Next generation sequencing for clinical diagnostics: Five year experience of an academic laboratory

Abstract

Clinical laboratories have adopted next generation sequencing (NGS) as a gold standard for the diagnosis of hereditary disorders because of its analytic accuracy, high throughput, and potential for cost-effectiveness. We describe the implementation of a single broad-based NGS sequencing assay to meet the genetic testing needs at the University of Minnesota. A single hybrid capture library preparation was used for each test ordered, data was informatically blinded to clinically-ordered genes, and identified variants were reviewed and classified by genetic counselors and molecular pathologists. We performed 2509 sequencing tests from August 2012 till December 2017. The diagnostic yield has remained steady at 25%, but the number of variants of uncertain significance (VUS) included in a patient report decreased over time with 50% of the patient reports including at least one VUS in 2012 and only 22% of the patient reports reporting a VUS in 2017 (p = .002). Among the various clinical specialties, the diagnostic yield was highest in dermatology (60% diagnostic yield) and ophthalmology (42% diagnostic yield) while the diagnostic yield was lowest in gastrointestinal diseases and pulmonary diseases (10% detection yield in both specialties). Deletion/duplication analysis was also implemented in a subset of panels ordered, with 9% of samples having a diagnostic finding using the deletion/duplication analysis. We have demonstrated the feasibility of this broad-based NGS platform to meet the needs of our academic institution by aggregating a sufficient sample volume from many individually rare tests and providing a flexible ordering for custom, patient-specific panels.

From cytogenetics to cytogenomics: whole-genome sequencing as a first-line test comprehensively captures the diverse spectrum of disease-causing genetic variation underlying intellectual disability

“…Chromosomal microarray analysis (CMA), either comparative genomic hybridization (CGH) or SNP arrays, has long been the first-tier test used to identify copy number variants (CNVs) in individuals with intellectual disability and neurodevelopmental disorders [10–12]. In recent years, MPS-based assays have been increasingly used in rare disease diagnostics, because of their high throughput and cost effectiveness in screening multi-gene panels for hereditary disorders [13]. Whole-exome sequencing (WES) is more and more widely used in clinical labs as a first-tier test, allowing detection of SNVs, INDELs, and CNVs covering multiple exons (typically > 2) [14–16].…”

Genetic Variants as Sudden-Death Risk Markers in Inherited Arrhythmogenic Syndromes: Personalized Genetic Interpretation

“…The filtration and annotation of genetic variants include multiple components, and the diagnostic decision depends on the data resources used. Recent studies recommended NGS coverage of at least 20× for diagnostic purposes [ 41 ]. Despite the high coverage obtained using gene panels, Sanger technology should be used to confirm all variants included in a genetic report and to discard false positives, especially concerning insertions/deletions (indels).…”

Functional characterisation guides classification of novel BAP1 germline variants

“…As of April 2020, 466 BAP1 germline variants of uncertain significance (VUS) have been reported on ClinVar, with 447 of them having single nucleotide variations and 404 of them resulting in missense mutations 22 . More of such BAP1 variants are expected to be discovered as clinical laboratories have increasingly adopted next-generation sequencing to diagnose hereditary disorders due to its costeffectiveness, high throughput and accuracy 23 . Given the increased risks and aggressiveness of cancer development and progression in patients with BAP1 germline variants, understanding how variants in BAP1 affect function will aid interpretation of uncertain variants.…”