Journal Article

Systematic Evaluation of Sanger Validation of Next-Generation Sequencing Variants

Biesecker, Leslie G, Mullikin, James C, Beck, Tyler F

2016

Overview

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. 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. 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. 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.

• Publisher: Oxford University Press
• Subject: Animals / Deoxyribonucleic acid / Design / DNA / DNA - blood / DNA - genetics / Funding / Genes / Genetic testing / Genetic Variation - genetics / Genomes / Genomics / Genomics - methods / Genotype & phenotype / High-Throughput Nucleotide Sequencing - methods / High-Throughput Nucleotide Sequencing - standards / High-Throughput Nucleotide Sequencing - statistics & numerical data / Humans / Models, Statistical / Mutation / Sequence Analysis, DNA - methods / Sequence Analysis, DNA - standards / Sequence Analysis, DNA - statistics & numerical data / Studies