Axiom Analysis Suite 4.0.1 Release Notes (For research use only. Not for use in diagnostic procedures.) Axiom Analysis Suite 4.0.1 includes the following changes/updates: 1. For library packages that support copy number analysis: a. Non-human diploid species copy number analysis is now supported. b. Copy Number Fixed Regions workflow now supports batches of fewer than 11 samples, although batches of at least one full plate are preferred. c. Samples that fail copy number quality control checks are now not used when detecting copy number cluster positions. 2. VCF genotype export format: a. INFO column reports the probeset s Call Rate (CR) and Conversion Type. b. FILTER column reports PASS if probeset_id has BestandRecommended=1, or FAIL if BestandRecommended=0. c. If probeset_id is NOT selected as the annotation to use for the ID field, the INFO field reports the probeset_id as well. d. New enforcement of Unique IDs if probeset_id is not selected as the annotation to use for the ID field: 3. Updates to SNP QC i. For probesets with duplicate reported ID, probeset with BestProbeset=1 is selected for export. ii. If there are still multiple probesets per ID, the first probeset_id sorted by name is selected. iii. Note that if probeset filtering is applied to enforce unique IDs in VCF, the log file will include the warning: "WARNING: Duplicate probeset for ID [value] not written to VCF". a. Chloroplast probesets are now handled as special SNPs. b. ps-metrics and ps-classification bug fixes and improvements include proper handling of data sets containing all male or all female samples. c. MT probesets that are in the special SNPs file will now be classified as MT. d. CR and Nclus now includes all non-nocall calls and all clusters regardless of genotype call. e. HomFLD has been updated for X, Y probesets so that any probeset for males and females with either n_aa==0, n_bb==0, or n_ab<clustermin has HomFLD value of NA. f. HomFLD_hap has been updated for X, Y probesets so that any probeset for males and females with either n_a<clustermin or n_b<clustermin has HomFLD_hap value of NA. 4. For library packages that support allele translation, like PharmacoScan: a. The alleles report fixes the reporting of haplotypes listed in the reportable and non-reportable sections. Axiom Analysis Suite 4.0.1 Release Notes Page 1 of 7
Axiom Analysis Suite 4.0 changes since 3.1 Changes to Analysis Setup and Dashboard 1. Create a new profile when running AxAS 4.0 for the first time. 2. Copy Number workflows are available for select Array Types. The workflows include: Copy Number Discovery, Copy Number Fixed Regions and Copy Number Reference Creation. The new workflows require an update to the library files for these Array Types. To get these library files, in the Preferences pane you need to either have the option checked for Check for Library File Updates at Start Up, or to click the Update button. 3. The Analysis Threshold files (like Human, Diploid, Polyploid) have been updated. To get these files, in the Preferences pane you need to either have the option checked for Check for Library File Updates at Start Up, or to click the Update button. a. New SNP QC parameter min-ychr-samples-cut is available to adjust the minimum number of samples of each gender needed to perform a specific QC test for Y chromosome probesets. Previously Y chromosome probesets would fail SNP QC if the analysis batch had too few samples of either gender. WARNING: The new parameter min-ychr-sample-cut in the Human, Diploid, and Polyploid threshold files is not recognized by versions of Axiom Analysis Suite earlier than 4.0. After downloading these files to your library folder, older versions of Axiom Analysis Suite will no longer be able to run new Genotyping or Best Practices workflows. b. If you are using a new library package, you may notice that the thresholds file includes a.v4 as part of the name (like Human.v4, Diploid.v4, Polyploid.v4). Older library packages will continue to use the existing Human, Diploid, and Polyploid thresholds files. Both versions of the thresholds files can be in the same library folder. c. For library packages that support Copy Number Discovery workflow, a new CN Region QC threshold section is visible. This section has parameters for changing the minimum reported copy number segment size. d. Threshold Settings selection continues to default to whatever was last run based on array type. If it is the first time the array will be run for the current user profile, the Threshold Setting Configuration menu will be blank. You will need to select the appropriate Threshold Setting prior to running your analysis. 4. For some newer library file packages, there is a General Analysis Section in the Analysis Settings Pane. The General Analysis Section contains Inbred File/Value, Hints and Gender file options. These inputs are no longer in the Sample QC and Genotyping Sections of associated workflows. In addition, the Hints and Inbred Penalty input fields will now be separated, allowing both to be used in one analysis. 5. Users can also import CEL files and Sample Attributes into New Analysis by selecting Import Samples and Attributes by Txt or by selecting ARR files. For Import Samples and Attributes by Txt, the header of the first column needs to be cel_files or sample filename. When the batch is loaded for the first time into the Viewer, the sample attributes from the analysis run will be present. 6. When running Reanalyze Selected Samples, results will be saved to a new batch. Users will be asked to provide a new batch name. 7. Users will no longer be able to browse for existing Suitcases in the Dashboard tab. To convert a suitcase file to a batch, users will need to run apt-suitcase-extract command line executable. Axiom Analysis Suite 4.0.1 Release Notes Page 2 of 7
Changes to SNP QC The following changes affect probeset metrics that appear in the ProbeSet Summary Table.This may change the reported ConversionType, which affects what probesets are included in the Recommended probeset list. 1. If no sample in the batch is assigned a gender, metrics for probesets in non-autosomal regions are reported for all samples. 2. If a library package does not have a specialsnps file that identifies probesets for special handling during genotyping, then probeset classification will assume all probesets are autosomal. 3. When determining ConversionType, the call rate of all samples is used for non-par X probesets. Previously only female sample call rate was used. 4. For Y chromosome probesets, the minimum number of called samples of each gender needed for the Euclidean distance test between male and female genotype clusters is configured by new parameter 'min-ychr-samples-cut'. 5. For non-pseudoautosomal X chromosome probesets, the check on difference in A-allele frequency between male and female samples will be done only if there are at least 15 male and 15 female samples. 6. For Y and non-pseudoautosomal X chromosome probesets, measured cluster variance reverts to the default value of 0.03 if the number of samples in the cluster is less than existing parameter clustermin. The new behavior is now similar to how variances are calculated when you select Regenerate SNP Metrics > Run PS Supplemental. 7. If no samples were genotyped for a probeset (either because all arrays suffered from blemishes, or because all samples report ZeroCN call code), then metrics will now be reported. This change allows the probeset to be assigned the appropriate ConversionType. Changes to the Batch Viewer 1. Concordance can now be measured for a subset of samples. Concordance can now be measured against multiple samples in a reference file. Concordance results can be viewed in table or matrix view. Low and Marginal thresholds can be set. 2. In the Sample Table tab, the existing operations Scatter Plot, Box Plot, Plate View, and Concordance are now available from the QC Analysis menu. 3. Plot and graph improvements include: ability to set threshold line on Box and Scatter plots, increase in number of plates allowed in Box plots on X axis, grouping plates by affymetrixscan-date on Box plots, and for 384 well plates there is an additional plate view which maps the 96 well source plates to the 384. For Cluster Plot and CN Regions Plots, hover the mouse over a data point to view a tooltip containing sample name, including Alternate Sample Name (if available). 4. There is a new table in the Summary Tab. The Marker Metrics Summary table contains details on number and percentage of markers (affy_snp_id) based on best and recommended in each ConversionType. 5. Whether an Inbred Penalty was applied can be found in the Sample Summary in the Summary tab. Inbred Penalty score is now a column in the Sample Table. Axiom Analysis Suite 4.0.1 Release Notes Page 3 of 7
6. Sample Table can show the new filtered_call_rate metric, which differs from existing call_rate in that it is restricted to genotyped probesets that are in the batch-specific Recommended probeset list. If calls are manually edited and/or the operation Regenerate SNP Metrics changes the Recommended probeset list, then filtered_call_rate will update. 7. Functionality of Sample Attributes file has been enhanced. If the sample attribute text file contains Alternate Sample Name column, samples can be exported using the Alternate Sample Name instead of Sample Filename. Inclusion of Plate Name column will allow Plate View plots to be labeled with plate names instead of barcode. 8. The name Probeset is used in more places instead of SNP. For example, ProbeSet Summary Table is the new name for the SNP Summary Table tab. 9. SNP List has been renamed to ProbeSet List, and includes improved handling such as ability to remove ProbeSet List from batch. 10. The location of existing Probeset Lists in menus also includes its folder. 11. Default ProbeSet Summary Table column order has changed when opening new batches. 12. Hyperlinked dbsnp RS ID values in the ProbeSet Summary Table simplifies searching for relevant TaqMan assays. 13. Two new columns are available to un-hide in the ProbeSet Summary Table: a. specialsnp_chr identifies the probeset group, which is one factor in the calculations that determine ConversionType. b. MMD is the minimum Mahalanobis distance between points in AA and BB clusters, in the plane created by the AB cluster. This metric is currently not used to determine ConversionType. 14. If annotations are imported into the batch, information such as NCBI and Genome Versions, will be visible in the bottom tool bar of the Sample Table and ProbeSet Table. 15. From the ProbeSet Summary Table, when doing Reanalyze > Regenerate SNP Metrics, selecting OTV posterior file if it exists will actually use the OTV posterior file. 16. Results from a subset of samples can now be exported from a batch. 17. Exporting genotyping data to VCF format: a. The metadata section now includes VCF version as well as genome database version. b. CHROM field uses the common name for the chromosome (i.e. X, Y, MT ) instead of only integers (annot.db s chromosome shortname instead of Chr_id ) c. Marker rows are sorted in genomic order. d. Position of deletion probesets is no longer off by one base. e. Ability to export subset of samples f. Use of Alternate Sample Name in place of Sample Filename. 18. Exporting genotyping data to PLINK format: a. Option to export letter call codes b. Ability to export subset of samples c. Use of Alternate Sample Name in place of Sample Filename 19. Exporting genotyping data to TXT format with Forward Strand Base Call option: if the genotype batch uses copynumber-aware call codes, then haploid calls (like C instead of diploid C/C ) are properly exported as a call without a / delimiter, instead of being changed to --- (NoCall). Axiom Analysis Suite 4.0.1 Release Notes Page 4 of 7
20. For analysis batches with copy number results a. Summary tab with content customized for copy number results b. New plate view visualization of MAPD sample QC metric, if batch doesn t contain genotype results. c. Ability to export copy number results to Variant Call Format (VCF), files compatible with the Broad Institute s Integrative Genomics Viewer (IGV), and files compatible with BioDiscovery s Nexus software. d. For Copy Number Discovery analysis: Ability to display for all or selected samples the new CN Discovery Segments Table, Loss of Heterozygosity Segments Table, and Principal Components Analysis visualization to group samples by similarity of copy number segment calls. e. For Fixed CN Region analysis: i. New Mega Region CN Plot for displaying results for multiple copy number regions of same chromosome in one plot. ii. New ability to export copy number calls for selected CN regions. iii. Export Copy Number Data has been relocated within the CN Summary Table. 21. For analysis batches of arrays that support star allele translation (like PharmacoScan), the Perform Allele Translation operation has changed. a. Allele translation reports can be restricted to a subset of selected samples. b. The Recommended probeset list filter is selected by default. c. Samples for genes with copy number gains include the duplication code xn as part of the reported diplotype name. Note: a library package update is needed for the phenotype report to assign a gene function other than unknown for xn alleles. d. New alleles report, listing all the star alleles in the translation library file that can and cannot be called for probesets supplied for allele translation. e. In the phenotype report, Gene Activity has been renamed to Gene Function. f. Haploid star allele calls now correctly report all allele possibilities if there is a NoCall among a probeset used for haplotyping. Software and Hardware Requirements 1. Axiom Analysis Suite only supports 64-bit systems: a. Windows 7 Professional SP1 (64-bit) b. Windows 10 Pro (64-bit) 2. Memory and CPU Recommended Requirements: a. Quad Core System, 2.83 GHz b. Recommended 16GB RAM Axiom Analysis Suite 4.0.1 Release Notes Page 5 of 7
The following are known issues and limitations in Axiom Analysis Suite 4.0.1: 1. An analysis batch viewed in Axiom Analysis Suite 4.0 or newer may not open with older versions of Axiom Analysis Suite. For example, if in Axiom Analysis Suite 4.0 you apply a filter to the probeset_id field of the ProbeSet Summary Table, close the batch, and try to view the batch in Axiom Analysis Suite 3.1, the batch will not open. 2. Copy Number workflows have larger memory requirements than genotyping workflows, so you will not be able to run as many samples with Copy Number workflows. This is why there is no single workflow that does both copy number and final genotyping. The maximum number of samples for Copy Number workflows is dependent on the array type and the available RAM on the computer. On a system with 16GB of memory, 500 samples per batch has been tested successfully. 3. If there are multiple versions of a library package for the same array in the current library folder, Axiom Analysis Suite may not auto-select the desired one. You will need to check that the correct package is selected prior to running a new analysis. It is recommended that you keep only the latest version of a library file package in the current library folder. 4. Axiom Analysis Suite will not error out if the inbred_het_penalty value in the Inbred Penalty File starts with a numeric character and includes invalid characters (i.e not [0-9.]). 5. Annotation information such as Genome and NCBI version will not be available for CN Batches. 6. When creating Box Plots, attributes with more than 36 characters will be truncated. 7. During Analysis Setup, clicking Restore will not restore when switching between analysis configurations. 8. After running OTV Caller, some calls may be changed to OTV, as expected. However in the Cluster Plot, when selecting the OTV posterior option, the OTV posterior ellipse is not displayed. 9. For supported arrays with predefined copy number regions, like CarrierScan: If you export unedited genotype calls using Export Genotyping Data operation, the calls may be different from the batch folder s AxiomGT1.calls.txt file. This is because AxiomGT1.calls.txt is a temporary file that does not include call changes that might be made by OTV Caller as part of the Genotyping and Best Practices workflows. CarrierScan users are advised not to use the AxiomGT1.calls.txt file. 10. For supported arrays with predefined copy number regions, like CarrierScan or PharmacoScan: If you manually select an optional CN Control CEL List File during analysis setup, the file must not be inside the library package folder being used. If it is, you will get an error message about being unable to read the file. 11. When genotypes are exported to TXT with the forward strand option, ZeroCN call codes (reportable with the PharmacoScan array type) are converted to the equivalent of NoCall ( --- ). A work-around is to export to a different format. 12. If the 'Regenerate SNP Metrics' operation doesn't complete, the ProbeSet Summary Table is not completely refreshed. A work-around is to remove all probeset filters. 13. If the threshold settings are modified during analysis set up and not saved, the manually entered values will stay despite selecting the same default setting in the pull down until the user selects Restore. 14. In the Marker Metrics Summary table of the Summary tab, the count for all the ConversionType sometimes won t match the total count for Number of Markers. This is because there are cases where some probesets don t have a ConversionType. For example, if a batch has only human female samples, no metrics are reported on chromosome Y (including ConversionType). Axiom Analysis Suite 4.0.1 Release Notes Page 6 of 7
15. Axiom Analysis Suite does not retain a copy of the original Confidence value for each call in a binary cache. Confidence values can be changed by running OTV Caller. If after running OTV Caller you select the option to revert calls to the original values, the original Confidence values will not be written back to the binary cache. This means that when you export genotype calls to TXT and select the option to include Confidence, the exported Confidence values are not necessarily consistent with the exported calls. The original Confidence values are available in the file AxiomGT1.confidences.txt in the results folder. 16. AxLE 1.2 will not accept AxAS 4.0 generated Copy Number batches as input. To include Copy Number, the workaround is: Run Best Practice Workflow, using those results, run the Axiom CNV Tool to output the BAF and L2R. AxLE will then include the BAF and L2R data in its output. 17. Sample attributes cannot be imported to the Sample Table if they were imported to the same analysis results in a previous instance of Axiom Analysis Suite Viewer. A workaround is to close Axiom Analysis Suite, remove the existing sample attributes by deleting the files [analysis results folder]\axiomanalysissuitedata\sampleattributes.bin' and SampleAttributes.header.bin, then re-open the analysis results and do a fresh import of sample attributes. 18. Custom Sample QC and SNP QC threshold settings created in Axiom Analysis Suite 2.0 or older versions cannot be used in Axiom Analysis Suite 3.0 or newer. 19. If you get the error message Axiom Batch Access Control Service is not running when you try to open an analysis result, see if there s a lock file inside the last batch folder you accessed. If so, delete the lock file and restart the computer. Then reopen the analysis result. 20. If for a new analysis the progress bar remains at 0% and no status messages appear for several minutes, then the Axiom Batch Access Control Service may not be running. The following procedure may fix the problem: A) Stop the workflow and quit Axiom Analysis Suite, including any open batches in the Viewer. B) Control click on the Windows Taskbar and open Task Manager. C) In the Services tab, search for a service with the Description Axiom Batch Access Control Service. D) Control click on this service row and select either Start or Restart. E) When the service says it is Running, close TaskManager, and retry using Axiom Analysis Suite. 21. The Sample Table will display a maximum of 7 digits for some data values. If the raw value is 1234567.123, then the displayed value will be 1234567. Axiom Analysis Suite 4.0.1 Release Notes Page 7 of 7