A multi-laboratory assessment of clinical exome sequencing for detection of hereditary disease variants: 4441 ClinVar variants for clinical genomic test development and validation

Abstract

Background and aimsWhole-exome sequencing (WES) technology has become an essential tool in the clinical diagnostic for rare genetic disorders, however, the issues that reduce testing precision, sensitivity, and concordance are not clear under routine testing conditions. The study is to systematically evaluate the comparability of clinical WES testing results in laboratories under routine conditions. MethodsWe designed a multi-laboratory study across 24 participating laboratories in China. We assessed sequencing quality across capture methods and sequencing platforms, benchmarked the impact of coverage and callable regions on detecting single nucleotide variants (SNVs), small insertions and deletions (Indels) under the same computational approaches, and compared the sensitivity, precision and reproducibility on detecting mutations across laboratories. ResultsHigh inter-laboratory variability on variants detection were found across participating laboratories. Sample DNA concentration and sequencing evenness are two major variables that lead to the coverage variation. The difference in bioinformatics tools and computational settings affect the sensitivity and precision of the final output. Besides, copy-number variants (CNVs) identification is less reproducible than SNVs and Indels in the WES testing. We also compiled a list of 4441 low coverage ClinVar variants of 1176 genes from this study, which can be used as a source for creating in silico and synthetic DNA reference materials for clinical genetic disorder detection. ConclusionsThe considerable inter-laboratory variability seen in both sequencing coverage evenness and variants detection highlights the urgent need to improve the precision, sensitivity and comparability of the results generated across different laboratories. The list of low coverage variants can have important implications for the development and validation of clinical genetic disorder tests by laboratories. This study also serves to best practice inform guidelines for detecting clinical genetic disorders by exome sequencing.