Complete Variant Assessment in VSClinical

Recent webcasts have been focused on our latest features in Pharmacogenomics and the IVDR announcement for our tools now marked as a medical device. While technology and market updates are incredibly important to our customers, it is also important to take a step back and revisit the fundamental purpose of our products. The Golden Helix product stack is tailored for comprehensive variant evaluation and facilitating a complete tertiary analysis from initial data import to final clinical report. The tertiary analysis process is comprised of various steps of variant prioritization that includes but is not limited to filtering on alternate allele frequency to isolate rare variants in the population and ontology to capture variants that are predicted to impact protein function. Variant assessment is only possible by referencing numerous databases that contain the necessary fields and evidence used for filtering and interpretation. VarSeq is our variant assessment platform and not only hosts a central access point to the numerous databases but also contains a dedicated interpretation interface designed to direct the user through the germline ACMG and somatic AMP guidelines for variant evaluation. Though VarSeq contains the necessary tools to streamline comprehensive variant assessment, there will likely never be a full replacement for the human component to variant assessment. The goal with VarSeq is to achieve the best of both worlds, a defendable degree of automation to improve analytical efficiency paired with upholding the necessary human touch required to render accurate variant interpretations.

The purpose of this webcast is to expose our audience to the fundamental annotation and interpretation capabilities of VarSeq. We will pay close attention to the required depth of assessing variant impact to demonstrate that simple record matching against variant annotation is only one step of the process and requires human review. Moreover, we will also look at examples of recent advancements in variant calling strategies that provide multiple variant outputs as evidence to support a singular biological impact and complete diagnostic outcome. Please join us as we explore how users can make the most out of the VarSeq software to streamline comprehensive variant assessments in a clinical utility.

1. Complete variant assessment in VSClinical July 24, 2024 Presented by Darby Kammeraad, Director of Customer Success

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3. Complete variant assessment in VSClinical July 24, 2024 Presented by Darby Kammeraad, Director of Customer Success

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 NIH SBIR Grant 1R43HG013456-01  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. Golden Helix at-a-Glace 5 Company Snapshot: Leading SaaS provider of tertiary genomic analysis solutions for NGS labs Golden Helix is a SaaS bioinformatics solution provider specializing in next-gen sequencing (“NGS”) data analysis  The Company’s software enables automated workflows and variant analysis for gene panels, exomes, and whole genomes  Key Clinical Applications Prenatal testing Hereditary disease testing Reproductive testing Oncology Marquee Global Clients Golden Helix’s solutions allow clients to increase throughput, ensure consistent quality, maximize revenue, and save time  1998 Company Founded Bozeman, Montana Headquarters Recognitions Government Research Pharmaceuticals Agrigenomics Testing Labs Translational Labs Human Genetics Research Hospitals Academia Publications Content & Resources Pharmacogenetics testing

6. 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… Medical Device Certification

7. ISO Certification 13485:2016 7 ISO 13485:2016 from TÜV SÜD ISO 13485:2016 is an international standard that specifies requirements for a quality management system (QMS) for organizations involved in the design, development, production, and servicing of medical devices. maintain a quality management system demonstrate sufficient risk management show consistent tracking of customer satisfaction and safety in the market demonstrate continued improvement efforts on the product and system level.  ISO 13485:2016 is designed to objectively document that we are holding ourselves to the highest quality standards as we are providing innovative solutions to hospitals, testing labs, and research institutions globally.

8. Recent webcasts 8 Pharmacogenomics https://www.goldenhelix.com/resources/webcasts/pgx-analysis-in-varseq-a-users-perspective/index.html https://www.goldenhelix.com/resources/webcasts/introducing-vspgx-pharmacogenomics-testing-in-varseq/index.html VarSeq Dx – Medical device certification in Europe https://www.goldenhelix.com/resources/webcasts/introducing-varseq-dx-as-a-medical-device-in-the-european-union/index.html TWIST Exome CNV calling with VarSeq https://www.goldenhelix.com/resources/webcasts/analyzing-performanc-of-the-twist-exome-kit-leveraging-vs-cnv/index.html Topics for today Fundamentals on variant analysis in VarSeq Searching for the variant: designing a filter strategy that is effective and concrete Evaluating the variant: comprehensive yet high-level impact assessment for reporting

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10. Step 1: Searching for the variant 10 Import spectrum of varian t types: Small variants = SNVs or small indels (typically <100bp) (SVs) CNVs = exon level or larger deletions or duplications (SVs) Breakends – Inversions, Translocations, etc. Designing the filter: user-defined workflow Variant quality: Read depth, genotype quality, Alt allele fraction Find rare variants: Alternate allele frequency (Clinical norm is <1%) Sequence ontology: Loss of function, missense, splice, downstream or upstream variants Diagnostic focus: dedicated gene panel or phenotype specific to case Variant classifier and clinical variant databases: ACMG auto classification, ClinVar , ClinGen , LOVD, and others Ultimate goal: validate filter efficacy and lock it down for routine application

11. Filtering with Clinical Databases: ClinVar , ClinGen , LOVD, etc 11 Leiden Open Variation Database LOVD v3.0 Designed to be a flexible tool for gene-centered collection and display of DNA variants. The design of the database follows the recommendations of the Human Genome Variant Society (HGVS) allowing for storage complete clinical assessments https://doi.org/10.1038/s41431-021-00959-x

12. Transitioning to Step 2: Evaluating a variant in VSClinical 12 Annotating the variant: Tabularized breakout of evidence. 1:1 record matching (much like traditional approach in excel) Visualizing the variant in GenomeBrowse Pro : quick review of evidence invites prioritization and deeper evaluation Con : not as efficient for full assessment Evaluation in VSClinical Interpretation hub designed for comprehensive review of variant descriptions Highly automated met with human review to handle exceptions Cataloged classification and interpretations Customizable reporting

13. Demo – Example variants 13 Loss of Function Splice variant in MKS1 Initial pathogenic assessment changed upon deeper review MET exon 14 skipping Multiple variant types lead to same high level assessment

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15. NIH Grant Funding Acknowledgments 15 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 NIH SBIR Grant 1R43HG013456-01  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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17. eBook Library 17 Prenatal Genetics – Learn the Following Existing approaches to prenatal WES, along with clinical indications for its use   How VarSeq and VSClinical can be utilized for its use A few interesting cases of variants and their classifications Pharmacogenetics – Learn the Following Foundations of Pharmacogenomics Genetic variability and drug response Pharmacogenomic test reporting nomenclature and terminology The Pharmacogenomic eco-system VSPGx - A pharmacogenomics application

18. Secured CE Mark for EU 18 VarSeq Dx VarSeq Dx is designed with compliance and reliability for your clinical analysis. VarSeq Dx is our flagship software, VarSeq , that is CE marked to meet the European In Vitro Diagnostic Regulation (IVDR 2017/746) requirements. VarSeq Dx satisfies the IVDR requirements within the European Economic Area (EEA). Verification CE MARK ISO Certification Our customers will work with our Field Application Scientist to verify the installation and ensure proper usage of the software. This can be used for ISO QMS software validation documentation.

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