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.

Clinical Validation of a Next Generation Sequencing Panel Test for Hereditary Colorectal Cancer

“…However, digging beyond the surface, the need to confirm all results seems to be an overly-Draconian approach to the real, yet limited risk of a false positive result. A recent publication suggests that within controlled parameters, specific alterations like SNVs might not need confirmation [15,16]. We agree and feel that our zero false positive rate in our validation data suggest this is a more realistic and evidence based position to take.…”

BRCA1 and BRCA2 sequence variations detected with next-generation sequencing in patients with premature ovarian insufficiency

“…To facilitate the implementation of NGS into routine laboratory practice several studies done such as the Dutch Society for Clinical Genetic Laboratory Diagnostics (VKGL) working group. And also, in a recent paper researchers have been emphasized that the necessity of Sanger confirmation of next-generation sequencing variants lower than 30x depth of coverage might need to be explored (25). As a conclusion, we did not detect an association between POI and the BRCA1 and BRCA2 gene variations.…”

Review of Clinical Next-Generation Sequencing

“…61 Supplementary testing may include genomic areas that are not reliably sequenced and confirmatory testing for certain variants that do not meet certain quality requirements. 4,85 There should be documentation of areas that cannot be reliably sequenced and a policy for addressing these areas (either as supplemental testing or a disclaimer in the report). 84 Like any test, NGS is subject to false positives, and the validation process should identify the metrics that require a confirmatory test to verify the presence of a variant identified by NGS.…”

“…84 Like any test, NGS is subject to false positives, and the validation process should identify the metrics that require a confirmatory test to verify the presence of a variant identified by NGS. 4,61,85 After the initial validation, any procedural changes require revalidation. Careful thought should be given to the initial design of an assay, as redesign requires revalidation.…”