title: Confirming Variants in Next-Generation Sequencing Panel Testing by Sanger Sequencing.
abstract: Current clinical laboratory practice guidelines for next-generation sequencing (NGS) do not provide definitive guidance on confirming NGS variants. Sanger confirmation of NGS results can be inefficient, redundant, and expensive. We evaluated the accuracy of NGS-detected single-nucleotide variants (SNVs) and insertion/deletion variants (indels) and the necessity of NGS variant confirmation using four NGS target-capture gene panels covering 117 genes, 568 Kbp, and 77 patient DNA samples. Unique NGS-detected variants (1080 SNVs and 124 indels) underwent Sanger confirmation and/or were compared to data from the 1000 Genomes Project (1000G). Recurrent variants in unrelated samples resulted in 919 comparisons between NGS and Sanger, with 100% concordance. In a second comparison, 762 unique NGS results (736 SNVs, 26 indels) from seven 1000G samples were found to have 97.1% concordance with 1000G phase 1 data. Sanger sequencing and 1000G phase 3 data confirmed the accuracy of the NGS results for all 1000G phase 1 discrepancies. In all samples, the depth of coverage exceeded 100× in >99.7% of bases in the target regions. In conclusion, confirmatory analysis by Sanger sequencing of SNVs detected via capture-based NGS testing that meets appropriate quality thresholds is unnecessarily redundant. In contrast, Sanger sequencing for indels may be required for defining the correct genomic location, and Sanger may be used for quality-assurance purposes.
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota. Electronic address: baudhuin.linnea@mayo.edu.
authors.forename: Linnea M
authors.initials: LM
authors.lastname: Baudhuin
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Susan A
authors.initials: SA
authors.lastname: Lagerstedt
authors.affiliation: Division of Biomedical Statistics and Informatics, Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota.
authors.forename: Eric W
authors.initials: EW
authors.lastname: Klee
authors.affiliation: Division of Biomedical Statistics and Informatics, Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota.
authors.forename: Numrah
authors.initials: N
authors.lastname: Fadra
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Devin
authors.initials: D
authors.lastname: Oglesbee
authors.affiliation: Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota. Electronic address: ferber.matthew@mayo.edu.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Ferber
mesh_terms: D014644:Genetic Variation / Q000235:genetics*; D023281:Genomics / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000592:standards*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*