Our last webcast introduced the newest features of VarSeq that will be included in our upcoming release. After a serious developmental effort, we are excited to announce one of these being the integration of ACMG Guidelines for CNVs! VarSeq is not only the tertiary environment to filter through your imported SNVs and indels from any VCF file, but also includes robust and proven capabilities of CNV detection and clinical variant interpretation. With our upcoming release, users will be able to leverage an automated CNV ACMG classification filter that is paired with the CNV evaluation in the VSClinical interpretation hub. This webcast will expose attendees to literature reinforcing the quality of our CNV caller, as well as showing examples of CNV analysis to demonstrate how this tool can streamline the analysis process. In this webcast, we will cover: Describing the new CNV guidelines and how Golden Helix tackles their complexity Assessing CNV impact on Gene and Gene Dosage Cited literature referencing the accuracy of VarSeq’s CNV calling Product demonstrations of VSClinical’s CNV interpretation/classification and final report functionalities Manually traversing the guidelines and classification process is fundamentally complex with multiple criteria considerations, collecting any necessary caveats, and bulk literature review just to name a few. It is our goal to simplify and streamline this process without losing user-control of final results or overlooking any crucial criteria components necessary for final classification. We hope you will join us to see how this is accomplished as we explore the ACMG CNV Guidelines within VSClinical from the user’s perspective!

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2. NIH Grant Funding Acknowledgments
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• Research reported in this publication was supported by the National Institute Of General Medical Sciences of
the National Institutes of Health under:
o Award Number R43GM128485-01
o Award Number R43GM128485-02
o Award Number 2R44 GM125432-01
o Award Number 2R44 GM125432-02
o 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.

3. Who Are We?
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Golden Helix is a global bioinformatics company founded in 1998

4. Cited in 1,000s of Peer-Reviewed Publications
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5. Over 400 Customers Globally
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6. When you choose Golden Helix, you receive
more than just the software
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Software is Vetted
• 20,000+ users at 400+ organizations
• Quality & feedback
Simple, Subscription-
Based Business Model
• Yearly fee
• Unlimited training & support
Deeply Engrained in Scientific
Community
• Give back to the community
• Contribute content and support
Innovative Software Solutions
• Cited in 1,000s of publications
• Recipient of numerous NIH grant and other
funding bodies

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Quick Poll
What Next-Generation Sequencing data are you working
with i.e. gene panels, exome, genome, a combo, or all?

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9. New ACMG and ClinGen Guidelines
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for the interpretation of Copy Number Variants
• First learned about the guidelines at ASHG 2019
• Development of clinical workflow began after obtaining
preprint
• Conversations with guidelines authors were instrumental in
helping design the workflow
• Product currently in beta testing with international customers
Riggs ER, Andersen EF, Cherry AM, et al. Technical standards for the interpretation
and reporting of constitutional copy-number variants: a joint consensus
recommendation of the American College of Medical Genetics and Genomics
(ACMG) and the Clinical Genome Resource (ClinGen). Genet Med. 2020;22(2):245-257.
doi:10.1038/s41436-019-0686-8
https://clinicalgenome.org/

10. New ACMG and ClinGen Guidelines
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For the Interpretation of Copy Number Variants
• Provides much needed framework for evaluation of small CNVs
detected in gene panels
• High level overview of CNV scoring system
o Scoring system with criteria broken down into 5 sections
o Each criteria adds to a total score, some with ranges
o Total score -1.0 to 1.0 maps from Pathogenic to Benign
o Separate criteria codes for gains vs losses
o Complex, 5 pages table with ~80 distinct criteria codes
• We designed a comprehensive workflow to score, classify,
interpret and report CNVs following these guidelines

11. CNV Guidelines Components
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1 Impact of a CNV on the gene
(Sections 1-3)
2 Evidence that the gene tolerates this type of impact (dosage sensitivity)
(Section 4)
3 Patient disorder and inheritance alignment to what is known for the
gene (Section 5)
Three major components of the scoring system (per gene)

12. CNV Impact on Gene
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CNV Guidelines Components
• Simple for whole gene
deletions/duplications
• Use protein level in-silico
analysis to evaluate
partial gene impact
• Partial gene duplications
often act like Loss of
Function variants

13. Example: Full Deletion of HI Gene
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Example:
• SHH Full Gene Deletion
• Gene classified as
Haploinsufficient
• Entire coding sequence
is deleted

14. Example: Full Deletion of HI Gene
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Example:
• SHH Full Gene Deletion
• Gene classified as
Haploinsufficient
• Entire coding sequence
is deleted

15. Example: Deletion within Benign Region
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Example:
• Partial deletion of PCSK9
• Dosage sensitivity
unlikely
• CNV is fully contained by
established benign
region

16. Example: Deletion within Benign Region
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Example:
• Partial deletion of PCSK9
• Dosage sensitivity
unlikely
• CNV is fully contained by
established benign
region

17. Example: Deletion of Last Exon
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Example:
• Partial deletion of ACSL4
• Haploinsufficient gene
• Deletion overlaps 3’ end
• Deletion only includes
last exon
• No other pathogenic LoF
variants in exon

18. Example: Deletion of Last Exon
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Example:
• Partial deletion of ACSL4
• Haploinsufficient gene
• Deletion overlaps 3’ end
• Deletion only includes
last exon
• No other pathogenic LoF
variants in exon

19. Gene Dosage Sensitivity
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CNV Guidelines Components
• ClinGen has rigorously evaluated genes for
haploinsufficiency and triplosensitivity
• If not in ClinGen, you can follow “Section 4” to score the
gene yourself, caving the result to an in-house “Gene
Dosage KB”
• A gene without either ClinGen or Section 4 dosage
sensitivity will never reach Likely Pathogenic or
Pathogenic scoring level

20. Clinical Evaluation for Sample
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CNV Guidelines Components
• Two important pieces of evidence evaluated at the
sample level:
o Inheritance and segregation of the CNV with
the phenotype and disorder
o The specificity of the phenotypes to the
specific gene
• CNVs in established dosage sensitive genes can be
scored Pathogenic without sample-specific scoring
• For novel genes, previous cases provide evidence
records for establishing whether that gene is
haploinsufficient / triplosensitive

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Quick Poll
How do you currently call CNVs: CMA, MLPA, NGS or
combination?

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23. VS-CNV Resources eBooks, Webcasts, Tutorials
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Head to goldenhelix.com to see our extensive list of webcasts, eBooks, blogs, tutorials and more to
guide you through your project design.

24. VS-CNV Example Publication References
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Validation of NGS-CNV approach
100% concordance with MLPA: M. A. Iacocca, J. Wang, J. S. Dron, J. F. Robinson, A. D. McIntyre, H. Cao and R. Hegele, "Use of next-generation sequencing to
detect LDLR gene copy number variation in familial hypercholesterolemia," Journal of lipid research, vol. 58, no. 11, pp. 2202-2209, 2017.
Detection/confirmation of large deletions with exome sequencing: J. S. Dron, J. Wang, A. J. Berberich, M. A. Iacocca, H. Cao and P. Yang, "Large-scale
deletions of the ABCA1 gene in patients with hypoalphalipoproteinemia," J Lipid Res., vol. 59, no. 8, pp. 1529-1535, 2018.
Exon deletion associated with maturity-onset diabetes of the young (MODY) confirmed with Sanger sequencing: A. J. Berberich, C. Huot, H. Cao, A.
D. McIntyre, J. F. Robinson, J. Wang and R. A. Hegele, "Copy number variation in GCK in patients with maturity-onset diabetes of the young," The Journal of Clinical
Endocrinology and Metabolism, vol. 104, no. 8, pp. 3428-3436, 2019.
A. J. Berberich, A. Mokashi, A. D. McIntyre, J. F. Robinson, H. Cao, J. Wang and R. A. Hegele, "Bioinformatic detection of copy number variation in HNF4A causing
maturity onset diabetes of the young," Clinical genetics, vol. 96, no. 4, pp. 376-377, 2019.
A.J. Berberich, J. Wang, H. Cao, A. D. McIntyre, T. Spaic, D. B. Miller, S. Stock, C. Huot, R. Stein and J. Knoll, "Simplifying Detection of Copy Number Variations in
Maturity Onset Diabetes of the Young," Canadian Journal of Diabetes, 2020.
R. Ho, J. Wang, H. Cao and R. A. Hegele, "The Identification of Novel CNVs in Patients with Inherited Lipodystrophy," Atherosclerosis Supplements, vol. 32, p. 10,
2018.
A. S. Geller, E. Y. Polisecki, M. R. Diffenderfer, B. F. Asztalos, S. K. Karathanasis, R. A. Hegele and E. J. Schaefer, "Genetic and secondary causes of severe HDL
deficiency and cardiovascular disease," Journal of lipid research, vol. 59, no. 12, pp. 2421-2435, 2018.

25. Example Variants
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Het Deletion: exon 23 of BRCA1 for Sample diagnosed with breast cancer
• Found filtering for familial breast and ovarian cancer panel
• BRCA1 classified in ClinGen as haploinsufficient (Focus on section 2)
• 57 pathogenic LOF variants downstream (region critical to protein
function).
• Novel – no presence in gnomAD CNVs nor DGV CNVs
Het Deletion: exons 4-6 of DSG2 for Sample with Arrhythmogenic right ventricular
cardiomyopathy
• Found filtering with PhoRank for high variant score with Right Ventricular
Cardiomyopathy
• DSG2 classified in ClinGen with little evidence for haploinsufficiency (Section 4)
• 49 pathogenic LOF in gene
• Novel – no presence in gnomAD CNVs nor DGV CNVs

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VarSeq Demonstration

27. COVID-19 Publications & Articles
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Investigating the Global Spread of SARS-CoV-2 Leveraging
Next-Gen Sequencing and Principal Component Analysis
European Journal of Clinical and Biomedical Sciences
Christiane Scherer, James Grover, Darby Kammeraad, Gabe Rudy, Andreas Scherer
Diagnosing and Tracking COVID-19 Infections Leveraging
Next-Gen Sequencing
The Journal of Precision Medicine Feature | July 8, 2020
Golden Helix: Enabling Precision Medicine with Cutting-
Edge NGS Technologies
Clinical OMICs Feature | July 15, 2020
Leveraging Next-Generation Sequencing Technology in the
Fight Against COVID-19
Clinical Lab Manager Feature | May 4, 2020
SARS-CoV-2 Global Spreading Investigation using Principal
Component Analysis of Sequence Variants
Journal of Genetics and Genome Research
Christiane Scherer, James Grover, Darby Kammeraad, Gabe Rudy, Andreas Scherer

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Inc 5000 Recognition
Golden Helix was named to the 2020 Inc 5000 List
of Fastest-Growing Private Companies
• Second year in a row receiving this award
• Thank you to our customers and partners for our
sustained success

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ClinGen Requirements
Genomic Analysis Software Platforms

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Any Questions?