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Systematic Evaluation of Sanger Validation of Next-Generation Sequencing Variants.

ABSTRACT: BACKGROUND:Next-generation sequencing (NGS) data are used for both clinical care and clinical research. DNA sequence variants identified using NGS are often returned to patients/participants as part of clinical or research protocols. The current standard of care is to validate NGS variants using Sanger sequencing, which is costly and time-consuming. METHODS:We performed a large-scale, systematic evaluation of Sanger-based validation of NGS variants using data from the ClinSeq® project. We first used NGS data from 19 genes in 5 participants, comparing them to high-throughput Sanger sequencing results on the same samples, and found no discrepancies among 234 NGS variants. We then compared NGS variants in 5 genes from 684 participants against data from Sanger sequencing. RESULTS:Of over 5800 NGS-derived variants, 19 were not validated by Sanger data. Using newly designed sequencing primers, Sanger sequencing confirmed 17 of the NGS variants, and the remaining 2 variants had low quality scores from exome sequencing. Overall, we measured a validation rate of 99.965% for NGS variants using Sanger sequencing, which was higher than many existing medical tests that do not necessitate orthogonal validation. CONCLUSIONS:A single round of Sanger sequencing is more likely to incorrectly refute a true-positive variant from NGS than to correctly identify a false-positive variant from NGS. Validation of NGS-derived variants using Sanger sequencing has limited utility, and best practice standards should not include routine orthogonal Sanger validation of NGS variants.

SUBMITTER: Beck TF

PROVIDER: S-EPMC4878677 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Systematic Evaluation of Sanger Validation of Next-Generation Sequencing Variants.

Beck Tyler F TF Mullikin James C JC Biesecker Leslie G LG

Clinical chemistry 20160204 4

<h4>Background</h4>Next-generation sequencing (NGS) data are used for both clinical care and clinical research. DNA sequence variants identified using NGS are often returned to patients/participants as part of clinical or research protocols. The current standard of care is to validate NGS variants using Sanger sequencing, which is costly and time-consuming.<h4>Methods</h4>We performed a large-scale, systematic evaluation of Sanger-based validation of NGS variants using data from the ClinSeq® pro ...[more]

PMID: 26847218

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Project description:OBJECTIVE:To validate the targeted next-generation sequencing (NGS) platform-Ion Torrent PGM for KRAS exon 2 and expanded RAS mutations detection in formalin-fixed paraffin-embedded (FFPE) colorectal cancer (CRC) specimens, with comparison of Sanger sequencing and ARMS-Scorpion real-time PCR. SETTING:Beijing, China. PARTICIPANTS:51 archived FFPE CRC samples (36 men, 15 women) were retrospectively randomly selected and then checked by an experienced pathologist for sequencing based on histological confirmation of CRC and availability of sufficient tissue. METHODS:RAS mutations were detected in the 51 FFPE CRC samples by PGM analysis, Sanger sequencing and the Therascreen KRAS assay, respectively. Agreement among the 3 methods was assessed. Assay sensitivity was further determined by sequencing serially diluted DNA from FFPE cell lines with known mutation statuses. RESULTS:13 of 51 (25.5%) cases had a mutation in KRAS exon 2, as determined by PGM analysis. PGM analysis showed 100% (51/51) concordance with Sanger sequencing (?=1.000, 95% CI 1 to 1) and 98.04% (50/51) agreement with the Therascreen assay (?=0.947, 95% CI 0.844 to 1) for detecting KRAS exon 2 mutations, respectively. The only discrepant case harboured a KRAS exon 2 mutation (c.37G>T) that was not covered by the Therascreen kit. The dilution series experiment results showed that PGM was able to detect KRAS mutations at a frequency of as low as 1%. Importantly, RAS mutations other than KRAS exon 2 mutations were also detected in 10 samples by PGM. Furthermore, mutations in other CRC-related genes could be simultaneously detected in a single test by PGM. CONCLUSIONS:The targeted NGS platform is specific and sensitive for KRAS exon 2 mutation detection and is appropriate for use in routine clinical testing. Moreover, it is sample saving and cost-efficient and time-efficient, and has great potential for clinical application to expand testing to include mutations in RAS and other CRC-related genes.

Dataset Information

Systematic Evaluation of Sanger Validation of Next-Generation Sequencing Variants.

Publications

Systematic Evaluation of Sanger Validation of Next-Generation Sequencing Variants.

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