title: Sanger Confirmation Is Required to Achieve Optimal Sensitivity and Specificity in Next-Generation Sequencing Panel Testing.
abstract: Next-generation sequencing (NGS) has rapidly replaced Sanger sequencing as the method of choice for diagnostic gene-panel testing. For hereditary-cancer testing, the technical sensitivity and specificity of the assay are paramount as clinicians use results to make important clinical management and treatment decisions. There is significant debate within the diagnostics community regarding the necessity of confirming NGS variant calls by Sanger sequencing, considering that numerous laboratories report having 100% specificity from the NGS data alone. Here we report our results from 20,000 hereditary-cancer NGS panels spanning 47 genes, in which all 7845 nonpolymorphic variants were Sanger- sequenced. Of these, 98.7% were concordant between NGS and Sanger sequencing and 1.3% were identified as NGS false-positives, located mainly in complex genomic regions (A/T-rich regions, G/C-rich regions, homopolymer stretches, and pseudogene regions). Simulating a false-positive rate of zero by adjusting the variant-calling quality-score thresholds decreased the sensitivity of the assay from 100% to 97.8%, resulting in the missed detection of 176 Sanger-confirmed variants, the majority in complex genomic regions (n = 114) and mosaic mutations (n = 7). The data illustrate the importance of setting quality thresholds for panel testing only after thousands of samples have been processed and the necessity of Sanger confirmation of NGS variants to maintain the highest possible sensitivity.
authors.affiliation: Ambry Genetics, Aliso Viejo, California.
authors.forename: Wenbo
authors.initials: W
authors.lastname: Mu
authors.affiliation: Ambry Genetics, Aliso Viejo, California.
authors.forename: Hsiao-Mei
authors.initials: HM
authors.lastname: Lu
authors.affiliation: Ambry Genetics, Aliso Viejo, California.
authors.forename: Jefferey
authors.initials: J
authors.lastname: Chen
authors.affiliation: Ambry Genetics, Aliso Viejo, California.
authors.forename: Shuwei
authors.initials: S
authors.lastname: Li
authors.affiliation: Ambry Genetics, Aliso Viejo, California. Electronic address: aelliott@ambrygen.com.
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Elliott
mesh_terms: D000483:Alleles; D014408:Biomarkers, Tumor; D005787:Gene Frequency; D005820:Genetic Testing / Q000379:methods / Q000592:standards; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods / Q000592:standards; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000175:diagnosis / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D014408:Biomarkers, Tumor