Systematic Evaluation of Sanger Validation of Next-Generation Sequencing Variants

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 62; no. 4; pp. 647 - 654
• Main Authors: Beck, Tyler F, Mullikin, James C, Biesecker, Leslie G
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.04.2016
• Subjects: 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
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2015.249623

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.