Release Note. Agilent Genomic Workbench 6.5

Transcription

1 Release Note Agilent Genomic Workbench 6.5 Associated Products and Part Number # G2566AA Agilent Genomic Workbench with Feature Extraction provided as part of the Agilent Scanner # G4460AA - G4462AA - Agilent Genomic Workbench with Feature Extraction Commercial License # G4463AA - G4465AA - Agilent Genomic Workbench with Feature Extraction Academic License # G3794AA G3799AA - DNA Analytics Software Modules New for the Agilent Genomic Workbench In addition to a set of improvements that facilitate an easy installation and system diagnosis, Agilent Genomic Workbench 6.5 release provides a great amount of new functionalities. Key features include: Ability to design custom SNP microarrays through earrayxd; Ability to use Agilent Literature Search to design custom microarrays around biological context of interest; Ability to handle CGH+SNP microarrays in Feature Extraction, Sample Manager and Workflow Manager; Ability to identify SNP genotypes and LOH in DNA Analytics for cytogenetic samples. Note: AGW 6.5 supports SNP and LOH analysis of constitutional cytogenetic samples but the algorithms are not designed yet to handle challenging samples. As a result, we will not be in a position to support the following samples: cancer (heterogeneity, polyclonality, ploidy, etc), WGA (whole genome amplification), ULS-labeling, and FFPE. Development is ongoing to be able to support this down the road. earray XD Support SNP Probe Search and Custom CGH+SNP Microarray Design Support creation of CGH+SNP custom microarrays in CGH application space. After downloading Agilent catalog SNP probe data set, SNP probes can be selected out specifically to cover genomic regions or SNPs of interest. The selection can be based on a preferred minimum allele frequency; total available space on array, or based on reference samples used in the experiments. Agilent Literature Search Within Cytoscape earrayxd plugin, Agilent Literature Search tool allows retrieving networks of genes involved in certain biological context within literature, saving such networks, and start custom probe/bait design.

2 On-demand Data Download Agilent Genomic Workbench 6.0 requires downloading of entire Agilent and Workgroup data set. In this new release, data set of interest can be selected to be downloaded on demand. The supported data set include Agilent catalog data for all available application types, especially the newly released Agilent SNP probe data set, which contains 118,726 SNP probes covering 64,367 SNPs throughout the genome. Agilent CGH/CHIP HD data set are not available for download. Agilent Genomic Workbench Core Utilities Feature Extraction (FE) Support new control type SNP. Support import of DyeNormLists downloaded from earray. Support DyeNormList automatic association with grid file from corresponding grid template. New changes in protocol options for using DyeNormLists during extraction. Default Dyenorm Gene List in grid template has been renamed to Dye Norm Gene List. External DyeNormList is only supported for Non-Agilent arrays. When creating grid file for Agilent arrays the Feat.CSV is no longer an output. The DyeNormList used during extraction and the number of probes that contains are shown in QC report, Output XML and tab text file. IsGridFile variable is added to output XML and tab text file. New QC report for photolith test slide is added. Log ratio distribution plot is added to ChIP QC report. FE PARAMS section is added to output XML. FE tab text output files can now be generated in zipped format. Gridding improvements are made for 30 micron feature size arrays. Support for post processing plug Ins. Improved Up/Down regulated feature plot. Target Enrichment Quality Analyzer Ability to run analysis in workflow mode. Support gzip and zip files (4G uncompressed size limit); support plain.txt formats (no size limit). Support AB output read file. Sample Manager Support SNP reference sample. Workflow Manager Ability to use SNP and LOH algorithms in a workflow.

3 DNAAnalytics 6.5 Some key new featutres that are offered in this new release of DNAAnalytics 6.5 SNP genotype and Copy Number detection LOH (Loss/Lack of Heterozygosity) detection. GC Content correction Genotyping of reference samples New visualization panels for SNP + LOH Updated aberration reports with LOH information & SNP Genotype report SNP specific QC Metrics

4 System Requirements for Agilent Genomic Workbench 6.5 server Windows XP or Windows Vista (Business, Ultimate) and Windows 7 (Enterprise) with Pentium 4 or later (server installation is supported on 64 bit configuration) Processor: Intel Core 2 Duo CPU Available Memory (RAM): min 8 GB Hard Disk Space: 100 GB - for MySQL 200 GB - for Common Storage Internet Connectivity: T1 or T3 connection (1.5 Mbps) If MySQL is already installed on the computer, make sure it is version 5.1 Pro. Otherwise MySQL will be installed with Genomic Workbench. MySQL server configuration is done automatically during the installation of Agilent Genomic Workbench Server software System Requirements for Agilent Genomic Workbench 6.5 Client Software Windows XP or Vista or 7 with Pentium 4 or later Processor: 2GHz CPU or greater, dual core CPU recommended Available Memory (RAM): 64-bit: 4GB RAM minimum (need more when working with 244K or higher microarrays) 32- bit: 2GB RAM minimum Hard Disk Space: 40GB Free Disk Space Monitor: 1280x768 Display minimum Internet connectivity: T1 or T3 connection (1.5 Mps) for use of earrayxd Tips on using Agilent Genomic Workbench Client Software 1. Need to download a design for running analysis : During the process of downloading/importing a catalog or workgroup design, the steps involved and icon changes in the left side navigation panel is confusing. Explanation: Downloading a design in Agilent Genomic Workbench is performed for one or both of the following two purposes: A) To obtain the most recent GEML file for the design for extracting an image and to prepare Agilent Genomic Workbench database to be ready to run data analysis within DNA analytics; B) To obtain the content (Probes, Probe Groups) of the design to view, copy, etc in order to design other custom microarray designs within earrayxd. Design download can be initiated by one of the following 3 methods: 1. In Agilent Genomic Workbench navigation panel, right-click the design AMADID (shows a yellow arrow) and choose Download from earray.com. Alternatively you can search an array design in earrayxd and click the icon for Download from earray.com.

5 2. Within FeatureExtraction, configure earray Preference to allow automatic download of designs. Either within FeatureExtraction standalone or Agilent Genomic Workbench workflow, start an extraction. 3. In FeatureExtraction, right-click in Grid Template Browser and choose Add. All of the above 3 methods will achieve purpose A); only method 1 and 2 achieve purpose B). Once design download is initiated by using one of the 3 methods above, download of GEML file immediately starts. After the GEML file is downloaded, the design appears in the Grid Template Browser in Feature Extraction. You can now use the GEML file/grid template to perform extractions. The completion of GEML download then triggers two simultaneous processes: Process 1 is an earrayxd process. This process downloads the content of the design, including its probes and probe groups. This process is not triggered if you use method 3 above to initiate download. Process 1 is monitored through a corresponding task item in Tasks -> Download Array Design from earray.com. The color change of the circle next to the task indicates progress. When download completes, the color of the arrow next to the AMADID and the circle next to the task both turn green, which indicates that you can now use the content of the design for custom array creation. Process 2 is a DNA analytics process. It populates the genome build information. This process is always triggered. The completion of process 2 is shown by the addition of a sub-node Design Samples Build build number underneath the design AMADID, which indicates that you can now run analysis using this design. Completing all three steps on a 1M feature CGH design usually takes about 3 hours. In the case of downloading a CGH+SNP microarray design, Process 1 will not start unless SNP data set is first downloaded by selecting Catalog SNP Data and click on Download button in Home->Data panel. If SNP data set is not downloaded, only Process 2 can complete, thus you will see a yellow arrow next to the AMADID with genome build sub-node underneath, indicates that you may use the design to do analysis. If you would like to have the full data set downloaded for designing custom microarrays, you may then right click the AMADID and select Download from earray.com to start Process 1. Completion of Process 1 is indicated by green arrow next to the AMADID. In some cases, the sub-node Design Samples and corresponding genome build label do not appear after a long interval. This problem may be solved by closing the application and re-launching. 2. Downloaded design shows yellow arrow instead of green arrow : After downloading a design, the sub-node Design Samples -hg19 appears, the circle next to the download task also turns green, but the arrow next to the AMADID is still yellow. Explanation: This may happen when you try to download a design for Gene Expression, CGH+SNP, SureSelect Target Enrichment and mirna applications. For these applications, by your selection, Agilent Genomic

6 Workbench brings in the entire Agilent data set to your installation. For CGH, ChIP and CH3 applications, this will not be an issue since Agilent Genomic Workbench brings down data on a per design basis. The appearance of Design Samples -hg19 sub node indicates that you can run workflow and analysis with this design. You may proceed without any issue if this is your only interest. The yellow arrow indicates the full probe content of the design is not downloaded to your Agilent Genomic Workbench server, thus you are not able to use the content for earrayxd custom design purpose. This is also reflected by the action options shown when you right click on the AMADID. If you need full probe content to be available for a design of the above mentioned application types, you need to first download the entire data set for this application by going to Home -> Data and clicking on Download button for the data set of interest. For example, if you are interested in designing custom CGH+SNP custom microarrays, you need to select Download for Catalog SNP probe data. After this download completes, in navigation panel, you then right click the design AMADID and select Download from earray.com. Another example is if you are interested in designing custom SureSelect libraries using some of the Agilent catalog library content, you will need to go to Home -> Data and select Download for Catalog Target Enrichment bait data. After this download completes, in navigation panel you can right click on an ELID and select Download from earray.com. In both examples above, after the design download completes, the yellow arrow will turn into a green arrow next to the design AMADID/ELID. You may then use the content of the design for making other custom designs. 3. Need to import a design for running analysis : I have a design that was made through either private collaboration with Agilent, or by another workgroup, so it doesn t show in earrayxd or earray web site. I don t know how to use Agilent Genomic Workbench to run analysis. Explanation: In this case, simply import the design through Home->Import->Design File. Once the design is imported and sub-node Design Samples Build build number appears underneath the AMADID, you can run analysis or workflow. 4. First time launch client software fails : Right after installing Agilent Genomic Workbench 6.5 server software, immediately install Agilent Genomic Workbench 6.5 client software and try to launch, pop-up message comes up and shows data download is not completed. Explanation: The installation of Agilent Genomic Workbench server software places a set of data download requests to retrieve the following information from earray web site: user account, control grids, formats, Agilent catalog design meta information, Workgroup design meta information and Workgroup probe/bait data set. Some of these download requests can be viewed in Job Queue console, some are not shown. It takes about 30 minutes for the data sets to be downloaded and you can then launch the client software. During this time, you will not be able to launch client software. The only data set that may take longer to

7 process is Workgroup probe/bait data set, which depends on the amount of custom data your workgroup has created on earray web site. However, you may launch the client before Workgroup probe/bait data set is fully downloaded. 5. Win 7/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. 6. Win 7/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. 7. 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. To change memory settings for running DNA Analytics In Notepad, open the file.../program Files/Agilent/Agilent Genomic Workbench Standard Edition 6.5/ Agilent Genomic Workbench Standard Edition 6.5.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 Workflow mode 1. In Notepad, open the file.../program Files/Agilent/ Agilent Genomic Workbench Standard Edition 6.5/config/config_workflow.properties. 2. Find the property, HEAP_SIZE=-Xmx1200m. 3. Change 1200 to your preferable memory setting. For example, if you have 2 GB RAM, change the property to HEAP_SIZE=-Xmx1400m. 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 6.5/config/config_FEImport.properties.

8 2. Find the property, HEAP_SIZE=-Xmx512m. 3. Change 512 to your preferable memory setting as you did for Workflow mode. 8. User may encounter an error message Could not create Java Virtual Machine Error : There is a known incompatibility between Agilent Genomic Workbench (AGW) versions 6.0 and 6.5 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 and Known Issues Known Issues in earray XD 1. When AGW backend service is restarted, any design writing tasks that are being processed will not finish If an Array design Writer job entry is in PROCESSING state, and the AGW backend service is restarted, the entry is not processed. Contact Agilent support to run a script to update the job_status from PROCESSING to PENDING. This behavior is seen once in a while and not always. (TT_142703) 2. Literature Search does not change focus correctly In the Literature Search window, when Search Probes button is clicked, the focus does not switch to the Search Probes page. (TT_140767) 3. earrayxd sync over locked probe groups created on earray web site Custom Probe groups that are used in Array designs completed or submitted on the earray web based-application, after AGW server installation, are synced over to earrayxd.

9 (TT_140134) 4. SNP search gives different total number of SNPs A specific case of SNP search, querying for Total Number of SNPs = 5000, returns more probes than expected (5001 probes). (TT_139913) 5. Request for quote HTTP 404 error In earrayxd, if a user clicks on the Request for Quote icon for an array design or library while an instance of a web browser is already open, the user is not directed to the Quote page of earray.com. The HTTP 404 Page Not Found error is displayed instead. If all the web browser windows are closed, the functionality works fine. (TT_138693) 6. HD data sync doesn t happen when HD data updated on production. HD data sync does not happen when HD data is updated on the earray website. User can perform HD searches again to get new HD data. 7. 'Import Custom Genome for Tiling' is not available in CH3 application space Workaround User will not be able to Import custom genome for tiling in CH3 application space. Users can import their custom genome in ChIP/ CGH/ SureSelect TargetEnrichment application. This custom genome will be available for tiling in CH3 application space. (TT_120513) 8. Array Layout Visualization: Slide view is not proper for 8 X 15 and 8 x 60 format If user selects a 8x15 or 8x60 array design for array layout visualization, the slide borders are not clearly visible in the visualization.

10 Bug Fixes in earray XD Using import genome, when file contains non-fasta format content, earrayxd does not give proper error message. Performance is slow in Probe Group download, Exon Interval Finder and Probe Download. Known Issues in Feature Extraction Workflow fails when design is partially loaded (TT ) Unable to download million feature array through FE (TT ) DyeNormList needs Cancel button (TT ) Design downloaded and inserted by earrayxd does not contain DyeNormlist when opened through FE (TT ) MySQL ODBC connector 5.1 is not supported (Legacy TT 2322). Sometimes the automation icon does not appear for extraction set (Legacy TT 2356). OK button on grid template disabled only during DyeNormList launch. This would allow users connected from other clients to delete grid template after DyeNormList is launched (Legacy TT 2360). Left out of the command line chapter, chapter 6 of the FE Reference guide is a warning that has been added to the software. All of the error and warnings the software can produce while extracting an array are listed at the end of that chapter. The warning we missed says Agilent does not support this configuration, please consult the support matrix in the Feature Extraction users guide for a supported configuration.the warning ID for this is Note that when refer users to the users guide, the configuration information is explained on page 19. Changing the color scale does not work while in grid mode. If user attempts to change color scale from All Channels to Red Channel or Green Channel, it does change (Legacy TT 2309). The 1-color QC report has page break problems causing one of the pages to break across a table when the QC report in put into PDF mode (Legacy TT 2299). Extraction automation does not work for Agilent cropped multiplex images. This is because we adorn the pack information on the barcode so it doesn t get lost when the image is cropped (Legacy TT 2287). The earrayxd integration feature is not quite complete in that after FE runs, the project explorer is not updated to reflect which grid template and protocol was automatically assigned. This can be

11 confusing to user after the project has finished as they do not get the feedback in the Project Explorer (Legacy TT 2236). Flip functionality fails with a poor error message if not enough disk space is available to complete the operation. The error message reads "Save as failed due to unknown reasons".(legacy TT 2210) After a scan is generated, the image should retain the parameters by which it was generated, and they should be retrievable from the Feature Extraction menu item, Image File Info..., which displays the Scan Image Properties window. Parameters that are currently missing include: -Single pass or Double pass -16-biit or 20-bit -Laser set points and/or actual laser power readings for each channel (this should reflect compensation, if invoked) -PMT compensation factor -PMT Calibration Error flag status (Legacy TT 2203) For new format scans, on 64 bit OS PCs, the first submenu option i.e. "Flip upper left to lower Right"(Landscape/portrait) and save" is not completely shown (TT 2190). For new format scans while setting color ranges, the ability to adjust colors in high fidelity mode can lead to errors namely if the user tries to set Auto Scale Image after unsetting this option. (Legacy TT 2166). If a user attempts to import a grid template during an extraction run, the grid template may fail to properly import into the software. The work around is to only import grid templates when a project is not running. Legacy TT 2112). earray automatic online protocol update can fail if the either the protocol or the metric associated with that protocol are missing (but not both) when the update occurs. It is possible to make this happen because even read only protocols and metric sets can be removed using FeNoWindows. The worst case is if the metric set is missing from FE, then the earray will fail to update the protocol due to a missing metric set (Legacy TT 2075). In rare instances, the switch to configure mode button will not be enabled after the extraction is complete. The extraction will have to be closed and re-opened to enable to config / run mode toggle (Legacy TT 2069). The grid template that is currently in use during an extraction, can be removed causing the extraction to fail (Legacy TT 2042). The earray configuration setting Automate FE extraction by automatically downloading design files from earray can cause unwanted behavior. If user wishes to extract an image using a grid template with an AMADID doesn t match the image s. Disabling the earray configuration setting temporarily causes FE to behave correctly in this case (Legacy TT 2040). Attempting to calculate spot size and centroids in manual grid mode using a high resolution scan of a third party array will cause FE to crash (Legacy TT 2032). In rare instances the QC report can fail to generate. This is true when 30u feature size 2-pack arrays are used in FE, using a CGH protocol that generates an old style CGH QC report. For the 2-pack 30- micron feature size arrays the new streamlined CGH QC report must be used (Legacy TT 2019). It is not recommended to run projects containing multiple extractions directly through FeNoWindows. Projects containing multiple 30-micron feature size 1 million feature array will run out of memory if run

12 directly using FeNoWindows. The work around is to either use the FE GUI to run these projects or to break up the project into multiple projects each containing 1 extraction (Legacy TT 2016). Do not remove the DBConnectInfo.ini file from the FE installation folder. If that file is not available the software cannot be removed via the install or Add/Remove programs (TT 1944). When viewing shape files, feature outliers are not visible until the image is zoomed or cropped (which effectively zooms) (Legacy TT 1955). Viewing the scan properties can cause the image to appear distorted. Minimizing followed by reopening the image will correct the issue (Legacy TT 1959). Scans of 2, 3 and even 5 micron resolution using full sized scan regions are quite large creating memory issues for the software. In order to address memory and performance issues the following restrictions are true about the imaging. The new view window feature (with Ctrl-N as the shortcut) that allows users to open one channel of the scan will not work for new format scans. When cropping a new format scan to view the image close up, what FE refers to as high fidelity mode, zoom out is disabled below 200%. When creating a grid file of Agilent arrays some annotation columns may be lost. Currently only the primary annotation columns are certain to make it into the grid file. Those annotation columns are Probe Name, Systematic Name, and Gene Name. Other annotation fields are not certain to be output. There is no workaround to this issue (Legacy TT 1917). For large grid templates using the dye normalization list editor can take a very long time. This is a function of the number of probes that have to read from the database and loaded into the editor. It is possible to create the lists outside of FE and load them into the software without using the list editor. Please consult the FE manual for details (Legacy TT 1913). Blank fields, namely blank annotation fields above and beyond the primary annotation fields of Probe Name, Systematic Name, Gene Name, used inside of the GEML xml design files will fail to load into the MySQL database (Legacy TT 1912 and 1853). Under Windows Vista, FE users may not have permission to write to certain directories or overwrite certain files. This will cause FE to fail to load grid templates, protocols and/or metric sets. Also it will cause FE to fail to process extractions. The solution to this is to make sure the User Access Controls are turned off and ensure that C:\Program Files\Agilent\MicroArray\FE\Temp is writable and empty (Legacy TT 1836). It is possible using the FE to remove a grid template while the software is processing extractions. This is not recommended as it could cause the project to fail if that grid template is used (Legacy TT 1826). Applying many different image manipulations, including Flip and Rotate on 5 micron 16-bit scans, may cause FE to run out of memory and yield the message "Failed to load bitmap". The workaround is to close the FE software, re-open it, and try the operation again (Legacy TT 1874, 1768, and 1766). When cropping an image using the new crop image dialog, the file name is appended with 1_1 although "Cropped multiplex" is not selected (Legacy TT 1679). When running an XDR extraction project, the summary report can give grid placement error The spot centers are shifted relative to their nominal grid.' But the QC report says the grid is normal. This is an indication that XDR failed to properly extract the low PMT scan and there may be an issue with the image registration (Legacy TT 1675).

13 When a two-channel tiff file is split into two single-channel tiff files (one for Red and one for Green) from the Agilent Microarray Scanner, On-Time project treats the split tiff files as two separate singlechannel files (Legacy TT 282). Protocol, DyeNormList, or GridTemplate cannot have special characters in the name or description. The character that is sure to break is"'"(legacy TT 652 & 657). FE is not designed to support concurrent users. The software will only allow one user to user FE at a time. If another user is logged into the same machine, only one will be allowed to run FE. You cannot open a.tiff image from a CDROM or DVD. Copy the image to a hard disk or network share. Bug Fixes in Feature Extraction XML output needs date/time (TT ) Manual Gridding Help UI: Step_5: Need Save instructions & re-title (TT ) Manual Grid: "Unplaced" not updated correctly (TT ) Preferences: Grid Setting: remove "Auto Detect" ( TT ) Create QC Report type for Pixel placement chip (TT ) Manual Gridding hot keys not working (TT ) Manual Gridding Help text: change step # 5 title (TT ) Manual Gridding not closing Project windows correctly (TT ) FeNoWindows Usage list is not correct (TT ) Cannot load a shape file to the low PMT XDR scan (TT ) GridMode SaveGrid doesn t allow Project to be run (TT ) GEML file drop into UI causes unexpected windows behavior( TT ) Mouse-over when shape files are applied inaccurate for "low fidelity" mode (TT ) No indication that a normalization list is used (TT ) Compatibility with Windows 7(TT ) Pack info not written to MAGE_ML output file for cropped images.( TT ) Missing columns when manual gridding (TT ) Call executable after FE run - Customer Request (TT ) Workflow extraction design not found error needs clarification (TT ) Customer request - Change to MAGEML output - include IsFeatPopnOL as means to "Fail" data (TT ) For CGH, and GE2 QC reports, the log ratio spatial plot has errors in sampling. Currently, if there are > 3500 significant Up or Down-regulated log ratios, then the code does an incorrect sampling. This leads to a confusing appearance of the number of up and down regulated features (Legacy TT 2207) Protocols that are loaded into the database by name are specifically not case sensitive but protocols specified in the project are case sensitive. (Legacy TT 1699). Known Issues in QC Tool Quality objects: can be deleted by non-owners (Legacy TT 1929). QC table: not sorting by ExtractionTime correctly (Legacy TT 2175). ExtractionStatus,QCReportType and ColorMode will be shown in integer format when exported to a file (Legacy TT 2497).

14 The queries, metric sets, and charts are not ordered in alphabetical order in the navigator panes (Legacy TT 2520). Known Issues in Workflow Manager 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 ). Bug fixes in DNAAnalytics 6.5 Workflow: Console Summary needs Workflow name field. (TT_115735) AGW workflow: Issue with "Use grid file option" on Feature extraction properties dialog. (TT_116475) Workflow extraction save FE has lost folder (TT_118784) 'Data Available For Number of Chromosomes' value in design properties dialog is incorrect for some catalog designs. (TT_123155) Workflow Password UI is incorrect (TT_135150) Common aberration should not run for CHIP or CH3 arrays. (TT_136013) Intervals crossing p/q split (TT_119056) Create a gene track at the end of differential aberration (TT_085613) View Preferences not stable (TT_135239) UI plot and table user choices are not stable (TT_140416) 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 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_075144) 2. Cyto Report keeps running for Use Workspace Settings on HMM Steps: 1. Create an Experiment and apply Aberration witth HMM Alogorithm. 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

15 (TT_075661) 3. If any gene list is applied, Aberration in all the views gets 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. Attribute values set by one user are not getting updated for the other users in the same session Array attribute values updated by one user are not visible to other users in the same session. Other users have to restart the application to see the updated values. Restarting the application will show the updated attribute values. (TT_084866) 5. Application becomes very slow after you apply lowess OR variance stabilization with intra array. Chip Context > Application becomes very slow after you apply lowess OR variance stabilization with intra array. If you remove intra array, the application again work properly. Another observation: In case all the normalizations are applied in one step, then it takes a very long time. Instead if you apply all the normalizations one by one, it takes much less time. (TT_115165) 6. 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) 7. Colored filled circle looks like a square. Colored filled circle symbols in gene view looks like squares in the scatter plot views (TT_117963) 8. Users are recommended to download the design files first before running workflow. If the design is not already downloaded and available to DNAAnalytics,

16 1. Sometimes workflow fails as FE cannot get the grid template for extraction. 2. For Analysis workflow sometimes is stuck in the design verification step Design needs to be fully downloaded with genome build node before starting a workflow The genome build is a sub-node under the design node 9. OutOfMemoryError while restoring auto created experiment from workflow with million feature data OutOfMemoryError while restoring auto created experiment from workflow with 100 million feature data (TT_118045) 10. Workflow for fusing catalog data fails if design level filter is applied. Create workflow to fuse catalog design data. Now in the run analysis settings apply design filter. Run the workflow. Actual result: Workflow will take too much time and it will fail. (TT_118809) 11. Some operations may take a long time when you use a catalog design for the first time. When catalog design data is used for the 1 st time in an experiment then some operations, which needs different column data for that design, may take a long time for certain operations. But the next time onwards, if you use any data set of that catalog design, the operations will be very fast. 12. 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 Common Aberration and also in UI. (TT_076245) 13. 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) 14. 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.

17 (TT_133074) 15. 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) 16. 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) 17. Sometimes workflow keeps in waiting state. After user starts the run of a workflow, it keeps in waiting state even though no workflow is already running. Switch to home tab and come back to Workflow tab. The workflow will show as running. (TT_138074) 18. White row added in Sample manager by one user is visible to all user connected to same server. White row added in sample manager by one user is visible to all the user connected to that server. Any user can modify and update thet white row. Workflow of the original user may throw some warning message or error message if he tries to run that workflow which might be using the same white row. No (TT_140374) 19. Sometimes the table view is missing. When application is launched 1 st time after fresh installation, sometimes the table view is missing and only 3 plot views are displayed. This may also happen after the application has been installed and used. Switching to Open Application, or any other tab, and then back to Genomic Viewer brings back the Table View. (TT_132357, TT_144158) 20. Workflow Console screen blank at Completion. Sometimes at the conclusion of a workflow run, the console log screen is blank. Switching to Summary Console, then back to the workflow console will show the log correctly.

18 (TT_130489) 21. DyeNormList search function missing Error message. Make new DyeNormList; make some changes; choose "Save"; make more edits. Now if you choose "Contains name" and then Probe search for a string in the wrong case (such as "nc", when there are only "NC" probes), then UI does nothing. It should give a message that no probes are found. The search behavior works as expected if Save is not done first.. (TT_145164) 22. Workflow extraction of XDR images fails. XDR workflow fails if both (High PMT: Image_H) and (Low PMT: Image_L) images are added in a single workflow Add only High PMT or Low PMT scan while running workflow. (TT_145456) 23. SNP plots not showing; have to change tabs. Sometimes SNP plots do not show data. Change tabs & come back, now the data are visible. (TT_145136) Important : DNAAnalytics CGH aberration boundaries will be different between AGW6.0 and AGW6.5. a) In the previous releases of AGW and DNAAnalytics, the start and stop of the aberration intervals are extended on either side by 200bp. With the increased density of our SurePrint G3 microarrays, this interval margin is no longer applicable and it is set to 0 bp in AGW 6.5. b) The stop position of the last probe is considered as the end of the interval. In the previous releases of AGW and DNAAnalytics, the start position of the last probe is considered and 200 bp is added to that to define the end of the interval. In AGW 6.5, the stop position of the last probe is considered as the end of the aberration interval. 2. In contrast to regular CGH QC metrics, the SNP QC metrics of a CGH+SNP microarray aren t calculated until after the SNP copy Number calculation is done. When an array is imported in the data node or added to an experiment, the SNP QC metrics for that array will not be available. SNP QC metrics are available only after the SNP Copy Number (CN) has been calculated. Just selecting the array will not make the SNP QC metrics available, but once the SNP CN has been calculated once, the metrics are available for that array even if it is not selected. 3. For CGH+SNP microarrays, it is mandatory to associate a reference sample with a microarray. In AGW6.5, it is now possible for the user to associate the red and green sample information for a workflow from the workflow panel.

19 Associating a reference sample can be done in three ways: 1. In the sample manager. 2. From the interactive mode. 3. From the workflow mode. The 3 rd option was added to the software after the manual was finalized; hence this option is not described in the manual. For the third method, the software assumes the array polarity is+1; thus, it is mandatory to add the reference to the Green sample in the workflow mode, if the workflow has SNP analyses. 4. Aberration results in AGW 6.5 may differ slightly from AGW 6.0 The method on how the chromosomes are split between the p and q arm has changed so the aberrations around the centromeres can differ slightly. 5. Aberration filter now has an option to split the nested aberrant intervals. This option is set TRUE in the software by default. This split happens when the child interval gets filtered out on height criteria (e.g. a small amplified interval within a bigger deleted interval) but the parent interval (bigger deletion) passes the criteria. 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 of the software. Also one may see aberrations with less probes than the aberration filter criteria. 6. Calculation of Call Ambiguity and Goodness of Fit SNP QC metrics may evolve in next release. These two SNP QC metrics calculations may be modified in next release and are currently under investigation. We want users to focus on Call Rate and Separability SNP QC metrics in AGW6.5