Towards clinical molecular diagnosis of inherited cardiac conditions: a comparison of bench-top genome DNA sequencers

Abstract

Background: Molecular genetic testing is recommended for diagnosis of inherited cardiac disease, to guide prognosis and treatment, but access is often limited by cost and availability. Recently introduced high-throughput bench-top DNA sequencing platforms have the potential to overcome these limitations.

Methodology/principal findings: We evaluated two next-generation sequencing (NGS) platforms for molecular diagnostics. The protein-coding regions of six genes associated with inherited arrhythmia syndromes were amplified from 15 human samples using parallelised multiplex PCR (Access Array, Fluidigm), and sequenced on the MiSeq (Illumina) and Ion Torrent PGM (Life Technologies). Overall, 97.9% of the target was sequenced adequately for variant calling on the MiSeq, and 96.8% on the Ion Torrent PGM. Regions missed tended to be of high GC-content, and most were problematic for both platforms. Variant calling was assessed using 107 variants detected using Sanger sequencing: within adequately sequenced regions, variant calling on both platforms was highly accurate (Sensitivity: MiSeq 100%, PGM 99.1%. Positive predictive value: MiSeq 95.9%, PGM 95.5%). At the time of the study the Ion Torrent PGM had a lower capital cost and individual runs were cheaper and faster. The MiSeq had a higher capacity (requiring fewer runs), with reduced hands-on time and simpler laboratory workflows. Both provide significant cost and time savings over conventional methods, even allowing for adjunct Sanger sequencing to validate findings and sequence exons missed by NGS.

Conclusions/significance: MiSeq and Ion Torrent PGM both provide accurate variant detection as part of a PCR-based molecular diagnostic workflow, and provide alternative platforms for molecular diagnosis of inherited cardiac conditions. Though there were performance differences at this throughput, platforms differed primarily in terms of cost, scalability, protocol stability and ease of use. Compared with current molecular genetic diagnostic tests for inherited cardiac arrhythmias, these NGS approaches are faster, less expensive, and yet more comprehensive.

Figure 1. Coverage of target genes.

a. The percentage of each gene that is captured and sequenced (at least one read) is shown for each platform (MiSeq in red, PGM in black), for 15 samples; Three genes were consistently fully sequenced. Coverage of KCNQ1 and KCNH2 was more variable: KCNQ1 and KCNE1 were fully covered in the best performing samples, while the best performance on KCNH2 covered >97% of the gene. b. Mean sequencing depth across each gene, for 15 samples. Quartiles are shown. There is significant intra- and inter- sample variability.

Figure 2. Coverage of KCNQ1 and KCNH2 for the two platforms.

Mean depth of coverage for 15 samples is shown for two genes on a log scale. Regions of no coverage therefore have negative values. The blue lines indicate local GC content (calculated with a 50 bp sliding window). Regions consistently missed have high GC content, with similar patterns for both platforms. KCNQ1 exons 1 & 8 and KCNH2 exons 1, 4 & 12 are difficult to sequence. A cartoon of the exon structure is shown beneath each panel. Plus (+) and minus (-) denote gene strand. Plots for all genes are shown in Supporting Information Figure S5. a.) MiSeq b.) Ion Torrent PGM.