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Session


Keywords: Diagnostics; Genome sequencing; Genetic testing; Triplet and other repeats; Copy number/structural variation

Authors:
A. Kaplun; N. Neerman; C. Stanley; G. Faust

Affiliation: Variantyx Inc., Framingham, Massachusetts.


Background:

WGS is well positioned to become the clinical diagnostic standard for rare genetic disorders due to the benefits inherent in PCR-free, genome-wide sequencing combined with its continually decreasing cost. Initial published reports are supportive of this paradigm shift, yet all WGS assays are not created equal. We’ve undertaken the challenge to develop and validate a clinical WGS pipeline that supports simultaneous analysis and interpretation of multiple variant types, resulting in a clinically available alternative to sequential testing.

Methods:

Clinical grade whole genome sequencing (WGS) is conducted on an Illumina platform using the Illumina TruSeq DNA PCR-Free Library Preparation Kit. With the PCR-free protocol, significantly less average coverage is required compared to panels and exomes. Specifically, we find that 30X mean mappable coverage provides a highly uniform sequencing depth with 97.3% of nucleotides covered at ≥8X and 99.4% of HGMD and ClinVar annotated variants covered at ≥8X - a depth that is sufficient for specific and sensitive variant calling. Following alignment of reads, a combination of open source and proprietary algorithms call small sequence change variants, structural variants, mitochondrial variants and tandem repeat expansions. Variants are subjected to annotation and filtering before being presented to our clinicians for interpretation within the context of the patient’s clinical symptoms and medical history.

Results:

In 2018, we obtained full CLIA accreditation and CAP certification for all four components of our WGS pipeline: small sequence changes, structural variants, mitochondrial variants and short tandem repeats. We will present a combination of secondary and tertiary validation results which demonstrate >99% sensitivity, specificity and PPV for single nucleotide variants; >95% sensitivity and specificity for indels up to 50 nucleotides; >96% clinical sensitivity for structural variants; the ability to detect mitochondrial heteroplasmy levels down to 5%; and >99% sensitivity for tandem repeat expansions spanning >20 known pathogenic loci. We will additionally present cases representative of those processed in the first 18 months that the full Genomic Unity™ test has been available for clinical use.