Affymetrix Expression Console Software v1.0

Transcription

manual.book Page i Thursday, October 5, 2006 12:21 PM
Affymetrix Expression Console™ Software
Version 1.0 — User Guide
P/N 702387 Rev. 1.0
manual.book Page ii Thursday, October 5, 2006 12:21 PM
ii
For research use only.
Not for use in diagnostic procedures.
Trademarks
®, GeneChip®, HuSNP®, GenFlex®, Flying Objective™,
Affymetrix®,
®
CustomExpress , CustomSeq®, NetAffx™, Tools To Take You As Far As Your Vision®,
The Way Ahead™, Powered by Affymetrix™, GeneChip-compatible™, and Command
Console™ are trademarks of Affymetrix, Inc.
All other trademarks are the property of their respective owners.
Limited License Notice
Limited License. Subject to the Affymetrix terms and conditions that govern your use
of Affymetrix products, Affymetrix grants you a non-exclusive, non-transferable,
non-sublicensable license to use this Affymetrix product only in accordance with the
manual and written instructions provided by Affymetrix. You understand and agree
that except as expressly set forth in the Affymetrix terms and conditions, that no
right or license to any patent or other intellectual property owned or licensable by
Affymetrix is conveyed or implied by this Affymetrix product. In particular, no right or
license is conveyed or implied to use this Affymetrix product in combination with a
product not provided, licensed or specifically recommended by Affymetrix for such
use.
Patents
Software products may be covered by one or more of the following patents: U.S.
Patent No's. 5,733,729; 5,795,716; 5,974,164; 6,066,454; 6,090,555, 6,185,561
6,188,783, 6,223,127; 6,228,593; 6,229,911; 6,242,180; 6,308,170; 6,361,937; 6,420,108;
6,484,183; 6,505,125; 6,510,391; 6,532,462; 6,546,340; 6,687,692; 6,607,887; and other
U.S. or foreign patents.
Copyright
©2006 Affymetrix, Inc. All rights reserved.
manual.book Page iii Thursday, October 5, 2006 12:21 PM
iii
Table of Contents
CHAPTER 1
Welcome
3
INTRODUCTION
3
WORKFLOW DIAGRAM
5
ABOUT THIS MANUAL
6
FAQS
7
CONVENTIONS USED IN THIS GUIDE
7
TECHNICAL SUPPORT
CHAPTER 2
CHAPTER 3
10
Installation and Setup
13
SOFTWARE REQUIREMENTS
13
MINIMUM HARDWARE RECOMMENDATIONS
13
INSTALLATION INSTRUCTIONS
14
SETUP PROFILE
14
LIBRARY FILES
17
Creating a Study
25
GETTING STARTED
25
ANALYZING FILES
32
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CHAPTER 4
CHAPTER 5
Affymetrix® Expression Console™ Software v.1.0 – User Guide
3’ Expression Array Analysis
ANALYSIS CONTROLS
35
ANALYSIS ALGORITHMS
35
ANALYZE DATA
36
RESULTS
38
Exon Array Analysis
SPECIFY CONTROLS
CHAPTER 6
CHAPTER 7
CHAPTER 8
35
QC Tables and Graphs
43
43
53
REPORTS
54
GRAPHS
58
Exporting Data
81
EXPORTING
81
SAVING IMAGES
86
Controls and Thresholds
89
REPORT CONTROLS
89
REPORT THRESHOLDS
95
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Contents
CHAPTER 9
CHAPTER 10
Appendix A
Appendix B
Advanced Analysis
v
101
3' EXPRESSION ARRAY CONFIGURATIONS
101
MASK FILES
109
MAS 5.0 CHP FILES IN THE COMMAND CONSOLE FORMAT
113
MAS 5.0 CHP FILES IN THE GCOS FORMAT
115
ADVANCED CONFIGURATION EXON ANALYSIS
116
Analysis Scripts
123
ANALYSIS SCRIPT CREATION
123
ANALYSIS SCRIPT DELETION
125
ANALYSIS SCRIPT EXECUTION
126
Algorithms
129
MAS 5.0 ALGORITHM
129
RMA ALGORITHM
130
PLIER ALGORITHM
131
COMPARISON OF ALGORITHMS
132
Algorithm Parameters and Outputs
MAS 5.0 COLUMN HEADINGS
135
135
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COLUMN HEADINGS FOR RMA AND PLIER
138
PROBE SET SUFFIXES
140
Index
143
manual.book Page 1 Thursday, October 5, 2006 12:21 PM
Chapter
1
Welcome
Chapter
1
3
Welcome
Welcome to the Affymetrix® Expression Console™ software (v.1.0)
User Guide. The Expression Console application provides signal
estimation and QC functionality for the GeneChip® Expression Arrays
(3’ Expression Arrays and Exon Arrays). The Expression Console
software allows users to:
• Generate probe set summarization (CHP) files from feature
intensity (CEL) files for both 3’ Expression Arrays and Exon
Arrays
• Capture a standard set of metrics for evaluating the success of the
individual hybridizations for both 3' Expression Arrays and Exon
Arrays
• Identify outlier samples in the data set
The Expression Console software is targeted for research personnel
(such as laboratory technicians, research associates, and scientists)
analyzing Affymetrix GeneChip® data.
Introduction
The Affymetrix® Expression Console™ software provides an easy way
to create summarized expression values (CHP files) for individual or
collections of 3' Expression Array and Exon Array feature intensity
(CEL) files. In addition to CHP writing, the Expression Console
application also produces a collection of QC metrics for evaluating the
success of hybridizations. The user defines thresholds for these metrics
and the software highlights the metrics that do not meet the defined
thresholds. In addition, the CHP files are highlighted in the study
table, if they have any metrics outside of the defined thresholds.
Individual QC metrics for each labeling technique are discussed in
Chapter 6, QC Tables and Graphs.
The Expression Console application contains graphic capabilities for
visual inspection of the hybridization results. To identify outliers, the
application is designed to display:
• Line graphs for individual or collections of metrics or probe sets
• Box plots for signal distributions before or after normalization
4
• MvA plots for signal distributions
• Heat maps for correlation matrices
The Expression Console application is not a secondary analysis
package. However, it does create the CHP files required for
secondary analysis packages from the Affymetrix GeneChip®
Compatible Program.
Chapter 1 | Welcome
Workflow Diagram
Figure 1.1
Affymetrix® Expression Console™ software workflow
5
6
About this Manual
This manual presents information about the Affymetrix Expression
Console™ software in the following chapters and appendices:
• Chapter 2, Installation and Setup: Describes how to install and
configure the Affymetrix® Expression Console™ v.1.0 software.
• Chapter 3, Creating a Study: Describes how to create a study to
analyze the array data.
• Chapter 4, 3’ Expression Array Analysis: Describes how to create
CHP files from CEL files for 3' Expression Arrays using either the
MAS5, RMA, or PLIER algorithm.
• Chapter 5, Exon Array Analysis: Describes how to create CHP files
from CEL files for Exon Arrays by applying either RMA or PLIER
algorithms.
• Chapter 6, QC Tables and Graphs: Describes how to run reports and
apply graphs for data interpretation.
• Chapter 7, Exporting Data: Describes how to export data using
PDF, TXT, and PNG file options.
• Chapter 8, Controls and Thresholds: Describes how to identify,
define, modify and/or remove controls and thresholds.
• Chapter 9, Advanced Analysis: Describes how to modify the default
algorithm parameters for either the 3' Expression Array or Exon
Array.
• Chapter 10, Analysis Scripts: Describes how to create a script that
automates the process of running the selected analysis algorithm
and creating a standard set of QC graphs and tables.
• Appendix A, Algorithms: Briefly describes the algorithms offered in
the Expression Console™ software with links to reference material
for further reading.
Chapter 1 | Welcome
7
• Appendix B, Algorithm Parameters and Outputs: Gives definitions for
report column headings, which represent MAS5, RMA, and
PLIER output data.
FAQS
A list of Frequently Asked Questions (FAQS) about the Affymetrix®
Expression Console™ software can be found on the Affymetrix website
at www.affymetrix.com; then go to /Support/Product/Software/Expression
Console Software.
Conventions Used in This Guide
This manual provides a detailed outline for all tasks associated with
Affymetrix® Expression Console™ software. Various conventions are
used throughout the manual to help illustrate the procedures
described. Explanations of these conventions are provided below.
STEPS
Instructions for procedures are written in a numbered step format.
Immediately following the step number is the action to be performed.
Following the response, additional information pertaining to the step
may be found and is presented in paragraph format. For example:
1. Click Yes to continue.
The Delete task proceeds.
In the lower right pane the status is displayed.
FONT STYLES
Bold fonts indicate names of commands, buttons, options or titles
within a dialog box. When asked to enter specific information, the
input is displayed in italics within the procedure being outlined.
For example:
1.
Click the Find toolbar button
; or
8
Select Edit
Find from the menu bar.
The Find dialog box opens.
2. Enter AFFX-BioB-5_at in the Find what box, then click Find
Next to view the first search result.
3. Continue to click Find Next to view each successive search result.
SCREEN CAPTURES
The steps outlining procedures are frequently supplemented with
screen captures to further illustrate the instructions given.
The screen captures depicted in this manual may not exactly match
the windows displayed on your screen.
ADDITIONAL COMMENTS
Throughout the manual, text and procedures are occasionally
accompanied by special notes. These additional comments and their
meanings are described as follows:
Information presented in Tips provide helpful advice or shortcuts
for completing a task.
The Note format presents supplemental information pertaining to
the text or procedure being outlined.
The Important format presents important information that may
affect the accuracy of your results.
Chapter 1 | Welcome
9
Caution notes advise you that the consequence(s) of an action may
be irreversible and/or result in lost data.
Warnings alert you to situations where physical harm to person or
damage to hardware is possible.
10
Technical Support
Affymetrix provides technical support to all licensed users via phone or
E-mail. To contact Affymetrix® Technical Support:
AFFYMETRIX, INC.
3420 Central Expressway
Santa Clara, CA 95051 USA
Tel: 1-888-362-2447 (1-888-DNA-CHIP)
Fax: 1-408-731-5441
[email protected]
[email protected]
AFFYMETRIX UK Ltd.,
Voyager, Mercury Park,
Wycombe Lane, Wooburn Green,
High Wycombe HP10 0HH
United Kingdom
UK and Others Tel: +44 (0) 1628 552550
France Tel: 0800919505
Germany Tel: 01803001334
Fax: +44 (0) 1628 552585
[email protected]
[email protected]
Affymetrix Japan K.K.
Mita NN Bldg. 16F
4-1-23 Shiba Minato-ku,
Tokyo 108-0014 Japan
Tel. 03-5730-8200
Fax: 03-5730-8201
[email protected]
[email protected]
www.affymetrix.com
Chapter
2
Chapter
2
13
The Affymetrix® Expression Console™ software is a stand-alone
application. It can be installed on computers that have GeneChip®
Operating System (GCOS) software, Affymetrix GeneChip®
Command Console™ (AGCC) software, or neither.
Affymetrix recommends that if you are using GCOS CEL files, you
should use the Data Transfer Tool (DTT) provided by Affymetrix to
move the CEL files out of the GCOS directory.
Software Requirements
The Expression Console software can be installed with the following
operating systems:
• Microsoft Windows 2000 Professional with service pack 4.0 or
higher
• Microsoft Windows XP with service pack 2.0 or higher
Minimum Hardware Recommendations
The minimum hardware recommendations are:
• Memory (RAM): 1 GB*
• Hard drive: 20 GB** (sufficient space should be available to
meet user data requirements)
• Processor: 2.0 GHz Intel Pentium or higher
* Due to the large number of datapoints produced by the Exon Arrays,
an additional 1GB of RAM is highly recommended.
**The larger file sizes associated with Exon Array data should be taken
into account when calculating the necessary free space requirement.
14
Installation Instructions
To install the Expression Console software:
1. Go to www.affymetrix.com and download the software from the
following location:
Support / By Product / Software / Affymetrix® Expression Console™
Software
Unzip the downloaded software package.
3. Double-click on setup.exe to install the software.
4. Follow the directions provided by the installer.
5. The setup process installs the required Microsoft components,
which includes the .NET 2.0 framework.
2.
If there is a previous version of Expression Console software
installed, the installation software prompts the user to remove it
before installing the new version.
Setup Profile
The Affymetrix® Expression Console™ software is ready for
configuration. Follow the steps below to configure the software for
sample analysis.
Create a Profile
A profile is used as a method to group options and parameters so that
those parameters can be used again. Once the software is started, the
profile can only be changed when there is no study is open, by clicking
, selecting Edit → Change User Profile from the drop-down
menu, or going to Toolbox → Configuration → Specify User
Profile.
To open the software and setup a profile, perform the following steps:
Chapter 2 | Installation and Setup
1.
15
Open the Expression Console™ application by selecting Start →
Programs → Affymetrix → Expression Console.
The Expression Console software window opens with the Profile
Information dialog box displayed.
Affymetrix® Expression Console™ software - Profile Information Window
Type in a name for your profile and click OK.
By entering a profile name, you retain the specific analysis settings
and quality control thresholds for each profile entered.
A list of previously created profiles, if any, are found in the drop-down
menu on the Profile Information dialog box.
2.
You can select a different profile without terminating the program,
but the current study must be closed to open another profile.
16
Delete A Profile
The list of previously created profiles are found in the drop-down
menu on the Profile Information dialog box.
To remove profiles no longer needed:
1.
2.
3.
4.
5.
Close the study window, if open.
Select File → Utilities.
Select the User Profile Management tab (Figure 2.1)
Highlight the profile to be removed.
Click Delete.
Figure 2.1
User Profile Management Tab
17
Library Files
Library Files – Download Option
The Expression Console software requires information stored in
library files (array types) to analyze the CEL files generated by
GCOS or Affymetrix GeneChip® Command Console™ (AGCC)
software. These files are available from Affymetrix and can be
downloaded within the Expression Console application.
When you click OK in the Profile Information window the first
time (Figure 2.1), a dialog box opens asking you to direct the
software to your GeneChip library files folder (Figure 2.2). You
can select any location for the library files folder
You can select any location for the library files folder. However,
once you direct the software to the folder location, do not place any
library files in a subfolder. The Expression Console application
cannot find library files in a subfolder!
If the Affymetrix GeneChip® Command Console™ (AGCC) software
is installed on your system, the Expression Console™ application
defaults to the files in C:\Command_Console\Library.
If the Affymetrix GeneChip® Operating System software (GCOS) is
installed on your system, Affymetrix recommends that you do not
select the GCOS library file directory as the library file directory for
Expression Console, to avoid confusion.
18
Expression Console downloads library files from NetAffx for
analysis, but these are not registered with GCOS and are not
sufficient to scan arrays.
Figure 2.2
Browse For Folder window to locate GeneChip library files folder
Library files can be downloaded from NetAffx™.
1.
Select File → Download Library Files.
A dialog box opens requesting your account information for
NetAffx (Figure 2.3).
To obtain a NetAffx account, go to www.affymetrix.com, click
Register at the top of the Affymetrix main page and follow the
instructions.
19
Figure 2.3
NetAffx Account Information dialog box
2.
Enter your registered email address and password.
The NetAffx Library Files window opens (Figure 2.4). This
window contains a complete list of Affymetrix library files that can
be downloaded. Library files previously downloaded are marked
currently installed.
20
Figure 2.4
NetAffx Library Files window - downloading a checked library file
3.
Check the library files needed and click the Download button.
The appropriate files are downloaded to the folder you previously
indicated. The green status bar at the bottom of the window
highlights during the download process.
If the Abort button is selected, the download process stops with a
message indicating which library files failed to download.
21
When you create a new study and add CEL/CHP files that do not
have corresponding library files loaded in the correct folder, you will
be prompted to download the appropriate library files.
Figure 2.5
Status Window - indicating missing library file
Library Files – Copying Files Manually
For computers that are not connected to the internet and therefore
cannot take advantage of the library file download option, it is possible
to manually copy the necessary files to the computer with Expression
Console:
Create a folder on the computer to hold the library files for the
Expression Console application.
2. Copy the necessary files from the CD or other removable media to
the library file folder. In order for the default report controls to be
identified for the array type, this should be done with the
application closed.
1.
For 3' Expression Arrays, only the .psi and .cdf files need to be
copied to the directory.
B. For Exon Arrays, minimally the .clf, .pgf, .bgp, and .qcc files need
to be copied to the directory.
A.
Do not create subdirectories within the library file folder. The
Expression Console software does not look at subdirectories.
22
To continue, proceed to Chapter 3, Creating a Study.
Chapter
3
Creating a Study
Chapter
3
25
Creating a Study
This section describes how to create a study with probe level intensity
files (CEL) and probe level summarization files (CHP) in the
Affymetrix® Expression Console™ software.
Getting Started
To get started using the Affymetrix® Expression Console™ software,
the user creates a study consisting of a collection of probe cell intensity
files (CEL) and/or probe level summarization files (CHP) and their
associated sample information for analysis and examination.
The study table contains the following:
• File names (CEL or CHP)
• Sample attribute information from either the .xml files for GCOS
(requires the use of DTT to transfer the data from the GCOS
database) or .arr files (sample/array) from AGCC
• An indication of whether or not CHP files pass the user-defined
tolerances for the array metrics. See Chapter 8, Controls and
Thresholds.
• Whether or not the results are on a linear or log scale
By default, the algorithms PLIER and MAS 5.0 are linear; the
RMA algorithm is in the log scale. Under the edit menu, the scale
can be changed, but this does not change the scale of the data in
the CHP file. See Appendix A, Algorithms.
Follow the steps below to create a study in the Expression Console
software.
GCOS users must use DTT v1.1, using the Flat File option, to
transfer files to be analyzed by the Expression Console software
from the GCOS database to an independent folder. More detailed
instructions can be found at www.affymetrix.com; then go to
Support/Technical/Tutorial/GCOS.
26
Study Window
Follow the steps below to create a study to analyze CEL files or view
CHP file data.
1.
Create a study by first selecting one of the following:
- The icon
- File → New Study
- Toolbox → Study → Create New Study.
For further analysis in an existing study, select
, or File → Open
Study, or Open → Existing Study from the Toolbox.
The Affymetrix Study window opens.
Figure 3.1
Affymetrix Study window
2.
To add CEL files from your data set of interest to the study
window, do one of the following:
Chapter 3 | Creating a Study
27
- Click the Add Intensity Files button
- Select File → Add to Study → Probe Cell Intensity Files
- In the workflow toolbox, select Study → Add Intensity Files.
The Select Probe Cell Intensity Files window opens. Sample
attributes from .arr or .xml files are displayed for the associated
CEL files, if available.
Figure 3.2
Affymetrix Study window with CEL files
28
Figure 3.3
Select Probe Cell Intensity Files window
3.
To add previously created CHP files to the study window , select
the Add Summarization Files button, click File → Add to
Study → Probe Level Summarization Files, or go to the
workflow toolbox and select Study → Add Summarization
Files.
The Select Probe Level Summarization Files window opens and
sample attributes from .chp files are displayed.
Use the Files of type menu selection at the bottom of the window
to filter the CHP files based on the selected algorithm type.
29
Figure 3.4
Select Probe Level Summarization Files window
After CHP files are added to the study, either at the end of an
analysis or directly by clicking the Add Summarization Files
button, the metrics and controls associated with that algorithm
run are compared against the user defined thresholds. Any files
that fail to meet any one of the criteria are highlighted.
On a system with AGCC, the Expression Console software uses the
AGCC index to locate the matching ARR file; otherwise, it pairs the
CEL/CHP and ARR files by matching the root names.
On a system with GCOS, the Expression Console application will
use the sample attribute information from the .xml files, if the .xml
files are in the same folder as the CEL or CHP files and have the
same root name (e.g., 12345.xml and 12345.cel).
4.
In the Select Probe Cell Intensity Files or Select Probe Level
Summarization Files window, click the column heading of
choice to sort the files based on that attribute.
30
5.
Select the files to be added and click Open to add the selected
CEL/CHP files to the study window.
Library Files
To analyze the selected study files, the Expression Console application
automatically locates the associated library files in the indicated
folder, when the software is first installed. If the selected library path
is not specified, Expression Console software defaults to the AGCC
library files folder, if it is present.
If the correct library files have not been downloaded to the selected
library file directory, the following error message displays when you
attempt to analyze your files:
Figure 3.5
Error message - opening CEL files for analysis
The status window at the bottom of the Expression Console
application window lists the missing library files. If you need to
establish a new path:
1.
Select Edit → Set library path and locate the new directory.
All studies must be closed to activate the Set library path menu
selection.
2.
Select the CEL files to analyze and click Open.
The CEL files you selected are populated in the Affymetrix Study
window with their accompanying attributes.
31
Figure 3.6
Affymetrix Study window with CEL files
Study Window Controls
The study window in the Expression Console application controls the
files that are analyzed by the software. Within the study window the
default sort order for the tables and graphs is determined. In addition,
a sample attribute can be selected that will be pre-pended to the array
names for display in the tables and graphs.
File Consolidation
A study can contain files that are located in multiple directories on the
computer. To aid in file management, the Expression Console
application can move all of the files associated with a study to a single
directory. Studies cannot be consolidated when a study is open.
1.
2.
3.
4.
5.
Select the File Consolidation Tab (Figure 3.7).
Browse
to find the study to be consolidated.
Select an output directory to hold all of the associated files.
Click Consolidate and then Close.
32
Figure 3.7
File Consolidation Tab
File consolidation moves the files to the selected directory.
Analyzing Files
To analyze a 3' Expression Array data set, proceed to Chapter 4, 3’
Expression Array Analysis.
To analyze an Exon Array data set, proceed to Chapter 5, Exon Array
Analysis.
Chapter
4
Chapter
4
35
Probe cell intensity data from 3' Expression Array analyses is further
analyzed in the Affymetrix® Expression Console™ software application
using the MAS5, RMA, and PLIER algorithms to create CHP files.
Follow the instructions below to analyze 3' Expression Array data.
Analysis Controls
Affymetrix microarrays contain the hybridization, labeling and
housekeeping controls that help determine the success of the
hybridizations. For more information on the interpretation of these
controls, see the whitepaper, Data Analysis Fundamentals, at for the
time www.affymetrix.com.
To aid in the examination of these controls, the Expression Console
application displays summarized probe information in tabular format.
In order for this feature to function properly, the controls must be
identified prior to analysis. Once the controls have been identified for
a particular GeneChip (probe array type), they are saved and only need
to be updated if the user wishes to modify the controls.
For most standard Affymetrix GeneChip® Arrays, a standard set of
defaults has been provided; however, it is recommended that the user
should verify all controls before initiating their analysis. For details on
defining the controls and their thresholds, refer to Chapter 8, Controls
and Thresholds.
Analysis Algorithms
By default, the 3' Expression Array workflows are set as follows:
• PLIER workflow is Quantile normalization with PM-MM
• RMA workflow is Quantile normalization and has a general
background correction
• MAS 5.0 workflow is set so that all probe sets are scaled to TGT =
500
For more details about the algorithms, see Appendix A, Algorithms and
Appendix B, Algorithm Parameters and Outputs.
36
Analyze Data
To analyze 3' Expressson Array data:
1.
Create a new study or open an existing study. See Chapter 3,
Creating a Study.
In the study window, check the CEL files for Analysis.
3. To analyze the selected CEL files in the study, select Analysis →
3' Expression Arrays and select the appropriate workflow
(MAS5, RMA, or PLIER) (Figure 4.1).
2.
You can also look at the controls alone to obtain a cursory look at
the success of the hybridization. This allows you to remove the
obvious outliers prior to the time investment of analyzing the full
array.
Figure 4.1
3’ IVT Expression Array - MAS5 Algorithm analysis
The suffix dialog box for the selected workflow opens (Figure 4.2).
Chapter 4 | 3’ Expression Array Analysis
37
Figure 4.2
Expression Console - MAS5 - File name suffix dialog box
For advanced analysis, select Analysis → Advanced Expression
Configuration and choose the appropriate Configuration or Mask
file menu (Figure 4.3).
Figure 4.3
Expression Console application - Advanced Analysis
For details on how to utilize the advanced analysis features, see
Chapter 9, Advanced Analysis.
If you want to run a script (an automated analysis with preset
parameters) to generate CHP files, select Analysis → Expression
Analysis Scripts and create a new script or open an existing script
(Figure 4.4). For details on how to set up an analysis script, see
Chapter 9, Advanced Analysis.
38
Figure 4.4
Expression Analysis scripts menu
4.
Enter a suffix to identify the specific samples after analysis.
When you click OK, analysis begins. The progress of analysis is
displayed in the Status window (Figure 4.5).
Figure 4.5
MAS5 Analysis files in the Status window
Results
Several minutes may be required for processing results, depending
on the number of arrays selected. Once processing is complete, the
study window shows a new group containing the newly
summarized data (CHP files). The new group is named with the
type of algorithm selected.
Chapter 4 | 3’ Expression Array Analysis
39
The files are listed in the status window at the bottom of your
screen as they are generated, and the Affymetrix Study window
(Figure 4.6) is populated with the corresponding CHP files.
Figure 4.6
Affymetrix Study window - MAS5 analysis results
Figure 4.7
Affymetrix Study window - MAS5 analysis results.
Highlighted CHP files (Figure 4.7) contain metrics outside of defined
thresholds.
40
Chapter
5
Exon Array Analysis
Chapter
5
43
Exon Array Analysis
Probe cell intensity data (CEL) from Affymetrix GeneChip® Exon
Arrays are analyzed in the Affymetrix® Expression Console™ software.
The application uses the RMA-sketch workflow for both Exon and
Gene level analyses to create CHP files. Follow the instructions below
to analyze Exon Array data.
Affymetrix® Exon Arrays include the following:
• Human – HuEx-1_0-st-v2
• Mouse – MoEx-1_0-st-v1
• Rat – RaEx-1_0-st-v1
Specify Controls
To analyze data from the Affymetrix Exon Arrays:
1.
Download the appropriate library files, File → Download
Library Files, (for example, human Exon Arrays require
HuEx-1_0-st-v2 library files).
To use custom library files for an analysis, see Chapter 9, Advanced
Analysis.
Create a new study or open an existing study. See Chapter 3,
Creating a Study.
3. Add the CEL files you want to use to create CHP files by clicking
the Add Intensity Files button.
2.
4.
Select the level of analysis by clicking the Run Analysis button in
the Affymetrix Study window to see the Available Analyses
menu box or select Analysis to display a drop-down menu
(Figure 5.1). Then perform one of the following:
44
Figure 5.1
Select the Run Analysis button in the Affymetrix Study window
- For the Gene level, select Analysis → Gene Level
(Figure 5.2), or select the Gene Level from the drop-down
menu (Figure 5.1). Then select the annotation level, Core or
Extended. These describe transcript structure confidence
levels (for the annotation used to define the transcript) that are
annotations broadly defined as follows:
– Core: limits analysis to exon-level probe sets that map to
BLAT alignments of mRNA with annotated full-length CDS
regions.
– Extended: limits analysis to transcripts that are defined by
exon-level probe sets that map to cDNA alignments and their
annotations based on cDNA alignments, in addition to
transcripts defined by the core category.
– Full: analysis includes transcripts that consist of exon-level
probe sets that map to sets of ab-initio gene predictions in
addition to transcripts defined by the core and extended
categories.
For further explanation of confidence levels, see the Affymetrix
whitepaper, Exon Probeset Annotations and Transcript Cluster
Groupings, at www.affymetrix.com; then go to Support/Technical
Documentation/White Papers.
Chapter 5 | Exon Array Analysis
45
Figure 5.2
Gene Level probe set with confidence modes: Core, Extended, Full
- For the Exon level, select Analysis → Exon Level, or select
one of the exon level analysis options from the drop-down
menu (Figure 5.2). Then select the confidence level, Core,
Extended, or All. These are gene confidence levels, which are
input transcript annotations broadly defined as follows:
– Core: limits analysis to exons that consist of BLAT
alignments of mRNA with annotated full-length CDS regions.
– Extended: limits analysis to cDNA alignments and their
annotations based on cDNA alignments, in addition to core
defined exons.
– Full: analysis uses exons derived from sets of ab-initio gene
predictions in addition to the core and extended exons.
– All: consists of all three confidence levels plus probe sets that
map to more than one gene or to genes that do not align to the
genome.
For further explanation of confidence levels, see the Affymetrix
whitepaper, Exon Probeset Annotations and Transcript Cluster
Groupings, at www.affymetrix.com; then go to Support/Technical
Documentation/White Papers.
- For Controls, select Analysis → Controls Only → Controls
(Figure 5.3).
A subset of probes, on all arrays, are identified as controls. This
option summarizes expression values for controls only.
46
You can look at the controls alone to obtain a cursory look at the
success of the hybridization. This allows you to remove the obvious
outliers prior to the time investment of analyzing the full array.
Figure 5.3
Exon analysis - Controls Only
The RMA-sketch workflow is automatically selected by the
software.
Figure 5.4
Available Analyses - Controls Only - Controls
5.
Click OK.
The suffix dialog box for the selected array and confidence level
opens.
47
Figure 5.5
Expression Console - Exon Core - File name suffix dialog box
You can add a suffix to the file name in order to further identify your
samples, or you can leave it blank. The summarization method is
automatically included in the file name.
Enter a suffix to identify the specific samples after analysis.
7. Click OK; analysis begins.
6.
The processing time for the CEL files depends on a number of
factors, which include the number of CEL files, level of analysis,
number of probes under consideration, amount of available RAM,
and computer processor speed. See Chapter 2, Minimum Hardware
Recommendations.
The progress of the analysis can be tracked in the status window
(Figure 5.6) (Figure 5.7). Since Exon Arrays are processed as a batch,
the CHP files are not added to the study until the completion of the
analysis. The files are listed in the status window as they are generated;
then the Affymetrix Study window (Figure 5.8) is populated with the
corresponding CHP files.
48
Figure 5.6
Exon Core level analysis files in the Status window
Figure 5.7
Gene Core level analysis files in the Status window
Figure 5.8
Affymetrix Study window - Gene core level analysis results
49
The CHP files are ready for QC analysis within the Expression Console
software or other compatible software. CHP files are located in the
same directory as the first CEL files in your input list. Outliers,
samples outside the threshold boundaries, are highlighted in orange
(Figure 5.9).
Figure 5.9
Affymetrix Study Window - Gene core level analysis results with outliers
50
Chapter
6
Chapter
6
53
To help researchers establish quality control processes for gene
expression analyses, Affymetrix has developed several controls.
Researchers are encouraged to monitor these controls on a regular basis
to assess assay data quality. Many of these control metrics are
generated as a product of the primary analysis by the respective
analysis algorithms. To help with monitoring these values, a collection
of tools for viewing and graphing metrics associated with the array and
the analysis are provided in the Affymetrix® Expression Console™
software. These metrics include but are not limited to:
• Hybridization controls
• Labeling controls
• Internal control genes (Housekeeping controls)
• Global array metrics
• Algorithm parameters
This section describes how to generate graphs and tables allowing the
quality of individual hybridizations included in a single study to be
easily assessed and gives some introductory guidance on their
interpretation. For more detailed information about graph and table
interpretation, refer to the whitepapers, Data Analysis Fundamentals
and Exon Arrays Quality Analysis, at www.affymetrix.com.
In general, Affymetrix highly encourages users to create a running log
of the parameters to monitor quality and potentially flag outlier
samples. Evaluation of particular samples should be based on the
examination of all sample and array performance metrics in light of
this history. A good, general rule for the examination of these types of
quality control data is to look for outliers when compared to other
highly related samples. For example, tissue A may have an overall low
level of gene expression so that the percent of probes detected may
normally be between 10–15%. Therefore, a %Present = 11 would not
be an indication of a problem. However, if tissue B normally has a
%Present between 45–50, and one sample has %Present = 11, that is an
indication of a problem. Examination of which metrics or controls are
outliers will provide insight into the source of the problem and
possible solutions.
54
The Expression Console software has the ability to store user defined
thresholds for the QC metrics and highlight, in the reports and study
tables, those metrics outside the thresholds and arrays that contain
them. Expression Console software comes with an initial set of defaults
for both metrics and thresholds, which the user can modify according
to individual preferences. For more information, see Chapter 9,
Advanced Analysis.
Reports
Reports available in the Expression Console software provide a tabular
view of all QC metrics, including:
• Algorithm parameters (See Appendix B, Algorithm Parameters and
Outputs for a list of parameters, outputs, and their definitions.)
• Global analysis metrics
• Corner +, Corner -, Center +, and Center -, if available
• Count and percentages of detection calls
• Average signal value for each detection call type
• Signals, detections, and 3'/5' ratio for spike and housekeeping
controls
Note that these metrics are algorithm specific. For details about the
MAS 5.0 algorithm parameters, see the GeneChip® Operating System
Software (GCOS) manual (PN 701439) and the Statistical Algorithms
Reference Guide. For details about the PLIER algorithm parameters, see
the Guide to Probe Logarithmic Intensity Error (PLIER) Estimation
technical note and the Quality Assessment of Exon Arrays whitepaper at
www.affymetrix.com.
To view the full set of report metrics, select Report → View Full
Report.
Chapter 6 | QC Tables and Graphs
55
Full Report
A Full Report (Report → View Full Report) displays a table of the
chosen algorithm parameters and the resulting array QC metrics
contained within the CHP files. The Threshold Test column displays
whether or not all the metrics are within or one or more metrics are
outside of the user-specified thresholds.
Figure 6.1
Full Report - 3' Expression Array with MAS5
56
Custom Report
Customization of the report to define a subset of columns (report
metrics) for the tabular display is possible by selecting Report →
New Report and checking the categories you want to display in the
table.
The list of available metrics to plot is based on the CHP files in the
study. For example, if you have MAS 5.0 CHP files open, then
parameters associated with PLIER are not visible.
Figure 6.2
New Report - 3' Expression Array with MAS5
57
The categories available for selection in the Report window depend
on the expression array selected and the algorithms used to
generate the CHP files in the study.
Delete A Custom Report
To delete a Custom Report:
Click File → Utilities.
2. Select the Parameter Management tab (Figure 6.3).
3. In the Custom Reports section, select the report to be deleted.
4. Select Delete and then Close.
1.
Figure 6.3
Parameter Management Tab
58
Graphs
In addition to the tabular display of the metrics, the Expression
Console software supports several different graphical views, including
signal histograms, box plots, line graphs, and heat maps depicting the
correlation between arrays. The graphical displays, especially the box
plots and heat maps, are useful in identifying outlier samples.
Figure 6.4
Graph menu items
Probe Cell Intensity View
The Probe Cell Intensity View (Graph → Probe Cell Intensity
View) is a view of the CEL intensities arranged by physical position
on the array. This view is used to quickly determine if any features on
the array do not have a signal. For in-depth image inspection and
gridding evaluation, it is recommended that customers use GCOS or
the manual grid application available within the Affymetrix
GeneChip Command Console (AGCC) software package (Figure 6.5).
59
Figure 6.5
Probe Cell Intensity View
The display area can be adjusted using the arrow keys, while the zoom
level is controlled by the + and - buttons. Moving the mouse over a
cell in the image displays the features XY coordinate and intensity
values. Selecting a region (click and drag the mouse) zooms the display
to show the selected region. Right clicking on the image enables the
following options:
• Select a gray scale palette (Gray)
• Select a color scale palette that goes from black to red to yellow to
white (Heat)
• Adjust the range for the color scale (Scale)
• Save the image in PNG format
• Copy the image to the clipboard (Copy to clipboard)
A particular file may appear solid black when you first open the
Probe Cell Intensity View. Use the View → Zoom feature to zoom
in on a particular area of investigation.
60
Signal Histogram
The signal histogram (Graph → Signal Histogram) is enabled when
the CHP files are selected in the study window (Figure 6.6). This
graph displays a histogram plot of the signal values for the selected
CHP files in a box or line graph. A legend of the input CHP files is
displayed in the upper right corner of the histogram.
Figure 6.6
Signal Histogram - 3' IVT with MAS5 algorithm
A right click popup menu provides the following options for the user:
• Save the graphic as a PNG file (Save as PNG)
• Save the underlying data used from the histogram to a TXT file.
(Save data as TXT)
• Use bars in the histogram to display the values (Bars only)
• Use lines in the histogram to display the values (Lines only)
• Use bars and lines to display the values (Bars + Lines)
• Edit the scale of the Y axis (Scale)
61
A slider just below the histogram allows you to adjust the zoom factor
of the X axis and a scroll bar allows you to adjust the visible region of
the graph.
Box Plot
The box plot graph shows a standard box plot of data from either CHP
or CEL files. A general rule to use when examining box plots is to look
for individual arrays that are dramatically different from the others and
most importantly from other replicates in the same group.
A right-click popup menu is provided for all of the box plots
containing:
• Save the graphic as a PNG file (Save as PNG)
• Save the data to a TXT file (Save data as TXT)
A slider is provided on the X axis to adjust the zoom factor. This is the
number of files to display on the X axis.
Box Plot – Probe Cell Intensity
The probe cell intensity box plots are generated from CEL file probe
cell intensity values. This graph is generated by selecting Graph →
Box Plot - Probe Cell Intensity. The probe cell intensity creates a
box plot of the probe intensity values for each array. Probe cell
intensities are prior to analysis/summarization and have not been
normalized; therefore, some differences in the distributions are to be
expected. At the feature intensity level, the Exon Arrays have about 6
million probes; some delays in the generation of these graphs are
expected.
62
Figure 6.7
Box Plot - Probe Cell Intensity - 3' IVT Expression Array
Relative Probe Cell Intensity
The distribution of the ratio of the intensity of each probe to the
median probe intensity across all of the selected arrays is summarized
in the Box Plot - Relative Probe Cell Intensity. Therefore, the plot
compares the distribution of intensities on each array to the median
probe intensity value for the group. As such, it is a good way to
identify arrays with divergent probe intensity distributions relative to
the other arrays in the study.
Figure 6.8
Box Plot - Relative Probe Cell Intensity - 3' IVT Expression Array
Figure 6.9
Box Plot - Relative Probe Cell Intensity - Exon Array
63
64
Box Plot – Signal
The Signal Box Plot is generated from summarized probe set signal
values in CHP files. This graph is generated by selecting the Graph
→ Box Plot - Signal menu item.
Figure 6.10
Box Plot - Signal - 3' IVT Expression Array
Figure 6.11
Box Plot - Signal - Exon Array
65
Box Plot – Relative Signal
The Relative Signal Box Plot summarizes the distribution of the ratio
of signal for each probe set to the median probe set signal across all the
selected arrays. Therefore, the plot compares the distribution of probe
set Signal values on each array to the median array for the group. As a
result, it is a good way to identify arrays with divergent signal
distributions relative to the other arrays in the study.
Figure 6.12
Box Plot - Relative Signal - 3' IVT Expression Array
MvA Plot
The MvA plot (Figure 6.13) is a comparison plot comparing M
(magnitude of change) on the Y-axis versus A on the X-axis, where
M = log(Signal array1) - log(Signal array2) and A (average
log(Signal)). The Y-axis is displayed on a log2 scale with green
threshold lines for +/- two-fold changes. The color coding of the plot
indicates the density of probes represented by that data point. The
following are displayed:
66
• The title of the graph, which indicates the two CHP files used to
create the MvA plot
• The Pearson’s correlation (r2) value
Figure 6.13
MvA Plot - 3' Expression Array with MAS5 algorithm
Correlations
The relationship between two variables is described by their
correlation. Two standard statistical measures of linear correlation
(Pearson’s and Spearman) are provided in the Expression Console
software. These are used to compare the signal estimates or detection
p-values between two arrays. To simplify the interpretation of this
data, the correlation values are presented as a heat map (Figure 6.14).
The Spearman test is used with fewer samples since it is a rankbased test. Pearson’s test is used when there are more data, which
allows normalcy to be confirmed.
67
For all of the heat maps, a right-click popup menu enables the
following:
• The graphic to be saved in PNG format (Save as PNG)
• The underlying r2 values to be saved as a TXT file (Save data as
TXT)
• The image to be copied to the clipboard (Copy to clipboard)
• A dialog box to adjust the min/max values to use to map values to
a color scale (Edit Scale)
In addition, a slider is provided on either axis to adjust the zoom factor
controlling the number of files to show on a given axis. A scroll bar is
provided to adjust the visible region of the graph. Clicking on a cell
in the graph displays the associated files in the lower left part of the
window. The display includes the file names and the value.
Pearson’s Correlation (Signal)
Signal concordance is evaluated using the Pearson’s correlation
coefficient (r2) value to compare the signal values in two CHP files.
The heat map contains a pairwise comparison of the signal values from
all the selected CHP files, where the r2 values have been converted into
a pseudocolor scale.
68
Figure 6.14
Pearson’s Correlation (signal) - 3' IVT Expression Array
Pearson’s Correlation (Detection P-Value)
P-value concordance is evaluated using the Pearson’s correlation
coefficient (r2) to compare the p-values from two CHP files. The
resulting heat map contains a pairwise comparison of the detection pvalues from all of the selected CHP files, where the r2 values have been
converted into a pseudocolor scale.
Only MAS5 and Exon level analyses calculate detection p-values to
generate the graph associated with them.
69
Figure 6.15
Pearson’s Correlation (detection p-value) - 3' IVT Expression Array
Spearman Rank Correlation (Signal)
This is the Spearman rank correlation of the signal values between two
CHP files. The heat map contains a pairwise comparison of the signal
values from all the selected CHP files, where the Spearman r2 values
have been converted into a pseudocolor scale.
70
Figure 6.16
Spearman Rank Correlation (signal) - 3' IVT Expression Array
Spearman Rank Correlation (Detection P-Value)
This is the Spearman rank correlation of the p-values between two
CHP files. P-values are only available in MAS5 CHP files and Exon
Arrays analyzed at the exon level. The heat map contains a pairwise
comparison of the detection p-values from all the selected CHP files,
where the Spearman r2 values have been converted into a pseudocolor
scale.
Only MAS5 and Exon level analyses calculate detection p-values to
generate the graph associated with them.
71
Figure 6.17
Spearman Rank Correlation (detection p-value) - 3' Expression Array
Line Graphs
Line graphs allow the user to graph metrics or signal values for
specified groups of probe sets for selected CHP files.
Report Metrics
The report metrics is a line graph of the selected metrics (Graph →
Line Graph - Report Metrics) calculated and stored in the header of
the CHP. These include the algorithm parameters, global array
metrics, and spike-in and housekeeping control values that are stored
in the CHP file. The available metrics for graphing depend on the
metrics available for the CHP files in the current study. Metrics not
present in the CHP file cannot be graphed.
72
Figure 6.18
Report Metrics - 3' Expression Array analyzed with MAS5
Probe List
The probe list is a line graph of probe sets defined in a probe list file).
Select Graph → Line Graph - Probe List, the Select the probe list
window opens (Figure 6.19). When you select the probe list of
interest, Expression Console software creates a line graph of the probe
sets (Figure 6.20).
Figure 6.19
Select the probe list window
Figure 6.20
Probe List Line Graph - 3' Expression Array
73
74
Figure 6.21
Probe List Line Graph - Exon Array
New Probe List
The New Probe List window (Graph → New Probe List) enables
users to create their own probe list by selecting the probe sets they
want to investigate (Figure 6.23).
Figure 6.22
New Probe List - 3' Expression Array
Figure 6.23
New Probe List - Exon Array
75
76
Open Probe List
To open a probe list:
1.
Select Graph → Open Probe List (Figure 6.24), and select the
probe list.
Figure 6.24
Select the probe list
2.
Click Open.
The Probe List of interest opens for viewing (Figure 6.25).
Figure 6.25
View the Probe List
Delete a Probe List
To delete a probe list:
1.
In the Probe List section, select the probe list to be deleted.
4. Click Delete and then Close.
3.
Figure 6.26
Parameter Management Tab - Delete a Probe List
77
78
Chapter
7
Exporting Data
Chapter
7
81
Exporting Data
The Affymetrix® Expression Console™ software enables the user to
export the data in tables and graphs to various text or pdf documents.
Exporting
Tables and Graphs to PDF
All of the currently opened/displayed tables and graphs can be
exported to a PDF format. Select Export → Export All Tables/
Graphs To PDF and browse to the chosen folder.
Figure 7.1
Tables and graphs in PDF format
82
Probe Set Results (pivot table) to TXT
The Export → Export Probe Set Results (pivot table) to TXT
menu item displays a table of probe set results from the CHP files in
the study.
1.
Select Edit → Probe Level Summarizations Report Options.
The Probe Level Summarizations Report Options dialog box
opens (Figure 7.2).
2.
Select the options to be displayed in the probe set results (pivot
table):
- Signal
- Detection p-value (MAS5 and PLIER workflows)
- Detection (MAS5)
- #Pairs (MAS5)
- #Used Pairs (MAS5)
Figure 7.2
Probe Level Summarization Report Options
P-values are only available when using MAS5 summarization for 3'
Expression Arrays and Exon Arrays analyzed at the exon level. No
p-values are available for the gene level analysis irrespective of the
summarization method chosen.
3.
Select Export → Export Probe Set Results.
Chapter 7 | Exporting Data
Figure 7.3
Export Probe Set Results (pivot table) to TXT
83
84
A Report to GCOS RPT File
The Expression Console software is capable of creating GCOS
formatted RPT files for MAS 5.0 CHP files. However, they cannot be
generated by the other applications. The summarization workflows
offered (i.e., RMA and PLIER-based workflows) are created with the
Expression Console software, but not other applications. Use the
Export → Export Report to GCOS RPT File menu item to save the
CHP file information for all checked files in the current study to RPT
files. Each CHP file creates a RPT file in the same directory using the
same base file name.
Figure 7.4
Export Report to GCOS RPT File
A Study to TXT File
To export the Expression Console study table to TXT, right-click on
the study window and select Export Study To TXT.
Figure 7.5
Export Study to TXT
Chapter 7 | Exporting Data
A Table as TXT File
After running an analysis, select Report → View Full Report (or
alternatively, a user customized report selection). When the report
displays (Figure 7.6), right-click on the report and select Export
Table As TXT.
Figure 7.6
Export table as text
85
86
Saving Images
Images of the graphs and plots in Expression Console can be exported
for use in other applications two ways:
• The image can be copied to the clipboard by right-clicking the
image and selecting Copy to clipboard.
• Save the image as PNG by right-clicking on the graph and
selecting Save As PNG (Figure 7.7) or select File → Save As
PNG. The application prompts the user for a name and location
to save the file.
The Expression Console software allows you to save a graph as a
PNG (Portable Network Graphic), which is an image format that
enables the graphic to be easily moved into another application or
document without loss of quality.
Figure 7.7
Save the histogram as a PNG
Chapter
8
Chapter
8
89
This section describes how to identify and modify the controls for 3'
Expression Arrays and how to set user-defined thresholds for both
Exon and 3' Expression Arrays. (Currently, modification of the
controls for Exon Arrays is not supported with the Expression
Console™ software.) For guidance on how to set thresholds for
individual metrics for 3' Expression Arrays, refer to the whitepaper
Data Analysis Fundamentals, and for Exon Arrays, refer to the
whitepaper Exon Arrays Quality Analysis.
Report Controls
To help researchers establish quality control processes for gene
expression analyses, Affymetrix has developed several controls which
allow researchers to monitor assay data quality. These include but are
not limited to:
• hybridization controls
• labeling controls
• internal control genes
• algorithm parameters
• algorithm outputs
The Expression Console software provides functionality to display and
highlight individual metrics and the arrays containing metrics outside
of user-defined tolerances.
In general, Affymetrix highly encourages users to create a running log
of these parameters to monitor quality and potentially flag outlier
samples. The QC functionality built into the Expression Analysis
Software can help with this. Evaluation of particular samples should
be based on the examination of all sample and array performance
metrics in light of the history of the metrics performance in an
individual tissue and array type.
When analysis results are viewed within the software, the metrics for
the report controls are compared to a set of user definable thresholds.
Any results identified as being outside of the selected thresholds are
tagged as Outside of Bounds.
90
For 3' Expression Arrays, the Report Controls window (Figure 8.1)
enables the user to modify the group of probe sets that constitute the
spike and housekeeping controls. The probe array type is selected from
the drop-down menu at the top of the window to modify the control
probes for that probe array type only. The list of control probe sets on
the array are displayed on the left side of the window. By default, the
probe sets are filtered to show only the AFFX controls using the Filter
button at the bottom. The filter is case sensitive.
In order for the report to contain control information, the controls
must be defined before the analysis is run. If additional or different
controls are required, the analysis must be rerun following
redefining the controls.
Figure 8.1
3' IVT Expression Array Report Controls window
Chapter 8 | Controls and Thresholds
91
Spike Controls
The 20x Eukaryotic Hybridization Controls are spiked into the
hybridization cocktail, independent of RNA sample preparation, and
are therefore used to evaluate sample hybridization efficiency on gene
expression arrays. The default spike controls are listed as:
• AFFX-r2-Ec-BioB
• AFFX-r2-Ec-BioC
• AFFX-r2-Ec-BioD
• AFFX-r2-P1-Cre
BioB is at the level of assay sensitivity (1:100,000 complexity ratio)
and should be called Present at least 70% of the time. BioC, BioD,
and cre should always be called Present with increasing signal
values.
Internal Control Genes (Housekeeping Genes)
Internal control genes, or housekeeping genes, are gene transcripts
that are constitutively expressed in most samples. These transcripts
serve as internal controls, are useful for monitoring the quality of the
starting sample, and are subject to any variability in the labeling of the
sample and hybridization to the array, for 3' Expression Arrays. For
Human, Mouse, and Rat 3' Expression Array types, β-actin and
GAPDH are used to assess RNA sample and assay quality.
Specifically, the signal values of the 3' probe sets for actin and
GAPDH are most informative and, therefore, as a general
recommendation, these should be compared to the signal values of the
corresponding 5' probe sets. The ratio of the 3' probe set to the 5'
probe set should generally be less than 3 for the One Cycle Labeling
protocol. For more details on interpreting the housekeeping genes, see
the whitepaper, Data Analysis Fundamentals at www.affymetrix.com.
The Housekeeping controls are:
• GAPDH
• β-Actin
92
Control probe set names are unique to each array design.
Labeling Controls
Poly-A RNA controls can be used to monitor the entire target labeling
process. Each eukaryotic GeneChip probe array contains probe sets
from several B. subtilis genes that are absent in eukaryotic samples (lys,
phe, thr, and dap). These Poly-A RNA controls are in vitro synthesized,
and the polyadenylated transcripts for the B. subtilis genes are
premixed at staggered concentrations. The Poly-A controls can be
spiked into a complex RNA sample, carried through the sample
preparation process, and evaluated like the internal control genes. The
GeneChip® Poly-A RNA Control Kit (P/N 900433) contains the
following four exogenous, premixed control spikes:
• Lys: AFFX-r2-Bs-lys (1:100,000)
• Phe: AFFX-r2-Bs-phe (1:50,000)
• Thr: AFFX-r2-Bs-thr (1:25,000)
• Dap: AFFX-r2-Bs-dap (1:6,667)
All of the Poly-A controls should be called Present with increasing
signal values in the order of lys, phe, thr, dap.
Identify / Remove Controls
Most 3' Expression Array types have a set of controls identified by
default. Follow the steps below to change or set the report controls for
the specified array type.
Select , or Edit → 3’ Expression Report Controls.
The Report Controls dialog box opens (Figure 8.2).
2. Choose the array type from the Select Array Type drop-down
menu at the top of the dialog box.
1.
93
Figure 8.2
3’ Expression Report Controls dialog box
In the probeset box , located on the left side of the window,
highlight a probe set ID and select the 5'> to make that probe set
the 5' probe, M> the middle probe, or 3'> the 3' probe.
The upper box is used to define the spike-in control probe set IDs
and the lower box the housekeeping control probe set IDs. For each
probe set ID, a minimum of two probesets must be identified (for
example, a 3' and a 5').
4. After you identify the 3', M, or 5' probe sets, select the >> button
to load it into the control list.
5. Add or remove probe set IDs by clicking on the appropriate
button
or
.
Control metrics for the probe set IDs contained in the two boxes of
the right hand side of the window will be included in the report
for any subsequent analyses performed.
3.
94
To view all available probesets, clear the text box next to the Filter
button, then click the Filter button. A list of all the probesets on the
array are displayed.
Or, to view specific probeset controls, type the identifying letters
and/or numbers in the text field and click the Filter button.
Delete Report Controls
To delete all of the controls for an array type:
3. In the Report Controls section, select the array type.
4. Click Delete to remove the controls for that array type.
1.
Figure 8.3
Parameter Management Tab - Delete Report Thresholds
95
Report Thresholds
The report thresholds (Figure 8.4) define boundary conditions against
a set of metrics that are computed during the analysis step. These
metrics include report controls and statistics based on probe set Signal
and Detection calls.
Fixed thresholds are applied to selected metrics in one of three ways:
• Compare metric 1 to threshold value – Example, compare the
Percent Present calls to a fixed value to confirm that the metric
passes a minimum or maximum value.
• Compare metric 1 to metric 2 – Example, compare two items,
such as the average signal value for the Cre control against the
average signal value for the bioD control to confirm that the
detected signals are consistent with the known relative abundance
in the sample.
• Compare metric 1 to average of metric 1 across arrays by
standard deviation – Example, compare the spread of values
such as scaling factors across all selected arrays of the same type.
The calculation uses the mean and standard deviation to describe
the spread of the data being compared. The final in-bounds range
is then determined as the mean plus and minus a user defined
factor times the standard deviation times the user selected
multiplier.
96
Figure 8.4
Report Thresholds window
Define / Modify Report Thresholds
The Report Thresholds listed are default settings and can be used
without further modification on the part of the user. Report thresholds
can be changed at anytime and the tables will be dynamically updated
to reflect the changes once they have been saved.
If a desired metric is not present in the report for a 3' Expression
Array type, the 3' Expression Controls must first be modified and
then the analysis must be run.
To set report thresholds:
1.
Select Edit → Report Thresholds,
, or Toolbox →
Configurations → Specify report thresholds to open the
window.
2.
97
Select the comparison type:
- Compare metric 1 to threshold value
- Compare metric 1 to metric 2
- Compare metric 1 to average of metric 1 across arrays by
standard deviation
Select the comparison operator – less than, greater than, or equal
to.
4. Select the item/value/range multiplier to compare to. The
availability of this option is dependent on the comparison type you
selected.
5. To delete a threshold item, click in the box so that it is highlighted
in yellow, then click the Remove button.
3.
To set thresholds for algorithm metrics, a CHP file analyzed with
that algorithm must be loaded into the study window.
If a CHP is not selected, the software is unable to determine the
metrics that are available for thresholding; therefore, one cannot
add thresholds for new metrics without first selecting a CHP file.
Delete Report Thresholds
To delete all of the thresholds for an array type:
3. In the Report Thresholds section, select the array type.
4. Click Delete to remove the report thresholds for that array type.
1.
98
Figure 8.5
Parameter Management Tab - Delete Report Thresholds
Chapter
9
Advanced Analysis
Chapter
9
101
Advanced Analysis
Each analysis workflow provided in the Expression Console™
application has several parameters, which can be changed from the
defaults provided by Affymetrix. This section describes how to change
the parameters and save them to a configuration file. The file is then
saved to the user profile for use in later analyses. Since Exon and 3'
Expression Arrays have different algorithm options available, each
array type has its own custom analysis configuration setup dialog.
Affymetrix
Power
Tools (www.affymetrix.com
Network/Analysis/Affymetrix Power
→ Support/
Tools) is a
command line application that provides the ability to further
customize algorithm parameters and is intended for use by more
experienced users.
Developers’
Affymetrix Power Tools is also available as source code and binaries
for several different computing platforms. These can be found at
the same location on the Affymetrix web site.
3' Expression Array Configurations
This section describes how to set the algorithm parameters for MAS
5.0, PLIER, and RMA for use with 3' Expression Arrays. For
additional information about setting MAS 5.0 parameters, see the
white paper, Data Analysis Fundamentals and the Statistical Algorithms
Reference Guide, on the Affymetrix web site. For explanations of the
different normalization and background correction strategies for
PLIER and RMA, see Appendix A, Algorithms.
102
New Configuration – MAS5
1.
Select Analysis → Advanced Expression Configurations.
The Custom Analysis Algorithms dialog box opens
(Figure 9.1).
Figure 9.1
Advanced Analysis - MAS5 Custom Analysis Algorithms dialog box
2.
Select the MAS5 algorithm and click OK.
The New MAS5 Parameter settings dialog box opens
(Figure 9.2).
Figure 9.2
New MAS5 Parameter Settings dialog box
3.
Select the settings and click Save.
Chapter 9 | Advanced Analysis
4.
103
To change the detection parameters or mask out a subset of probes,
click the Advanced button (Figure 9.2).
The window expands to include Detection Parameters
(Figure 9.3).
Figure 9.3
New MAS5 Parameter Settings - Advanced window
5.
Figure 9.4 Scale to select sets
Enter new parameters and click Save.
The Save window opens.
If you select the Scale to select sets radio button, the Probe Set
MSK file field is highlighted (Figure 9.4). Use the browse button
to find the appropriate MSK file.
104
To create a new MSK file, see Create a New Mask (MSK) File, on page 109.
6.
Enter a name for this collection of parameters (Figure 9.5), and
click Save.
Figure 9.5
Name the new configuration
7.
To analyze array data with the new parameters, select Analysis →
Execute Advanced Configuration, and select the appropriate
file name (Figure 9.6).
105
Figure 9.6
Execute Advanced Configurations
A dialog box opens to announce analysis initiation (Figure 9.7).
Figure 9.7
Advanced Analysis Suffix Identifier
8.
Enter a suffix for run identification, if needed, and click OK to
start the analysis.
106
New Configuration – PLIER Workflow
The Custom Analysis Algorithms dialog box opens (Figure 9.8).
1.
Figure 9.8
Advanced Analysis - PLIER Custom Analysis Algorithms dialog box
2.
Select the PLIER workflow and click OK.
The New custom workflow for PLIER dialog box opens
(Figure 9.9).
Figure 9.9
Advanced Configuration for PLIER
3.
107
Figure 9.10
4.
start the analysis.
New Configuration – RMA
1.
The Custom Analysis Algorithms dialog box opens
2.
Select the RMA algorithm and click OK.
The New custom configuration for RMA dialog box opens
(Figure 9.11).
Figure 9.11
Advanced Configuration for RMA
108
The RMA Background Correction field is automatically applied to the
PM-only probes when selecting RMA.
Since RMA has its own background correction algorithm,
Expression Console software does not allow this to be modified.
3.
Figure 9.12
4.
start the analysis.
Delete an Advanced Configuration
To delete an advanced configuration:
3. In the appropriate Configuration section, select the configuration
to be deleted.
1.
4.
Click Delete.
109
Figure 9.13
Parameter Management Tab - Delete Configuration
MASK Files
Create a New Mask (MSK) File
The Expression Console application uses MSK files for either setting
the scaling factor or excluding or masking certain user-selected probe
pairs from an expression analysis. The Selected Probe Sets scaling
option, in the advanced configuration for the MAS5 algorithm, adjusts
the trimmed mean signal of the selected probe sets on a probe array to
the user-specified target signal value. Expression Console software
utilizes user-selected probe sets (specified by a Scale Factor mask file)
to calculate the trimmed mean signal and derive the scale factor for the
probe array so that Target Signal = Scale Factor x Trimmed Mean
Signal of selected probe sets. For a selected set of arrays, predefined
msk files are available from Affymetrix at www.affymetrix.com. The
other use for mask files is to allow user-selected probe pairs to be
excluded or masked from an expression analysis.
110
For human, mouse, and rat, a list of 100 normalization genes is
provided to the user.
To create a MASK file:
1.
Open Analysis → Advanced Expression Configurations and
select New Mask (MSK) File (Figure 9.14).
Figure 9.14
Advanced Expression Configurations - New Mask File
The Select the Array Type dialog box opens.
Figure 9.15
Select the Array Type
2.
Select the array type and click Open.
Figure 9.16
New MASK (MSK) File dialog box
A window opens displaying all probe sets for that array type
(Figure 9.17).
111
112
Figure 9.17
Probe Sets for Expression Array types
Double-click on the probe sets of interest to highlight each set.
When you click Save, an MSK file of the selected probe sets is
created.
4. Make changes if necessary and click Save, or click the
box to
exit.
3.
Open Mask (MSK) File
Mask Files are specific for the probe array type. The Expression
Console software will not open a mask file that is incompatible with
the currently selected probe array type.
To view or edit an existing MSK file:
1.
Select Analysis → Advanced Expression Configurations →
Open Mask (MSK) File.
The Select the MSK file dialog box opens.
113
Figure 9.18
Select the MSK File dialog box
Select the MSK file and click Open.
The selected MSK file opens displaying the selected probe sets,
which are highlighted.
3. Make changes if necessary and click Save, or click the
box to
exit.
2.
MAS 5.0 CHP Files in the Command Console Format
By default, the Expression Console software stores the MAS5 analysis
results (Signal, Detection, Detection P-Value, # of Probe Pairs, and #
of Probe Pairs Used) in a CHP file using the new Affymetrix
GeneChip® Command Console™ (AGCC) format. This new format
provides additional features such as :
• An embedded unique file identifier
• A copy of the CEL and DAT headers (if the input CEL/DAT files
are also in the AGCC file format)
114
The new file features provide the ability to trace the file’s lineage
independent of the file name.
To configure the software to write the MAS5 CHP files in the
newer AGCC format:
• Select Analysis → Advanced Expression Configurations →
Save MAS5 CHP files in Command Console format
(Figure 9.19).
Figure 9.19
Save MAS5 CHP files in Command Console format
115
MAS 5.0 CHP Files in the GCOS Format
To support the transition of software applications to use the newer
Affymetrix file parsers (those capable of reading both the older CHP
file format as well as the newer AGCC CHP file format), the
Expression Console software has the ability to write the MAS5 analysis
results to the older GCOS format CHP file.
To configure the software to write MAS5 CHP files in the older GCOS
format:
• Select Analysis → Advanced Expression Configurations →
Save MAS5 CHP files in GCOS format (Figure 9.20).
Figure 9.20
Save MAS5 CHP files in GCOS format
For more information about the Command Console (AGCC) file
format and C++/Java parsers that read both GCOS and Command
Console files, see www.affymetrix.com; then go to Support/Developer/
Fusion.
Analysis results from PLIER and RMA cannot be stored in GCOS
formatted CHP files.
116
Advanced Configuration Exon Analysis
1.
Select Analysis → Advanced Exon Configurations. The
Advanced Exon Setup dialog box opens (Figure 9.21).
Figure 9.21
Advanced Exon Setup dialog box
A.
Select the Analysis Type:
The setup box in the upper left-hand corner enables users to
select their analysis level for the configuration. For details on
the different analysis levels, see the whitepaper,
Exon Probeset Annotations and Transcript Cluster Groupings, at
www.affymetrix.com.
In the Expression Console software, each level is paired with a
default set of library files for that analysis and array type
provided by Affymetrix. To use other versions of the probe
group file, the intensity layout file, background probes, or the
117
QC probeset file, browse to the desired file on the appropriate
line of the library file section at the bottom of the screen.
It is always possible to reload the default files for an analysis by
clicking the Default Files button. To use a different metaprobeset file for Gene level analysis or probeset file for Exon
analysis, select Other and browse to the file you wish to use in
the library file section.
B. Select the Summarization Method. For more information
about the summarization methods, see the algorithm details in
Appendix A, Algorithms and the whitepapers, Gene Signal
Estimates from Exon Arrays and Guide to Probe Logarithmic
Intensity Error (PLIER) Estimation, at www.affymetrix.com.
C. Select the Background Correction. For more information
about background correction, see the whitepaper, Exon
Background Correction, at www.affymetrix.com.
D. Select the Normalization Method. For more information
about normalization, see the whitepaper, Gene Signal Estimates
from Exon Arrays, at www.affymetrix.com.
E. Confirm the library file selections in the bottom portion of the
window. Note that selecting a confidence level in the Analysis
Type automatically selects the appropriate file from
Affymetrix.
If you want to use your own custom meta-probeset file or
probelist file, select Other in the analysis level to activate the
browse feature for that line.
2. Click Save.
118
Figure 9.22
Name the new configuration
Enter a name for this configuration (Figure 9.22), and click Save.
4. To analyze array data with the new parameters,
3.
Select Analysis → Execute Advanced Configuration.
B. Select the appropriate file name.
A.
119
Figure 9.23
Execute Advanced Configurations
5.
To delete an advanced configuration:
A.
1) Select the Parameter Management tab (Figure 9.24).
Figure 9.24
Parameter Management Tab - Delete Configuration
2)
In the appropriate Configuration section, select the
configuration to be deleted.
120
3)
Click Delete.
A dialog box opens to announce analysis initiation.
Figure 9.25
B.
start the analysis.
Chapter
10
Analysis Scripts
Chapter
10
123
Analysis Scripts
To streamline the analysis workflow within the Expression Console™
software, the user can create an analysis script. In the analysis script,
the user selects and saves a specific analysis configuration and a set of
graphs and tables that are automatically generated at the end of each
analysis. Expression Console analysis scripts include the following
features:
• Data table can be exported as a tab-delimited TXT file
• Selected graphs and tables can be exported as PDF files
• User-defined scripts are associated with a user profile
Scripts are associated with the user profile open at the time the
script is created.
Analyses that have array-specific details contained within them,
such as custom MAS 5.0 and custom Exon analysis scripts, are
array-type specific.
Analysis Script Creation
To create an analysis script:
1.
Select Analysis → Exon Analysis Scripts → New Script.
The New Script dialog box (Figure 10.1) displays.
124
Figure 10.1
New Script dialog box
Select the workflow from the drop-down menu. If a study is open,
then the analysis methods in the list are restricted by the array type
in the study.
3. Select the table and graphs to be generated after the completion of
the analysis from the relevant sections.
4. If desired, select the probe set results table for a tab-delimited
export of the expression data.
2.
5.
If desired, select the PDF option to save a PDF of the selected
graphs and tables.
The application prompts the user to name the exported files at the
time of the analysis script execution.
Chapter 10 | Analysis Scripts
Analysis Script Deletion
To delete an analysis script:
3. In the Scripts section, select the script to be deleted.
4. Click Delete.
1.
Figure 10.2
Parameter Management Tab - Delete Analysis Script
125
126
Analysis Script Execution
To execute an analysis script:
Open a study.
2. Select the CEL files for analysis.
3. Select Analysis → Execute Analysis Script.
The Select script menu box display (Figure 10.3).
1.
Figure 10.3
Select Script menu
If the script calls for any exports, the user is prompted for names
and locations for saving files.
Appendix
A
Algorithms
Appendix
A
129
Algorithms
This appendix briefly describes the MAS 5.0, RMA, and PLIER
algorithms as they are used to analyze data in the Affymetrix®
Expression Console™ software. References and links to various
publications, which describe the MAS 5.0, RMA, and PLIER
algorithms in detail, are given. See a Comparison of Algorithms for a sideby-side comparison of the assumptions, advantages and disadvantages
of using each individual algorithm.
MAS 5.0 Algorithm
The MAS 5.0 algorithm uses the Tukey’s biweight estimator to
provide a robust mean Signal value and the Wilcoxon’s rank test to
calculate a significance or p-value and Detection call for each probe set.
Background estimation is provided by a weighted average of the
lowest 2% of the feature intensities. Mismatch probes are utilized to
adjust the perfect match (PM) intensity. Linear scaling of the feature
level intensity values, using the trimmed mean, is the default to make
the means equal for all arrays being analyzed.
The MAS 5.0 algorithm (also known as the Statistical Algorithm)
analyzes each array independently. As a result, individual probespecific affinities can not be considered and the ability to detect small
changes between experiment and control samples is reduced in
comparison to either RMA or PLIER.
The primary use of the MAS 5.0 algorithm is to obtain a quick report
regarding the performance of the arrays and to identify any obvious
problems before submitting the final set of arrays to one of the
multichip analysis methods (RMA, PLIER).
For a more detailed description of the MAS 5.0 algorithm, see the
Statistical Algorithms Reference Guide at www.affymetrix.com; then go
to Support/By Support Type/Technical Documentation/Technical Notes/
Software.
130
RMA Algorithm
The Robust Multichip Analysis (RMA) algorithm fits a robust linear
model at the probe level to minimize the effect of probe-specific
affinity differences. This approach:
• Increases sensitivity to small changes between experiment and
control samples.
• Minimizes variance across the dynamic range, but does compress
calculated fold change values.
RMA consists of three steps:
1. Background adjustment
2. Quantile normalization
3. Summarization
This is a multi-chip analysis approach. Therefore, all arrays intended
for comparison should be included together in the summarization
step.
For a more detailed description of the RMA algorithm, see the
publication, Exploration, Normalization, and Summaries of High Density
Oligonucleotide Array Probe Level Data, Biostatistics, April 2003; Vol. 4;
Number 2: 249–264.
Appendix A | Algorithms
131
PLIER Algorithm
The Probe Logarithmic Intensity Error Estimation (PLIER) algorithm
method produces a signal by accounting for experimentally observed
patterns in probe behavior and handling error at the appropriately low
and high signal values.
Similar to RMA, the PLIER algorithm also utilizes data across all
arrays submitted for analysis to minimize the effect of probe-specific
affinity differences. Unlike RMA, which uses a simple global
background correction approach, the standard PLIER configuration
uses a probe-specific background providing a higher degree of
accuracy at the cost of increased signal variance. Exon Arrays use a
more advanced and efficient background correction method utilizing
surrogate mismatched probes. (See the technical note, GeneChip® Exon
Array Design at www.affymetrix.com; then go to Support/By Support
Type/Technical Documentation/Technical Notes/Software). Signal variance
can be addressed following the PLIER analysis by applying a
variance-stabilizing data transformation. A simplistic but effective
approach is to add a value of 16 to each and every signal value
(PLIER + 16 algorithm).
This is a multi-chip analysis approach. Therefore, all arrays intended
for comparison should be included together in the summarization
step.
For a more detailed description of the PLIER algorithm, see Guide to
Probe Logarithmic Intensity Error (PLIER) Estimation at
www.affymetrix.com; then go to Support/By Support Type/Technical
Documentation/Technical Notes/Software).
132
Comparison of Algorithms
Table A.1 A comparison of the MAS5, RMA, and PLIER algorithms used with the Expression Console™
application
Algorithm
MAS5
RMA
PLIER
Advantages
Disadvantages
Single-array algorithm is
independent of other data in the
dataset:
Not as sensitive as either RMA
or PLIER to small changes in
target abundance:
•
•
Less computationally intensive
than RMA or PLIER
•
Conservative
•
Smooth downweighting of
outliers
•
Positive output values
•
Minimal bias
Minimizes the variance seen
across the arrays
•
Higher reproducibility of signal over
singlearray analyses
•
Good differential change detection
•
Variance stable on log scale
Assumptions
Limited ability to adjust for probe
specific affinity
differences
•
Singlearray analysis
•
Multiplicative error
•
Unstable variance at low end
•
•
Lower precision in signal
calculation
Signal is adjusted by the mis
match probes (PM–MM)
•
Background imputed to handle
negative differences
•
Multiplearray analysis
•
Multiplicative error
•
PM (perfect match) only
•
Single background is used to
adjust each intensity
Not as sensitive as PLIER in
the ability to detect small fold
changes
•
In cases where feature intensities
disagree, may have more than one
solution (mitigated by median pol
ish)
•
Positive bias contributed to signal
values
•
Compresses fold changes for low
intensity probe sets
Ability to detect small fold
changes:
More variance in indivdual
signals than seen with RMA
•
Higher reproducibility of signal
(lower coefficient of variation) with
out loss of accuracy
•
Computationally intensive
•
Multiplearray analysis
•
Mixed error model PM–MM,
PM only, etc.
Higher sensitivity to changes in
abundance for targets near back
ground
In cases where feature intensities
disagree, may have more than one
solution (mitigated by median
polish)
•
•
•
Multiple background options
•
Performance relative to amount of
model data provided
•
Smoothly handles intensities
below background
•
Variance not stable on log scale
•
Dynamic weighting of the most
informative probes in an experi
ment to determine signal
•
High degree of accuracy for
signal and fold change calculations
•
Lack of bias
Appendix
B
Appendix
B
135
The Expression Console™ software displays column headings for
individual parameters and outputs in the Report window (Report →
View Full Report), for each of the 3' Expression Array algorithms
(MAS 5.0, RMA, PLIER). This appendix gives column heading
definitions.
MAS 5.0 Column Headings
Threshold Test
The minimum number of probe pairs a probe set
must have in order for the probe set data to be
included in the calculation of the report statistics
HZ
Number of horizontal zones used in background
subtraction
VZ
Number of vertical zones used in background
subtraction
BG
Minimum, maximum, average, and standard
deviation of the background intensity calculated
for the probe array
Alpha1 (α1)
Alpha2 (α2)
Tau (τ)
Noise (Raw Q)
Significance level for the detection p-value in an
analysis – Alpha1 is a user-modifiable parameter
that is set in the New MAS5 Parameter Settings
dialog box (Analysis → Advanced Expression
Configurations → New Configuration).
If the probe set detection p-value is < alpha1, the
call is present.
Second significance level for the detection p-value
in an analysis and a user-modifiable parameter set
in the same dialog box as Alpha1 – If the probe set
detection p-value is greater than or equal to
alpha2, the call is absent. If alpha1 is less than or
equal to the Detection p-value < alpha2, the call is
marginal.
A user-modifiable parameter, ideally set to a value
that is a little larger than the median of the
discrimination scores of the probe sets whose
targets are absent, to avoid false detected calls
The degree of pixel-to-pixel variation among the
probe cells used to calculate the background
136
Scale Factor (SF)
The scale factor specified in the Scaling tab of the
Expression Analysis Settings dialog box or
computed by the algorithm
Norm Factor (NF)
The normalization factor computed by the
algorithm
Scale Mask
Contains the name and location of the mask file if
one was used for the analysis
RawQ
Baseline Noise – The degree of pixel-to-pixel
variation among the probe cells used to calculate
the background in the baseline probe array
BG Avg
The average background intensity calculated for
the probe array
BG Std
The standard deviation background intensity
calculated for the probe array
BG Max
The maximum background intensity calculated for
the probe array
BG Min
The minimum background intensity calculated for
the probe array
Noise Avg
The average noise calculated for the probe array
Noise Std
The standard deviation noise calculated for the
probe array
Noise Max
The maximum noise calculated for the probe array
Noise Min
The minimum noise calculated for the probe array
Corner+ Avg
The average cell intensity for the sense probe cells
used in the grid alignment process
Corner+ Count
The corner count for the sense probe cells used in
the grid alignment process
Corner- Avg
The average cell intensity of the antisense probe
cells used in the grid alignment process
Corner- Count
The corner count of the antisense probe cells used
in the grid alignment process
Central- Avg
The average cell intensity for the nine probe cells
that comprise the cross at the center of an
antisense probe array
Central- Count
The central count for the nine probe cells that
comprise the cross at the center of an antisense
probe array
Appendix B | Algorithm Parameters and Outputs
137
#Probe Sets Exceeding
Probe Pair Threshold
The number of probe sets that exceed the probe
pair threshold
Probe Pair Threshold
The minimum number of probe pairs a probe set
must have in order for the probe set data to be
included in the calculation of the report statistics
Control Direction
The direction (sense or antisense) of the target
(sample)
#P
The number of probe sets present
%P
The percent of probe sets present
Signal(P)
The average signal for the probe sets defined as
present
#M
The number of probe sets whose detection is
marginal
%M
The percent of probe sets whose detection call is
marginal
Signal(M)
The average signal for probe sets whose detection
call is marginal
#A
The number of probe sets absent
%A
The percent of probe sets called present that are
absent
Signal(A)
absent
Signal(All)
The average signal for all probe sets on the array
138
Column Headings for RMA and PLIER
Metrics are dependent on the analysis algorithm, array type, and the
level; therefore, not all of the metrics are always present in the Full
Report.
Raw Corner+ Avg
The average cell intensity prior to any background for
the sense probe cells adjustment used in the grid
alignment process
Raw Corner+ Count
The count of the number of sense probe cells used in
grid alignment
Raw Corner- Avg
The average cell intensity prior to any background for
the antisense probe cells adjustment used in the grid
alignment process
Raw Corner- Count
The count of the number of antisense probe cells used
in grid alignment
Raw Central- Avg
The average cell intensity before background
adjustment for the sense probe cells that comprise the
cross at the center of the array
Raw Central- Count
The average cell intensity before background
adjustment for the anti sense probe cells that
comprise the cross at the center of the array
Spike-probeID-signal
The signal for the probe sets that correspond to the
labeling and hybridization spike controls
pm_mean
The mean signal for all of the PM probes on the array
mm_mean
The mean signal for all of the MM probes on the array
bgrd_mean
The average signal for the probes used to calculate
the background
Appendix B | Algorithm Parameters and Outputs
139
pos_vs_neg_auc
The area under the curve (AUC) for a
receiver operator curve (ROC) comparing the intron
controls to the exon controls by applying a threshold
to the probe set summary – The ROC curve is
generated by evaluating how well the probe set
summary separates the positive controls from the
negative controls (e.g., exon from intron). The
assumption (which is only valid in part) is that the
negative controls are a measure of false positives and
the positive controls are a measure of true positives.
An AUC of 1 reflects perfect separation whereas as an
AUC value of 0.5 would reflect no separation. Note
that the AUC of the ROC curve is equivalent to a rank
sum statistic used to test for differences in the center
of two distributions.
Signal(A)
absent (Exon-level DABG > 0.01)
Signal(All)
apt-opt-cdf-file
The cdf file used for the analysis
apt-opt-probe-count
The total number of probes (features) on the array
whether or not the probes are used
apt-opt-qc-groups-file
The file used to identify the control probe and probe
sets for the analysis run
#P
The number of probe sets present – For exon level
analysis, the DABG p-value is less than or equal to
0.01.
%P
The percent of probe sets present – For the exon level
analysis, this is defined as the DABG probe level pvalue is less than or equal to 0.01.
Signal(P)
present (Exon-level DABG <= 0.01)
#A
The number of probe sets absent – For exon level
analysis, the DABG p-value is greater than 0.01.
%A
The percent of probe sets called present absent – For
exon level analysis, the DABG p-value is greater than
0.01.
Signal(A)
absent (Exon-level DABG > 0.01)
Signal(All)
140
Probe Set Suffixes
A number of standard metrics are created for particular groups of
probe sets on the arrays. The key below describes the suffix and
descriptions for the following groups of probe sets:
- all_probeset (all probe sets within a group)
- bac_spike (bacterial spikes and hybridization controls)
- polya_spike (labeling controls)
- neg_control (negative control probes)
- pos_control (positive control probes)
KEY: Suffix for Output Definition
_probesets
From the QC Report, the number of probe sets actually analyzed
_atoms
From the QC Report, the number of PM–MM or PM–GCBG probe pairs
actually analyzed
_mean
The mean signal value for all the probe sets
_stdev
The standard deviation for all of the probe sets analyzed
_mad_residual_mean
The mean absolute deviation of the residual for a chip versus all chips in
the data set
_mad_residual_stdev
The standard deviation of the residual for a chip versus all chips in the data
set
_rle_mean
The mean absolute relative log expression (RLE) – This metric is generated
by taking the probe set summary for a given chip and calculating the
difference in log base 2 from the median value of that probeset over all the
chips. The mean is then computed from the absolute RLE for all the probe
sets for a given CEL file.
_rle_stdev
The standard deviation of the relative log expression (RLE) – This metric is
generated by taking the probe set summary for a given chip and calculating
the difference in log base 2 from the median value of that probeset over all
the chips. The standard deviation is then computed from the absolute RLE
for all the probe sets for a given CEL file.
_percent_called
The percent of probe sets from the exon analysis called present (DABG <= 0.01)
Index
143
Index
Symbols
% Present call 53
Numerics
3’ Expression Array 3
configurations 101
probe set names 92
Expression Console™
advanced analysis 37, 101
analysis controls 35
analysis results 38
configuration file 101
controls only analysis 45
create profile 14
custom report 56
data analysis 36
delete profile 16
detection 54
Exon level analysis 43
exporting data 81
full report 55
Gene level analysis 43
graphs 58
hardware requirements 13
hybridization 46
installation instructions 13
library files 17
report metrics 56, 71
reports 54
signal 54
software requirements 13
specify controls 43
status window 38
study window 26
workflow 5
control probes
negative 140
positive 140
controls 35, 89
correlation matrices 4
custom report
delete 57
A
advanced configuration
delete 108
advanced expression configuration 37
AFFX controls 90
Affymetrix Power Tools 101
Affymetrix technical support
10
D
DABG 140
Data Transfer Tool 13
detection
calls 95
p-values 66, 68
documentation
conventions used 7
AGCC software 13, 29
algorithm
comparisons 132
parameters and outputs 135
algorithm parameters 53, 89
algorithms 35
analysis results 38
array metrics 25
B
bacterial spikes 140
b-Actin 91
box plot 61
relative signal 64, 65
signal 64
box plots 58
C
CEL files 3
CHP files 3
confidence levels 44, 46
control
metrics 53, 93
DTT 25
E
Exon Array 3, 43
advanced configuration 116
background correction 117
human 43
mouse 43
normalization method 117
rat 43
summarization method 117
F
FAQS 7
Exon level analysis 43
all 45
core 45
extended 45
full 45
exporting
data 81
probe set results to TXT 82
report to GCOS RPT file 84
study to TXT file 84
table as TXT file 85
tables and graphs to PDF 81
expression analysis scripts 37
expression arrays 3
G
GAPDH 91
GCOS
library file directory 17
RPT files 84
software 13, 29
gene expression 53
Gene level analysis 43
core 43, 44
extended 44
full 44
GeneChip® 3
144
global array metrics 53
graphs 53
grid evaluation 58
parameter settings 102
new 110
open 112
scale factor 109
heat maps 4, 58, 67
housekeeping controls 53, 71,
90
hybridization 3, 36
controls 53
multi-chip analysis 131
MvA plot 4, 65
identify controls 92
interanl control genes 91
internal control genes 89
NetAffx
account 18
account information dialog
box 19
library files 20
L
labeling controls 53, 89
library files 30
copying files manually 21
custom 43
default location 17
download 17
error message 30
folder 17
NetAffx 17
set library path 30
line graphs 58, 71
linear correlations 66
linear scale 25
log scale 25
probe list 72
probe set suffixes 140
profile information dialog box
16
p-values 82
concordance 68
N
I
intensity files 25
summarization files 25
summarization report options 82
create 109
metrics 53
MSK file
H
probe level
MASK files
Q
QC 3
analysis 49
functionality 89
metrics 54
O
R
outliers 3, 49, 58
outside of bounds 89
relative probe cell intensity 62
report
controls 89, 90
define and modify thresholds 96
delete controls 94
delete thresholds 97
metrics 71
thresholds 95
P
parameter management 94,
119
Pearson’s Correlation (detection p-value) 68
Pearson’s correlation (Signal)
67
RLE 140
RMA 25, 35, 101, 130
background correction algorithm 108
column headings 138
configurations 107
sketch workflow 46
Pearson’s test 66
PLIER 25, 35, 54, 101, 131
column headings 138
configurations 106
relative log expression 140
M
MAS 5.0 25, 35, 101, 129
background estimation 129
CHP files in AGCC format
113
CHP files in GCOS format
115
column headings 135
configuration 102
PM–GCBG 140
PM–MM 140
PNG
format 59
images 86
Poly-A RNA controls 92
probe cell intensity view 58
S
screen captures 8
signal
distributions 4
estimates 66
histogram 60
Index
Spearman Rank Correlation
detection p-value 70
signal 69
Spearman test 66
spike controls 90, 91
study window
file consolidation 31
sort order for tables and
graphs 31
suffix 47, 105
T
technical support 10
threshold 39, 89
boundaries 49
test 55
U
user profile management tab
16
145
146

Affymetrix Expression Console Software v1.0

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