Dataset Information

Performance comparison of bench-top next generation sequencers using microdroplet PCR-based enrichment for targeted sequencing in patients with autism spectrum disorder.

ABSTRACT: Next-generation sequencing (NGS) combined with enrichment of target genes enables highly efficient and low-cost sequencing of multiple genes for genetic diseases. The aim of this study was to validate the accuracy and sensitivity of our method for comprehensive mutation detection in autism spectrum disorder (ASD). We assessed the performance of the bench-top Ion Torrent PGM and Illumina MiSeq platforms as optimized solutions for mutation detection, using microdroplet PCR-based enrichment of 62 ASD associated genes. Ten patients with known mutations were sequenced using NGS to validate the sensitivity of our method. The overall read quality was better with MiSeq, largely because of the increased indel-related error associated with PGM. The sensitivity of SNV detection was similar between the two platforms, suggesting they are both suitable for SNV detection in the human genome. Next, we used these methods to analyze 28 patients with ASD, and identified 22 novel variants in genes associated with ASD, with one mutation detected by MiSeq only. Thus, our results support the combination of target gene enrichment and NGS as a valuable molecular method for investigating rare variants in ASD.

SUBMITTER: Koshimizu E

PROVIDER: S-EPMC3774667 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Performance comparison of bench-top next generation sequencers using microdroplet PCR-based enrichment for targeted sequencing in patients with autism spectrum disorder.

Koshimizu Eriko E Miyatake Satoko S Okamoto Nobuhiko N Nakashima Mitsuko M Tsurusaki Yoshinori Y Miyake Noriko N Saitsu Hirotomo H Matsumoto Naomichi N

PloS one 20130916 9

Next-generation sequencing (NGS) combined with enrichment of target genes enables highly efficient and low-cost sequencing of multiple genes for genetic diseases. The aim of this study was to validate the accuracy and sensitivity of our method for comprehensive mutation detection in autism spectrum disorder (ASD). We assessed the performance of the bench-top Ion Torrent PGM and Illumina MiSeq platforms as optimized solutions for mutation detection, using microdroplet PCR-based enrichment of 62 A ...[more]

PMID: 24066114

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Project description: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.

Project description:The development and innovation of next generation sequencing (NGS) and the subsequent analysis tools have gain popularity in scientific researches and clinical diagnostic applications. Hence, a systematic comparison of the sequencing platforms and variant calling pipelines could provide significant guidance to NGS-based scientific and clinical genomics. In this study, we compared the performance, concordance and operating efficiency of 27 combinations of sequencing platforms and variant calling pipelines, testing three variant calling pipelines-Genome Analysis Tool Kit HaplotypeCaller, Strelka2 and Samtools-Varscan2 for nine data sets for the NA12878 genome sequenced by different platforms including BGISEQ500, MGISEQ2000, HiSeq4000, NovaSeq and HiSeq Xten. For the variants calling performance of 12 combinations in WES datasets, all combinations displayed good performance in calling SNPs, with their F-scores entirely higher than 0.96, and their performance in calling INDELs varies from 0.75 to 0.91. And all 15 combinations in WGS datasets also manifested good performance, with F-scores in calling SNPs were entirely higher than 0.975 and their performance in calling INDELs varies from 0.71 to 0.93. All of these combinations manifested high concordance in variant identification, while the divergence of variants identification in WGS datasets were larger than that in WES datasets. We also down-sampled the original WES and WGS datasets at a series of gradient coverage across multiple platforms, then the variants calling period consumed by the three pipelines at each coverage were counted, respectively. For the GIAB datasets on both BGI and Illumina platforms, Strelka2 manifested its ultra-performance in detecting accuracy and processing efficiency compared with other two pipelines on each sequencing platform, which was recommended in the further promotion and application of next generation sequencing technology. The results of our researches will provide useful and comprehensive guidelines for personal or organizational researchers in reliable and consistent variants identification.

Dataset Information

Performance comparison of bench-top next generation sequencers using microdroplet PCR-based enrichment for targeted sequencing in patients with autism spectrum disorder.

Publications

Performance comparison of bench-top next generation sequencers using microdroplet PCR-based enrichment for targeted sequencing in patients with autism spectrum disorder.

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