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Best practices for the analytical validation of clinical whole-genome sequencing intended for the diagnosis of germline disease.

ABSTRACT: Whole-genome sequencing (WGS) has shown promise in becoming a first-tier diagnostic test for patients with rare genetic disorders; however, standards addressing the definition and deployment practice of a best-in-class test are lacking. To address these gaps, the Medical Genome Initiative, a consortium of leading healthcare and research organizations in the US and Canada, was formed to expand access to high-quality clinical WGS by publishing best practices. Here, we present consensus recommendations on clinical WGS analytical validation for the diagnosis of individuals with suspected germline disease with a focus on test development, upfront considerations for test design, test validation practices, and metrics to monitor test performance. This work also provides insight into the current state of WGS testing at each member institution, including the utilization of reference and other standards across sites. Importantly, members of this initiative strongly believe that clinical WGS is an appropriate first-tier test for patients with rare genetic disorders, and at minimum is ready to replace chromosomal microarray analysis and whole-exome sequencing. The recommendations presented here should reduce the burden on laboratories introducing WGS into clinical practice, and support safe and effective WGS testing for diagnosis of germline disease.

SUBMITTER: Marshall CR

PROVIDER: S-EPMC7585436 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Best practices for the analytical validation of clinical whole-genome sequencing intended for the diagnosis of germline disease.

Marshall Christian R CR Chowdhury Shimul S Taft Ryan J RJ Lebo Mathew S MS Buchan Jillian G JG Harrison Steven M SM Rowsey Ross R Klee Eric W EW Liu Pengfei P Worthey Elizabeth A EA Jobanputra Vaidehi V Dimmock David D Kearney Hutton M HM Bick David D Kulkarni Shashikant S Taylor Stacie L SL Belmont John W JW Stavropoulos Dimitri J DJ Lennon Niall J NJ

NPJ genomic medicine 20201023

Whole-genome sequencing (WGS) has shown promise in becoming a first-tier diagnostic test for patients with rare genetic disorders; however, standards addressing the definition and deployment practice of a best-in-class test are lacking. To address these gaps, the Medical Genome Initiative, a consortium of leading healthcare and research organizations in the US and Canada, was formed to expand access to high-quality clinical WGS by publishing best practices. Here, we present consensus recommendat ...[more]

PMID: 33110627

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Project description:Next-generation sequencing (NGS)'s crucial role in supporting genetic diagnosis and personalized medicine leads to the definition of Guidelines for Diagnostic NGS by the European Society of Human Genetics. Factors of different nature producing false-positive/negative NGS data together with the paucity of internationally accepted guidelines providing specified NGS quality metrics to be followed for diagnostics purpose made the Sanger validation of NGS variants still mandatory. We reported the analysis of three cases of discrepancy between NGS and Sanger sequencing in a cohort of 218 patients. NGS was performed by Illumina MiSeq® and Haloplex/SureSelect protocols targeting 97 or 57 or 10 gene panels usually applied for diagnostics. Variants called following guidelines suggested by the Broad Institute and identified according to MAF <0.01 and allele balance >0.2 were Sanger validated. Three out of 945 validated variants showed a discrepancy between NGS and Sanger. In all three cases, a deep evaluation of the discrepant gene variant results and methodological approach allowed to confirm the NGS datum. Allelic dropout (ADO) occurrence during polymerase chain or sequencing reaction was observed, mainly related to incorrect variant zygosity. Our study extends literature data in which almost 100% "high quality" NGS variants are confirmed by Sanger; moreover, it demonstrates that in case of discrepancy between a high-quality NGS variant and Sanger validation, NGS call should not be a priori assumed to represent the source of the error. Actually, difficulties (i.e., ADO, unpredictable presence of private variants on primer-binding regions) of the so-called gold standard direct sequencing should be considered especially in light of the constantly implemented and accurate high-throughput technologies. Our data along with literature raise a discussion on the opportunity to establish a standardized quality threshold by International Guidelines for clinical NGS in order to limit Sanger confirmation to borderline conditions of variant quality parameters and verification of correct gene variant call/patient coupling on a different blood sample aliquot.

Dataset Information

Best practices for the analytical validation of clinical whole-genome sequencing intended for the diagnosis of germline disease.

Publications

Best practices for the analytical validation of clinical whole-genome sequencing intended for the diagnosis of germline disease.

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