Abstract

We have developed a comprehensive set of next-generation sequencing tests for detecting single-nucleotide variants (SNVs), insertions and deletions (INDELs) and deletions and duplications (Del/Dups) in all inherited diseases.

The 214 panels contain 2053 clinically actionable genes that span 2243 conditions in 14 medical specialities. We adopted Oligonucleotide-Selective Sequencing (OS-Seq) method for targeted sequencing and implemented the flow-cell capture using the Illumina NextSeq500 sequencing system. We generated six OS-Seq assays that targeted 374 to 585 genes’ coding exons, 15 adjacent bases of the splice regions and pathogenic intronic variants. Analytic validity of the NGS assays was demonstrated for SNVs, INDELs and Del/Dups using cohorts of reference samples with high-quality variant calls.

Analytic validation results showed that the NGS assays achieve, on average, 0.993 sensitivity, 0.999 specificity, 0.993 positive predictive value for detecting SNVs and 0.961, 0.885 and 0.668 sensitivity for detecting INDELs of 1-10, 11-20, and 21-30 bases, respectively. Longest detected INDEL was 46 bases. Repeatability and reproducibility of the OS-Seq assays were 0.994 and 0.998, respectively. 99.6% of the target regions were covered with over 15x sequencing depth and mean sequencing depth at nucleotide level was 234x. We demonstrated the assays’ sensitivity to detect different size (1-4 exons) Del/dups: 0.715, 0.952, 0.990 and 0.999. Del/dup detection was demonstrated to detect >316bp deletions and >544bp duplication and clinical sensitivity of the del/dup detection was 92.42%.

Our results demonstrate the analytic validity of the Blueprint Genetics’ sequencing panels and show that the technology is well-suited for clinical diagnostics of inherited disorders.

Keywords: Next-generation sequencing; inherited disorders; validation

Authors: S. Myllykangas, P. Salmenperä, M. Gentile, S. Bruce, M. Valori, M. Muona, A. Sarin, J. Koskenvuo