• The VS-CNV algorithm has been upgraded with new options, filters and quality flags for the NGS Exome use case. Newly added CNV algorithms will use some of these options, but project templates will preserve the previous algorithm defaults. For more details see our NGS Exome CNV Calling tutorial and the documentation CNV Caller on Target Regions.
• There is a new Create Low Quality Targets wizard accessible from the Manage Reference Samples dialog that produces a set of targets from a selected panel that fail various quality metrics, including average depth across all reference samples, extreme GC content and too short spans.
• Previously when importing external CNVs from a text file, the text file containing the CNVs had to be associated with a sample to successfully complete import. Now, the CNV site files can be imported without a sample association.
• When multiple CNV VCF files were imported into a project with multiple samples, the mapping of samples to VCF was not respected when the imported file order did not match the project sample order. This has been fixed.
• When importing external CNVs, merge behavior settings can now be saved and incorporated into project templates.
• In the CNV table and in the details panel, the ACMG Sample CNV Classifier algorithm now displays the CNV classifications correctly with spaces. Previously, the classifications were displayed as a single word without a space.
• VarSeq now supports editing of the sample names in the project without triggering a re-running of the CNV and LOH algorithms.
• If the coverage table contains extra chromosomes with lowercase letters in their names, a crash would occur in the code used to call whole chromosome CNV events. These issues have now been resolved.

VSClinical ACMG

• Novel splice sites will now be considered by VSClinical when computing the recommended ACMG scoring criteria. Variants that have 3 or 4 out of the 4 splice site algorithms agree that a novel splice site is introduced by the variant will have “No” recommendation for BP7 and will recommend PP3 (instead of potentially BP4), allowing variants that would otherwise be filtered out as likely benign to have some weak pathogenic evidence. Ultimately, review of literature and clinical case history is required to get a novel splice site to a Likely Pathogenic classification. The Computational Evidence section and recommendation text has been enhanced with details about the effect of novel and canonical splice sites, distinguishing between events that cause a frameshift or stop gain versus those that preserve the reading frame.
• We have changed the recommended criteria for predicted splice disrupting variants from PVS1 (with modifiers) to PP3_Strong and PP3_Moderate depending on the predicted impact. This change was made to reflect the exact language of the 2018 PVS1 guideline paper in conjunction with a discussion with one of the authors of that paper.
• The following strength modifiers have been added to the ACMG/ACGS Guideline workflows: PS1_Moderate, PS1_Supporting, PM1_Strong, and PS3_VeryStrong.
• The ACMG Classifier algorithms within VarSeq projects were updated to more strictly apply PM5 only when ClinVar variants are contained by the current mutated codon. This prevents the case of larger deletions that overlap the current variant from triggering a PM5 recommendation. It also now applies it consistently for in-frame insertions and deletions.
• The ACMG Classifier algorithms now applies the ACGS guidelines-specific criteria rules, just like the recommendation in VSClinical. This includes PVS1 for stop-loss variants, PVS1_Moderage and PS1_Moderate (potentially) for start losses and PS1_Supporting (potentially) for splice disrupting variants.
• The VarSeq ACMG Classifier algorithm now recommends PM6 (Unconfirmed Denovo allele) versus PS2 (Confirmed Denovo Allele) for variants that are in a de Novo genotype state in the project. The guidelines require the confirmation of maternity and paternity to use PS2, and PM6 if otherwise assumed.
• In regions with poor multi-species alignment, such as the mitochondrial, BP4 was sometimes being recommended because the algorithm mistook the lack of data as evidence of “Not Conserved. The ACMG Classifier algorithms now only recommend BP4 when there is evidence in both GERP and phyloP that the positions are not conserved.
• Various rare edge cases in variant classification have been harmonized to have consistently suggested criteria and reasons between the ACMG Classifier algorithms within VarSeq and the VSClinical ACMG workflow recommendations.
• The ACMG Classifier algorithms improve handling of multi-allelic variants that require normalization (such A/AA/-) to correctly annotate the normalized form of each alternate allele (the insertion of a A, and deletion of A in this example).
• The ACMG Classifier algorithms had issues when the same sub-population names appeared in multiple custom control population sources. The algorihtm can now handle these duplicate names.
• The ACMG Classifier algorithms had the default options for including population frequency sources to double the homozygous count. This made sense when computing the number of alleles, but was being done unnecessarily in this context, causing the recommendation based on homozygous counts to have incorrect numbers in the recommendation text. Note that the recommendation code itself was correct and not impacted.
• When the ACGS guidelines are chosen as the scoring system, PP5/BP6 will not be recommended as per the following recommendation: “The ClinGen Sequence Variant interpretations group recommends that this criterion is not used (Biesecker and Harrison, 2018).”
• VSClinical now allows adding and annotating variants up to 1000bp upstream and downstream of an interpreted transcript.
• VSClinical now supports adding and interpreting variants in non-coding RNAs. For example, adding NR_003051.3(RMRP):n.-19_-13dupTCTGTGA will now detect the upstream non-coding RNA and detect and display the Pathogenic ClinVar assertion and recommend PP5 along with PM2.
• Mitochondrial variants can now be manually added to VSClinical with NC_012920.1:m.9035T>C where the HGVS notation uses “m” instead of “g” as the prefix.
• PM1 will now be recommended in VSClinical for larger variants spanning multiple amino acids when the nearby pathogenic variant is within 6 amino acids of either the start or stop of the affected amino acids. Previously this computation only compared the distance between the amino acid starts of the compared variants.
• When saving variant interpretations, there is now a “Save & Next” button that will take you to the next unsaved variant in the evaluation. If all variants are saved, the button will say “Save & Review” and take you back to the variant table in the Evaluation tab.
• When using a project opened over a network share, doing quick and repetitive saves of variant assessments in VSClinical had the potential to back up the save queue of the project workflow state and cause a crash. This was only observed in a relatively slow network when the project workflow states were over 10MB, but the issue was addressed by forcing the serialization of these save requests.
• When classifying variants with the ACGS guidelines, now two very strong (VS) criteria will result in a Pathogenic auto-classification.
• The Splice Predictions criteria now considers disruptive novel splice site predictions as well as changes to existing splice sites. The wording of the criteria and responses has also been updated to reflect these changes.
• The splice sites view in VSClinical displays various details on hover. The “Delta Raw” metric has been added to the splice site prediction tool tip for GeneSplicer and MaxEntScan predictions.
• The VarSeq 2.2.2 release introduced a regression that removed the ability to switch genes in the transcript selection dialog when a variant overlapped multiple genes. This capability has now been restored to allow the evaluation of the variant to switch to a different overlapping gene or even non-coding RNA.
• Multi-allelic variants in the 1000 Genomes catalog would in some situations be picked up incorrectly in the ACMG recommendation engine, impacting criteria like BS2. This has been fixed to always pick the correct allele frequency records from the 1000 Genomes catalog.
• The system Word report templates have been updated to have different formatting and output for each of the Test Result values of Positive, Negative, Inconclusive, Not Evaluated
• An option to “Open Systems Filters Location” has been added to the drop-down menu for the Word template output selector that displays the system filters file that may be useful when learning the programming environment for Word template filter functions.

VSClinical AMP

• The first two tabs of the VSClinical interface have been combined into an Evaluation tab. This makes it easy to see the tumor type before adding mutations to the evaluation. A “Changelog History” section was added as well. Similarly, a fly-out menu is available from the top-right of the interface, similar to VSClinical ACMG, that provides an overview of the evaluation and quick access to actions like close and finalize.
• A Commercial Labels field has been added to the reportable drugs section. This field fills in automatically from DrugBank’s provided “Product” field for drugs and drug combinations.
• The variant scoring card now has an auto generated Oncogenicity Interpretation based on the scored criteria that can be added to the variant interpretation with a single button.
• Spice acceptor variants that are predicted to cause exon skipping of an in-frame exon are optionally interpreted as exon deletions instead of LoF variants. This allows cancer variants like NM_007297.4:c.5327-1G>A to be interpreted as a MET exon 14 deletion as it is commonly characterized in the literature.
• You can now add Loss of Heterozygosity events to the AMP workflow from the CNV project table or manually. Per-gene LOH events can be provided with biomarker interpretations and added to clinical reports with AMP tier evidence levels.
• When using a project that has not been configured with VSClinical ACMG, loss of function germline variants showed an error message and not a recommendation for the ACMG PVS1 criteria. These variants can now be added without issue.
• When saving somatic or germline variant interpretations, there is now a “Save & Next” button that will take you to the next unsaved variant in the evaluation. If all variants are saved, the button will say “Save & Review” and take you back to the variant table on the Mutation Profile tab.

VSPipeline

• Liftover can now be enabled in the import command with the additional “liftover=true” parameter, allowing the import of VCF files with different coordinate systems than the genome assembly specified in the project template.
• Attempting to import a VCF file with a detected genome assembly different than that of the selected assembly for the project template will produce an error with a description of the mismatched coordinate systems.
• The update_cnv_import command caused an import error when provided more than one CNV VCF file as input. It now works with both single VCF file and per-sample VCF files.
• To support the above capabilities, a task_wait command is required before exporting or closing the project to ensure the import and all dependent algorithms are run. See Waiting For Task Completion for more details.
• VSPipeline will now provide a more informative error message for license_activate if the program does not have permissions to write the license file.
• There is now a download_annotations.sh script that invokes vsipeline with the request to download the reference sequence for the given genome assembly (or GRCh37 by default).

VarSeq Annotations and Algorithms

• A new “Gene List Exon Coverage” algorithm is available that will compute coverage statistics on a list of genes instead of requiring a user-provided target region annotation file. For each gene, the clinically relevant transcript is selected, and coverage is computed for the coding region of each exon. See Gene List Coverage Statistics.
• The Coverage Statistics Algorithm (and Binned Coverage Statistics) now have a “Count Duplicate Alignments (filtered by default)” that when check, turns off the filtering of reads marked as duplicates in the BAM.
• Most VarSeq algorithms, such as the ACMG Classifier and the Annotate Transcripts Algorithm, that have user defined setting options now have a right-click menu option allowing the algorithm options to be edited from the variant or CNV table directly.
• ACMG CNV Site Classifier: A new algorithm that is computed in the CNV Table. This algorithm computes classifications for each CNV based on the ACMG CNV Guidelines and does not require sample designation, or the CNVs to have been called with the VarSeq CNV algorithm. Additionally, this algorithm can be used with CNVs imported from an external caller.
• Updates have been made to the Annotate Transcripts algorithm to better support annotating non-coding RNAs along with a new option to include non-coding RNA, mircoRNA, and mRNA transcripts. Another option has been added to allow the annotation of upstream and downstream variants. These options are on by default for newly added gene annotations.
• Our shipped project templates have been updated to match the default options for gene annotation, including the annotaiton of non-cding RNAs, the inclusion of upstream and downstream variants and the detection of splice sites. Splice site predictions no longer require a VSClinical license.
• The transcript annotations algorithm novel splice site predictions for insertions and deletions now considers the canonical splice site and the novel splice site in conjunction to not predict an introduced splice site that is just the shifted canonical site.
• There are RefSeq transcript alignments to the human genome that have to compensate for differences in the reference sequence to the canonical RNA sequence. When these differences are one or two missing or additional bases, the transcript alignment introduces a one or two base “intron” (alignment gap). The transcript annotations algorithm will no longer classify variants in these gapped introns as canonical splice site mutations, but simply as splice region variants. This results in fewer benign polymorphisms from being classified and considered incorrectly as loss of function variants.
• The RefSeq shipped gene track for GRCh37 was replaced with a version that contained the same transcripts but has updated gene names and aliases to match the latest genes names from NCBI. Specifically, the gene names for the MT chromosomes were using older naming conventions and not the “MT-” prefix used by the latest gene tracks.

VarSeq Projects and General

Note

There have been fairly large changes in the system gene preferences file (under Data/GenePreferences.gene-pref) in VarSeq 2.2.2 and now in VarSeq 2.2.3. These changes were prompted by the incorporation of the MANE transcripts in our most recent gene tracks and the large increase in the number of available transcripts in those tracks. In a high-profile example, BRAF went from single transcript to multi-transcript status, switching the long-established default transcript. Our heuristic for selecting clinically relevant transcript has been stable, but we use the system gene preferences to override the heuristic when there is a clear community preference for a given transcript. For example, BRAF NM_004333 has 563 ClinVar submissions referencing this specific transcript, yet the “MANE Select” mRNA transcript NM_001374258 has 0. In VarSeq 2.2.3, we have used the ClinVar Assessments counts to ensure these clear preferences are the default. Of course, you can switch transcripts used in VSClinical at any time, and saving a variant interpretation also saves your transcript preference for all future variants in a given gene. Using this updated strategy, in VarSeq 2.2.3 there are 850 genes with preferred transcript defined.

• The shipped gene preferences file has been updated to select default transcripts based on data from the ClinVar Assessments track and the gene track. Transcripts that have more than 10 ClinVar assessments and more than double the assessments than the next most interpreted transcript will be set as the default transcript, overriding the MANE transcript in about 90 genes. The remaining 750 genes with manual transcripts are ones without MANE transcripts, resulting in the clinically interpreted transcript replacing the transcript that would have been selected by VarSeq’s default heuristics.
• VarSeq now runs on Mac with the “Dark Mode” setting enabled. Previously, the setting caused a mix of inverted and regular colors. While VarSeq does not have a separate dark color palette, it now will consistently use the default colors and remain usable in dark mode.
• Attempting to import a VCF file with a detected genome assembly different than that of the selected assembly for the project template will produce a warning on the select sources screen and prompt with a final warning message when completing the wizard.
• The HGVS output for variants before and after transcript sequences has been improved to be able to use the “dup” syntax for insertions. For example: NR_003051n.-22_-15dupTACTCTGT.
• The log messages for algorithm computation completion now list the relevant axis (table types) in the output. For example, annotating a NV table will now have an event name of “Task Finished” (CNVs).
• On import, there is a new default option to “Use Default Chromosome Names” that will rename alternative chromosome names to the primary names used in VarSeq. For example, a VCF with NC_000009.11 in the CHR field will be imported as 9. Similarly a VCF with “MT” for a GRCh38 project will be renamed to “M” (see following note).
• Annotating assessment catalog variants with an “M” chromosome name on GRCh38 would fail due to the assembly primary chromosome name being “MT”. We changed a couple of things in this regard. First, we made changes to the annotation code to allow project variants with non-primary chromosome names to be read and saved to assessment catalogs. Second, we changed the default mitochondrial chromosome name on GRCh38 to be “M”, matching the reference sequence naming provided by NCBI and commonly used for alignment to GRCh38. Finally, we translate non-primary chromosome names to the primary chromosome names of the assembly on import.

Note

There that GRCh38 assessment catalogs with previously saved “MT” chromosome entries will not be seen in new projects that now have “M” mitochondrial chromosome names on import. Reach out to support and we will help you update your historical assessment catalogs to rename all “MT” interpretations to match the new “M” naming convention going forward.

• VarSeq projects templates will now remember preferences about mapping per-VCF fields like QUAL into sample fields when saving project templates. This also works for imported CNV VCF files as part of the project template.
• GenomeBrowse: Improved handling of plotting mitochondrial chromosome variants regardless of “M” or “MT” being used as the chromosome name.
• GenomeBrowse: When the version of an annotation source is updated in the variant and CNV tables, if the source is plotted in GenomeBrowse, the plot will now update to the same annotation version that was used in the table.
• GenomeBrowse: The Feature List when displaying all features of filtered clinically relevant GRCh38 gene tracks will now display all features, including those in the mitochondrial chromosomes
• GenomeBrowse: Newly created BAM plots will now have the “Filter Duplicate Alignments” option turned on, to match the coverage statistics algorithms in VarSeq.

Note

If you are interested in adding the new VSClinical ACMG or VS-CNV workflow product to your VarSeq license, please contact your account manager or info@goldenhelix.com.

• The VSClinical ACMG Guidelines workflow now has an additional CNV interpretation framework based on the ACMG/ClinGen guidelines. This product supports interpreting CNVs detected with VS-CNV or imported CNVs alongside variants and requires both a VSClinical ACMG license and a CNV license.

Release notes are organized by product. Each product section starts with new features, then polish items and bug fixes for that product or functional area.

VS-CNV

• A new flag was added to the CNV table output which denotes deletions that contain two or more heterozygous variants. This flag is called “Deletion Contains Heterozygous Variants”.
• Probability/Segregation: A new algorithm has been implemented, “Copy Number Probability/Segregation”, which runs on the CNV table and computes the expected copy number of each called CNV. If parental information is provided for a sample, the algorithm also computes the probability that the CNV is present in the mother and the father. This is useful for having a computed confidence that a CNV is de Novo.
• Latest Assessment: A new algorithm has been implemented, “Latest CNV Sample Assessments”, which annotates the CNV table with the latest assessments from a selected CNV assessment catalog.
• Matching Sample: Two new algorithms has been implemented, “Annotate CNVs Matching Current Sample” and “Annotate Regions Matching Current Sample”, which annotate the CNV or region table with the assessments from a selected CNV assessment catalog that contain the current sample name in a specified “Samples” field.
• The default value of “Size of the Reference Sample Subset” in the Advanced Parameters for the CNV Caller on Target Regions has been increased from 100 samples to 10,000 samples. This returns VS-CNV to match the 2.2.0 release behavior in most cases.NoteThe 2.2.1 update added a feature to subset the considered reference samples to handle extremely large reference sample sets. The default value of 100 used in 2.2.1 resulted in selecting sub-optimal reference samples and thus changed the performance of the CNV caller negatively for some customers. To maintain the same normalization behavior as Version 2.2.0, we have updated the default to a very large number, namely 10,000.
• The CNV output of a karyotype value has been adjusted to only report a karyotype on CNVs larger than at least one cytoband. CNV flags will also be reported for large CNVs along with karyotypes.
• The Binned CNV Caller algorithm was running slowly due to processing the Variant Allele Frequencies (VAF) values, even though they were not incorporated into the calling algorithm. The VAF values are no longer processed during Binned CNV Caller computations.
• The CNV caller algorithm (Target or Binned) would get triggered to re-run when any sample table field was edited. Now it only re-runs when the “Samples” name field changes.
• The “Annotate Overlapping Genes” algorithm has been enhanced to support the match mode of features that are “Within” or “Overlap” the current record. This is in addition to the current matching based on a similarity (Jacard) index.
• The “Annotate Overlapping Regions” and “Annotate Overlapping CNVs” algorithms now have the additional options of annotating records that are completely “Within” another annotation record or annotating records that completely “Contain” other annotation records.
• The CNV gene annotation algorithm, when run on a large number of CNVs, would produce an “Algorithm Error”, reporting that it “exceeds the maximum number of unique strings”. The algorithm has been updated to have the correct type for the “Region (Clinically Relevant)” field and to no longer produce this error.
• When adding a CNV annotation algorithm through Add > Computed Data, then selecting Annotate Overlapping CNVs (or Regions) from the CNV table, afterwards selecting a warehouse source, the software would report that only local sources are supported. This has been updated to support setting parameters for annotating warehouse-based catalogs.
• The sample sex detection for the CNV caller has been enhanced to use the heuristic of a normalized Y mean depth ratio of < 0.05 for females.
• The Target CNV Caller dialog has new “Advanced” parameters:
  • The checkbox “Signal Scaling for High Percent Difference” allows manually (enabling or) disabling normalization scaling performed on samples with a high percent difference to the reference samples.
  • The new parameter “Percent of Targets to Force Aneuploid Call” forces an event to be called a whole chromosome event if the percentage of targets in a given state exceed the selected percentage (95% default). In other words, if the percentage of deleted or duplicated targets in a chromosome exceed the selected threshold, then the entire chromosome will be called as an aneuploid deletion or duplication event.
  • The new parameter “Sample Type” can be set to either “Auto”, “Gene Panel”, or “Exome” for inferring the sample type. If the sample type is “Exome”, the algorithm is permitted to call whole chromosome events and report these events in the Karyotype output at the sample level as being whole chromosome events. You may force this option if you desire to call whole chromosome aneuploidy events, especially in the X or Y chromosomes.
  • The option “Subset normalization targets in exomes to those containing het variants” will cause the algorithm to only use targets containing variants with a heterozygous VAF when computing the mean coverage for normalization. This is recommended only for highly mutated tumor samples. In previous releases this action always took place for “exome” sized target lists, but now must be turned on explicitly as an option as most samples do not benefit from this normalization technique.

VSClinical ACMG

• As noted above, the VSClinical ACMG Guidelines workflow now has an additional CNV interpretation framework based on the ACMG/ClinGen guidelines. This product supports interpreting CNVs detected with VS-CNV or imported CNVs alongside variants and requires both a VSClinical ACMG license and a CNV license.
  • The existing ACMG variant scoring workflow has been reorganized into a single view under the Variants tab.
  • The new ACMG CNV scoring workflow has been created under the CNV tab.
  • The Gene tab now contains coverage statistics at the target level as well as the ability to provide a gene list for summary statistics and reporting.
  • The Phenotype tab allows selecting phenotypes and disorders for the patient as well as entering patient notes that result in automatically extracted terms.
  • The Reports tab summarizes all reportable information and the ability to render that information into a customizable Word and PDF report output.
• The management of annotation sources used by the ACMG (and AMP) guidelines has been updated to allow selecting specific versions to be used in the evaluation process. The versions selected when creating an evaluation will be locked. This means that regardless of what new or updated sources are downloaded, an evaluation will continue to use and display the versions of sources selected on creation. The ACMG workflow allows these versions to be locked to the project template so all new evaluations are created with a specific version list. The name and version date of each source is available to display in the Word-based report system.
• The VSClinical ACMG catalog schema has been expanded with fields “Omim ID”, “Mondo ID”, “Report Section”, “Criteria Comments”, “Citations Data”, “Interpretation Citations”, “Exon Number” and “Interpretation Notes”. There is a useful button in the ACMG configuration dialog to upgrade your catalog to include these fields. Once upgraded, all newly saved interpretations will include data in these fields from the evaluation.
• The population catalogs using the scoring criteria BA1, BS1, and PM2 can now be configured in the options dialog under the “ACMG Frequency Sources” tab. The catalogs for controls (BS2) can be controlled under the “ACMG Control Sources” tab.
• The PM2 criteria recommendation is now based on a very low allele frequency instead of an absolute total allele count to be more robust to population catalogs with small sample sizes. In rare cases, the previous behavior resulted in both PM2 and BA1 being recommended. These settings are tunable, but by default the PM2 threshold is less than 0.02% for recessive genes and 0.01% for dominant genes. The threshold will be evaluated on the sub-population with the largest allele frequency that meets the specified minimum allele count.
• The ACMG guidelines have been updated to incorporate the Recommendations for interpreting the loss of function PVS1 ACMG/AMP variant criterion. This allows PVS1 to be recommended at lower strength levels (Strong, Moderate, Supporting) based on LoF variants with less definite evidence of pathogenicity.
• The Gene Constraints track used by VSClinical has been switched from ExAC to gnomAD. You can expect the constraint values used in PVS1 and PP2 to evaluate the tolerance of loss-of-function variants and missense variants (respectively) to be different. Additionally, the LoF value now being used is based on the computations of the observed/expected ratio (O/E) and specifically the upper bound of the 90% CI computed on this value as suggested by the gnomAD team in the gnomAD v2.1 blog post.
• A special consideration warning is now given if “PVS1” and “PP3” are both scored.
• The ACMG criteria have had the evidence strength modifiers expanded to include newly published papers’ recommendations.
• Variants reported in the Gene Region and Mutation Profile table for forward-strand genes are now displayed in their right-aligned position. In most cases, this should match the HGVS descriptions reported by ClinVar for these variants.
• Now, adding the ACMG Classifier algorithm will always use the existing project’s version of the gene track for nearby variant criteria in order to stay consistent with the current project. Previously, the algorithm always selected the most recent locally downloaded gene track version.
• The ACMG classifier algorithm now ensures that previous classifications are detected and counted even when the transcript name has changed, such as when a gene track has been updated.
• The ACMG classifier algorithm now matches any gene annotation algorithm in the project to define the splice region variant for the purpose of the variant sequence ontology.
• Comments that were added to different classification sections in VSClinical were not being correctly added to the variant interpretation when selecting the “Add Text to My Interpretation” option. This has now been fixed.
• Comments that were written in the “Criteria List Table” were not being saved to the evaluation. This has now been fixed.

VSClinical AMP

• The AMP workflow has now adopted the concept of work being done in “Evaluations” similar to the VSClinical ACMG workflow. This allows for the creation of potentially multiple evaluations per samples, and also allows for wiping the slate clean by deleting an evaluation. Existing projects are upgraded to show a single evaluation where work was done for a sample.
• The AMP workflow report templates have been updated to use the new customizable “filters” technique also used in the ACMG workflow. This means the input data has been re-organized to be less redundant while also providing more details.NoteDue to these changes, Word report templates based on the previous version may not fully render. We suggest you re-create custom reports starting with the two default report templates. Please contact support if you have a more complex custom template and we will be happy to assist in transitioning it to the new system.
• A “Somatic Catalog” was added to the AMP workflow. This allows saving and auto-filling previously saved oncogenic scoring and interpretations at the variant level for somatic variants. This is the equivalent of the “Classified Germline” catalog for the ACMG workflow and germline variants in the AMP workflow. You can save/load variants into this catalog in the “Variants” tab, while still using the “Cancer Interpretations” catalog in the “Biomarkers” tab.
• The updated management of annotation source versions now used in VSClinical is used in the AMP guidelines as well. See the note in the ACMG section for more details.
• A new Genes tab is available with the details and plots about the target region coverage. You can now provide virtual gene panel lists at the sample or project level to report coverage details.
• The cancer tumor type options have been expanded to have a top-level type for each tissue type. For example, “Lung Carcinoma” can now be selected instead of just one of its sub-types.
• When scoring somatic variants, the cancer hotspot criteria is now available to manually score for any variant type, not just missense and in-frame indels. The auto-scoring system will continue to only recommend the hotspot criteria for missense changes.
• When searching for clinical trials, there is now a filter option for trials that contain specific Diseases in their summary.
• Manually adding germline variants to the AMP Guidelines was producing an error. It is now supported again.
• There was an SSL handshake error on Windows when using the cloud convert Word to PDF feature. This has now been fixed.

VSReports

• APIs that allow for reading input data from other samples have been added to VSReports.
• VSReports now allows the creating of reports in projects that contain only variant sites without associated samples.
• VSReports now allows selecting record sets for Coverage and CNV sections from any of the tables in the project. Previously only a single table of a given type was supported. This allows, for example, reports that may select failed target regions from different coverage tables.

VSPipeline

• VSPipeline now has a “update_cnv_import” command that allows the setting of input files for importing external CNVs into the project from VCFs.

VarSeq Annotations and Algorithms

• Latest Assessment: A new algorithm has been implemented, “Latest Variant Sample Assessments”, which annotates the variant table with the latest assessments from a selected variant assessment catalog.
• Matching Sample: A new algorithm has been implemented, “Annotate Variants Matching Current Sample”, which annotates the variant table with the assessments from a selected assessment catalog that have the current sample name in a specified “Samples” field.
• Transcripts: The novel splice site predictions in the transcript annotation algorithm now has a “N of 4 Changed To Predicted Splicing” field to improve filtering options for interesting novel splice sites.
• Transcripts: Previously the novel splice prediction algorithm reported any change in splicing predictions, but now it reports only changes from not-splicing to splicing for a novel splice site.
• Transcripts: For multi-allelic variants, it is possible that each alternate allele has a different sequence ontology and impact on the overlapping transcript. The “(Clinically Relevant)” output fields, including the HGVS descriptions, have been updated to prioritize reporting the allele with the highest impact on the transcript.
• Transcripts: The “Distance to Splice Site (Clinically Relevant)” field in the transcript annotation algorithm has been updated to account for all combinations of a variant being before or after the splice site and on reverse strands. For clarity, this distance calculation is now the number of bases that would be required to move the variant to overlap the canonical splice site. This is generally one greater than the previously reported number.NoteThe output of the “N of 4 Predicted Splice Disrupted” outputs may also change due to changes related to this fix. We expect the change to be in the direction of an additional algorithm predicting the variant as splice disrupting.
• Transcripts: The HGVS format for frame-shifts that introduce stop-gains has been changed to not include the “fs” suffix. Thus, for instance, we have “p.Leu103*” instead of “p.Leu103*fs” (as preferred by ClinVar). This matches the format expected by other tools such as Mutalyzer.
• Target Region Coverage: The option to “Collapse overlapping regions” was added to the coverage statistics algorithm so that, if this option is selected, target regions that overlap do not get counted more than once.
• Target Region Coverage and Binned Region Coverage: These algorithms now allow users to optionally include multiple additional depth threshold calculations outside of the default 1x, 20x, 100x, and 500x options.
• Gene List: The Match Genes List Per Sample algorithm was creating an error if there were certain missing fields in the input source. This has been changed to run even if some fields are missing.
• Phenotype Linked Genes: The Match Genes Linked to Phenotypes algorithm will now show the linked genes as well as the selected HPO terms used in the project Log.
• PhoRank: The Human Phenotype Ontology and Gene Ontology tracks used by PhoRank and other phenotype-based features have been updated to the latest versions.
• PhoRank: The PhoRank algorithm was previously producing ‘nan’ values for the score when all of the genes involved receive a score of zero. They are now filled in with ‘?’ values.
• PhoRank: Running the Variant PhoRank Gene Ranking algorithm with HPO terms did not recognize the HPO terms if “Enhance with OMIM Phenotypes” was selected. This has now been fixed.
• Mendel Error: The Mendel Error algorithm now outputs the fields “Inherited from Mother Count” and “Inherited from Father Count” to the sample table.
• Import: There is now improved support for importing CNVs in VCFs from more external callers such as CANVAS.
• Import: The import system used to add additional sample level fields was made robust to detect and handle non-UTF-8 text encoded using the Latin-1 codec in addition to continuing to support the default UTF-8 Unicode.
• Import: The way in which multi-allelic variants have their alternate alleles represented on import was dependent on file list order (if different files used different allele orderings), but this has been changed to be file order agnostic by forcing all multi-allelic variants to be in lexicographic order (A/C/G/T).
• Import: The “Match Variants to Affected Individuals’ Genotypes” import option had an out of bounds error with the trio date when samples were excluded on import. This has been resolved to allow sample subsetting and variant matching on import.
• Import: When importing files to VarSeq, the default import selections were not preserved if the Advanced Options checkbox was checked and a project template was being used. This has now been fixed.
• Import: Importing multiple CNV files for different samples was not correctly mapping CNVs to the assigned sample. This has now been fixed.
• Import: The 23andMe import tool has been updated to set the genotypes for insertions/deletions to missing, as the provided data in their text file does not inform genotypes.

VarSeq Projects and General

• The shipped gene tracks have been updated:
  • GRCh37: RefSeq Genes 105.20190906 v2, NCBI with the 0.91 release of MANE transcript status.
  • GRCh38: RefSeq Genes 109.20200815 v1, NCBI, with the 0.91 release of MANE transcript status.
• Network The following “timeouts” have been increased:
  • Loading remote source info: from 5 seconds to 1 minute.
  • Querying remote annotation source: from 1 minute to 3 minutes.
• Sources: In a multi-user or clinical environment, it may make sense to not use the latest version of sources published to the public and secure annotation sources when adding new algorithms or using VSClinical but rather depend on the latest downloaded sources. This fundamental behavior can be changed for all users launching VarSeq by adding a “hosts.json” file to the install directory. This file also allows setting the default VSWarehouse URL so that each user does not need to configure that internal property the first time they run VarSeq. See Installing and Initializing for more details
• Sources: VarSeq now prompts when a requested download action will create a second copy of an annotation source. This prevents the problem of a shared annotations folder collecting multiple fully downloaded copies of updated annotation sources.
• Projects: The shipped templates now have the common annotation sources unlocked so that the most recent versions of these annotations will be used automatically.
• Projects: Certain complex templates would run to completion but not display the per-sample data in the coverage table. The visibility of these fields has been resolved.
• Projects: When using a saved project template which used LiftOver in the import process, changes to the LiftOver options on new projects were not recognized. New projects can now change the assembly of input files on project templates saved with LiftOver enabled.
• Projects: The project loading performance was improved for projects with multiple overlapping filters and algorithms.
• Projects: On the latest Mac and Linux operating systems, complex projects could crash due to low default resource limits applied to applications. VarSeq now raises the allowed file handles and will no longer crash due to this cause.
• Annotation Sources: You will now be prompted if you select an annotation source to download that is already present in your annotations folder or is in the process of being downloaded. This situation may happen when you are using a shared annotation folder or you are running multiple instances of VarSeq.
• Tables: In VarSeq tables, the Change Column Visibility dialog now makes it easier to disable a group and not lose the state of an individual column’s visibility.
• GenomeBrowse: On GRCh38, the X and Y chromosomes would only display if there was plotted data present in them. This has been updated to always display these chromosomes, which matches the behavior for GRCh37.
• GenomeBrowse: The handling of gene tracks with malformed coordinate systems has been improved.
• GenomeBrowse: Plotting certain tracks that used chromosome identifiers such as NC_000001.10 failed to display the zoomed-out coverage. This was an impact from recent versions of dbSNP. These recent versions of dbSNP are now supported.
• GenomeBrowse: In rare circumstances, closing the GenomeBrowse view would cause the program to crash. Changes were made to the cleanup strategy to prevent further crashes upon closing the GenomeBrowse view.

• A new feature, Genotype > Quality Assurance and Utilities > LD Score Computation and Binning, has been added that
  • Determines an LD Score for every marker,
  • Finds the minor allele frequency (MAF) for every marker, and
  • (Optionally) categorizes (or “bins”) every marker according to its LD Score, its MAF, or a combination of LD Score and MAF.The base pair window size for computing LD Scores may be selected, as well as MAF thresholds for binning (if MAF binning has been selected).
• A new feature, Genotype > Compute GBLUP Using Bins, has been added which performs Genomic Best Linear Unbiased Predictors (GBLUP) using markers that have been binned into categories, with a separate Genomic Relationship Matrix (GRM) being created and used for every category.The outputs of this feature correspond with the outputs of Genotype > Compute Genomic BLUP (GBLUP):
  • If computed dynamically, a GBLUP Genomic Relationship Matrix (GRM) for every bin (category), and a Genomic Relationship Matrix List of the GRM spreadsheet numbers.
  • Binned GBLUP estimates by sample This spreadsheet includes the total estimated random effects, the estimated random effects corresponding to each GRM, and (optionally) the predicted phenotype value for each sample.
  • GBLUP fixed effect coefficients This spreadsheet shows the intercept value and the coefficients for any fixed-effect covariates you may have specified. (This corresponds to a new spreadsheet now created by Genotype > Compute Genomic BLUP (GBLUP).)
  • Binned GBLUP estimates by marker This spreadsheet delineates each marker’s bin (category) name and the value of its allele substitution effect (ASE).To categorize your markers into bins, you can
  • Use the new Genotype > Quality Assurance and Utilities > LD Score Computation and Binning feature (mentioned above),
  • Use the chromosome name as the binning category,
  • Run DNA-Seq > Variant Binning by Frequency Source from the spreadsheet menu, or
  • Any other method you wish to use for categorizing markers.
• The Genotype > K-Fold Cross Validation (for Genomic Prediction) feature has a new option, Predict Only Once per Iteration. This effectively allows a mode of creating sample subsets using “sampling with replacement”.
• A new feature, Edit > Recode > Clean Up Missing Genotype Data, has been added which will convert partially-missing genotypes (such as “A_?”, ”?_B”) to the proper missing-value form, which is ”?_?”.
• A new Tools > CPU and (GenomeBrowse) Memory Settings dialog has been added to allow control over memory used for reading annotation files and for GenomeBrowse scene rendering, and also control over the number of CPU processors used. Note: This feature is distinct from the Memory Usage section of Tools > Global Product Options, which allows control of memory for dataset caching, transposing of spreadsheets, and analysis.
• Three new capabilities have now effectively been added to SVS as a result of changes (as noted in the sections below) to existing features:
  • The liftover functionality of Import > Import VCFs and Variant Files is now available.
  • Genotype > Predict Phenotypes from Existing Results may now be used on results from Genotype > Compute Genomic BLUP (GBLUP).
  • Dominant-model or Recessive-model data (as well as Additive-model data) may now be PCA-corrected, then used as input to either Genotype > Mixed Linear Model Analysis or Genotype > Mixed Linear Model Analysis with Interactions.

Polishes

• Genome assemblies have been added and updated, including Chicken (Gallus gallus, GRCg6a), Cowpea (Vigna unguiculata, JGI_UCRv1.0), Water Buffalo (Bubalus bubalis UOA_WB_1), and Barley (Hordeum vulgare IBSC_v2).
• Several SVS tutorials have been updated to account for newer features (such as SKAT-O) and other improvements in SVS, to use newer annotation data, or to make the explanations more clear.
• The Data Source Library window now automatically refreshes after a track finishes downloading.
• Target Region CNV options and Whole Genome CNV options may now be saved as project options or global options.
• The final screen for Import > Import VCFs and Variant Files has been greatly improved to show all import parameters clearly.
• The family data feature of Import > Import VCFs and Variant Files now imports the data as a pedigree spreadsheet.
• The HapMap importer (Import > HapMap) has been updated to be able to incorporate IUPAC single-letter encodings.
• The instructional prompts, error messages, and the log messages for Edit > Recode > Recode AGCT Alleles to AB were modified to increase clarity for the user.
• The names of the menu items in the Affymetrix CNV import options have been improved to clarify what type of data is supported.
• The following improvements have been made to the Genotype > Compute Genomic BLUP (GBLUP) feature:
  • The intercept value and the beta values for the covariates are now output as a separate spreadsheet, using the same format as does Genotype > K-Fold Cross Validation (for Genomic Prediction). This means that Genotype > Predict Phenotypes from Existing Results may now be used with outputs from the Genotype > Compute Genomic BLUP (GBLUP) feature.
  • Predict random effects for samples with missing phenotypes may now be used with the Average Information algorithm (and thus with features requiring the Average Information algorithm).
  • The user interface for Genotype > Compute Genomic BLUP (GBLUP) (and for Genotype > Quality Assurance and Utilities > GBLUP Genomic Relationship Matrix) now detects whether or not you have an X Chromosome in the marker map, and changes its defaults (and other behavior) accordingly.
  • In Genotype > Genetic Correlation of Two Traits using GBLUP, Exclude the residual covariance from computations has been changed to be the default, since this invokes a version of this algorithm which more realistically represents the problem, and also is more likely to converge.
  • For the Average Information REML algorithm, both for this feature and for the Genotype > Genetic Correlation of Two Traits using GBLUP feature, SVS will always strive to give useful output, even if the algorithm “did not converge”.
  • The Variance/Covariance matrix from the Average Information REML algorithm is now output as an SVS spreadsheet.
  • Calculation of allele substitution effects (ASE) for multiple GRM’s has been optimized.
  • A few error messages (and error notes for the node change log) for the EMMA algorithm, as well as messages to note collinearity of covariates, have been clarified. In addition, many messages have been reformatted to improve their appearance.
  • Integer columns are no longer allowed as gene-by-environment category variables. (This feature works best using only a limited number of categories. Integers with only a limited number of distinct values may always be converted to “strings” (categorical variables) in Edit > Edit This Spreadsheet.)
• The following improvements have been made to Genotype > Predict Phenotypes from Existing Results:
  • Dosage Compensation and Individual Normalization may now be specified as inputs.
  • The checking for whether the covariates listed in the fixed-effect spreadsheet are all re-specified as covariates when using this feature has been made more precise.
  • Random effects will now be predicted for all samples. This is in addition to the current output of predicting phenotype values for samples with valid covariate values.
  • The overall flow of dialog and execution has been improved.
• The run log for Genotype > Bayesian Genomic Prediction is now updated more often, so it is less likely that the software will appear to have stopped running when it hasn’t.
• For Numeric > CNV on NGS Target Regions, there is now a progress dialog for creating the state spreadsheet, and an optimization for creating the state spreadsheet when showing a categorical CNV state. Thus, the software will not appear to have stopped running at this step when it hasn’t.
• For Genotype > Mixed Linear Model Analysis with Interactions, outputs are now documented for the Linear Regression (fixed effects only) with Interactions sub-feature.
• The mathematics for Genotype > Mixed Linear Model Analysis with Interactions has been more fully documented.
• For the Genotype > Mixed Linear Model Analysis Multi-locus mixed model GWAS (MLMM) feature, how the p-value for a “covariate marker” is found is now documented more clearly.
• Other documentation for the Mixed Linear Model Analysis features has been polished.
• For Genotype > K-Fold Cross Validation (for Genomic Prediction), Dosage compensation is available as an option when using GBLUP.
• Genotype > Quality_Assurance > Mendelian Error Check will now handle partially-missing genotypes.
• The link to the documentation for the Illumina Genome Studio SVS plug-in, which is no longer supported, has been removed.
• The Formulas and Theories documentation sections on Linear Regression for Association Testing and on Multiple Linear Regression have been improved.
• A more specific explanation of what constitutes the “number of tests” to be used for a Bonferonni correction has been given for each of many different tests.
• For BEAGLE imputation, the Allelic R-squared and Dosage R-squared outputs are better documented.
• The hard limit for the number of samples that can be processed by Principal Component Analysis (PCA) has been removed. Additionally, the number of samples for PCA analysis that triggers a question about memory usage has been increased to 8,000.
• Internal code changes to Principal Component Analysis (PCA) have been made to slightly optimize performance and to make future changes more reliable.
• The Secure Annotation source MedGenome OncoMD is no longer supported.

Bugs Fixed

• The Genotype > Quality Assurance and Utilities > Fixation Index Fst feature was hanging up and stalling while computing confidence intervals. This has been fixed.
• The user is now prevented (with an error message) from trying to simultaneously import both VCF files that have samples and other VCF files that do not have samples. Before, SVS would try to do this, but crash.
• The liftover functionality of Import > Import VCFs and Variant Files has been fixed for SVS.
• The SVS CNV caller now overwrites older versions of reference samples that already exist (rather than erroring out).
• The Sample Summary Table spreadsheet from SVS CNV now shows the Percent Difference field as a true percentage.
• Logging out of SVS now clears information about the current licensed machine.
• The license flags are now kept when running .tsf convert, including from the stand-alone GenomeBrowse.
• SVS installations on servers that generate a different machine ID every time a user connects to the server now work correctly.
• The bundled unzip library for Linux has been updated to work with SVS marker maps.
• For Genotype > Mixed Linear Model Analysis and Genotype > Mixed Linear Model Analysis with Interactions, real or double precision data will now work when the Dominant or Recessive model is selected. This will allow taking Dominant-model or Recessive-model data, PCA-correcting it, then using those results as input to either of these Mixed-Model features.
• When adding a plot item to a GenomeBrowse plot from the current SVS project, the project view now always shows the spreadsheet column selection window on the right. Previously, that window would sometimes be absent.
• When adding a plot item to a GenomeBrowse plot from a different freshly-generated spreadsheet (that is in the current SVS project), the plot item will always appear and use the proper scaling (possibly after a fresh computation of mapping and scaling parameters takes place). Previously, sometimes a new plot item would either not appear or be of an incomplete scaling that did not cover its range properly.
• An occasional hanging up when exporting from SVS on Linux has been fixed.
• The following X-Chromosome-related issues have been fixed in the Genotype > Compute Genomic BLUP (GBLUP) and Genotype > Quality Assurance and Utilities > GBLUP Genomic Relationship Matrix features:
  • For Overall Normalization, female X-Chromosome data is now more realistically scaled with respect to male X-Chromosome data and with respect to male and female non-X-Chromosome data.
  • X-Chromosome data is now normalized and centered using the overall major and minor allele frequencies, rather than by separate male and female allele frequencies.
  • Allele substitution effects (ASE) that are computed for the X-Chromosome now use data from both genders. Before, male ASE values were computed only with male data, and female ASE values were computed only with female data.
• Other issues fixed in the Genotype > Compute Genomic BLUP (GBLUP) and Genotype > Quality Assurance and Utilities > GBLUP Genomic Relationship Matrix features are:
  • Normalizing a GBLUP Genomic Relationship Matrix using Individual Normalization now accounts for the monomorphic markers that were scanned.
  • When a separate GRM is used for the X Chromosome, the allele substitution effect (ASE) for any marker is now calculated based only on which GRM to which that marker contributed. Thus, there will be only one pair of ASE value columns now output for this use case.
  • Since by-marker effects calculated based on gene-by-environment GRM matrices are not really allele substitution effects, as such, SVS no longer uses this label for these values. (The label “Allele Activity” is used instead.)
  • Some internal code changes were made that will eliminate “Future warning” messages from being written to the Python shell window.
• The following issues have been fixed in the Genotype > Predict Phenotypes from Existing Results feature:
  • This feature now uses the correct allele substitution effect (ASE) data as input. Before, the first 350 ASE results were used in a repeating loop, and the skipping of monomorphic markers was inconsistent.
  • This feature will now work even if a fixed-effect coefficient is exactly zero. Before, this situation would cause this feature to error out with a spurious error message.
  • This feature will no longer crash if Correct For Gender is selected.
• The following issues have been fixed in the Genotype > K-Fold Cross Validation (for Genomic Prediction) feature:
  • Selecting Correct For Gender no longer crashes the script.
  • The Area Under the Curve test has been corrected. It used to show values that were sometimes very slightly above one.
  • The genotypic and error variances for the GBLUP estimate spreadsheets are now displayed in their node change logs.
• For Genotype > Mixed Linear Model Analysis and Genotype > Mixed Linear Model Analysis with Interactions, canceling from Linear regression (fixed effects only) no longer crashes the script.
• The add-on script for converting binary and integer values to genotypes now applies the marker map correctly, even if there are unmapped fields in the spreadsheet.
• SVS anotations for categorical arrays are now correctly encoded in SVS spreadsheets.
• Building reference panels for BEAGLE imputation can now handle larger datasets without crashing.
• Annotation with CADD and annotating CNV’s have both been fixed to not crash SVS.
• File > Create Marker Map from dbSNP no longer crashes if there are no matching rsIDs.
• DNA-Seq > Variant Binning by Frequency Source has been fixed to accommodate an empty alternate frequency list in the Allele Frequency Annotation track.
• All of the DNA-Seq > Collapsing Methods > … options have been updated to accommodate transcript lists in the newer versions of the RefSeq track.
• The progress bar for DNA-Seq > Collapsing Methods > Count Variants per Gene now shows progress realistically.
• The progress bar for Numeric > Fishers Exact Test for Binary Predictors now shows progress more smoothly.
• Unnecessary dividing by zero in DNA-Seq > Collapsing Methods > SKAT-O has been removed. The main effect of this change is to clean up extraneous messages from being shown in the Python shell window.
• For Import > Third Party (from the project view), the nonsense file type “0 (*)” has been removed.
• The screen layout of DNA-Seq > Collapsing Methods > Mixed-Model KBAC has been improved to work properly under Linux.

• The new VSClinical AMP workflow has been updated with a new Drugs & Trials section to support the inclusion of details on relevant therapies and clinical trials in the produced report.
• A new field has been added to the Import Wizard so the Sample’s Sex can be specified on import.
• There is now a remote staging server that contains annotation sources that are in development but are not yet ready for regular release. You can add it in the Manage Data Sources dialog by selecting Remote and Staging Annotations from the New locations dialog.
• Saving projects as templates from active projects now gives users the option to automatically update annotation sources on new project creation for select sources, or to preserve the annotation versions. See the Annotation Options tab of the Save Project as Template dialog.
• There is now a new command option in vspipeline to update annotation sources in the project template to the most current annotation source before importing data.
• A new CNV Probability/Segregation algorithm was added for trio models to show the expected copy number for each called CNV as well as the probability that the CNV is present in the father or mother samples.
• Annotating CNVs with CNV catalogs or other interval types now includes a new Overlaps Type field. This enables filtering of whether the current project region or CNV is fully contained within the annotation or fully contains the annotation.
• For CNVs annotated against genes, the HGVS and Sequence Ontology are provided as additional fields for CNVs that are contained within a gene. This provides useful details about whether the deleted or duplicated exons cause frameshift or in-frame changes to the protein sequence.

Bugs Fixed

• On VCF import, the default behavior to “fill-in” fields at the same genomic location across normalized records in specific situations overwrote values that were present from other VCF lines for the same samples. The information from the VCF is now consistently conserved.
• Samples that were manually renamed within an active project were keeping their original names in the VSWarehouse upload. This has been changed to preserve the changed name.
• Subsetting down samples to be uploaded to VSWarehouse was causing a crash. This has been changed to allow sample subsets to be uploaded.
• When lifting over data on import or in data curation, some data was not correctly adjusting for when the reference and alternate allele switched. This has been fixed.
• There were issues accessing MySQL databases for storing assessment catalogs on Mac platforms. The specific database driver was updated to fix this issue.
• In CNV tables, the Region column was not able to be changed to different display modes such as Chr, Start and Stop without being hidden. This has been fixed.
• Setting AppData folder file paths to top level network drives was causing an issue, but this is now supported.
• The Kinship fields produced from running the Sample Statistics algorithm on a family trio were not respecting the family relationship if the proband was not the first sample. This has been fixed, and also parental relationships can be changed, and the Sample Statistics will rerun in an open project to reflect any changes.
• VarSeq would sometimes fail to load the annotation repositories when connected to a warehouse annotation server that caused a timeout. This issue was resolved, and the network timeout was raised to two minutes for loading remote annotation sources.
  
• The Sample PhoRank Gene Ranking algorithm was incorrectly linking phenotypes to subsequent samples. This has been changed to not propagate previous gene sample scores.
• The following changes have been made for VSClinical AMP Guidelines:
  • In the Biomarker Summary Assessments tab, CIViC records under the same CIViC Variant ID will be collapsed into one assessment entry with the highest star rating assessment displayed.
  • After revoking a Report sign out in the AMP Guidelines, the Report field for Small Variants on the Mutation Profile tab could not be adjusted. This has been changed so Small Variants can have the Report field changed after revoking a report.
    
  • The Allele Ratio bar section in the sidebar of somatic variants was not showing the ratio amounts for project variants.
    
• The following changes have been made for VSClinical ACMG Guidelines:
  • The genotypes of the parent samples in a trio project were not being read correctly when added to VSClinical. Now the genotypes and inheritance pattern is filled in from the project.
    
  • Leaving VSClinical and the Available Downloads dialog open for an extended period could use more computer memory while searching for download dependencies. This has been fixed to not grow memory usage over time.
  • When a variant is not present in gnomAD, a link to open the region in the gnomAD coverage browser was not working with the latest gnomAD website. The hyperlink has been updated to generate the right types of intervals.

Polishes

• Altering the location of an AppData Path or Folder in VarSeq used to require a refresh in the Manage Data Sources… window to locate the new file path, but now this is updated automatically when a new instance of VarSeq is opened.
• The Match Gene List function now includes an option to load gene name values to auto complete list entries and to confirm that the gene name is recognized. This help ensures that all entered genes are valid gene symbols. This feature leverages the new Aliases field present in the updated RefSeq genes tracks.
• After running, the Match Genes List function will list gene names that were not found in the gene source list in the run log and the source information details. Additionally, the gene or alias association is displayed in the curation notes section.
• The HGVS notation for a variant for insertions edge-case have been improved. Duplicate codon insertions are now described in their short form (such as T599dup versus T599_T599dup) and insertions that include a stop codon now match the updated HGVS specification to describe in the inserted sequence (i.e. p.E858_L859insVP* instead of p.L859_W1755delinsVP).
• The dialog listing out of date sources that may be updated now additionally lists which table those sources are on when they are not on the variant table.
• The RefSeq Genes annotation tracks now include a MANE Status field to indicate if the gene has a MANE identified transcript. If so, that transcript will be selected as the clinically relevant transcript.
  
• Importing data into an assessment catalog from a file, the Last Edit field had very rigid data format requirements. This has been adjusted to support different date formats.
• Improvements were made to an edge case when logging and the software remembers being activated for a different machine. This case is handled without requiring explicitly logging out or going outside the software to delete the user properties file.
• For the annotation Convert Wizard, liftover has been improved to better handle variant deletions and a new option was added to append the reference allele if missing on liftover.
• There is now an extra option to export the exon segments of downloaded gene tracks in a BED file format. This is useful for creating target panel lists for specific genes that reflect the coding regions of genes.
• Now the Log tab will denote the name of the template used in the project creation.
• The Convert Wizard options are now displayed in a tab style to condense the view.
• The browse for BAM path dialog has been updated to utilize the native Windows explorer dialog.
• Now the “Launch Another VarSeq Instance” option is available on all platforms and can be found under the File menu. (Previously only on Mac)
• The default VSWarehouse URL can be configured using a “hosts.json” file for deploying VarSeq to large number of users.
  VarSeq now supports VCF.BGZ file extensions in the import process as an expected extension.
• VarSeq now allows users to specify a maximum number of processors to be used during computation. This feature can be found under Tools > Memory and CPU Settings.
• Now unicode characters can be used in sample names and in assessment catalog that save records per Sample, such as the ACMG classification catalog.
• The following improvements were made for the CNV algorithm and its outputs:
  • The CNV Reference Sample selection algorithm has been adjusted to favor the most recently added samples with the most similar mean sampling depth. This prevents over-fitting when many reference sequence samples are available.
  • When the CNV Caller was run using samples with an undefined percent difference or a missing BAM file path it would error out. Now it completes and skips the absent samples by filling in the corresponding output with missing values. A flag of “No coverage information” is placed in the Samples table for any skipped samples.
  • Now the Annotate Overlapping Genes algorithm for CNVs will list all of the overlapped transcripts where before only partially overlapping transcripts were listed.
  • The LoH Caller algorithm now provides the ability to change the Minimum Variant GQ value for calling LoH regions. Also, the default value for the minimum variant GQ value is set to 30 (it was previously 80).
  • The icon to Add CNV Annotations was missing from tables of imported CNV and has now been added to match other tables.
    
• The following polishes have been made for VSClinical:
  • A new field called “Other Transcript Effects” was added to the ACMG Guidelines table output that denotes effects present in neighboring transcripts for the same gene as the selected variant.
  • Some language in our “recommendations” used one-letter HGVS and others three-letter HGVS for variants. This was changed to one-letter representation uniformly.
• The following polishes have been made for VSClinical AMP Guidelines:
  • Reference genotype variants from callers like Freebayes can now be added as somatic variants to VSClinical projects.
    
  • Now clinical trials are listed in the evidence tab for Biomarkers.
  • Germline variants now have the Inheritance/Variant Type filled in for the Variants tab for germline mutations.
  • Users will now be notified that a tumor type has not been selected when first selecting one of the add variants options in the Mutations Profile tab.
  • Now the GenomeBrowse variant view will adjust to zoom and highlight the variant or biomarker selected.

• When running the Annotate Transcripts algorithm, a new field “Coding Diff” displays the amino acid coding change. For example, cAg/cGg for a Gln/Arg missense.
• Samples and their associated variants will now be removed from a project on import if the sample is unchecked in the Import Wizard and the variants do not exist in any other sample set. This works regardless of whether the import files are per-sample or multi-sample variant files.
• A revert/undo icon was added to the top of the filter chains to allow the previous filter move to be reverted.
• The Count Alleles algorithm now has the option to list the names of a limited number of samples that contain heterozygous or homozygous genotypes.
• The assessment catalogs used to store the Cancer Interpretations for the VSClinical AMP Guidelines can now be created and stored on VSWarehouse.
• The Plot button in GenomeBrowse has been upgraded to a dropdown menu that allows users to quickly access commonly plotted annotation tracks and update plotted tracks if more current track versions are available.
• A new algorithm called Annotate Variants Matching Sample was introduced that allows sample specific variant annotating and is especially useful when annotating with sample specific assessment catalogs.
• VarSeq has added standard project templates for the GRCh38 genome assembly.

Bugs Fixed

• The latest version of gnomAD 2.1.1 splits out multi-allelic variants into their own individual records, not preserving the multi-allelic form. This meant sites with two common non-reference alleles were not being annotated by gnomAD 2.1.1 and filtered out. A new multi-allele aware variant annotation mode was added to support this representation and multi-allelic variants will now match records for the individual alleles in gnomAD.
• Editing the sample table and modifying the parent fields did not update all dependent algorithms, but now modifying the parent relationships between samples will update algorithms that rely on these relationships like Mendel Error.
• Adjusting the color of an already established variant set did not adjust the color of the variant set column immediately but does now.
• Switching the filter chain between table types (like from Variants to Coverage Regions) was disabling the previous filter type.
• The project link created when using the Project Name as an assessment catalog field was broken.
• Exporting multiple sample files to XLSX file format caused a crash. This has been fixed to allow multiple samples to be exported to XLSX format at the same time.
• The import wizard layout has been updated on multiple pages and especially the finalize page to better handle the many options on different size screens and platforms. The many import options are displayed in expandable and collapsible menus to conserve space. Also, a few help links to the manual on advanced options were fixed.
• When annotating coverage regions and CNVs with genes, the percent CDS coverage field was incorrectly computing the percent of the entire gene instead of the coding regions of the gene.
• In the assessment catalog import data wizard, the layout of the fields has been fixed so that it cannot be minimized beyond comprehension.
• Certain tracks being plotted in GenomeBrowse with missing file paths were taking time to error out, but this has been updated to error out immediately and inform the user of the missing file path.
• In GenomeBrowse, when coverage was being computed for a BAM file and the user pauses the computation, one file is created on resuming and not multiple versions.
• Using the Data Annotation Convert Wizard with the left-align feature, we require a local copy of the reference sequence but did not handle the case when it was not present. Now the Convert Wizard shows a message to the user to download the required reference sequence if needed.
• The following changes have been made for VSClinical:
  • When adding multi-allelic variants from the table, only the alleles present in the current sample are added to the evaluation. In general, a lot of work went into improving the complex cases of handling multi-allelic variants in this release.
  • When switching which transcript a variant is based on, the Variant Summary and Auto Classification will now update to reflect the changed HGVS.
  • Previously, VSClinical always requested CADD annotations remotely, even when there was a local copy of CADD. This has been fixed to recognize the downloaded track.
  • Similarly, locally downloaded Multiple Sequence Alignment sources were not being used in favor of remote access. They will now be used.
  • Malformed ACMG records were causing errors. These may have been set by manually adding variants to an ACMG catalog. This has been fixed to more robustly handle malformed records.
  • The option to manually add variants in the ACMG Guidelines workflow now prevents the user from selecting a variant that is not in a gene for analysis.
  • VSClinical now does a much better job of handling projects with annotation sources that are in are incomplete or have missing input.
  • The ACMG Classification algorithm would not consider variants saved in the configured catalog as “Uncertain Significance”. It now overwrites the auto-recommendation with the last saved user classification, including a VUS classification.
  • The Variant Evidence card in the ACMG Guidelines allows users to switch between GRCh37 and GRCh38 assemblies and if the liftover can’t be performed, an error message is now shown.
  • Certain missense variants that lacked a missense badness score were showing errors in the ACMG Workflow. This has been corrected to allow these variants to be evaluated.
• The following changes have been made for VSClinical AMP Workflow:
  • In some cases, the thresholds for the Coverage Regions plots were reset to the default values on project open. This has been changed to keep the preferred values.
  • In some cases, intronic variants were not reported as intronic in the “Location” field in the reports tab.
  • If a specific tumor type had an interpretation saved for a Gene Summary, it was not being preferred over an interpretation for all cancers for new Biomarkers.
  • The Wild-Type variant dialog was not showing the most up-to-date variant count for the selected gene, and now updates correctly.
  • In the Null Variants in Oncogenic Gene section of the Variants tab, the card displaying supporting evidence for LoF variants in the gene did not update to match the current gene.

Polishes

• Performance of using VarSeq assessment catalogs with VSClinical or directly has improved for the use case of multiple users using an SQLite file-based catalog on a shared networking drive, especially during concurrent access.
• When an algorithm or annotation source is updated, new fields will be hidden in existing tables by default. You can right-click on the table source header or open the Visibility dialog to adjust visible fields.
• Empty columns that were created when using the Compute Fields algorithm on a subsequent source are now removed from the computation preview.
• The overlapping transcripts algorithm and the annotate transcript algorithm both utilize the same gene preferences file for continuity.
• When naming new variant sets, numbers can now be used in the second position of the naming Initials.
• When opening a custom project template where an input annotation source is not found, there is now an option to delete the algorithm that uses the missing annotation source on import.
• VarSeq more efficiently authenticates with VSWarehouse when performing assessment catalog operations.
• The Convert Wizard has a new option when lifting over variant tracks to adjust counts on known fields when the reference and alternate alleles are flipped in the new assembly. This feature is checked by default to match the previous behavior.
• On Linux, opening external programs such as the web browser or folder explorer now works on a large variety of Linux flavors and systems.
• Now when associating BAM files to samples, the file name closest to the sample name will be selected first.
• Tracking the current connected warehouse is now more robust to differences in inclusion of trailing slash in URL.
• The ability for users to control memory settings has been adjusted to provide Low, Medium, High, and Custom options.
• In GenomeBrowse, coverage statistics do not automatically start computing until the user requests it. Also, a recent regression in performance of computing coverage in the previous VarSeq version has been fixed.
• The table icon on GenomeBrowse plots now acts as a toggle to show and hide un-docked table views of the current plot data.
• Switching the assembly in the assembly dropdown menu in GenomeBrowse used to switch the assigned assembly for the project. This caused confusion and has changed so that assembly switching in GenomeBrowse is independent of the project assigned assembly.
• The following polishes have been made for VSClinical:
  • You can now start an evaluation even without running the ACMG Sample Classifier algorithm. In general, less dependencies are made on the variant table to ensure the latest algorithms and state of previous assessments are always represented.
  • Warnings and notifications have been added to ACMG Guideline classifications to show when previous classifications were last saved, if any scoring criteria has changed since the previous classification, if the ClinVar classification record has changed, and if the maximum population frequency has changed since the previous classification was made.
  • The ACMG Guidelines will also show a Special Considerations notification when a variant overlaps multiple genes or transcripts or scoring seems to have been left in an incomplete state regarding clinical or functional evidence.
  • The Blinded Interpretation option in the ACMG Guidelines has been updated to fully blind previous classifications and interpretations to the current user. The resulting evaluation starts off as if the variant was being seen for the first time.
  • The Gene Region and Mutation Profile table has been updated to include the HGVS amino acid description and have a “Sources” column that makes it clear which entries are coming from ClinVar, your internal catalog or both. Similarly, tooltips on each column break out the details coming from each source. In the AMP workflow, CIViC records are integrated into this table as another unique source. Optionally configured Consortium Sources will similarly be integrated.
  • The Related or Nearby Variants section of the Studies tab in the ACMG Guidelines has been enhanced so variants previously seen can be clicked to show that variants Historical Interpretations dialog.
  • The Download Dependencies screen for the AMP and ACMG Guidelines does not appear every time a new tab is opened if initially skipped or all required dependencies are downloaded.
  • The ACMG criteria that were scored during a previous classification in the ACMG Guidelines will now be set as scored when a new evaluation for the previously scored variant is opened.
  • The ACMG Options dialog now displays the assessment catalog database type and location with a destination link where just the file path to the assessment catalog was displayed before.
  • The Variant Finalization screen in the ACMG Guidelines now has an ‘Update Previously Classified’ option to request the latest saved classifications information from the assessment catalog for the current variants in the evaluation.
  • Now when switching between variants in the ACMG Guidelines, the view of each expanded section is preserved to more easily compare sections between variant analysis.
  • Variant cards that allow users to switch between GRCh37 and GRCh38 views will now automatically open on the default assembly of the project.
  • The Historical Interpretations dialog now displays more details about the previously scored variants in the list.
  • The Historical Interpretations section of previous variant classifications in the ACMG Guidelines now shows the most recent classification first and is sorted in reverse chronological order.
  • The Variant Summary for variants that extend beyond gene boundaries has been improved to better emphasize the upstream and downstream impact.
  • For variants that overlap 2 or more genes, only 1 gene will be shown in the Variants to Evaluate list at the start screen of the ACMG Guidelines. Once the evaluation is created, the variant can change which gene is being used in the evaluation from the change transcript dialog. The fact that the variant overlaps two genes is placed in the new Special Considerations section.
  • Phrasing has been adjusted in the ACMG Guidelines to reserve the term, “Finalize” for samples and evaluations. Before, individual variants could be finalized, but now they are only, “Saved”.
  • Saving a variant in the current should not change the number of “Previous Samples” reported seen for that variant in various contexts. We ensure that the current sample is never counted in this count.
• The following polishes have been made for VSClinical AMP Guidelines:
  • The version of COSMIC used in the Somatic Catalogs section of the Variant Annotation tab is now displayed.
  • When adding a variant that is missing a protein notation, the coding notation is used.
  • A warning message now exists to remind users that the tumor type needs to be set before adding biomarkers to the AMP Guidelines.
  • Now when adding variants to the mutation profile, the Oncogenicity/Pathogenicity classification computed informs how by default the variant will be reported. Benign/Likely Benign variants will be set to “Don’t Report”.

• VSClinical AMP Guidelines work-flow now available as an add-on licensed product to VarSeq! This product supports the evaluation of somatic variants, CNVs and fusions following the AMP guidelines for cancer.

New Features

• There is now an algorithm called, Variant Site ACMG Classifier, that allows users to use the ACMG Guidelines at the project level versus the sample level. This is useful for catalog projects and projects without sample genotype information.
• The coverage stats algorithms now have additional mapping quality output.
• Samples can now be removed during import by un-checking the box next to the sample name.
• The Assessment Catalog feature to import from a project table can now loop over all samples in the project. For example, you can now export filtered CNV entries from multiple samples into an assessment catalog.

Bugs Fixed

• When merging 3 or more VCFs for a give genomic position, if the first file contained no data for the position and the subsequent files contained more than one alternate allele, the samples with the second observed alternate allele would get their variant updated to be the first alternate. This resulted in incorrect variants for some samples. In our testing, this situation is very rare and very unlikely to occur in the wild.
• The HGVS protein notation for frameshifts containing the length of the extended protein in some cases was off-by-one by not counting the stop codon in the length. For example we reported NP_056153.2:p.G646Wfs*11 instead of NP_056153.2:p.G646Wfs*12. This has been fixed as well as similar edge cases.
• Similarly the HGVS protein notation for deletion/insertions sometimes contained an extra “Ter” description. Now NM_001232.3:c.1195_1196insTGA will have the HGVS of NP_001223.2:p.Glu399_Ter400delinsValLys
• Un-checking the ‘Match CNV Type’ when using the Annotate Overlapping CNVs algorithm no longer causes a crash.
• Variants with little to no evidence of pathogenicity are now classified as VUS and not missing in the ACMG Sample Classifier VarSeq algorithm.
• The order of the list of transcript outputs in the Sequence Ontology are now in the same order as the Transcript Names when using the Annotate Transcripts algorithm.
• The project history and activity log files were not updating during use of the ACMG Guidelines. This has been fixed so they update accordingly.
• Annotating against an assessment catalog now lists multiple records at the same genomic positions in most-recently added order in the table.

Polishes

• The download manager for VSClinical ACMG and AMP allow you to “Skip for Now” updated annotations tracks such as ClinVar if your prefer to use out of date local copies. This may be useful when performing evaluations over the course of days and not wanting to update annotation sources used mid-stream.
• The left-align algorithm is turned on by default for new projects. This ensures variants are in the canonical position required for annotation.
• There is now a Tools > Memory Settings that allow you to increase the memory used by VarSeq’s algorithm and data processing pipeline. This can improve performance in certain situations.
• The Dashboard view has been removed from the VSClinical tab
• When merging VCF files that have the same field (such as DP) defined in both the INFO and FORMAT (samples) section with VCF files that have it only defined the INFO section, we now allow the up-casting of the INFO level field to be sample-level so that it can be merged with the sample-level fields in other VCF files. This is helpful when merging samples from multiple VCF callers where some put sample-specific data in the INFO fields.
• Annotating against COSMIC produces 1:1 annotations and no longer creates a “Unique Counts” summary fields. This matches the updated curation format of COSMIC.
• HPO terms that were tagged as “Obsolete” no longer appear in the auto-complete in the PhoRank and genes for phenotype inputs.
• The Count Alleles algorithm now has the additional output: # Samples that count the number of samples which contain the allele.
• The binned coverage algorithm now by default does not take into consideration coverage over bases of the reference genome containing missing ‘N’ bases.
• Support for importing CNVs from VCF files has improved by handing more representations of a CNV variant.
• The Mendel Error algorithm now denotes from which parent the variant was inherited in a Trio analysis.
• Left-aligning variants can now be accomplished by either moving the variants that have been left-aligned, or by creating a duplicate entry at the left-aligned position.
• Gene tracks plotted in GenomeBrowse can now be filtered to show only the Clinically Relevant transcript.
• When transcript annotations are run with novel splice site detection, the output names are more intuitively name “N of 4 Predicted Splicing”
• The HGVS notation reported in gene annotation now supports reporting a “dup” at the amino acid level NP_000483.3:p.Glu56_Asp58dup.
• Now when running the CNV algorithm, the reference samples are provided with a unique checksum description so that reference samples cannot be included more than once in the reference set. Additionally reference samples keep track of preferences that influence their values so that they are used only with other samples of the same configuration.
• Users are now alerted when the project does not contain any samples while attempting to access VSClinical outputs.
• You can now update a project to use a newer version of an annotation track before downloading and running with the older version.
• All enumerated categories are kept when applying an annotation source to a project. This for example makes it easier to create a project template with a smaller set of variants that may not match all possible categories of an annotation source such as ClinVar.

New Product Add-Ons for SVS

• Golden Helix SVS now includes in-silico splice site, functional prediction and conservation methods available with the Clinical Variant Scoring add-on. This includes 4 splice site algorithms, SIFT/PolyPhen2 predictions that run on 100-way multiple species alignments, and GERP++ and PhyloP methods to calculate conservation scores.

New Features

Additional 2.0.1 Notes

Bugs Fixed

• Creating projects from existing templates unintentionally added the splice prediction algorithm option to existing gene annotations. This would then stop the algorithm from running if the current user was not licensed for VSReports or VSClinical. This has been fixed so newly created projects from existing templates will be created without the splice predictions and run to completion. When adding gene annotations to a project, the choice to use the splice site prediction algorithms will be available if that feature is licensed.
• VSClinical recommendations for BS1 are now aware of quality flags so as not to recommend scoring BS1 when the population catalog data may not be reliable.