Towards Clinical Molecular Diagnosis of Inherited Cardiac Conditions: A Comparison of Bench-Top Genome DNA Sequencers

Abstract

BackgroundMolecular 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 FindingsWe 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/SignificanceMiSeq 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.

Fast clinical molecular diagnosis of hyperphenylalaninemia using next-generation sequencing-based on a custom AmpliSeq™ panel and Ion Torrent PGM sequencing

“…The average GC content in these amplicons was 79.1%. As other reports have confirmed [5], overly high GC content influences the efficiency of amplification. In cases in which Ion Torrent PGM is unable to identify the pathogenic mutation, there is still a need to use other methods to make up for the challenges of sequencing high-GC content segments.…”

“…It has already been adapted for clinical molecular diagnosis, from specific genes such as CFTR [3], BRCA1, and BRCA2 [4] to disease-causing gene panels such as inherited arrhythmia syndromes [5], long QT syndrome [6], and cardiomyopathies [7]. In this study, we aimed to construct and evaluate the efficiency of a fast and comprehensive assay based on a custom AmpliSeq™ panel and the Ion Torrent PGM sequencer for the detection of mutations in HPArelated genes (PAH, PTS, QDPR, GCH1, and PCBD1).…”

Technological advances in precision medicine and drug development

“…The platforms leverage different sequencing technologies: Illumina utilizes sequencing-by-synthesis, measuring the signal as fluorescently-tagged nucleotides bind [131], while Ion employs semiconductor sequencing, in which the shift in pH caused by the release of hydrogen ions during DNA extension is measured [132]. While each platform and associated sequencing method has its own advantages and disadvantages, it has been repeatedly demonstrated that performance is relatively comparable for similar applications [133,134], and they uniformly allow for high throughput and multiplexing, low sequencing costs, and low error rates [130]. When comparing these two NGS platforms against each other, the main differences are the turnaround time and upfront cost; the elimination of optics in Ion semiconductor sequencing has allowed for a faster runtime and lower upfront cost than Illumina, but Illumina ultimately offers a lower cost per base pair [133,134].…”

Translating Sanger-Based Routine DNA Diagnostics into Generic Massive Parallel Ion Semiconductor Sequencing

“…ent in the raw data but could not be called except with settings that also generated many additional false-positive calls (23 ), we conclude that the false-negative detection rate is largely due to suboptimal data analysis, a problem that might be solved by testing or developing alternative data software tools. Notwithstanding, it might be worthwhile to test alternative NGS-based sequencing machines such as the Illumina MiSeq (19,20,24 ) to further im- prove performance, because the approach we used here is not restricted to ISS. In particular, the use of alternative platforms could reduce the number of false-positive variants that occur mainly in homopolymer stretches, which is an inherent weakness related to the principle of sequencing by synthesis (25 ).…”