Release Note. Agilent Genomic Workbench Lite Edition 6.0. New functionalities in Agilent Genomic Workbench Lite Edition

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1 Release Note Agilent Genomic Workbench Lite Edition 6.0 Associated Products and Part Numbers # G3794AA G3799AA - DNA Analytics Software Modules New functionalities in Agilent Genomic Workbench Lite Edition The new release of Agilent Genomic Workbench, version 6.0, offers many new functionalities: Agilent Genomic Workbench Lite 6.0 provides for the first time, SureSelect Target Enrichment Quality Analyzer, which allows quality assessments for SureSelect Target Enrichment experiments; Agilent Genomic Workbench Lite 6.0 offers new algorithms in analysis modules and also improvements of existing analysis for ChIP, CGH and Methylation. Agilent Genomic Workbench Core Utilities The Agilent Genomic Workbench Core Utilities contained Sample Manager and Workflow Manager as part of the 5.0 release. New to the 6.0 Lite release is enhanced functionality for Sample Manager and Workflow modules. Specific additional functionalities are: Target Enrichment Quality Analyzer In the 6.0 release, as part of the SureSelect Target Enrichment application space, you have access to the Target Enrichment Quality Analyzer. The Quality Analyzer is used to characterize the performance of a sequencing run in comparison with the targeted regions. Key features and functionality: Generate QC reports with a set of standard metrics; Provides report on enrichment level for target intervals. Sample Manager The Sample Manager enables you to easily associate meta data (patient/sample information) with individual microarray slides. The meta data is then persisted through the analysis process, making it easier to follow trends and analyze data. Key functionality: Associate sample information (Array Naming)

2 Enable sample information to show up in reports Workflow Manager The workflow manager enables you to automate and manage the data analysis process from a single user interface. Using the Workflow Manager, you will be able to: Analyze Tiff, FE txt, UDF file or already loaded experiment; Create new analysis method or use a already created one; Associate sample attributes loaded from sample manager; Create experiment at the end to view the results in the interactive mode; Create aberration report for each sample and save the result database; During a workflow run, support the ability of selecting a grid file (only if AGW Lite is pointing to FEv10.9, not FEv10.7); the ability to automatically determine grid template the protocol; the ability to configure FE output file generation and output location. DNAAnalytics 6.0 Some key new features that are offered in this new release of DNAAnalytics 6.0 Batman Implementation The Batman (Bayesian Tool for Methylation Analysis) implemented to be able to assign methylation calls to probes in Methylation arrays. Cancel Undo functionality Whenever an operation can be safely cancelled and system restored to a previous valid state, a Cancel button is available in the corresponding progress bar. Upon clicking on this Cancel button, the system goes back to the previous valid state. If such a valid state is not available and the operation cannot be safely cancelled, the Cancel button is not available. CoC Lowess and variance stabilization Lowess (LOcally WEighted polynomial regression) algorithm normalizes the channels within each array using a nonlinear polynomial fit to the data. The variance stabilization method2 is an alternative to Lowess normalization that fits a regression curve to signal intensities after applying an arcsinh(x) transform to each channel rather than a log(x) transform. The arcsinh(x) transform attempts to adjust the variance so that it is more consistent across the range of the data set. CoC visualization: option of Linear or log scale With the current release, the data and results can now be visualized in both linear and Log scales. The user has an option to configure and choose one or both of the scales. Handling of Non Unique Probes In DNAx Analytics 5.0, the user had to specifically expand designs to be able to visualize or analyze non unique probes. In the current release, all probes are imported by default. The non unique probes are highlighted by a blue color in the table view and by a different symbol in the graphical view. The user can choose to filter all or subset of non unique probes using Design level filters. Design level Filter

3 With this release, a new filter, namely Design filter is available. This filter can filter probes based on homology, number of perfect match hits that a probe has, whether it is in a pseudoautosomal region, or based on the performance score of the probe. This filter can be used to get various subsets of probes (including previous unique, non expanded probes ). Configurable colors and ranges for the views In this release, users can choose the color schemes, what quantity to color by and different ranges which should be displayed in different colors. Create Tracks from Methylation Scores Functionality of Creating tracks from methylation scores is added. In the earlier version (DNA Analytics 5.0), this functionality was available only for CGH scores. Cytoband information display in Gene View Displaying cytoband information in Gene View is added as an option. Pre-defined Peak-shape Detection v2.0 This method uses the expected distribution of sheared DNA fragments to model the shape of increased probe signal ( peaks ). Rendering Z-Score in Methylation context Any one of the combined Z score, the methylated Z score, and Unmethylated score, as well as Batman algorithm results, can now be chosen for display. Earlier, only the first option was available. Select Scale For Rendering The scale ranges can now be selected from the View Preferences dialog. Set Global and experiment context Array Name from SAF Row This functionality enables one to: 1. Change the names of the arrays displayed inside an experiment 2. Change the names of the arrays displayed in the data node globally for a set of arrays 3. Have different names for a same set of arrays in different experiments; i.e different names for same arrays that are included in multiple experiments 4. Be able to name the arrays, after the experiment is created, by a specific attribute chosen View Tab Reorganized In DNA5.0, many of the view options/menus are present in the Preferences dialog plus View tab. For AGW 6.0 release, these have been reorganized, so that all the view options are managed from one place, the View Preferences dialog. Workflow Changes Users are able to create, save, edit, and delete workflow and analysis methods. Reports and Output options are detached from analysis method and are now part of workflow. Aberration Filter Changes Aberration filter now has an option to split the nested aberrant intervals. This split happens when the child interval gets filtered out on height criterion (e.g. a small amplified interval within a bigger deleted interval) but the parent interval (bigger deletion) passes the criterion. In this case the parent interval is split into two separate intervals and these two splits are reported as separate intervals. In such cases the results of aberration filter would be different from previous version.

4 Intra Array Replicate Combine Changes In previous version of DNAAnalytics, we were considering probes as a replicate probes if the start positions were matching. In DNAAnalytics 6.0, 2 probes are considered as replicate probes if both start and stop positions match. Preloaded Data in DNAAnalytics 6.0 These are the datasets that are preloaded into the DNA Analytics 6.0 application: Type AMADID Genome Build # Probes (Size) FE Files CGH Hg US _ _S01_CGHv4_95_Feb07_1_1 2. US _ _S01_CGHv4_95_Feb07_1_2 3. US _ _S01_CGHv4_95_Feb07_1_1 4. US _ _S01_CGHv4_95_Feb07_1_2

5 System Requirements for Agilent Genomic Workbench Lite 6.0 Windows and Linux System Requirements: Operating System: Windows XP or Vista with Pentium 4 or later ; Red Hat Linux 5 Processor: 2GHz CPU or greater, Intel Core 2 Duo or better Available memory (RAM): 64-bit: 4GB RAM minimum (need more when working with 244K or higher microarrays) 32- bit: 2GB RAM Hard disk space: 40 GB (For analysis of large datasets, more space is required) Display resolution: 1280x768 Display minimum Internet connectivity: T1 or T3 connection (1.5 Mps) for use of earray Mac System Requirements: Operating System: Processor: Available memory (RAM): Hard disk space: Display resolution: Internet connectivity: Mac OS X v10.5.x (Leopard or higher) 3 GHz Intel Core 2 Duo CPU or better 4 GB 40 GB (For analysis of large datasets, more space is required) 1280 x768 or higher 1 Mbps or faster Tips on using Agilent Genomic Workbench Lite Software 1. Vista: need to change install directory : The software will not install correctly on Vista machines without the correct install directory. Workaround: User needs to make an install directory, with full permissions. And user should have administrative rights on the machine. 2. Vista: user.home property not set correctly : Sometimes, the user home directory value is wrongly set on Vista. Due to this, many processes like workflow will not function as they require user settings file which is located at user home directory. Workaround: User needs to get the user home directory corrected by authorized system administrator. 3. Change memory settings, if necessary : Sometimes, the user can change the memory settings to speed up the processing. Workaround: If you want to maximize the speed of processing, you can change the memory setting for the heap size of several processes. The heap size is controlled by two flags, Xms<size> and Xmx<size>. JVM starts with -Xms amount of memory and can grow to a maximum of -Xmx amount of memory. The 32-bit machine JVM does not support over 1400MB.

6 To change memory settings for running DNA Analytics In Notepad, open the file.../program Files/Agilent/Agilent Genomic Workbench Standard Edition 6.0/ Agilent Genomic Workbench Standard Edition 6.0.lax. 2. Find the line lax.nl.java.option.additional=-xms1024m -Xmx1400m - Dsun.java2d.noddraw=true. 3. Change the flags to your preferable memory setting. For example, if you have 2 GB RAM, change the line to read lax.nl.java.option.additional=-xms1400m -Xmx1400m - Dsun.java2d.noddraw=true. Note: Make sure the letter m is present at end of size and there is no space between the number and m. To change memory settings for running workflows: 1. Using Notepad (or any text editor program), open the config_workflow.properties file, located in the config folder of the program installation folder. By default, this location is C:\Program Files\Agilent\Agilent Genomic Workbench Lite Edition <version>\config. 2. Find the property, HEAP_SIZE=-Xmx1200m. 3. Change 1200 to the desired memory setting. This value sets the amount of RAM (in MB) that is allocated to the program in Workflow mode. Example: For a 32- bit machine with 2 GB RAM or higher, change the property to HEAP_SIZE=- Xmx1400m. This is the maximum allowed for a 32- bit machine. Example: For a 64- bit machine, to increase this setting to 3,000 MB (the maximum allowable setting if you have 4 GB RAM installed on your machine), change the property to: HEAP_SIZE=-Xmx3000m. To change memory settings for the background process for importing FE data files 1. In Notepad, open the file.../program Files/Agilent/ Agilent Genomic Workbench Standard Edition 6.0/config/config_FEImport.properties. 2. Find the property, HEAP_SIZE=-Xmx512m. 3. Change 512 to your preferable memory setting as you did for Workflow mode. 4. User may encounter an error message Could not create Java Virtual Machine Error : There is a known incompatibility between Agilent Genomic Workbench (AGW) versions 5.0 and 6.0 and Microsoft's Application Compatibility Toolkit (ACT) Version 5.5 (MS ACT). Installing MS ACT will result in an error, "Could not create the Java virtual machine", which is displayed when attempting to launch AGW. Workaround: Uninstalling MS ACT 5.5 will return functionality to AGW.. Bug fixes in DNAAnalytics 6.0 Cursor is shown at different position in the table & UI. Log Ratio - "Select Scale of Rendering". Color code of Signal Intensities does not make sense.

7 Common aberration/filter issue needs change in interval tree structure and alg. Table coloring not getting refreshed after applying/removing aberration filter. CH3 & ChIP Ribbons: accelerator keys. Workflow: FE Pref's needs to be version neutral & not needed as icon. Switch Application boxes. Scatter plot not re-drawing correctly after signal filter added. Switch Application functionality. Workflow Report: "Overwrite = False" bug. AnalysisMethod: SaveAs should start with current name. Sample Manager Array Data navigation pane: improper use of Demo node. Save Workflow dialog should be Save Analysis Method. ChIP QC_Report & QC_Metric need re-naming. Attribute file: non-editable fields inconsistent. Attributes File Importer: Incorrect Header logic Running cyto report by run by importing option gives error. Application gives error if you keep the application ideal for 3-4 hours. Error will occur, if you apply expand after applying moving avg on expt. EXP design is missing in the default CGH-EXP expt. ADM2/HMM getting applied. Expand is not working for catalog data. Create gene list option is not showing the selected row chromosome. Workflow: need to be able to Save and have persistent file. Import Attribute File with incorrect predefined fields yields incorrect import. Attribute file export incorrect when multiple Genome Builds are present. Track order does not change in the navigator. UserPref dialog: click Cancel should clear previous changes. Attribute File with replicate namesdoes not import correctly. Aberration disappears from the UI after applying Moving avg on experiment. When you aberration filter the table few is not always updated. Axon files should have design type as CGH/ChiP depending on context and not axon. In the open application tab, earrayxd application button is always disabled. Aberration filter should be modified to fix a couple of existing issues. Import Filter menu is missing. Empty Probe report is getting generated for single design experiment. Sample Manager table does not stay in sync. chrm is labeled wrongly in chromosome view. HMM results are not generated for all the arrays in the experiment. Sample information not passed to QC report from a workflow. Experiments created from workflow that failed. Wrong chromosome displayed in chromosome view. ADM-2 does not work on combined dye flip array. When applying the default feature filters, the moving average of the first subarray is disappearing on chromosome Y. Known Issues in DNAAnalytics Combine Track logic In this release, operations for combining tracks work by assuming the track to be sets of intervals but do not perform any operations on intervals themselves. For example if a track consists of intervals e1, e2 and another of interval e2, e3 then the logic of intersection would result in a track with just e2. However if a track has interval e1 and another

8 track has interval e2 where e2 is contained in e1 then the intersection would not give common part e2 but rather result would be empty and a message no annotations found would be shown (TT_865672) 2. Cyto Report keeps running for Use Workspace Settings on HMM Steps: 1. Create an Experiment and apply Aberration with HMM Algorithm. 2. Create a Cyto Template with option "Use Workspace Settings" selected in Analysis Settings tab. 3, Run the same cyto report. The report keeps running (TT_075661) 3. If any gene list is applied, Aberrations in all the views get updated except in the table Apply gene list functionality is not working on the data table. The gene list only gets applied on the aberrations in the views. Apply gene list only influences the display and not the actual aberration result. (TT_076300) 4. Issues with high density ChIP designs: If a million feature ChIP-on-chip design has most of the probes on a few chromosomes only, then analysis may have certain issues. 1. With those designs, ChIP workflow also may fail. The probe report may not be generated and application may yield Out of Memory error. 2. Fusing two or more such designs will cause analysis to fail. (TT_118009) 5. Colored filled circle looks like a square. Colored filled circle symbols in gene view looks like squares in the scatter plot views (TT_117963) 6. Number of probes in the report (except interval based text aberration report) includes failed probes. If feature filter is applied, then filtered probes are considered in the reported aberration intervals. (TT_119967) 7. Aberrations which are not common are also seen in the Text Summary of Common Aberration and also in UI. Aberrations which are not common are also seen in the Text Summary of

9 Common Aberration and also in UI. (TT_076245) 8. Workflow with 100 Million feature data with Z-score and penetrance reports fails. Workflow with 100 Million feature data with Z-score and penetrance reports fails. (TT_118044) 9. Cyto report header has incorrect text. Cyto report header has incorrect text if some special characters are present in the report header section in the cyto report template. (TT_133074) 10. ManualQCFlag status not reflected in Sample Manager immediately. If ManualQCFlag is set for any array from QC Metric table, it is not updated in the Sample Manager in same session Quit application and restart it. (TT_133163) 11. If Overwrite if file exists option is not checked in the workflow for reports, workflow will fail if we run more than one workflow continuously. If Overwrite if file exists option is not checked in the workflow for reports, workflow will fail if we run more than one workflow continuously. Cause: when 1 st workflow is running, the 2 nd workflow is in waiting state. When 1 st workflow completes and 2 nd one starts, the report file is already present. (TT_132376) 12. Analysis results might differ between AGW5.0 and AGW6.0. Analysis results might differ between AGW5.0 and AGW6.0 cause there are changes in Intra Array replicate combining and Aberration Filter functionality mentioned in AGW Lite 6.0 section above in the document. 13. When "Output location same as image" option is chosen then output files are generated in the same level as image, rather than in a workflow-generated folder on the same level as the image. When "Output location same as image" option is chosen then output files are generated in the same level as image, rather than in a workflow-generated folder on the same level as the image (TT_118784)

10 14. When doing an extraction in the workflow mode, you may get a security alert concerning Java binary files. When doing an extraction in the workflow mode, you may get a security alert concerning Java binary files. When prompted, choose "Unblock" to allow the workflow to continue. You may resolve this by changing your firewall security options (TT_115733) 15. Feature Level Filter and Design Level Filters don t filter out signal intensities for the filtered out probe. When Feature or Design level filter is applied on an experiment, the log ratios for the filtered out probes are grayed out in the table but signal intensity values are not. (TT_134112) 16. Probe based reports doesn t get generated with HMM if large number of probes are filtered out due to feature or design filter. Probe based reports (viz. probe aberration report, probe penetrance, graphical probe penetrance) doesn t get generated with HMM if large number of probes are filtered out due to feature or design filter. (TT_134112) 17. Signal Intensity Bar Chart plotting shows a line besides the regular bars. Signal Intensity Bar Chart plotting shows a line besides the regular bars. The issue is because of the third party software (jfreechart) being used in the application 18..Workflow has Genome Build which is not obvious to user. If a design has multiple builds, the last one that was imported will be the build that is used by Workflow with input: Extraction of an image; Or import of an array Import or Update the Design Genome Build last that you want to be used in workflow (TT_134290) 19. Axon data doesn t get plotted in the first run. After importing new GAL and GPR files, if new experiment is created with this data, sometimes the data does not show in the table or in views. Quit and reopen the application. Select the experiment and data gets plotted in views as well as table.

11 (TT_134654) 20. Hg19 issue in workflow with input = imported array. If an imported array has two genome builds and you select this array for a workflow, the genome build choice may not be stable; may switch to the other genome build.. (TT_133596) 21. NonUnique shading choice has incorrect behavior. De-select to highlight NonUnique probes. Quit & restart software. Sometimes this highlight choice is not stable. Other times the probes will not highlight when the choice is selected. Quit and reopen the application. (TT_135240) 22. Workflow Export/Import issues. Export a workflow from one instance of software. Import this into a second instance of software. Some parameters are not kept; such as, image choice for extraction or aberration report parameters. Need to manually add those parameters into the newly imported workflow. (TT_135298)