Analytic Validation of Whole Exome Sequencing for Clinical Diagnostics of Inherited Disorders


Keywords: whole exome sequencing; diagnostics; validation

Authors: M. Muona, M. Gentile, J. Koskenvuo, S. Myllykangas; Blueprint Genetics, Helsinki, Finland.

Abstract: Utility of Whole Exome Sequencing (WES) in clinical diagnostics has been limited by the non-uniform sequencing coverage across exons, leaving typically 5-10% of the regions with shallow coverage that prevents accurate variant detection. We have implemented a WES assay that enables wide breadth of coverage resembling high-coverage gene-panel based assays and provides high sensitivity in variant detection. We have performed analytic validation of the WES assay, which utilizes Agilent SureSelect Human All Exon V6 hybrid capture selection to capture 60 Mb of exonic targets in high coverage. Sequencing was performed using an Illumina HiSeq sequencing system. Analytic validity of the WES assay was demonstrated for single nucleotide variants (SNVs) and insertions and deletions (INDELs) using reference samples from The Genome In a Bottle Consortium with high-quality variant calls obtained. In the analytic validation, the WES assay achieved 99.5% sensitivity, 99.9% specificity and 99.4% positive predictive value for detecting SNVs and 97.2%, 95.7% and 97.0% sensitivity for detecting INDELs of 1-10, 11-20, and 21-30 bases, respectively. Longest detected INDEL was 35 bases. Repeatability of the WES assay was 99.4%. Of the target regions, 98.86% were covered with over 15x sequencing depth and the mean sequencing depth at nucleotide level was 149x. The results demonstrate that comprehensive sequencing coverage allowing high variant calling sensitivity makes this WES assay well-suited for clinical diagnostics of inherited disorders.


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