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. 2018 Jan 4;19(1):5.
doi: 10.1186/s12859-017-1991-3.

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

Aditya Deshpande  1   2 , Wenhua Lang  3 , Tina McDowell  3 , Smruthy Sivakumar  1   4 , Jiexin Zhang  5 , Jing Wang  5 , F Anthony San Lucas  1 , Jerry Fowler  1 , Humam Kadara  6 , Paul Scheet  7   8   9
Affiliations

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

Aditya Deshpande et al. BMC Bioinformatics. .

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.

Keywords: Ion Reporter; Ion torrent; MuTect; Next-generation sequencing; Variant calling strategies; Varscan2.

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Conflict of interest statement

Ethics approval and consent to participate

The malignant and normal samples were acquired from early-stage (stages I-III) NSCLC patients evaluated at The University of Texas MD Anderson Cancer Center. The study was approved by the MD Anderson Cancer Center institutional review board (IRB) and all participants provided written informed consents.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Variant Inspection Workflow. We used the depicted workflow to organize the variants called by the four software packages. We determined validity of variants via visual inspection after having established the categorization criteria described in the lower three boxes by consensus (Review and Decision, following independent inspections by individuals 1-4)
Fig. 2
Fig. 2
Venn Diagram of Number of Variants Grouped by Method Combinations. Listed under each method name are that method’s total called variants. (Note, areas are not scaled according to set size.)
See this image and copyright information in PMC

References

    1. Meyerson M, Gabriel S, Getz G. Advances in understanding cancer genomes through second-generation sequencing. Nat Rev Genet. 2010;11:685–96. doi: 10.1038/nrg2841. - DOI - PubMed
    1. Chmielecki J, Meyerson M. DNA sequencing of cancer: what have we learned? Annu Rev Med. 2014;65:63–79. doi: 10.1146/annurev-med-060712-200152. - DOI - PubMed
    1. Toyota T, Kudo M, Abe K, Goto Y. Plasma C-peptide response to oral glucose in low insulin responders, subdiabetic, and diabetic subjects. Tohoku J Exp Med. 1977;121:99–104. doi: 10.1620/tjem.121.99. - DOI - PubMed
    1. Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA, Kinzler KW. Cancer genome landscapes. Science. 2013;339:1546–58. doi: 10.1126/science.1235122. - DOI - PMC - PubMed
    1. Imielinski M, Berger AH, Hammerman PS, et al. Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing. Cell. 2012;150:1107–20. doi: 10.1016/j.cell.2012.08.029. - DOI - PMC - PubMed

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