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Agilent Feature Extraction Software (v9.5)

Quick Start Guide

What is Agilent Feature Extraction software?

Agilent Feature Extraction software extracts microarray scan data for images produced in three different situations:

• Agilent microarrays scanned on an Agilent scanner

• Agilent microarrays scanned on a GenePix scanner

• Non-Agilent microarrays scanned on an Agilent scanner

Feature Extraction handles each of these images differently. These differences are described throughout the documentation.

If you are a new user...

Read “Find out more about...” on page 3.

“Start the software” on page 9.

If you intend to analyze...

Saved Agilent images, read the “Instructional overview” on page 12, and go to “Exercise 1: Set up and run batch extractions on Agilent images” on page 17.

Saved non-Agilent images, read the “Instructional overview” on page 12, and go to “Exercise 2: Set up and run batch extractions on non-Agilent images” on page 26.

Agilent images in real time, read the “Instructional overview” on page 29, and go to “Exercise 3: Set up to run batch extractions on Agilent images in real time” on page 30.

Agilent Technologies

If you are an experienced user...

Read “What’s new in version 9.5?” on page 4.

Perform the exercises again if you want to sharpen your skills.

If you want to download updates, go to this website...

Agilent provides current protocols, grid templates and QC metric sets with the software. These sets are installed when the Feature Extraction software is installed.

If you want to download any other available protocols, grid templates or QC metric sets, including updates, go to these Agilent websites.

For FE protocols: http://www.agilent.com/chem/feprotocols

For grid templates: http://www.agilent.com/chem/designfile

For QC metrics: http://www.agilent.com/chem/feqcmetrics

For monthly updates on Agilent products, subscribe to E-Notes: http://www.agilent.com/chem/registration

2 Agilent Feature Extraction Software (v9.5) Quick Start Guide

http://www.agilent.com/chem/feprotocols

http://www.agilent.com/chem/designfile

http://www.agilent.com/chem/feqcmetrics

http://www.agilent.com/chem/registration

Find out more about...

...How to use the software

See the User Guide for detailed instructions for using the Feature Extraction software and for definitions and explanations of the fields and buttons on the screen.

Chapter 1– Working with Feature Extraction

Chapter 2– Extracting Microarrays Automatically

Chapter 3– Creating Grid Files and Templates

Chapter 4– Changing Protocol Settings

Chapter 5– Changing Image Displays

...Details about protocols, QC reporting, results and algorithms

See the Reference Guide for detailed information on protocols, results, reports and algorithms.

Chapter 1 – Default Protocol Settings

Chapter 2 – QC Report Results

Chapter 3 – Text File Parameters and Results

Chapter 4 – MAGE-ML Results

Chapter 5 – How Algorithms Calculate Results

Chapter 6 – Command Line Feature Extraction

...How to monitor QC metrics and do run charting

See the QC Chart Tool instructions for help with creating QC metrics to associate with FE protocols and with producing QC charts for your data.

To get a copy of the QC Chart tool and its user guide, go to the Web site:


For more information on the QC Chart Tool, see “Using the QC Chart Tool to monitor QC metrics (statistics) and do run charting” on page 14.

See Chapter 2, “Extracting Microarrays Automatically” in the User Guide to learn how to import QC metric sets and use them with your extraction.

Agilent Feature Extraction Software (v9.5) Quick Start Guide 3


What’s new in version 9.5?

Overview

Agilent Feature Extraction (FE) Version 9.5 includes the following new and changed functions:

• Ability to extract Agilent micro RNA (miRNA) microarrays

• Improved gridding and workflow for multiplex (formerly known as multipack) microarrays

• Updated FE protocols for all formats

• New and changed algorithms

• Ability to view (not extract) additional third-party images

• QC Report as pdf file

• Ability to pick whole chromosomes in the Dye Norm Editor

• Ability to save regular expression searches in the Dye Norm Editor

• Ability to load and remove Dye Norm files from the database via FENoWindows (command line version of FE)

• Three QC metric sets available with the FE software: CGH, GE 1-color and GE 2-color (with thresholds)

• QC report changes for CGH

• Automatic notice of new versions of FE

See the Release Notes installed with the Feature Extraction software for more information on version 9.5 (Start > Programs > Agilent > Feature Extraction > Documentation > Release Notes).

Functions added

Feature Extraction for microRNA (miRNA) microarrays

The miRNA analysis is still in development; check the Agilent website for the latest information.

Here is a list of FE functions added to support the extraction of Agilent miRNA microarrays, which are in the 8-pack microarray format:

• New FE protocol


The protocol takes advantage of the eXtended Dynamic Range function (XDR) of the Agilent scanner and Feature Extraction. The overall signal of miRNA images varies significantly from gene to gene, requiring scans with a high dynamic range. Because the GenePix Scanner does not support dynamic ranges above 4 orders of magnitude, GenePix scans are not really appropriate for this assay. You can download this protocol by going to http://www.agilent.com/chem/feprotocols.

See the MicroRNA Analysis System Guide for more information on how to change the default values for this protocol.

New algorithms have been added to the protocol to work with some of miRNA microarray analysis requirements.

See Chapter 5, “How Algorithms Calculate Results” in the Reference Guide for information on how they work.

• New output file

An miRNA feature extraction generates a new type of output file, called the GeneView file, and entitles it BarCode_Snn_pack_GeneView.txt. This file gives a list of all the miRNA genes, plus the new data output for each gene.

See Chapter 3, “Text File Parameters and Results” in the Reference Guide for the new data output for microRNA analysis.

• New QC Report

This report borrows metrics from the current 1-color gene expression QC reports.

See Chapter 2, “QC Report Results” in the Reference Guide for an example of the MicroRNA Analysis QC report.

Ability to view (not extract) additional third-party images

With FE 9.1 and FE 9.5 you can both view and extract the images from the GenePix 4000 A and 4000B scanners. With FE 9.5 only you can now view, but not extract, images (16 bit Tiff, either 1 or 2 channels) from these scanners:

Packard

InnopSys

GenePix other than 4000A and 4000B

Tecan

Also, FE 9.5 adds to its support of the Little Endian scan format with support for the Big Endian scan format.


http://www.agilent.com/chem/feprotocols

QC report as pdf file

At present, the QC report is generated as an html file linked from the onscreen Project Run Summary report. With a full license for FE 9.5 the QC report is generated as only a pdf file. The pdf file is linked from the Project Run Summary report. If you are using a demo license, the QC Report is in only html format.

Dye Norm Editor enhancements

• Ability to pick whole chromosomes in the Dye Norm Editor

• Ability to save regular expression searches in the Dye Norm Editor

See Chapter 2, “Extracting Microarrays Automatically” in the User Guide to learn how to use these functions to create a dye norm list.

Dye Norm File enhancements

• Easier loading of a Dye Norm file from the grid template properties page

See Chapter 2, “Extracting Microarrays Automatically” in the User Guide to learn how to load a dye norm list.

• Ability to load a dye norm list and associate it with a grid template via FENoWindows (command line version of FE)

• Ability to remove a Dye Norm list from the database via FENoWindows

See Chapter 6, “Command Line Feature Extraction” in the Reference Guide to learn how to initiate these functions via the command line.

Three QC metric sets available with the FE software

CGH, GE 1-color and GE 2-color metric sets are available with the FE 9.5 version of software. Only the GE 2-color metric set has thresholds.

You can also go to this website to download other metric sets: http://www.agilent.com/chem/feqcmetrics.

Automatic notice of new versions of FE

• Message letting you know if a new version is available for download and if your license entitles you to use the new version or not

• Message letting you know how to purchase the software upgrade if your license does not entitle you to use the new version




New algorithms

MicroRNAAnalysis

Calculates the TotalGeneSignal and TotalGeneSigError for miRNA microarrays

Q-test More accurately flags population outliers when the number of replicates is below the minimum population specified

Robust NegC_SD Calculates a standard deviation (SD) that is more robust to outliers, using an algorithm similar to the IQR algorithm

Enhanced Grid Testing

Results in fewer false negative and false positive warnings for gridding because Agilent has added more metrics to test for gridding accuracy

See Chapter 5, “How Algorithms Calculate Results”, in the Reference Guide to learn how the algorithms work.

Functions changed

Improved gridding and workflow for multiplex microarrays

• Improvement of the evaluation of the rotation of the slide in the slide holder

A new algorithm has been written that is more robust in the presence of noisy faint data and missing packs than the old algorithm.

• Recognition of missing packs and appropriate identification

• Pack labeling based on absolute location on glass slide

Updated FE protocols for all formats

• New gridding failure criteria

A default maximum difference between the grid centroid and the spot centroid is specified in the Calculate Metrics part of the protocol but is not visible nor changeable.

• Outlier rejection method for pixels and statistical method for spot value from pixels are independent of format, now visible and changeable in the Find Spots part of the protocol.

• New Q-test option for population outlers

• New robust method for calculating the standard deviation of the negative controls


See Chapter 4, “Changing Protocol Settings”, in the User Guide to learn more about the changes made to the protocols, and see Chapter 5, “How Algorithms Calculate Results” in the Reference Guide to learn how the changes affect the calculations.

Improved QC reporting for CGH

Wording changes for “amplified” and “deleted” have been implemented in the QC Report for CGH version 9.5. They are now “positive” and “negative”, respectively.

See Chapter 2, “QC Report Results” in the Reference Guide to view an example of the new CGH QC report.

Data migration change

If you install FE 9.5 on top of FE 9.1 or earlier versions, FE 9.5 offers these options:

• Update existing database to FE 9.5

This updates the existing design files in your current FE database. It installs new protocols and metric sets. This does not remove your modified protocols but does remove old FE 9.1 protocols. This option does NOT install the high density grid templates.

• Replace database with FE 9.5

This option installs the design files for the Agilent High Density (HD) microarrays, new protocols and metric sets. This removes all old protocols and grid templates by replacing the database.

Changes to text parameter and result files

See Chapter 3, “Text File Parameters and Results”, in the Reference Guide for the changes to the text parameter and result files.

New parameters in FEPARAMS table For descriptions of these parameters see “Parameters/options (FEPARAMS)” on page 113 of the Reference Guide.

New results in STATS table For descriptions of these statistical results, see “Statistical results (STATS)” on page 133 of the Reference Guide.

New results in FEATURES table For descriptions of these feature results, see “Feature results (FEATURES)” on page 147 of the Reference Guide.


Start the software

Start the software to set up batch extraction

• Double-click the Feature Extraction shortcut on your desktop.

This shortcut was created when you installed the Feature Extraction software application.

Figure 1 Feature Extraction software window with integrated Project Work Window

Note that the Feature Extraction software automatically creates the first project for you. Every time you start the software the Project 1 Work Window appears with an empty Extraction Configuration tab window.

Project Toolbar

Grid Template Browser Pane

FE Protocol Browser Pane

Project Explorer Pane

QC Metric Set Browser Pane


When you click the Project Properties tab, the following defaults appear for Project Properties.

Figure 2 Default project properties when the software is started

You can run Feature Extraction for one or more Agilent images scanned with either an Agilent scanner or with a GenePix 4000A or 4000B scanner with these default project properties. You can also set up and run multiple extractions with a combination of Agilent and non-Agilent images.

You can change where and how the Project Work Window and the Browser panes appear when you start the software. To learn more about working with this interface, see “Move windows and panes” on page 240 in the User Guide.

To learn more about setting up and running batch extractions with the Project Work Window, see the exercises in this guide.

NOTE If you do not see the QC Metric Set Browser when you first open the Feature Extraction software, select View > QC Metric Set Browser.


Start the software to view the image or set up a grid

• Drag and drop an image file to the Feature Extraction shortcut on your desktop.

The Feature Extraction (FE) software window appears with the dropped image. Note that the Project window does not open when FE is opened this way.

Figure 3 Feature Extraction software window with separated Image Work Window

To learn more about interactively finding spots and creating grid files for the image, see Chapter 3, “Creating Grid Files and Templates”, in the User Guide.

To learn more about changing the image display, see Chapter 5, “Changing Image Displays”, in the User Guide.

Image Toolbar

Work AreaImage Window


Set up and extract saved Agilent and non-Agilent image files

Instructional overview

Follow the roadmap below and the instructions on the next pages to get started.

See Chapter 2, “Extracting Microarrays Automatically” in the User Guide for details on this task.

Agilent Feature Extraction software works with the three image types (see page 1) using projects, extraction sets, grid templates or files, and protocols. The images to be run are organized into extraction sets containing the grid and method information to run the extraction. The extraction sets, in turn, are organized into projects.

Figure 4 Project Explorer with expanded extraction sets

Start Here!

Set up and run saved

image files

3 Add image files (extraction sets)

1 Create a “standard” project

2 Enter project properties

4 Check extraction configuration and save project

5 Add/change grid files/ templates (optional)

7 Run the extraction sets in the project

6 Add/change protocols (optional)

Extraction Set

Image File

Grid Template

Protocol

Grid File

12
Agilent F eature Extraction Software (v9.5) Quick Start Guide

project A grouping of information that contains one or more extraction sets and on which Feature Extraction is run as a whole.

extraction set Each grouping of microarray image file, grid template (or file) and protocol within a project. This term is used both before and after extraction.

grid template Grid information from Agilent design files or grid files stored in the database. A grid template includes feature annotation and general geometry about the microarray (number of rows, columns, subgrids, feature spacings), which is used to locate spots before data analysis takes place. Although not specific to any microarray image, grid template information is usually applied to the image for which the template was designed.

grid file You can also extract using a grid file, which has the nominal and “found” spot locations specific to the image from which the grid file was created. Grid files are created in the Feature Extraction program using a microarray image file and a microarray layout file (such as an Agilent Design file or a .GAL [GenePix Array Layout] file). The grid file contains the locations of the “found” spots if the “Calculate spot size and centroids” option is run before the file is saved. You can create grid templates from grid files, although the templates have only geometry information, no found spots.

protocol A list of steps and parameter values that define the data analysis algorithms and calculations used for Feature Extraction of a microarray image or images.

Setting up extraction sets

Every extraction set must have an image file, assigned grid template/grid file and protocol. Grid templates and protocols may be loaded automatically by the software. If they are not, or you want to use a different grid template or protocol, you can add them manually. Any extraction set that is missing one of the three components--tiff, grid template/grid file, or protocol--shows a red X in Project Explorer and a ? in the icon of the missing component and cannot be extracted.

Assigning a grid template/grid file to the extraction set

AMADID is the Agilent MicroArray

Design IDentifier.

Agilent microarray images scanned with an Agilent scanner For Agilent microarray images with barcode information embedded in the image, the software selects a grid template from the database using a portion of the barcode (AMADID) associated with the image file. If there are grid templates for that AMADID that come from multiple design files, the software selects the grid template from the most recently created Agilent design file. To add a barcode if the image has none, see “Add or change the New Array Identifier” on page 211 in the User Guide.


For this automated assignment to work, the grid template must exist in the database. If it does not, you can interactively associate a grid file with the extraction set.

Agilent uses barcode information for many workflow automation and data tracking steps. Inclusion of the Agilent barcode information in the TIFF file helps ensure that the data is quickly, easily and accurately processed and that informatics programs know how to analyze the results.

Non-Agilent images scanned with an Agilent scanner Non-Agilent image files from an Agilent scanner do not point to grid information that Agilent Feature Extraction software can read. Therefore, when you load the non-Agilent image file, you must add a grid file that you have already created.

See Chapter 3, “Creating Grid Files and Templates”, in the User Guide to learn how to create grid files.

Assigning a protocol to the extraction set

Automatically added protocols can come from two sources: a default protocol associated with the grid template, or a project level default protocol. You can select which of these options has the higher priority. You can also add a protocol to the extraction set manually.

Feature Extraction can run analyses on Agilent 1-color gene expression (GE), 2-color GE and CGH microarray images, whether scanned on an Agilent scanner or a GenePix scanner, and on non-Agilent 2-color GE images scanned on an Agilent scanner. Agilent includes FE protocols for extracting each of these image types.

Using the QC Chart Tool to monitor QC metrics (statistics) and do run charting

At times Feature Extraction software is used in a production environment, where the microarray processing protocols are similar and monitoring run-to-run consistency is an important goal. Feature Extraction can help monitor this consistency with the optional QC Chart Tool.

The QC Chart Tool lets you:

CAUTION These FE Protocols were optimized using data from Agilent catalog arrays, which have many replicated probes and validated Negative Control probes. If custom arrays without enough replicated probes are used, or arrays with custom probes designated as Negative Control probes are used, the default FE Protocols may not be optimal.


• Import summary statistics from a large number of extracted Agilent microarrays

• Import additional annotation information for each barcoded microarray

• Query the database to filter the imported data

• Generate a metric set that can use existing FE metrics (statistics) as well as metrics mathematically derived from FE metrics

• Graphically display the metrics as applied to imported microarray data sets

• Set upper, lower and range value thresholds for each metric

• Name, save, export and import sets of metrics.

For more information on the QC Chart Tool and where to download the software and the instructions for use, see “...How to monitor QC metrics and do run charting” on page 3.

QC metric sets Three metric sets are loaded into the database with FE 9.5. Check the web at http://www.agilent.com/chem/feqcmetrics for updates of metric sets and thresholds.

You can also create QC metric sets with the QC Chart tool and then import and associate the metric sets with FE projects. Only one metric set can be assigned to an FE project. When that project is run, the Feature Extraction program summarizes the metric statistics on each microarray’s QC report and shows if the thresholds (if any) were exceeded. It also reports this information in the output files in the statistics table.

For example, if a value for a selected result is outside the range of values specified in the QC metric set associated with the FE project, you get a message in the QC Report that says “Out-of-Range” for that metric.

See “Exercise 3: Set up to run batch extractions on Agilent images in real time” on page 30 to learn how to associate a QC metric set with an FE project.

Run charting In addition, at the end of the project, an optional QC chart can be opened to display graphically what the results are for each metric for each microarray.

An example of this chart is displayed below, highlighting how it can be used to identify microarrays that warrant further investigation. See Figure 5.

Even if no thresholds are present in the metric set, viewing how each extraction performs relative to the other extractions in the FE project for each metric on the QC chart is still very useful.




l

Figure 5 QC chart created for 78 extractions using a QC Metric Set with thresholds (green). Extraction values within range are blue. Those outside the range are red.


Exercise 1: Set up and run batch extractions on Agilent images

The following exercise takes you through the setup, analysis and reporting steps of Feature Extraction with multiple Agilent images that are installed with the software. See “Set up to install the example images” on page 4 of the Agilent Feature Extraction Installation Guide.

With this exercise you learn how to:

• set up and extract a 244k CGH image and produce a CGH QC report

• set up and extract eXtended Dynamic Range (XDR) paired images and produce a 1-color gene expression QC report with spike-ins

• set up and extract high density (244k) multiplex images as one image and produce one shape file and four QC reports with spike-ins on four images.

Try the Steps on the left before reading the Detailed Instructions.

Task 1: Add an extraction set for a CGH image.

Steps Detailed Instructions Comments

1 Add the Human_244k_CGH example image file installed with the FE software to Project 1.

a Click the Add Extraction Set(s) button on the toolbar, or right-click Project Explorer, and select Add Extraction....

b Select the Human_244k _CGH file, and click Open.

c Expand the extraction set that appears in Project Explorer.

• Note that usually the software automatically assigns a grid template to the extraction set if the image contains a valid Agilent microarray barcode.

• This example Agilent image requires that you create a grid template from its design file.

• You must assign both a grid template and a protocol to this extraction set.

2 Create a grid template from the design file and assign it to the extraction set.• The design file for this image is

14584_D_20060509.xml.

a Right-click the Grid Template Browser, and click Add.

b Go to the directory where the image file is stored.

c Select the design file (14584_D_20060509.xml), and click Open.

d Drag and drop the new grid template to the first row after the image name.

•


3 Assign the CGH FE protocol to the extraction set.

• From the FE Protocol Browser, drag and drop the CGH_v4_95 FE protocol onto the CGH extraction set.

• You can download _91 FE protocols to the Browser if you want to compare data run on v9.1 vs v9.5. These protocols use v9.5 image processing and v9.1 data processing during extraction.

• Download from the web site, http://www.agilent.com/chem/ feqcmetrics

Task 1: Add an extraction set for a CGH image.


Task 2: Add an extraction set for two 1-color gene expression (GE1) images produced with XDR range turned on.


1 Add the Rat_44k_1color_ expression example image file installed with the FE software to Project 1.• Note that this is a set of XDR

paired images. You only need to add one; the other is automatically added.


b Select the first Rat_44k_1color_ expression file, and click Open.


• Version 9.5 lets you extract images produced with the scanner extended dynamic range (XDR) turned on. The “H” image was scanned with a PMT gain of 100%, and the “L”, with a gain of 10%.

• Note that by default, the software automatically adds both the high and low XDR files to the extraction set even though you selected one.

• You must assign a protocol to this extraction set, at least initially.

2 Create a grid template from the design file and assign it to the extraction set.• The design file for this image is

013162_D_F_20070207.xml.



c Select the design file (013162_D_F_20070207.xml), and click Open.




3 Assign the GE1-v5 FE protocol to the extraction set.

• From the FE Protocol Browser, drag and drop the GE1_v5_95_Feb07 FE protocol onto the extraction set.

• You can enter a default protocol in the FE Grid Template Properties dialog box for future extractions.

• When the image and grid template are loaded to produce the extraction set, the default protocol is automatically loaded as well.

• The 1-color GE protocol for this extraction set has spike-ins turned on as the default setting. The QC report will show all the spike-in results.

Task 2: Add an extraction set for two 1-color gene expression (GE1) images produced with XDR range turned on.


Task 3: Add an extraction set for a 4x44k GE1 multiplex (4) and review the extraction configuration for the batch.


1 Add the Human 4x44k_1color_ expression multiplex image file installed with the FE software to Project 1.


b Select the first Human_4x44k_1color_expression multiplex file, and click Open.


• Version 9.5 lets you extract the multiplex images as one image and produces one shape file for the four images in the multiplex.

• Note that for this image you must add both a grid template and a protocol.

2 Create a grid template from the design file and assign it to the extraction set.• Hint: The design file,

014466_D_20060526.xml, resides in the same directory as the image file.



c Select the design file (014466_20060526.xml), and click Open.



3 Assign the GE1-v5 FE protocol to the extraction set.

• From the FE Protocol Browser, drag and drop the GE1_v5_95_Feb07 FE protocol onto the multiplex extraction set.

4 Check the Extraction Set Configuration tab sheet • Make sure that the automated

assignments are OK.• Save the project to the name

iiiAgilentsets, where “iii” are your initials.

a Expand the columns in the Extraction Set Configuration tab sheet to see all the entries for the two extraction sets.

b Select File > Save As.c Go to or create the directory for your

projects.d Enter iiiAgilentsets, and click Save.

“iii” are your initials.

• Note that the output file name is the name of the extraction set plus the protocol name.

• You must save the project before the software will start Feature Extraction.

• After you save the project, the project name changes in both the title bar and Project Explorer.

Task 3: Add an extraction set for a 4x44k GE1 multiplex (4) and review the extraction configuration for the batch.



Task 4: Run Feature Extraction on the batch and review the reports.


1 Run Feature Extraction on Project 1.• View the progress of each

extraction at the bottom of the page.

a Click the Project Run mode On/Off button, or select Project > Start Extracting.

The project extraction starts to run.

• Two extraction sets are run simultaneously. Note the two running monitors at the bottom of the screen.

b Click Running Monitor 2 to view the second extraction.

• Feature Extraction runs the third set only after one of the other sets is complete and then displays it in the monitor of the completed set.


2 Close the monitors and view the Project Run Summary.

• Click the Close icon twice to close the monitors and view the report.

• Below you see the summary reports for each of the three extraction sets.




• Note that two of the four images have problems with the linearity of the spike-in results.




3 Open the QC report for image 1 and image 2 of the human_4x44k_ 1-color expression extraction set, and confirm that the spike-in results seem reasonable or not.

See Chapter 2, “QC Report Results” in the Reference Guide for information on how to interpret the QC Report.

a In the Project Run Summary onscreen report, double-click the first link to the QC report for image 1, Or do the following:• Go to the directory where the .tif file

is saved.• Double-click the .pdf file for the QC

report.b View the spike-in plot on the third page

and confirm that the plot is linear.c In the Project Run Summary onscreen

report, double-click the second link to QC report for image 2,

d View the spike-in plot on the thrid page and confirm that the plot is not linear.

• You can find the QC Report in the same directory as the image file as a pdf file.

• See Chapter 2, “Extracting Microarrays Automatically” in the User Guide to learn to print this file.

• Note that the error for Image 2 is reflected in the spike-ins plot for that image.

Figure 6 Spike-ins plot for Image 1 (left) and Image 2 (right)




4 Print the latest Project Run Summary report for the project.

a Go to the results directory where Project 1 is saved.

b Open and print the _LastBatchReport .rtf file with MS Word.

• All Summary Reports are saved to the directory containing the project associated with the report.

• Note that there may be two reports with the .rtf extension.

• The Computer Name_LastBatch Report.rtf file contains the report for the last extraction.

• If an image was extracted previously, the previous .rtf files are named as Project Name_Date and Time.rtf.




Exercise 2: Set up and run batch extractions on non-Agilent images

This exercise shows you how to create a new project, change project properties and run Feature Extraction for the non-Agilent image shipped with the software.

Repeat these instructions to add non-Agilent images of your own to create a batch. You can also set up and run a combination of Agilent and non-Agilent images in one project.

Feature Extraction does not recognize non-Agilent images scanned on a GenePix scanner.

Try the Steps on the left before reading the Detailed Instructions.


1 Create a new standard project. • Select File > New > Standard Project, OR

• Click the New Project icon,

• A standard project is one where extraction sets are set up from existing scan files to run automatically as a batch.

• When you start the software, FE Project1 automatically appears on the screen. When you open the next project, the name is FE Project2. The name of every project that you open after that follows in numerical order, whether you save FE Project N or not or close FE Project N or not. Each time you restart the software, the first new project is FE Project1.

2 Change the following Project Properties:• Highest Priority Default

Protocol: Project Default• Project Default Protocol:

GE2-nonAT_95_Feb07• Use Grid file if available: True

a Click the Project Properties tab.b Click the cell next to Highest Priority

Default Protocol, and select Project Default.

c Click the cell next to Project Default Protocol, and select GE2-nonAT_95_Feb07.

d Click the cell next to Use Grid file if available, and select True.

• In the Project Properties tab screen, you can enter a number of variables that pertain to the project as a whole, rather than to individual extraction sets.

• Because some of these properties, such as Project Default Protocol, affect how the extraction set is constructed, you may want to enter these before you add the image files (extraction sets).


See Chapter 2, “Extracting Microarrays Automatically” in the User Guide for instructions on how to change other project properties.

• Note that any changes from the default settings are shown in orange.

3 Add the non-Agilent image shipped with the software to Project 2.• Add any other of your

non-Agilent images that you want in the batch.

a Click the Add Extraction Set(s) button, or right-click anywhere in Project Explorer, and select Add Extraction....

b Go to the directory containing the non-Agilent image, and click Open.

c Expand this extraction set.

• The grid was automatically assigned because its name is the same as the image name and you said to use the grid file if available.

• The protocol was automatically assigned to the extraction set because you set the highest priority default protocol to be the one for the project and then selected the nonAgilent protocol as the default project protocol.



4 Save the project to the name iiinonAgilentset, where “iii” are your initials.

a Select File > Save As.b Go to or create the directory for your

projects.c Enter iiinonAgilentset, and click Save.

• For non-Agilent images you must create a grid file (grid.csv) for use in the extraction set. See Chapter 3, “Creating Grid Files and Templates”.

5 Run Feature Extraction on iiinonAgilentset.• View the Summary Report.• View the QC report

a Click the Project Run mode On/Off button, or select Project > Start Extracting.

b Double-click the blue line containing a link to the QC report.

Note that the report says to Evaluate Grid.

See “Add a grid file to an extraction set” on page 69 of the User Guide to learn how to add a grid file to an extraction set. To create or edit a grid file, see Chapter 3, “Creating Grid Files and Templates”.

• The Summary Report shows that there are too many population outliers in this image.

• When you see error messages relating to incorrect gridding, whether they apply to non-Agilent or Agilent images, you then must either edit the existing grid file or create a new one (grid.csv) in the extraction set for that image.

• To edit a grid file, right-click the grid file associated with the unextracted extraction set in Project Explorer, and select Edit grid.

• To create a new grid file, right-click the empty spot for the grid file under the unextracted extraction set in Project Explorer, and click Create grid file.



Set up and extract Agilent image files in real time

Instructional overview

An On-Time project lets you set up the Feature Extraction software to automatically extract Agilent microarray image files as they are transferred from the Agilent scanner to a folder of your choosing.

Follow the roadmap below and the instructions on the next pages to get started.

Start Here!

Set up and run Agilent files in real

time

3 Enter the results path and formats

1 Create an “on-time” project

2 Enter the incoming image path

4 Enter the FE termination criteria

5 Enter other properties and save the project

6 Run Feature Extraction

NOTE On-Time projects do not work with GenePix-scanned images.

Agilent Feature Extraction Software (v9.
5) Quick Start Gui de 29

Exercise 3: Set up to run batch extractions on Agilent images in real time

In this exercise you set up the software to run a batch of Agilent microarray images in real time as they arrive from the Agilent scanner to a folder of your choosing. You also set up this batch to run with a QC metric set. This exercise does not include running Feature Extraction; you can do that on your own.


1 Create an on-time project. • Select File > New > On-Time Project. • Note that you do not add extraction sets to on-time projects.

2 Enter the incoming image path.• Use whatever path you would

normally select for your scanner.

a Click the cell next to Incoming Image Folder, and select the path for your scanned images.

b Click OK.

• The software assigns the appropriate grid template to each of the Agilent microarrays and the appropriate protocol included in the Grid Template Properties sheet.

• If the Agilent scanner scans a microarray whose grid template is not in the database, it is not processed in the on-time project.


3 Enter termination criteria.• Enter 16 hours and 47 minutes

for the duration of the project extraction.

1 Click the cell next to Use Run Duration Time Out, and select True.

2 If not expanded, expand the Duration Time folder.

3 Use the sliders to enter 0 for Days, 16 for Hours and 47 for Minutes.

4 Add the QC metric set, GE2_QCMT_Feb07, assuming the images to be extracted are all 2-color gene expression images.

a In Project Properties, click the cell next to QC Metric Set.

b Click the down arrow, and select GE2_QCMT_Feb07 from the list.

• The QC metric sets should have been installed with the FE software.

• To see the available metric sets, select View > QC Metric Set Browser, if not already visible.

• If a metric set you want to use is not available in the Project Properties list, right-click the QC Metric Set Browser to import either Agilent metric sets or metric sets created with the QC Chart Tool.

• Only one QC metric set can be used for a standard project or an on-time project.



5 Save the project as iiiontimeproject, where “iii” are your initials.

a Select File > Save As.b Go to or create the directory for your

projects.c Enter iiiontimeproject, and click Save.

6 Make sure that the scanner is ready and run Feature Extraction if you want.

• Click the Project Run mode On/Off button, or select Project > Start Extracting.

• The image files are saved to the Incoming Image Folder as they complete scanning and are then extracted automatically.

• You can view the progress of the extractions, the Summary Report and the QC Report as you did for the saved batch file extractions.

• The Summary Report notes how many metrics are within range for each extraction.

• The QC report for each image contains the values for the metrics and an evaluation of whether they were within (True) or exceeded (False) the threshold limit.

CAUTION Agilent-supplied QC metric sets are intended to assist users in monitoring microarray processing issues. They were not developed to detect microarray manufacturing issues.



Agilent Technologies

© Agilent Technologies, Inc. 2007

Printed in USA Fourth Edition, February 2007

www.agilent.com

In this book

This book contains brief instructions to help you get started with your Agilent Feature Extraction 9.5 software. This book shows you:

• What is new for version 9.5

• How to set up and run batch extractions of existing Agilent and non-Agilent image files

• How to set up and run batch extractions of Agilent image files in real time

agilent feature extraction software (v9.5) · agilent feature extraction software (v9.5) ... plus...

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