Analytical Validation of Short-Read Genome Sequencing for Diagnostic Panel and Exome Testing

Genome sequencing is commonly used for genetic disease research; however, most clinical laboratories use enrichment-based targeted sequencing for diagnostic panels and exome testing. To facilitate the implementation of clinical genome-based panel testing, short-read genome sequencing (≥40×) was subjected to analytical validation, including single-nucleotide variant (SNV), insertion/deletion (indel), and copy number variant (CNV) accuracy and reproducibility/repeatability, and clinical specimen types. Recall and precision for SNVs/indels (<50 bp) within nondifficult genome regions across Genome in a Bottle/National Institute of Standards and Technology reference materials were >99.9%, and nonreference genotype concordance for SNVs/indels within nondifficult coding sequence (CDS) regions across all reproducibility/repeatability assessments was >99%. In addition, genome sequencing of CNV (1.1 to 7270 kilobase) control specimens had sensitivity, specificity, and accuracy for deletions (n = 14) and duplications/triplications (n = 11) of >99.9%. Concordance for deletions and duplications (≥50 bp) within nondifficult genome regions across all reproducibility/repeatability assessments were >97% and >81.0%, respectively. Contamination and mosaicism were assessed, which determined that >99% SNV/indel accuracy was maintained up to contamination levels of approximately 9% and that 95% of small variants were detected at a level of approximately 30% mosaicism. Taken together, these data indicate that germline SNV/indel and CNV detection by short-read genome sequencing (≥40×) is accurate and robust, which supports its use as a platform for diagnostic genome-based panel and exome testing.