Dataset Information

Determining Performance Metrics for Targeted Next-Generation Sequencing Panels Using Reference Materials.

ABSTRACT: The National Institute of Standards and Technology has developed reference materials for five human genomes. DNA aliquots are available for purchase, and the data, analyses, and high-confidence small variant and homozygous reference calls are freely available on the web. These reference materials are useful for evaluating whole-genome sequencing methods and also can be used to benchmark targeted sequencing panels, which are used commonly in clinical settings. This article describes how to use the Genome in a Bottle samples to obtain performance metrics on any germline-targeted sequencing panel of interest, as well as the limitations of the reference materials. These materials are useful for understanding the limitations of, and optimizing, targeted sequencing panels and associated bioinformatics pipelines. Example figures are presented to illustrate ways to access the performance metrics of targeted sequencing panels, and a table of best practices is included.

SUBMITTER: Cleveland MH

PROVIDER: S-EPMC6172655 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Determining Performance Metrics for Targeted Next-Generation Sequencing Panels Using Reference Materials.

Cleveland Megan H MH Zook Justin M JM Salit Marc M Vallone Peter M PM

The Journal of molecular diagnostics : JMD 20180626 5

The National Institute of Standards and Technology has developed reference materials for five human genomes. DNA aliquots are available for purchase, and the data, analyses, and high-confidence small variant and homozygous reference calls are freely available on the web. These reference materials are useful for evaluating whole-genome sequencing methods and also can be used to benchmark targeted sequencing panels, which are used commonly in clinical settings. This article describes how to use th ...[more]

PMID: 29959024

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Project description:BackgroundWith the expanded availability of next generation sequencing (NGS)-based clinical genetic tests, clinicians seeking to test patients with Mendelian diseases must weigh the superior coverage of targeted gene panels with the greater number of genes included in whole exome sequencing (WES) when considering their first-tier testing approach. Here, we use an in silico analysis to predict the analytic sensitivity of WES using pathogenic variants identified on targeted NGS panels as a reference.MethodsCorresponding nucleotide positions for 1533 different alterations classified as pathogenic or likely pathogenic identified on targeted NGS multi-gene panel tests in our laboratory were interrogated in data from 100 randomly-selected clinical WES samples to quantify the sequence coverage at each position. Pathogenic variants represented 91 genes implicated in hereditary cancer, X-linked intellectual disability, primary ciliary dyskinesia, Marfan syndrome/aortic aneurysms, cardiomyopathies and arrhythmias.ResultsWhen assessing coverage among 100 individual WES samples for each pathogenic variant (153,300 individual assessments), 99.7% (n = 152,798) would likely have been detected on WES. All pathogenic variants had at least some coverage on exome sequencing, with a total of 97.3% (n = 1491) detectable across all 100 individuals. For the remaining 42 pathogenic variants, the number of WES samples with adequate coverage ranged from 35 to 99. Factors such as location in GC-rich, repetitive, or homologous regions likely explain why some of these alterations were not detected across all samples. To validate study findings, a similar analysis was performed against coverage data from 60,706 exomes available through the Exome Aggregation Consortium (ExAC). Results from this validation confirmed that 98.6% (91,743,296/93,062,298) of pathogenic variants demonstrated adequate depth for detection.ConclusionsResults from this in silico analysis suggest that exome sequencing may achieve a diagnostic yield similar to panel-based testing for Mendelian diseases.

Dataset Information

Determining Performance Metrics for Targeted Next-Generation Sequencing Panels Using Reference Materials.

Publications

Determining Performance Metrics for Targeted Next-Generation Sequencing Panels Using Reference Materials.

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