Next generation diagnostics in inherited arrhythmia syndromes : a comparison of two approaches

Abstract

Next-generation sequencing (NGS) provides an unprecedented opportunity to assess genetic variation underlying human disease. Here, we compared two NGS approaches for diagnostic sequencing in inherited arrhythmia syndromes. We compared PCR-based target enrichment and long-read sequencing (PCR-LR) with in-solution hybridization-based enrichment and short-read sequencing (Hyb-SR). The PCR-LR assay comprehensively assessed five long-QT genes routinely sequenced in diagnostic laboratories and "hot spots" in RYR2. The Hyb-SR assay targeted 49 genes, including those in the PCR-LR assay. The sensitivity for detection of control variants did not differ between approaches. In both assays, the major limitation was upstream target capture, particular in regions of extreme GC content. These initial experiences with NGS cardiovascular diagnostics achieved up to 89 % sensitivity at a fraction of current costs. In the next iteration of these assays we anticipate sensitivity above 97 % for all LQT genes. NGS assays will soon replace conventional sequencing for LQT diagnostics and molecular pathology.

Fig. 1

Summary of target selection designs for the two target enrichment strategies. LQT long QT syndrome, bp base pairs

Fig. 2

For a given target enrichment design, the percentage of bases reaching variant-calling criteria increases with increasing sequencing depth. For each sample, the percentage of target bases callable is plotted against the mean sequencing depth achieved for that sample. The Hyb-SR reached saturation when run with low multiplex, suggesting that a further increase in sequencing depth would not improve coverage. PCR-LR has not reached saturation: the dotted line shows maximum achievable coverage with a simulated sample generated by pooling reads from all samples, equivalent to ×660 depth

Fig. 3

Coverage of the three genes most commonly causing long QT syndrome. Sequencing depth is plotted base by base across the protein-coding portions of three genes for a single sample sequenced on both platforms. Coverage varies widely for the Hyb-SR approach. PCR-LR yields more even coverage within amplicons, but there remains significant inter-amplicon variability. The first exons of KCNQ1 and KCNH2 are poorly captured by both techniques. The proportion covered sufficiently for variant calling ranges from 78 % (KCNH2, Hyb-SR) to 100 %

Fig. 4

Target enrichment is strongly dependent on GC content. The distribution of GC content for the target region is shown, together with the GC content of bases consistently missed across all samples for each platform. Regions missed by Hyb-SR have a high GC content, while regions missed by PCR-LR may have a GC content at either extreme