Development of a Comprehensive Sequencing Assay for Inherited Cardiac Condition Genes

Abstract

Inherited cardiac conditions (ICCs) are characterised by marked genetic and allelic heterogeneity and require extensive sequencing for genetic characterisation. We iteratively optimised a targeted gene capture panel for ICCs that includes disease-causing, putatively pathogenic, research and phenocopy genes (n = 174 genes). We achieved high coverage of the target region on both MiSeq (>99.8% at ≥ 20× read depth, n = 12) and NextSeq (>99.9% at ≥ 20×, n = 48) platforms with 100% sensitivity and precision for single nucleotide variants and indels across the protein-coding target on the MiSeq. In the final assay, 40 out of 43 established ICC genes informative in clinical practice achieved complete coverage (100 % at ≥ 20×). By comparison, whole exome sequencing (WES; ∼ 80×), deep WES (∼ 500×) and whole genome sequencing (WGS; ∼ 70×) had poorer performance (88.1, 99.2 and 99.3% respectively at ≥ 20×) across the ICC target. The assay described here delivers highly accurate and affordable sequencing of ICC genes, complemented by accessible cloud-based computation and informatics. See Editorial in this issue (DOI: 10.1007/s12265-015-9667-8 ).

Keywords: Diagnostics; Genetics; Inherited cardiac conditions; Targeted sequencing; Whole exome sequencing; Whole genome sequencing.

Fig. 1

Stringent heat map showing the percentage coverage of ICC disease genes commonly used to inform clinical practice (n = 43) at 20× read depth using M3 (MiSeq, 150 bp PE), M4 (NextSeq 500, 150 bp PE), deep whole exome sequencing (WES; HiSeq 2500, 125 bp PE), WES (HiSeq, 125 bp PE) and whole genome sequencing (WGS: HiSeq X, 150 bp PE) (gene coverage at 20×: dark red ≤98 %; dark green = 100 %)

Fig. 2

Percentage coverage of all TTN exons (ENST00000589042/NM_001267550.1) at 20× read depth across methods (top four panels). Mappability score (score* [28]) and GC content in the TTN gene (bottom two panels). Error bars represent standard deviation