VarSeq 2.4.0: VSClinical ACMG Workflow from the User Perspective

Earlier this year, we released VarSeq 2.3.0 which brought massive updates to our VSClinical AMP interface, such as enhanced capabilities for automation and analysis of structural variants in the cancer context. Naturally, we wanted to follow that up shortly with similar advancements to our VSClinical ACMG interface, and also make our customers doing germline variant analysis happy.

Our latest software release, VarSeq 2.4.0, was therefore focused on the advancements in VSClinical ACMG, namely support for importing and clinically evaluating structural variants, long read sequencing, advanced automation with evaluation scripts in VSClinical ACMG and end-to-end automation of ACMG workflows with VSPipeline. These new and improved features were discussed in a great webcast by our VP of Product and Engineering, Gabe Rudy, last month.

This upcoming webcast by our FAS team will be a user’s perspective on the new features in VarSeq 2.4.0 and VSClinical ACMG and how our tools can precisely and efficiently enable the full spectrum NGS analysis for Mendelian disorders.

1. VarSeq 2.4.0: VSClinical ACMG Workflow from the User Perspective June 7, 2023 Presented by Rana Smalling, PhD, Field Application Scientist and Solomon Reinman, Technical Field Application Scientist

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3. VarSeq 2.4.0: VSClinical ACMG Workflow from the User Perspective June 7, 2023 Presented by Rana Smalling, PhD, Field Application Scientist and Solomon Reinman, Technical Field Application Scientist

4. NIH Grant Funding Acknowledgments 4 Research reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under: Award Number R43GM128485-01 Award Number R43GM128485-02 Award Number 2R44 GM125432-01 Award Number 2R44 GM125432-02 Montana SMIR/STTR Matching Funds Program Grant Agreement Number 19-51-RCSBIR-005 PI is Dr. Andreas Scherer, CEO of Golden Helix. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

5. Who Are We? 5 Golden Helix is a global bioinformatics company founded in 1998 Filtering and Annotation ACMG & AMP Guidelines Clinical Reports CNV Analysis CNV Analysis GWAS | Genomic Prediction Large-N Population Studies RNA-Seq Large-N CNV-Analysis Variant Warehouse Centralized Annotations Hosted Reports Sharing and Integration Pipeline: Run Workflows

6. Cited in 1,000s of Peer-Reviewed Publications 6

7. Over 400 Customers Globally 7

8. The Golden Helix Difference 8 FLEXIBLE DEPLOYMENT On premise or in a private cloud BUSINESS MODEL Annual fee for software, training and support CLIENT CENTRIC Unlimited support from the very beginning SINGLE SOLUTION Comprehensive cancer and germline diagnostics SCALABILITY Gene panels to whole exomes or genomes THROUGHPUT Automated pipeline capabilities QUALITY Clinical reports correct the first time

9. Today’s Presenters 9 Rana Smalling, PhD Field Application Scientist VarSeq 2.4.0: Structural Variants and Advanced Automation in VSClinical ACMG Solomon Reinman Technical Field Application Scientist

10. Content Overview 10 Streamlined NGS workflow Efficient and precise NGS analysis of germline disorders with Golden Helix software Evaluation of multiple variant types VSClinical ACMG supports the spectrum of variant types for Mendelian disorder analysis Demonstration Product demo with PacBio long read example  Review fusion analysis in new ACMG workflow

11. NGS Clinical Workflow Golden Helix provides comprehensive data analytics software that scales across gene panels, whole exomes, and whole genomes DNA Extraction in Wet Lab and Sequence Generation Interpretation and Result Reporting Primary Read Processing and Quality Filtering Alignment and Variant Calling Secondary *Golden Helix provides Secondary Analysis through a reseller agreement Tertiary Golden Helix’s software and primary focus Comprehensive secondary and tertiary analysis solutions for primary data aggregated by all commercially available sequencers Type Size Gene Panel Small (100MB) Whole Exome Medium (1GB) Whole Genome Large (100GB) Cancer use case Hereditary use case Process Analysis … and scales across multiple data set sizes for cancer and hereditary use cases Filtering and Annotation Data Warehousing Workflow Automation Golden Helix works with all major sequencers…

12. Role of Long Read Sequencing in NGS analysis 12 Nature Methods journal declared long-read sequencing to be 2023 “Method of the Year” Fulfilled wish lists for genomics labs doing large scale projects Long-read sequencing enabled the Telomere-to-Telomere Consortium (T2T) and Vertebrate Genomes Project (VGP) Better precision and accuracy with lower coverage More accurate coverage in difficult to capture regions Regulatory sequences, pseudogenes, centromeres, Alu elements, short tandem repeats, LINE1 elements and long repeats More confident SV and CNV calling Fewer gaps in genome assemblies mean more accurate assessment of deletions and duplications with multi-species implications Long-read is better at detecting complex genome rearrangements and structural variants often seen in cancer. The affordability and quality of long-read sequencing continues to improve 95-99% accuracy and rising in 2023 compared to 30-40% in 2010 PacBio read length histogram https://www.pacb.com/technology/hifi-sequencing/how-it-works/

13. Sentieon's DNAscope is optimized for long-read data 13 Literature from PacBio and Sentieon support DNAscope as the optimal variant caller Pre-built, robust pipelines from Sentieon to process SNPs, indels, and SVs for PacBio and Oxford Nanopore data and a growing list of other sequencers DNAscope LongRead from Sentieon is accurate, fast and efficient 4 hours on a 16-core machine for 30x HiFi samples >99.83% precision and recall on most recent GIAB benchmark dataset  Compared to other machine learning approaches to long-read variant calling, Sentieon is user-friendly and fast

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15. More than one type of genetic mutation can drive disorders Mutations that activate genes: Missense In-frame insertions/deletions Fusions Copy number amplifications Functions that inhibit or disable genes: Gene deletions Loss of function nonsense, frameshift indels Disabling fusions, structural variants Genomic Signatures that describe overall state of the mutated genome Comprehensive Genomic Profiling For Germline Disorders 15 Copy Number Rearrangements Base Substitutions Deletions Insertions Genomic Signatures

16. Increased Adoption of Structural Variants in NGS 16 Use in both Oncology and Germline testing SNPs/indels, CNVs and SVs are implicated in genetic disorders Structural variants relevance to cancer is well established; extended to ACMG germline scoring Rationale for including SVs in NGS tests for Mendelian disorders Increased affordability and accuracy of long-read technology (PacBio, Nanopore, etc.) Kits that simplify and integrate RNA detection with DNA Adoption of whole genome sequencing which enables structural variant calling Increased diagnostic yield from comprehensive tests covering multiple variant types.

17. Multi-Variant Type Analysis Workflow 17 Import Wizard Variant types identified on import based on VCF info Variants, CNVs, Break-end Pairs One VCF/multiple VCFs merged by name in header Import long read sequencing VCFs and alignment files from any secondary caller into VarSeq ( PacBio, Oxford Nanopore, Element Biosciences, Illumina , other) Variant Type Specific Tables and Filters Annotate and filter variant types individually Gene impact analysis Type-specific annotations and algorithms VSClinical Analysis Import from VarSeq tables or use Evaluation Script Only analyze filtered or “marked” variants, CNVs, fusions Import sample QC details, phenotype, clinical features Integrated Reporting Report sections: Primary, Secondary, VUS All variant types can be reported in any section

18. Interpreting Break-ends or fusions in VarSeq 18 Break-end Location Rearrangements within and in between chromosomes Coding genes, non-coding genes, introns Orientation and SV Type Translocations Deletions Duplications Inversions Effect on Gene In-frame Fusion Frameshift fusion Transcript ablation, frameshift, start loss Transcript fusion Non-functional rearrangement Intronic, intergenic, upstream, downstream, start gain

19. VarSeq Suite: Automate Inputs and Outputs 19 VarSeq is built for flexibility and automation Modular set of capabilities Support all records in VCF 4.3 Built-in support of custom pipelines   VCFs with all types of variants Custom callers output such as ArcherDx Sample or patient info VSPipeline automation of VSClinical input and outputs Reduction or elimination of custom work 

20. Examples for Product Demonstration 20 PACBIO long read example (Microcephaly indicated by gene panel screening) Small variants STAG1, OCA2, AP3B2,MKS1 Copy number variants APC6+1247 genes HNF1B+413 genes Complex structural variant RAB3GAP1::CDNF VSClinical ACMG guidelines evaluation Evaluation and report on 3 variant types

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22. NIH Grant Funding Acknowledgments 22 Research reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under: Award Number R43GM128485-01 Award Number R43GM128485-02 Award Number 2R44 GM125432-01 Award Number 2R44 GM125432-02 Montana SMIR/STTR Matching Funds Program Grant Agreement Number 19-51-RCSBIR-005 PI is Dr. Andreas Scherer, CEO of Golden Helix. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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24. 25 Licenses for 25 Months 24 Celebrating 25 Years in Business Limited quantity Licenses are 25-month license periods Available to new customers only Orders must be received by June 15, 2023 Visit goldenhelix.com/forms/25-for-25 or scan the QR code below

25. Conferences 25 European Human Genetics Conference, Booth #566 June 10 – 13, 2023 Glasgow, UK Monday, June 12, 12:00 - Corporate Satellite Talk (ALSH 1, Level 0) Achieving Economic Success as an NGS Lab: Strategy and Implementation AMP Europe, Milan, Italy, Booth #14 June 18 – 20, 2023 Milan, Italy Monday, June 19, 1:00 – Industry Symposium  Achieving Economic Success as an NGS Lab: Strategy and Implementation

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