Release Notes. Agilent CytoGenomics 2.5. Product Number. Key new features. Overview

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1 Release Notes Agilent CytoGenomics 2.5 Product Number G1662AA CytoGenomics Client 1 year named license (including Feature Extraction). This license supports installation of one client and server (to host the CytoGenomics database) on one machine. For additional client only installations- which connect to the same database on the central server- additional copies of this license are needed. Overview CytoGenomics is a cytogenetics research software tool designed to streamline your workflow for processing, analyzing and reporting of cytogenetic measurements. This includes copy number changes and copy neutral Loss of Heterozygosity and Uniparental Disomy data, generated with human samples on Agilent SurePrint G3 CGH-only and CGH+SNP microarray platforms. With CytoGenomics 2.5 we now offer an even more streamlined workflow with a highly sophisticated user interface. CytoGenomics is designed to: (1) import raw TIFF images generated from the Agilent microarray scanner as well as some non-agilent scanners (2) run Feature Extraction and perform analysis using customizable workflows. Utilizing the built in database, Cytogenomics allows the user to store and query samples and aberrations by classification information or location information. In addition, users can connect from aberration annotations to OMIM, DGV and Entrez public databases to analyze sample aberrations. During sample review, multiple users can annotate, edit and classify aberrations with full record traceability and generate signed-off reports on processed samples. CytoGenomics supports an enterprise server/client model for concurrent usage and collaboration. With strong analysis algorithms, data visualization, data management and reporting functionality, CytoGenomics software extends the Agilent microarray based product offerings into a complete cytogenetic research solution. Key new features A redesigned and improved workflow with completely new user interface Use of images from some non-agilent scanners (e.g.: Nimblegen MS 200) Ability to change moving average window size Support of triploidy and unbalanced tetraploidy samples

2 Improved aberration filter: Independent thresholds can now be set for amplifications and deletions New LOH aberration filter The use of ISCN nomenclature for aberrations Do not use probe list provides an easy way to filter out consistently bad probes or a list of probes of interest, without making changes to the design file cnloh fraction, a new QC metric to detect fraction of LOH in a sample Default and preloaded content The Hg19 version of the design file for the SurePrint G3 Human CGH Microarray 4X180K (022060) design is provided as part of CytoGenomics 2.5 installation. Hg18 and Hg19 version design files for the below listed commonly used designs can be downloaded from the CytoGenomics download page ( SurePrint G3 Human CGH+SNP Microarray 2X400K (028081) SurePrint G3 Human CGH+SNP ISCA Microarray 4X180K (029830) SurePrint G3 Cancer CGH+SNP Microarray 4x180K (030587), hg19 only SurePrint G3 Human CGH Microarray 4X180K (031748) SurePrint G3 Human CGH Microarray 2X105 (031750) SurePrint G3 Human CGH Microarray 8X60K (031746) SurePrint G3 Human CGH Microarray 4X180K (022060) SurePrint G3 Human CGH Microarray 8X60K (021924) SurePrint HD Human CGH Microarray 4X44K (014950) SurePrint HD Human CGH ISCA Microarray 4X44K (031747) The following frequently used tracks are provided upon installation: CNV-DGC_hg19_v2 CpG-Islands_hg19_v2 Cytoband_hg19_v2 Genes_hg19_v2 mirnas_hg19_v2 Pseudo Autosomal Regions_hg19_v2 CNV-DGC_hg18_v2 CpG-Islands_hg18_v2 Cytoband_hg18_v2 Genes_hg18_v2 mirnas_hg18_v2 Pseudo Autosomal Regions_hg18_v2 The tracks with _v2 are with updated annotations.

3 Agilent recommended Feature Extraction protocol for CGH microarrays (CytoCGH_0205_Oct12) and QC metric set for CGH microarrays (CytoCGH_QCMT_Oct12) are provided as default. Agilent recommended CGH analysis settings (Default Analysis Method - CGH v2), CGH+SNP analysis settings (Default Analysis Method CGH + SNP v2) and Mosaic Analysis Settings are provided as default. Agilent Feature Extraction for CytoGenomics Overview Agilent Feature Extraction (FE) for CytoGenomics is a component within CytoGenomics. FE performs TIFF image processing, background subtraction and normalization of microarray data. The user has the option of using this component as part of a CytoGenomics workflow or as a standalone tool. New features for Agilent Feature Extraction for CytoGenomics: Feature Extraction for CytoGenomics will now support PostgreSQL database: 1. Feature Extraction now only uses PostgresSQL for its database. 2. MSSQL, MySQL databases are no longer supported FE now uses Apache PDF maker for QC report generation. Administrator permissions are no longer required for Feature Extraction. The software now stores program information in the file system instead of the registry. The licensing information is now written to file system. These files, along with the other data previously created in the installation folder, are now located where a user without administrator permission has access. A new project setting enables the software to crop multipack images into individual arrays. When this setting is set to True, the multipack image is cropped after extraction is completed and stored in the output folder, along with the other results. The original multipack image is kept intact. A new command for performing extractions was added to FENoWindows. This command takes input as TIFF file and performs extraction using the protocol specified as <protocol_name>, and creates an output xml file <output_file> describing the status of the result of extraction Additional scan image information is displayed in the Scan Image Properties for images that were generated using the Agilent Scanner. For Agilent D scanner the following fields will be added: Laser set points and/or actual laser power readings for each channel (this reflects compensation, if invoked) PMT Calibration Error flag status Scan Control Version For the Agilent B and C Scanners the following fields will be added: Scan Control Version BSPScan Version Runtime Version Scan DSP Version VC DSP Version

4 AF DSP Version Boot ROM Version Extraction of Agilent arrays scanned on following third party scanners is enabled: 1. NimbleGen 2. Innopsys o Note 1: In case of NimbleGen and Innopsys scanned images, it is necessary to have Read and Write permissions on the directory where the images are stored, as FE will convert these images to the required format first and store the converted image into the same directory as the original image. o Note 2: Results from third party scanned images are not validated by Agilent. System Requirements for Agilent CytoGenomics computers Minimum Requirements for Windows Operating system 32-bit Windows (XP-SP2, Windows 7 Enterprise) Any program that enables you to open PDF files (for example, Adobe Reader ) Processor > 2 GHz Working memory (RAM) 4 GB Hard disk space 500 GB Display Resolution 1280 x 768 or higher Recommended Requirements for Windows Operating system 64-bit Windows 7 Enterprise Any program that enables you to open PDF files (for example, Adobe Reader ) Processor > 3 GHz Working memory (RAM) 8 GB Hard disk space 500 GB Display Resolution 1280 x 768 or higher Minimum Requirements for Macintosh Operating System Macintosh OS X Lion and OS X Mountain Lion Any program that enables you to open PDF files (for example, Adobe Reader ) Processor 3 GHz Intel Core 2 Duo CPU or better Working memory (RAM) 4 GB Hard disk space 40 GB (For analysis of large datasets, more space is required) Display resolution 1280 x 768 or higher Recommended Requirements for Macintosh Operating System Macintosh OS X Lion and OS X Mountain Lion Any program that enables you to open PDF files (for example, Adobe Reader ) Processor 3 GHz Intel Core 2 Duo CPU or better Working memory (RAM) 8 GB Hard disk space 40 GB (For analysis of large datasets, more space is required) Display resolution 1280 x 768 or higher

5 Known Issues for CytoGenomics 2.5 Users need administrative rights to install CytoGenomics on Windows 7 OS. (TT ) Sometimes the Application is unresponsive when user tries to run a workflow with CGH+SNP TIFF image and design file. This only happens when user provides the design file from a folder where user has many design files along with XML files which are not design files. (TT ) Sometimes, the text aberration report contains -infinity as a P-value, if the Z score algorithm is used for aberration calling. (TT ) If an exported.bed file type is not associated with any text editor, then the exported track BED file does not open when prompted to open. (TT ) Changing the default genome build doesn't get updated in Content if an AMADID has any default version already set and if the Genome Build constraint Allow using genome build other than default genome build flag is ON. (TT ) The application currently does not show a warning message for images which are not supported (for example from a non-supported Non-Agilent Scanner). (TT ) CytoGenomics License on a Mac has a cross sign shown multiple times in the End Users License Agreement text. (TT ) When the Mac client application points to a server on windows, database connection error will not be shown if the server machine is not on. (TT ) There is a License validation error on the Mac when logged on to the Mac machine using credentials other than the one with which the application is installed. (TT ) Sometimes SNP fit graph is not displayed, if valid SNP analysis is not selected. (TT ) Importing arrays with the same global display name is allowed when there is simultaneous access by different clients pointing to same server. (TT ) Uninstaller removes both client and server without asking any confirmation if same version is installed again in same location. (TT ) Workflow Describe Sample needs copy/paste (TT 19373) SAF import UI needs drop-down list corrected (TT ) 'Sample Review' tab does not refresh immediately when the record is deleted from the 'Supporting Files'. (TT ) Workflows failing if we try to run a job with the 2.0- Analysis Method with the Aberration filter imported in v2.5. (TT ) Message displayed is confusing when auto process stopped and user tries to switch database immediately (TT ) Allows user to run the workflow with same global display name run by other user. (TT ) Navigator gets collapsed when we change default for any design file. (TT ) Multi sample view is not displayed properly if Horizontal orientation is ON. (TT ) Failed/Aborted jobs are not getting removed from the 'sample Review' tab when sample associated with them is deleted from the 'Supporting Files' tab. (TT ) Auto Process workflow output needs folders consolidated (TT )

6 Some invalid images are shown for the drop down combo box in the application. (TT ) When only client is installed, after uninstalling client, nothing is getting installed and scripts are falling. (TT ) Sometime some of the gene view images are not displayed in the signed off PDF report. (TT ) For an only Extraction (no analysis) job ran in 2.0, on upgrade the job entry in the 'Sample Review' shows it as 'Job Failed'. (TT ) French OS-In one particular case two common storage folders are getting generated, so image workflows are failing. (TT ) Sharing for common storage sometime is not getting set properly for Everyone. (TT ) Old version's analysis method when imported in 2.5, do not populate same settings as in old version for aberration filter (TT ) For old analysis method containing Nesting filter condition, should be part of Aberration filter in 2.5 (TT ) Mac-When dock out and open probe id list dialog on cancel dock out view gets dock in. (TT ) Sorting gets removed in the classification count column in the original record after we switch to query result track. (TT ) In supporting files data tab, sorting needs for chromosome column search criteria. (TT ) Triage: Copy All has artifact in pasted picture (TT ) When tried to unlock record having per chromosome probe based report, then it gives wrong message. (TT ) In case of upgrade, in one case redundancy for the user in database (TT ) Points to note for upgrade from older version of Cyto to Cyto 2.5: Until v2.0, for LOH intervals probe count included Non-NaN probes but in v2.5 it is count of Non- NaN & Non-NN probes. So while migration, the probe count may change in 2.5. For upgrading from 1.5 to 2.5, if split parent property is ON in 1.5 then some of the aberrant intervals may not be reported in 2.5 for same record as this property is OFF in 2.5. If the record form earlier version is manually reassigned without diploid peak change, then the intervals in the manually reassigned record will be shown as flat. If the analysis method from 2.0 is imported in 2.5 then the aberration filter is shown with no values for parameters and nesting level filter is not saved as it was part of aberration algorithm parameters in previous versions. For failed and aborted jobs of v2.0, the global display name and analysis method can t be determined so it will be shown as NA (old version) after migration to v2.5.

7 Known issues for Feature Extraction for CytoGenomics 2.5 Sometimes the automation icon does not appear for the extraction set. (Legacy TT 2356) 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 not change. (Legacy TT 2309) For new format scans, while setting color ranges, the ability to adjust colors in high fidelity mode can lead to errors. This occurs if the user tries to set Auto Scale Image after unsetting this option. (Legacy TT 2166) 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 configure/run mode toggle. (Legacy TT 2069) 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. (Legacy TT 1944) When viewing shape files, feature outliers are not visible until the image is zoomed or cropped (which effectively zooms the image). (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) 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) In rare instances, the QC report can fail to generate. This is true when 30 micron 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) 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 {Probe Name, Systematic Name, and Gene Name}. Other annotation fields are not certain to be in the 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) 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 single-channel files. (Legacy TT 282)

8 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) Calculating 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) It is not recommended to run projects, containing multiple extractions, directly through FeNoWindows; that is, projects containing multiple 30-micron feature size 1 million feature arrays. FE will run out of memory if run 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) 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%. For XDR images, when we try to add them for extraction in FE we get an error and hence cannot run the extraction. (TT #198141) Workaround: The extraction for XDR images can be done using the CytoGenomics workflow.