Combined Impact: New Tools to Assess Complex and Compound Heterozygous Variants with VarSeq

The value of complex variant analysis has become increasingly apparent as long-read sequencing technologies provide us with more detailed data. However, the need for sophisticated tools to fully harness this data is crucial. VarSeq 2.6.2 addresses this need by introducing new support for analyzing complex variants, including advanced visualizations and algorithms that enhance our ability to assess the combined clinical impact of these variants. Join us for the upcoming webcast where we will:

Examine complex variants alongside their allelic primitives.
Utilize variant phase information to explore potential compound het variants and the combined effect of nearby variants.
The long-awaited release of our SNP and CNV compound het algorithm in a trio workflow.
And last, a sneak-peek at our upcoming Methylation analysis capabilities in VarSeq.

This session will provide valuable insights into taking advantage of these new tools to advance your analysis capabilities.

1. Combined Impact! New Tools to Assess Complex and Compound Heterozygous Variants with VarSeq September 18, 2024 Presented by : Julia Love, Associate Director of Product & Quality Jennifer Dankoff, PhD, Field Application Scientist

2. 2

3. Combined Impact! New Tools to Assess Complex and Compound Heterozygous Variants with VarSeq September 18, 2024 Presented by : Julia Love, Associate Director of Product & Quality Jennifer Dankoff, PhD, 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 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. Secured CE Mark for EU 7 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.

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 Integrating Long and Short Read Sequencing for Comprehensive NGS Analysis https://www.goldenhelix.com/resources/webcasts/integrating-long-and-short-read-sequencing-for-comprehensive-NGS-analysis/ Topics for today Overview of new VarSeq tools and algorithms for analyzing the following variant types: Complex variants and corresponding allelic primatives , collapsed phased variants, their combined impact, and the compound het condition. Small var iant and CNV compound het status in trios. A look at the current state of Methylation analysis with the VarSeq software

9. 9

10. Introducing the need for more complex variant tools 10 New capabilities in VarSeq v.2.6.2 Advanced sequencing technologies are producing massive amounts of data, opening-up avenues for more diverse complex variant analysis. Genetic variants come in many shapes, sizes, and structures. Contextualizing variants and complex variant interactions for more thorough classifications. These contribute to improved patient treatment options .

11. Long-Read Sequencing The Wholistic NGS Test 11 Clinically relevant properties of long read-sequencing: Better precision and accuracy with lower coverage across the whole genome. This is in particular helpful, identifying very rare variants with high confidence. Improved calling of Structural Variants and CNVs . Higher yield capturing regulatory sequences (UTRs), pseudogenes (SMN1, SMN2), centromeres, Alu elements (SINE), short tandem repeats , LINE1 elements, and long repeats. Other improvements include Ability to provide methylation calls . Genotype phasing allows compound het analysis to be completed on a single proband.  PacBio read length histogram https://www.pacb.com/technology/hifi-sequencing/how-it-works/ 2023 “Method of the Year” – Nature Methods journal

12. Phasing 101 Unphased example : Chr17:56350190 G>A Likely Pathogenic GT: 0/1 Chr17: 56350196 G>A Pathogenic GT: 0/1 Unclear if the gene is in compound het. Phased example : Chr17:56350190 G>A Likely Pathogenic GT: 0|1 Chr17: 56350196 G>A Pathogenic GT: 1|0 Both copies of the gene are affected. Phasing is the ability to infer haplotypes from genotype data. A phased variant will be shown as 0|1 or 1|0 . Phasing information can come from short-reads or long reads. ? ? 1|0 0|1

13. Case study: Singleton Analysis Analysis of complex variants in an immune compromised individual. Examples: Likely Pathogenic Multiallelic Variant in HLA- DRB5 Merged In-Phase Variants in NOD2 Evaluate Phased Compound Het Variants in the recessive IL10RA gene

14. Introducing the Complex Variant Table Analyze complex variant calls alongside variants split into allelic primitives. Annotations and algorithms can be applied to both tables. Set up independent filter strategies for SNVs and MNVs. Analyze variants in VSClinical in either configuration. Example: A complex variant in HLA-DRB5 that is Benign when split into Allelic Primatives but Likely Pathogenic when conserved as a complex variant . New capability in VarSeq v.2.6.2

15. Introducing Collapsed Phased Variants Merges variants that are in-phase and within a certain distance threshold together. Identify groups of variants and their compound effect . Can be used for both short and long read phased data. Annotate and evaluated collapsed variants in VSClinical . Example: Two variants in-phase are collapsed together, resulting in a Likely Pathogenic classification for the Dominant NOD2 gene. New Algorithm in VarSeq v.2.6.2

16. Introducing Phased Compound Het Detection Identify potential compound heterozygous variants in a single sample analysis ! For variants to be considered compound het from a single sample the following need to be true: The variants are in the same gene The variants are in the same phase set The variants are not in phase Example: Two Likely Pathogenic and Pathogenic variants in the Recessive IL10RA gene are not in-phase and found to be on opposing chromosomes . This results in a compound het status. New Algorithm in VarSeq v.2.6.2

17. Case Study: Trio Analysis Analysis of compound SNP and CNV variants in BRCA2 and breast cancer risk. Heterozygous deletion of exons 2-8 of BRCA2 inherited from the father . Pathogenic NM_000059.4:c.3812C>G variant in exon 11 inherited from the mother . Compound het for Pathogenic variants found in the proband.

18. Introducing the CNV Variant Compound Het Algorithm Compound heterozygosity often occurs when CNVs and SNVs are located in the same gene. This is an essential analysis for autosomal recessive disorders. Identifying compound het SNVs and CNVs aids the understanding of disease diagnosis and disease mechanisms. New Algorithm in VarSeq v.2.6.2

19. What is Methylation ? 5-mC CpG From ThermoFisher Sci DNA methylation is a chemical epigenetic modification of the DNA sequence. 5-methyl-cytosine is considered the ‘5 th base’ because of its unique properties. DNA methylation, especially in CpG islands is a cellular mechanism used for precise regulation of gene expression In cancer, there is a departure from this state. Hypermethylation of CpG islands is linked to gene silencing in tumor suppression genes. Hypomethylation is associated with genomic instability A Sneak Peek at an Up-and-Coming Research Field

20. Up-and-Coming Clinical Applications of DNA Methylation in Cancer https://cancerci.biomedcentral.com/articles/10.1186/s12935-023-03074-7 Research is showing that there are several methylation patterns which are relevant to a broad range of cancers . Lung cancer (LC), colorectal cancer (CRC), gastric cancer (GC), hepatocellular carcinoma (HCC), and esophageal cancer (EC). Using cfDNA in blood/serum to detect hypermethylation status of certain TSGs is useful as an early diagnostic marker for several cancers as well as prognostic indicator. VarSeq is your research assistant by importing long-read derived methylation data for viewing and filtering. A Sneak Peek at an Evolving Research Field

21. Current VarSeq Capabilities with Methylation Data In VarSeq you can import differentially methylated regions files . In your project, plot methylated regions in GenomeBrowse . Filter the methylation region table for differentially methylated regions. Annotate with custom databases. MethMarkerDB to be released later in 2024! Make your own interpretations in CancerKB for clinical reporting. Reach out to Golden Helix for more information about your workflow! A Sneak Peek at an Evolving Research Field

22. 22 Product Demo

23. In Summary Higher resolution sequencing has brought upon a need for more sophisticated complex variant analysis tools. Our VarSeq v2.6.2 introduces a number of tools to analyze: Complex Variants In-Phase Variants Compound Het Variants not In-Phase SNV and CNV Compound Het in Trios We are also developing new tools for the up-and-coming field of methylation analysis . In VarSeq you can import your DMR long-read data Plot and filter Create custom biomarkers to import into reports Reach out to Golden Helix if you would like to talk more about your methylation analysis needs.

24. NIH Grant Funding Acknowledgments 24 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.

25. 25

26. eBook Library 26 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

27. Secured CE Mark for EU 27 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.

28. 28