Dataset Information

Strategies for identification of somatic variants using the Ion Torrent deep targeted sequencing platform.

ABSTRACT:

Background

'Next-generation' (NGS) sequencing has wide application in medical genetics, including the detection of somatic variation in cancer. The Ion Torrent-based (IONT) platform is among NGS technologies employed in clinical, research and diagnostic settings. However, identifying mutations from IONT deep sequencing with high confidence has remained a challenge. We compared various computational variant-calling methods to derive a variant identification pipeline that may improve the molecular diagnostic and research utility of IONT.

Results

Using IONT, we surveyed variants from the 409-gene Comprehensive Cancer Panel in whole-section tumors, intra-tumoral biopsies and matched normal samples obtained from frozen tissues and blood from four early-stage non-small cell lung cancer (NSCLC) patients. We used MuTect, Varscan2, IONT's proprietary Ion Reporter, and a simple subtraction we called "Poor Man's Caller." Together these produced calls at 637 loci across all samples. Visual validation of 434 called variants was performed, and performance of the methods assessed individually and in combination. Of the subset of inspected putative variant calls (n=223) in genomic regions that were not intronic or intergenic, 68 variants (30%) were deemed valid after visual inspection. Among the individual methods, the Ion Reporter method offered perhaps the most reasonable tradeoffs. Ion Reporter captured 83% of all discovered variants; 50% of its variants were visually validated. Aggregating results from multiple packages offered varied improvements in performance.

Conclusions

Overall, Ion Reporter offered the most attractive performance among the individual callers. This study suggests combined strategies to maximize sensitivity and positive predictive value in variant calling using IONT deep sequencing.

SUBMITTER: Deshpande A

PROVIDER: S-EPMC5753459 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Strategies for identification of somatic variants using the Ion Torrent deep targeted sequencing platform.

Deshpande Aditya A Lang Wenhua W McDowell Tina T Sivakumar Smruthy S Zhang Jiexin J Wang Jing J San Lucas F Anthony FA Fowler Jerry J Kadara Humam H Scheet Paul P

BMC bioinformatics 20180104 1

<h4>Background</h4>'Next-generation' (NGS) sequencing has wide application in medical genetics, including the detection of somatic variation in cancer. The Ion Torrent-based (IONT) platform is among NGS technologies employed in clinical, research and diagnostic settings. However, identifying mutations from IONT deep sequencing with high confidence has remained a challenge. We compared various computational variant-calling methods to derive a variant identification pipeline that may improve the m ...[more]

PMID: 29301485

Similar Datasets

Project description:Targeted sequencing of PCR amplicons generated from bisulfite deaminated DNA is a flexible, cost-effective way to study methylation of a sample at single CpG resolution and perform subsequent multi-target, multi-sample comparisons. Currently, no platform specific protocol, support, or analysis solution is provided to perform targeted bisulfite sequencing on a Personal Genome Machine (PGM). Here, we present a novel tool, called TABSAT, for analyzing targeted bisulfite sequencing data generated on Ion Torrent sequencers. The workflow starts with raw sequencing data, performs quality assessment, and uses a tailored version of Bismark to map the reads to a reference genome. The pipeline visualizes results as lollipop plots and is able to deduce specific methylation-patterns present in a sample. The obtained profiles are then summarized and compared between samples. In order to assess the performance of the targeted bisulfite sequencing workflow, 48 samples were used to generate 53 different Bisulfite-Sequencing PCR amplicons from each sample, resulting in 2,544 amplicon targets. We obtained a mean coverage of 282X using 1,196,822 aligned reads. Next, we compared the sequencing results of these targets to the methylation level of the corresponding sites on an Illumina 450k methylation chip. The calculated average Pearson correlation coefficient of 0.91 confirms the sequencing results with one of the industry-leading CpG methylation platforms and shows that targeted amplicon bisulfite sequencing provides an accurate and cost-efficient method for DNA methylation studies, e.g., to provide platform-independent confirmation of Illumina Infinium 450k methylation data. TABSAT offers a novel way to analyze data generated by Ion Torrent instruments and can also be used with data from the Illumina MiSeq platform. It can be easily accessed via the Platomics platform, which offers a web-based graphical user interface along with sample and parameter storage. TABSAT is freely available under a GNU General Public License version 3.0 (GPLv3) at https://github.com/tadkeys/tabsat/ and http://demo.platomics.com/.

Project description:BackgroundMassively parallel sequencing (MPS) technologies have the capacity to sequence targeted regions or whole genomes of multiple nucleic acid samples with high coverage by sequencing millions of DNA fragments simultaneously. Compared with Sanger sequencing, MPS also can reduce labor and cost on a per nucleotide basis and indeed on a per sample basis. In this study, whole genomes of human mitochondria (mtGenome) were sequenced on the Personal Genome Machine (PGMTM) (Life Technologies, San Francisco, CA), the out data were assessed, and the results were compared with data previously generated on the MiSeqTM (Illumina, San Diego, CA). The objectives of this paper were to determine the feasibility, accuracy, and reliability of sequence data obtained from the PGM.Results24 samples were multiplexed (in groups of six) and sequenced on the at least 10 megabase throughput 314 chip. The depth of coverage pattern was similar among all 24 samples; however the coverage across the genome varied. For strand bias, the average ratio of coverage between the forward and reverse strands at each nucleotide position indicated that two-thirds of the positions of the genome had ratios that were greater than 0.5. A few sites had more extreme strand bias. Another observation was that 156 positions had a false deletion rate greater than 0.15 in one or more individuals. There were 31-98 (SNP) mtGenome variants observed per sample for the 24 samples analyzed. The total 1237 (SNP) variants were concordant between the results from the PGM and MiSeq. The quality scores for haplogroup assignment for all 24 samples ranged between 88.8%-100%.ConclusionsIn this study, mtDNA sequence data generated from the PGM were analyzed and the output evaluated. Depth of coverage variation and strand bias were identified but generally were infrequent and did not impact reliability of variant calls. Multiplexing of samples was demonstrated which can improve throughput and reduce cost per sample analyzed. Overall, the results of this study, based on orthogonal concordance testing and phylogenetic scrutiny, supported that whole mtGenome sequence data with high accuracy can be obtained using the PGM platform.

Dataset Information

Strategies for identification of somatic variants using the Ion Torrent deep targeted sequencing platform.

Background

Results

Conclusions

Publications

Strategies for identification of somatic variants using the Ion Torrent deep targeted sequencing platform.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets