between
individuals. The independent and paired T-test p values indicate that there is no
significant difference in expression of protein 1142 from pre- to post-drug
treatment groups (p=0.066 and p=0.160, respectively). The observed decrease in
protein expression between experimental groups is not due to a consistent
abundance change between individuals.
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C.2
Example of a two condition experiment
Experimental objective
An experiment is designed to investigate changes in protein expression in two
E.coli bacterial strains incubated at 37 oC over a 90 minute time period.
Experimental design
Bacterial cultures derived from each strain were incubated at 37 oC. Cell sample
aliquots were taken immediately prior to incubation then 30, 60 and 90 minutes
after commencing incubation.
The cell samples were lysed, labelled with CyDye DIGE Fluor minimal dye, then
equivalent amounts of protein were subjected to 2–D gel electrophoresis (as
described in the table on the following page – standards on individual gel not
shown). Triplicate gels were run and analyzed to account for experimental
variation. Each gel contained a CyDye DIGE Fluor Cy2 minimal dye labelled
internal pooled standard with CyDye DIGE Fluor Cy3 and Cy5 minimal dye
labelled test samples.
Assigning groups and conditions
There are two conditions present in this experiment:
•
Condition 1: two bacterial strains represented by 1 and 2.
•
Condition 2: Four sampling time points: Pre-incubation 30, 60, and 90
minutes incubations are represented by 1, 2, 3, and 4, respectively.
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Appendix C Experimental examples
C.2 Example of a two condition experiment
The table below shows the designation of groups, conditions and individuals for
the spot maps generated from the 12 experimental gels. The Cy2 dye labelled
internal standard samples are omitted from the table below.
Item
Gel
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
1
2
3
1
2
3
4
5
6
4
5
6
7
8
9
7
8
9
10
11
12
10
11
12
CyDye DIGE Fluor
minimal dye
Cy3
Cy3
Cy3
Cy5
Cy5
Cy5
Cy3
Cy3
Cy3
Cy5
Cy5
Cy5
Cy3
Cy3
Cy3
Cy5
Cy5
Cy5
Cy3
Cy3
Cy3
Cy5
Cy5
Cy5
Strain
(Condition 1)
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
Time
(Condition 2)
1
1
1
2
2
2
3
3
3
4
4
4
1
1
1
2
2
2
3
3
3
4
4
4
Group
1
1
1
2
2
2
3
3
3
4
4
4
5
5
5
6
6
6
7
7
7
8
8
8
The above parameters can be designated either in the DeCyder 2D Software
Batch Processor or from DeCyder 2D Software BVA. The resultant Spot Map
Table is illustrated below.
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The experiment assesses two conditions (strain and time), consequently a TwoWay ANOVA analysis is applicable.
The Two-Way ANOVA check box must therefore be selected in the Protein
Statistic dialog box to perform the statistical test.
Examples of the statistical outcome for selected spots are illustrated on the
following pages.
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Appendix C Experimental examples
C.2 Example of a two condition experiment
Example proteins
Fig C-3. Statistical outcome for protein 545.
The 2-ANOVA-Strain and 2-ANOVA-time values are not significant for protein
545. Therefore there is no strain-to-strain or duration of incubation effects on
the expression of this protein.
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Fig C-4. Statistical outcome for protein 162.
The 2-ANOVA-Strain and 2-ANOVA-time values are statistically significant for
protein 162. Therefore there are strain specific changes in expression of this
protein. (i.e. one strain consistently has higher expression of this protein).
Furthermore, the expression of this protein increases significantly in both strains
over time. However, there is no significant interaction between strain and time
of incubation, since the 2-ANOVA-interact is not significant.
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Appendix C Experimental examples
C.2 Example of a two condition experiment
Fig C-5. Statistical outcome for protein 721.
The 2-ANOVA-Strain and 2-ANOVA-time values are statistically significant for
protein 721. Therefore there are strain specific changes in expression of this
protein. The expression of this protein also changes significantly in at least one
timepoint. Moreover, there is a significant interaction (2-ANOVAInteraction<0.01) between strain and time, whereby there is a decrease in
expression of protein 721 over time that is specific to one strain.y
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Importing protein data Appendix D
Appendix D
Importing protein data
It is possible to import protein data such as protein ID:s and protein names from
an external source using the menu option File:Import Protein Data. The protein
data to import must be associated with picked spots in the currently active BVA
workspace. It is common for protein database identification search events to
return a number of protein candidates that are likely to be associated with the
corresponding picked protein spot and the Import Protein Data feature in BVA
can handle importing up to ten protein candidates for each protein spot. This
makes it possible to link to a suitable database according to section 5.13 for
each of the different protein candidates.
The XML format used for this type of import is called EttanProteinIdentification
and is described in detail in the documents EttanProteinIdentification.pdf and
EttanProteinIdentification.xsd, included in the "Documents\Formats" folder
where the DeCyder 2D program is installed, (Usually C:\Program Files\GE
Healthcare\DeCyder 2D\Documents\Formats\).
This way of importing protein data to BVA is suited for an environment where the
picking-identification process has been automated to some level. Picking and
protein identification can be performed in many different ways and this process
is currently out of scope for the DeCyder 2D software so it is up to the user of the
DeCyder 2D system to implement such an automated picking-identification
process suitable for the laboratory in question, using the provided XML format
to interface to the BVA application.
In such an automated process it is necessary to make sure the master spot
numbers exported in the pick list from BVA are conserved throughout the
process all the way to the corresponding EttanProteinIdentification file, (i.e.
spotId attributes in the SpotIdentification elements of the file). This is needed to
make sure that the identification results imported to the BVA workspace are
associated with the correct spot in the workspace.
A rudimentary validation of the information in the imported file is performed
during import to make sure that all the spots represented in the imported file
correspond to spots that have pick assignments. Any deviations are reported to
user before the import is completed.
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Appendix D Importing protein data
D.1 Import procedure in BVA
D.1
Import procedure in BVA
It is only possible to import protein data using the File:Import Protein Data
menu option if a valid pick list is defined in the current BVA workspace.
1
Select File:Import Protein Data.
2
Use the displayed Open File dialog to browse to the
EttanProteinIdentification XML file that contains the protein data to import
and select Open.
3
The imported data is validated.
If all the protein data for the spot in the imported file corresponds to picked
spots in the current BVA workspace, the protein data is added to the
corresponding spots and show up in the protein table in Protein mode.
If there are spots in the imported file that do not have pick assignments in
the current BVA workspace or if there are picked spots in the current BVA
workspace that are not represented in the imported file, a dialog is
displayed (see example below) informing the user that there are
differences. It is possible to continue with the import by pressing the “Yes”
button, but it is not recommended.
364
4
Sort the Protein table on Protein ID to get all the protein spots with imported
data at the top of the table.
5
Select a suitable row in the table corresponding to an identified protein and
click the row with the right mouse button.
6
Select the desired database to search from the displayed context menu. If
no database is defined, refer to section 5.13 for how to define database
links.
7
If multiple candidates are available for the current protein, the list of
candidates is displayed in a new sub-menu. Select the appropriate protein
candidate and the search result is displayed in a separate browser window.
DeCyder 2D 7.0 User manual 28-9414-47 Edition AA
Keyboard shortcuts Appendix E
Appendix E
E.1
Keyboard shortcuts
DIA module keyboard shortcuts
Spot Controls
Alt + C
Alt + P
Alt + N
Alt + I
Alt + X
Alt + K
Alt + D
Alt + M
Workspace
Alt + F
Alt + E
Alt + V
Alt + R
Alt + H
File menu
Ctrl + N
Ctrl + O
Ctrl + S
Ctrl + P
Edit menu
Ctrl + C
View menu
F7
F8
F9
Ctrl + B
Ctrl + A
Ctrl + R
Help menu
Shift + F1
DeCyder 2D 7.0 User manual 28-9414-47 Edition AA
Confirm spot
Previous
Next
Protein of Interest
Exclude
Pick
Protein ID
Comment
Open File menu
Open Edit menu
Open View menu
Open Process menu
Open Help menu
Create new workspace
Open workspace
Save workspace
Show print dialog
Copy
Zoom In
Zoom Out
Fit to window
Contrast - Brightness
View Area in 3D
Rotate 3D
Help What's This?
365
Appendix E Keyboard shortcuts
E.2 BVA module keyboard shortcuts
E.2
BVA module keyboard shortcuts
Workspace
Alt + F
Open File menu
Alt + E
Open Edit menu
Alt + V
Open View menu
Alt + R
Open Process menu
Alt + H
Open Help menu
File menu
Ctrl + N
Create new workspace
Ctrl + O
Open workspace
Ctrl + S
Save workspace
Ctrl + P
Print
View menu
F7
Zoom In
F8
Zoom Out
F9
Fit to window
Ctrl + B
Contrast - Brightness
Ctrl + A
View Area in 3D
Ctrl + R
Rotate 3D
Alt + Enter
Properties
Alt + B
Browse and sort Gel images
Modes
Alt + S
Spot Map Mode
Alt + M
Match Mode
Alt + P
Protein Mode
Alt + A
Appearance Mode
Help menu
Shift + F1
Help What's This?
Protein Related functions
Alt + ↓
Select next row in current table
Alt + ↑
Select previous row in current table
Alt + Page Down
Scroll down current table
Alt + Page Up
Scroll up current table
Alt + →
Select next Spot Map/protein in A mode
Alt + ←
Select previous Spot Map/ protein in A mode
Alt + C
Confirm/Unconfirm protein or match
Alt + D
Add/Break match
Alt + K
Pick protein
Alt + I
Protein of Interest
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Glossary Appendix F
Appendix F
Glossary
2SD value. 2 standard deviation of the spot ratio distribution, 95% of the spots
lie within this ratio for normally distributed data.
Abundance. The relative volume among all spots representing a particular
protein in a BVA data set. The weakest spot is taken as 1.00 and the others are
displayed relative to this.
ANOVA. ANalysis Of VAriance is a family of methods used to perform statistical
analysis on experimental results.
ANOVA 1 way. One-Way ANOVA test (assigns statistical significance to
differences in standardized protein abundance between experimental groups).
ANOVA 2 way. Two-Way ANOVA test, (assigns statistical significance to both
separate and mutual effects of two experimental conditions on standardized
protein abundance).
Appearance Mode. Region within a BVA workspace where users can track all
information on a particular spot from all the gel images.
Area of Interest. User defined region outside of which any detected protein
spots are excluded from analysis.
Artifact Rejection. Filtering out of non proteinaceous signal.
Auto Level. The stage in the algorithm in which spots are matched.
Bandwidth. The transmission capacity of a communications channel, usually
expressed in bits or bytes per second.
Batch Processor. DeCyder 2D Software module capable of performing fully
automated co-detection, quantification and matching of multiple spot maps.
BVA. Biological Variance Analysis module of DeCyder 2D Software.
BVA Batch list. The spreadsheet in the batch processor depicting which spot
maps are to be matched.
Centre of Volume (CoV). Central tendency of the spot volumes on a spot map.
Co-detection. Simultaneous detection of labelled protein spots from two in-gel
images.
Comment. User defined text string that can be linked to specific protein or spot
map.
Database Administration Tool. Tool for DeCyder 2D database management.
DeCyder 2D 7.0 User manual 28-9414-47 Edition AA
367
Appendix F Glossary
DIA. Differential In-gel Analysis module of DeCyder 2D Software capable of the
fully automated co-detection, quantification and matching of an in-gel image
pair.
DIA Batch List. Spreadsheet in the batch processor depicting which image pairs
are to be detected and quantified.
Exclude Filter. Filter used to remove non proteinaceous artifacts such as dust.
Exclude Area. User defined region inside of which any detected protein spots
are excluded from analysis.
Groups. The collection of spot maps relating to image pairs whose sample
images have undergone exactly the same experimental conditions (e.g. 3 spot
maps of samples from disease patients treated with drug A at a specific dose is
a single group).
Histogram Selections. User adjustable parameters that alter the appearance
of the histogram in the DIA module.
Image Loader. Image Loader module of DeCyder 2D Differential Analysis
Software securely imports gel images with important experimental information
into the DeCyder 2D database.
Item. A term used in the Batch module, denoting gels in the DIA batch list and
spot maps in the BVA batch list.
Independent Data. Data sets which have no effect on one another.
Label. Indicates the fluor used to label the protein.
Landmarking. Process of manually matching spots or sets of spots to the
master image before automated matching to aid the matching algorithm. The
manually matched spots are not affected by re-matching.
Linking. Linking via master or template is performed in EDA to identify spots that
are the same in different BVA workspaces. See DeCyder 2D EDA User Manual for
more information.
Master Image. Spot map to which all others are matched to.
Match confirmation. Manual verification by the user of matched spots. The
confirmed spots are not affected by re-matching.
Matching. Automatic linking of spots on selected images to the corresponding
spot on the master image.
Match Mode. Area within a BVA workspace where spot match data is displayed.
Maximum Peak height. Pixel value at the X,Y position of the spot. This is the
actual detected peak height value compensated for background level.
Maximum Volume. The highest volume from two co-detected spots.
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Glossary Appendix F
Normalization. Process that allows the direct comparison of spot data derived
from different gels.
Null hypothesis. Posits that there is no difference between variables being
tested. To reject it is to infer “statistical significance”.
Organizer. Tool for handling files in the DeCyder 2D database.
Paired Data. Data sets with an association between data point in every group.
Population. A group of spot maps on which to perform statistical analyses. One
or several experimental groups can be included in a population.
Primary image. Refers to the image with a blue title bar in the Image View of the
DIA module.
Protein filter. Function that enables the user to designate spots for picking
and/or spots as protein of interest based on user defined parametric values.
Protein ID. Unique protein identifier that can be used to search.
Protein Mode. Area within a BVA workspace where all the statistical data from
the analytical gels is located.
Protein Statistics. Function in the BVA module that applies statistical tools to
the protein data (providing that an experimental design with an internal
standard was used).
Repeated Measures. ANOVA Statistical ANOVA test applied to paired data.
Scatter Parameter. The spot data type used to display the spot ratios in the
histogram. This is shown in the right y-axis on the histogram and can be
maximum slope, volume, peak height or area.
Secondary image. Refers to the image with a green title bar in the Image View
of the DIA module.
Slope. Maximum gradient associated with the 3 dimensional attributes of a spot
map pair.
Spot Controls. Allows user to control, assign or input data to the workspace or
individual protein spots.
Spot Map. A gel image that have been processed in the DIA module.
Spot Map Mode. Area within a BVA workspace where all the spot map images
and their associated assignments are located.
Spot Number. Unique identifier for a spot in DIA or a spot set in BVA.
Standard Image. Image relating to the internal standard from each gel.
Standardization. Process of quantifying spot map data in relation to the
corresponding standard spot map data.
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369
Appendix F Glossary
Standardized Abundance. Abundance relative to the standard image displayed
as a ratio.
Student’s T-test. Statistical test that assesses differences between two
populations.
Template spot map. Spot map with user defined information, i.e. protein ID/AC,
name, pI, Mw; which can be imported into another workspace.
Threshold Mode. Value above or below which spots are classed as being
differentially expressed.
TIFF Image. Flexible image format used to exchange files between platforms
and software applications.
User Administration Tool. Tool for definition of users and their access rights.
Volume. Sum of the detected pixel values above background within a spot
boundary.
Volume Ratio. Refers to the ratio of the normalized volumes of a pair of spots
from a spot map pair. A value of 2.0 represents a two-fold increase while -2.0
represents a two-fold decrease, whilst a value of 1.00 represents an unchanged
spot.
Workspace. Environment where all the experimental data is stored.
XML (Extended Markup Language). Structured universal tagged language.
XML Toolbox. Shell, housing modules used for the extraction of data from
DeCyder 2D Software XML files in the form of tab separated text or web tables.
370
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Index
Symbols
.*.dmp format 232
Numerics
3D View
in BVA 86, 94
in DIA 50, 55
A
Access 30
Access to project 29
Add
DIA batch item 210
DIA workspace 189
DIA workspace to BVA batch list 210
gel files in Image Loader 39
gel files to import list in Image Loader 39
images to DIA batch list 199
protein databases 169
spot to master 135
Template/DIA workspace to BVA workspace 112
Add match 131
Add+Break Match 131
Administration of DeCyder database 223
Algorithms 347
Analysis spot map/image 113
Annotation 93
ANOVA 148
one-way 103, 142, 150
two-way 103, 142, 153
two-way, example 155, 357
Appearance 103
Appearance mode 88
Appearance Table
in BVA 106
Archive DeCyder database 236
Archived data
retrieve 239
Area of interest
in Batch Processor 202
in DIA module 73
remove 74
Artifact 75
Assign
function in Batch Processor 203
group in Batch Processor 203
group in BVA 142
picking references 187
picking references in DIA 176
spots for picking in BVA 191
spots for picking in DIA 180
Automatic detection of reference markers 176, 187
Automatic file grouping and naming 35
Average ratio 142, 145
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B
Background 345
Backup
restore data 226
Backup of DeCyder database 224
Batch processor 16, 197, 329
Batch status 216
Break Match
Add+Break Match 131
Break match 131
Brightness 306
BVA 16, 85
BVA Batch list 203
BVA Module 85
C
Calculate Mw and pI 165
Change access to projects 29
Change password
in Organizer 29
in User Administration Tool 221
Change Pick List 162, 192
Check settings at image import 46
Co-detection 69
Color overlay 117, 119, 127
check matching 119
Commenting matches 131
Conditions in BVA 156
Confirm match set 131
Confirm single match 131
Confirmation in BVA 168
Contour 93
Contrast 306
Contrast and Brightness 52
Copy to Master 135
Copy to Match 135
Create
BVA workspace 110
DIA workspace 66
new project 28, 37
user 218
Crop gel image 43
Customizing display colors 65, 108
CyDye DIGE Fluor minimal dyes 8
CyDye DIGE Fluor saturation dyes 8
D
Data View Control Panel 86
Database
DeCyder 16, 223
protein 169
Database Administration Tool 21, 223
Open 224
Database options 25
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DeCyder 2D 7
DeCyder database 16, 223
Deep Purple 303
Default user 217
Delete user 221
DIA 16, 49, 257
DIA Batch List 198
DIA Module 49
DIA Processes 49
Digital image 339
Display Mw and pI values 166
Dump files 232
Dust particles 75
Dye chemistry settings 46
Dyes 8
Dynamic range 344
E
Edit
gel information 31
match 128
options 129
pick locations in BVA 193
pick locations in DIA 181
picking references 188
project information 30
user account 220
workspace information 31
Edit in Batch Processor
DIA settings 206
Protein Filter settings 208
Protein statistics settings 207
Enter Mw and pI 164
Estimated Number of Spots 72
Ettan
DALT electrophoresis unit 72
DIGE system 8
Spot Handling Workstation 49
Spot Picker 49
Example
Experiments 353
Independent test 144
Paired analyses 353
Paired test 144
Two-Way ANOVA 155, 357
Exclude area
in Batch Processor 202
in DIA module 74
Exclude Filter 75, 308
Experimental design 10
Experimental Design View
in BVA 86, 97, 115
Experimental group 114, 142, 203
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373
Export
data from DeCyder database 229
data from DIA 84
from BVA 174
image 32
pick list in DIA 182, 274
pick list, in BVA 195
Extract data 244
F
False discovery rate (FDR) correction 142
Filter proteins 160
First login 218
Floating master 90, 91, 288
Frequency distribution 62
Function assignment
in Batch Processor 203
in BVA 113
G
Gel artifact removal 328
Gel ID 47
Gel image overlay 117
Gel overlay 117
Generate pick list
in BVA 195
Generate pick list in DIA 182, 274
Glossary 367
Graph View 86, 96
Graphical User Interface
BVA 86
DIA 50
Group
assignment 114
defining 142
in BVA 114
H
Help 8
Histogram 62
Histogram View 50, 62
I
Identify Protein of Interest
in BVA 184
in DIA 178
Image acquisition 339
Image Editor 42
as stand-alone module 45
crop 43
other functionalities 45
Image file names
recommended 35
Image Loader 16, 35
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Image View
in BVA 86, 89
in DIA 50, 51
Immobiline DryStrip 72
Import
data into DeCyder database 232
DIA workspaces into DeCyder database 232
gel files 37, 48
workspace, into DeCyder database 232
Import list in Image Loader
add gel files 39, 40
Independent analyses 143
Internal standard 10
Isoelectric point calculation 164
L
Landmarking
without prior matching 132
Licence agreement 25
License file 25
License options 25
Load XML files 244
M
Main 21
Main window of the software 25
Manual spot exclusion 78
Master
image 117
spot map 113
Match
check using color overlay 119
check using match vectors 121
confirmation 128
editing 128
editing options 129
Mode 88
quality score 139
Table 101
Matching 117
optimize using warping 118
theory 124
Max
peak height 58
volume 58
Merge spots 133
Model curve 62
Modes in BVA 88
Molecular weight calculation 164
Move selected 138
Multiple
Gel Views in BVA 89, 122, 132
image processing 329
pick lists 183, 190
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Multiple-selection of spots 131
N
New Spot 136
Noise 345
Non-protein spots 75
Normal workflow
Batch Processor 197
BVA 85
DIA 49
Image Loader 36
Spot picking in BVA 183
Spot picking in DIA 175
Null hypothesis
ANOVA 149
Student’s T-test 146
O
One-Way ANOVA 103, 142, 150
Online help 8
Open
Database Administration Tool 224
Open Workspace
BVA 111
DIA 67
Optimize matching
using warping 118
Organizer 21, 26
Overlay 117
P
Paired analyses
example 353
Paired analysis 143
Paired data 354
Password
change 29
Peak height 56
Pick
list, export in BVA 195
list, export in DIA 182, 274
list, multiple 190
locations in BVA 193
locations in DIA 181
spot map 113
Pick gel 187
Picking reference 93, 176
edit 188
Pixel 342
Post stain 303
PostStain 46
Post-translational modification (PTM) 80, 169
Primary spot map 55
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Print
BVA batch list 216
DIA batch list 216
in Batch Processor 216
in BVA 173
in DIA 83
Process multiple images 329
Profile settings 220
Project
create new 28, 37
Project sub-folders 27
Properties
files in DeCyder database 32
Properties, Batch Processor 209
Properties, BVA
3D View 94
Appearance Table 107
Colors 108
Graph View 96
Image View 92
Match Table 102
Protein database 169
Protein Table 105
Spot Map Table 100
Properties, DIA
3D View 57
Colors 65
Image View 53
Spot display 74
Table View 60
Workspace 68
PROSITE 170
Protein
AC 102
confirmation in BVA 168
database 169
Filter 160
ID 102
Mode 88
statistics 140
Table in BVA 102
Protein database
linking 169
Protein Filter 160
Protein of Interest
in BVA module 168, 178, 184
in DIA module 80
review in BVA 191
review in DIA 180
PTM 80, 169
Public access 30
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Q
Quality score 139
Quantitation
in BVA 109
in DIA 72
R
Recommended image file names 35
Reference
manual 7
markers 176, 187
Reference markers
automatic detection 176, 187
Release workspace 240
Remove
from Master 135
from Match 135
Remove area of interest 74
Remove DIA or BVA item in Batch 211
Repeated measures 150
Resolution 343
Restore DeCyder database data from backup 226
Retrieve archived DeCyder database data 239
Run Batch Processor 214, 335
S
Save
as Template 172
Batch workspace 212
BVA workspace 172
DIA workspace 83
Scatter parameter 62
Secondary spot map 55
Slope 61
Software structure 16
Split
merged spots 134
Split selected 137
Spot
annotation in BVA 108
area 56
confirmation in DIA 78
detection 69
detection, perform 71
exclusion 73
for picking in BVA 190
for picking in DIA 180
merging 133
number 56
picking, in DIA 175, 183
position 56
ratio 72
statistics 64
volume 56, 72
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Spot Control Panel 50
Spot Info dialog
in BVA 95
in DIA 59
Spot Map
attribute in BVA 113
function in BVA 113
Mode 88
sorting 90
Table in BVA 99
Table mode 113
Start the software 25
Statistical analysis in BVA 140
Statistical tests
ANOVA 148
Average ratio 142, 145
False discovery rate (FDR) correction 142
Independent analyses 143
One-Way ANOVA 142, 150
Paired analysis 143
Student’s T-test 145
Two-Way ANOVA 142, 153
Status in Batch Processor 215
Student’s T-test 142, 145
Sub-folders in projects 27
SwissProt 170, 171
T
Table View
in AT mode in BVA 106
in BVA 86, 95
in DIA 50, 58
in MT mode in BVA 101
in PT mode in BVA 102
in ST mode in BVA 99
parameters 61
Table View as Spot Info dialog in BVA 95
Table View as Spot Info dialog in DIA 59
Tagged Image File Format (TIFF) 341
Template 172
Template Spot Map 113
Threshold 63
Total protein stain 303
TrEMBL 170
Tutorials 7, 255
Two-Way ANOVA 103, 142, 153
example 357
Typhoon Variable Mode Imager 8
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U
User
administration tool 217
create 218
default 217
delete 221
manual 7
User account
edit 220
User Administration Tool 21
User-defined protein labelling 168
V
View
protein data in BVA 102
spot data 51
spot data in BVA 88
spot info in BVA 95
spot info in DIA 59
Views
3D View in BVA 86
3D View in DIA 50
Experimental Design View in BVA 86
Graph View in BVA 86
Histogram View in DIA 50
Image View in BVA 86
Image View in DIA 50
Table View in BVA 86
Table View in DIA 50
Volume 72
W
Warping 117
optimize matching using 118
theory 125
visualize using color overlay 127
Workspace
create 66
import 232
open in BVA 111
open in DIA 67
properties 68
properties, in Batch Processor 209
release 240
X
XML 244
Tag definitions 247
XML Toolbox 17, 244
Z
Zoom 52
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380
For contact information for your local
office, please visit
www.gelifesciences.com/contact
GE Healthcare Bio-Sciences AB
Björkgatan 30
751 84 Uppsala
Sweden
www.gelifesciences.com/decyder
GE, imagination at work and GE monogram are trademarks of General
Electric Company.
Cy, CyDye, DeCyder, Ettan, Immobiline and Typhoon are trademarks of
GE Healthcare companies.
DeCyder includes an Oracle® 10g database. Copyright © 1995 – 2007
Oracle Corporation. All rights reserved.
All third party trademarks are the property of their respective owners.
Third Party Credit statements:
Oracle is a registered trademark of Oracle Corporation and/or its affiliates.
This product includes the Xerces XML parser. Copyright © 2000 The Apache
Software Foundation, http://www.apache.org . All rights reserved.
This product includes parts developed using ITK, Copyright © 1999-2003
Insight Software Consortium, for licensing information see,
http://www.itk.org
Parts of this product implement the libTiff library, Copyright © 1988-1997
Sam Leffler, Copyright © 1991-1997 Silicon Graphics, Inc,
http://www.libtiff.org
Parts of this product include NMath components from CenterSpace
Software LLC, Copyright © 2002-2008. All rights reserved.
http://www.centerspace.net
Parts of this product include components from Farpoint Technologies, Inc.
Copyright © 1991-2007. All rights reserved. http://www.fpoint.com
Parts of this product include the TeeChart component from Steema
Software SL. Copyright © 1995-2008. All rights reserved.
http://www.teechart.com
Parts of this product include the open source MD5 implementation by L.
Deutsch. Copyright © 1999, 2000, 2002 Aladdin Enterprises. All rights
reserved.
Parts of this product include Cool Scrollbar Library.
Copyright © 2001 J. Brown.
CyDye: 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE)
technology is covered by US patent numbers US6,043,025, US6,048,982,
US6,127,134, and US6,426,190 and foreign equivalents and exclusively
licensed from Carnegie Mellon University.
CyDye: This product or portions thereof is manufactured under license from
Carnegie Mellon University under US patent number US5,268,846 and other
patents pending.
The purchase of CyDye fluors includes a limited license to use the CyDye
fluors for internal research and development, but not for any commercial
purposes. A license to use the CyDye fluors for commercial purposes is
subject to a separate license agreement with GE Healthcare.
© 2008 General Electric Company—All rights reserved.
First published Oct. 2008.
All goods and services are sold subject to the terms and conditions of sale
of the company within GE Healthcare which supplies them. A copy of these
terms and conditions is available on request. Contact your local GE
Healthcare representative for the most current information.
GE Healthcare UK Ltd
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Munzinger Strasse 5, D-79111 Freiburg, Germany
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imagination at work
28-9414-47 AA 10/2008
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