title: 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.
authors: National Human Genome Research Institute, NIH, Bethesda, MD;
authors: Tyler F
authors: TF
authors: Beck
authors: National Human Genome Research Institute, NIH, Bethesda, MD; NIH Intramural Sequencing Center, Rockville, MD.
authors: James C
authors: JC
authors: Mullikin
authors: National Human Genome Research Institute, NIH, Bethesda, MD; lesb@mail.nih.gov.
authors: Leslie G
authors: LG
authors: Biesecker
mesh_terms: D000818:Animals; D004247:DNA / Q000097:blood / Q000235:genetics; D014644:Genetic Variation / Q000235:genetics*; D023281:Genomics / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D006801:Humans; D015233:Models, Statistical; D017422:Sequence Analysis, DNA / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data
chemical_list: D004247:DNA