Integrating Long and Short Read Sequencing for Comprehensive NGS Analysis

The advent of long read sequencing has opened the door to a more complete NGS pipeline. The current usage paradigm that we are observing is rather than an absolute migration from short read to long read sequencing, users are leveraging both methodologies to build comprehensive and reliable NGS analysis workflows. Short read sequencing has a heavy market share and well-established pipelines, and is still the standard for NGS, but long read sequencing has opened avenues to solving unsolved cases and finding clinically relevant variants that could not be found using short read. Long read sequencing gives you more power to migrate to a complete NGS pipeline, enabling detection of complex structural variants such as CNVs and fusions without relying on microarray and MLPA for further validation, and accounting for phased genotypes, for example.

This webcast will delve into the benefits and use cases of each sequencing methodology, highlighting how they complement each other. We will demonstrate an example use case in which short read and long read data is available for the same individual and how easy it is to analyze both data types in one VarSeq project, yielding clinically relevant results.

1. Integrating Long and Short Read Sequencing for Comprehensive NGS Analysis August 21, 2024 Presented by : Rana Smalling, Field Application Scientist

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3. Integrating Long and Short Read Sequencing for Comprehensive NGS Analysis August 21, 2024 Presented by : Rana Smalling, 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 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 Comprehensive NGS workflow with short and long read approaches Review of short and long read sequencing and the benefits of each modality Considerations for simplifying and validating this comprehensive strategy

9. Outline 9 Comprehensive NGS workflow with short and long read approaches:  Benefits and uses for each sequencing modality An approach to validation of comprehensive NGS workflow  Software Demonstration – A combined long and short read workflow for analysis of aniridia in a family 2 4 1 3

10. Differences Between Long and Short Read Sequencing 10 Short read sequencing requires DNA fragmentation and amplification with subsequent bioinformatic assembly Read Length: 10s-100s of base pairs Sequence gaps, sample bias Examples: Illumina, IonTorrent – massively parallel sequencing by synthesis L ong read seq uencing enables the sequencing of long DNA strands in a single continuous process Read Length: 1,000s-100,000s of base pairs Large span, comprehensive variant detection Works in real time – adjust experiment at any stage Examples: PacBio - High fidelity (HiFi) circular consensus sequencing – rolling loop sequencing of circular DNA molecule by fluorescent signature Oxford Nanopore – DNA sequence deciphered by current disruption signature as it passes through a nanopore

11. Benefits and Limitations of Each Sequencing Modality 11 Short-read sequencing is the NGS workhorse, while long-read sequencing opens the door to more comprehensive NGS analyses Short Read Benefits Rapid high depth sequencing at low cost Long history of use Standardized workflows Long Read Benefits Single molecule direct sequencing eliminates amplification bias, sequence gaps Capture complex structural variants, phasing Capture difficult regions (pseudogenes, STRs, HLA regions) Accurate CNV detection at shallow depth Metagenomics, methylation, direct RNA-Seq Short Read Limitations Inability to sequence long DNA fragments Difficult to capture structural variants (copy number variants, fusions), phasing Difficult to capture pseudogenes, STRs, HLA regions Long Read Limitations Higher cost Shorter history of use Novel with fewer standardized workflows

12. Comprehensive NGS Workflows with both approaches 12 Gradual adoption of long read where users are leveraging the capabilities of both data types for comprehensive NGS analyses. Conventional short read sequencing is the workhorse for NGS analyses, while rising star long read is complementary for resolution of unsolved cases . Long read sequencing is particularly useful where short read fails to find causative variants such as complex structural variants and difficult to sequence regions. Example use cases in literature : “Long-read genome sequencing identifies cryptic structural variants in congenital aniridia cases” "Long-Read Sequencing Resolves a Complex Structural Variant in PRKN Parkinson’s Disease" "Application of long-read sequencing to the detection of structural variants in human cancer genomes“ “Resolving complex structural variants via nanopore sequencing”

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14. Simplify and validate a comprehensive NGS workflow  14 An approach to simplifying a comprehensive NGS workflow   Long read offers a simpler paradigm one pipeline to generate all relevant data, but while the field adapts, incorporating both short and long read pipelines can be complex Simplify with: Less software for comprehensive analysis PacBio and ONT pipelines have built-in callers for detection of small variants, CNVs an SVs ( DeepVaraint and NextFlow ) Sentieon also has the capacity to call CNVs and SVs from long read) May generate multiple VCFs from different callers, but VarSeq can consolidate them all into one project VarSeq does filtering, annotation, data visualization and clinical reporting on short and long read OTHER

15. Simplify and validate a comprehensive NGS workflow  15 An approach to validating a comprehensive NGS workflow   Additional non-NGS assays are often used to validate complex variant calls such as copy number alterations because of the low accuracy of short read for these difficult to sequence events. Incorporating long read allows you to simplify with: Less assays for validation Avoid MLPA or microarray for validation of your CNVs with long read Long read sequencing can detect CNVs accurately even at shallow sequencing depth Initial analytic validation would be done with your simplified pipeline using publicly available data (GIAB, other) Deletion Duplication

16. Demo Outline 16 Demo – Combined long and short read workflow A family analysis (trio) with long and short read data on the family in the same project Phasing, complex variants/fusions that were not detected on short read Accuracy and confirmation of CNVs with shallow WGS Clinical evaluation and report 2 4 1 3

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18. NIH Grant Funding Acknowledgments 18 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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20. eBook Library 20 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

21. Secured CE Mark for EU 21 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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