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.

Molecular epidemiology of Staphylococcus aureus bacteremia in a single large Minnesota medical center in 2015 as assessed using MLST, core genome MLST and spa typing

“…Bletz et al suggested that extraction of spa types from WGS with an optimized de novo assembly may enable nearly full compatibility with PCR/Sanger sequencing-based spa typing for MRSA [28]. However, assembly of repetitive areas based on short Illumina NGS read data may be prone to misassembling, resulting in failure or errors [29], as illustrated herein. Although it is not possible to compare data directly since different DNA extracts were used for PCR/Sanger sequencing- and WGS- spa typing herein, spa typing discrepancies between WGS data and traditional spa typing caution against WGS usage for spa typing.…”

Consensus Statement on next-generation-sequencing-based diagnostic testing of hereditary phaeochromocytomas and paragangliomas

“…Special attention should be given to limitations of NGS methods for sequencing and detection of variants in specific regions of the genome, including homopolymer repeats, indels, AT-rich regions and GC-rich regions 36 . Some NGS techniques, such as Ion Torrent (Life Technologies/ThermoFisher, Waltham, Massachusetts, USA), rely on single-nucleotide additions and can have a high error rate for indel detection (1%) 37 .…”

A comprehensive assessment of Next‐Generation Sequencing variants validation using a secondary technology

“…Depth coverage and heterozygous ratio are the essential quality control parameters for NGS variants. Many important studies used these quality parameters because of the flexibility (Baudhuin et al, ; Mu et al, ). In our present study, 6,939 variants with at least 35 × depth coverage and more than 35% heterozygous ratio were confirmed by a secondary methodology, which included 5,775 heterozygous variants, 760 homozygous variants, and 404 hemizygous variants.…”

“…Several commercial and academic laboratories have reported with small amounts of validated samples that clinical NGS variants meeting threshold quality metrics were unnecessarily validated (Baudhuin et al, 2015;Judkins et al, 2015;Sikkema-Raddatz et al, 2013;Strom et al, 2014). Recently, another two studies have been done with thousands of variants (5,800 and 7,845, respectively), by using a unique NGS sequencing platform with small gene sets (Beck, Mullikin, Program, & Biesecker, 2016;Mu, Lu, Chen, Li, & Elliott, 2016). The prior studies were not sufficient to conclude that variants validation will be insignificant or less significant for a large scale NGS application in clinical diagnosis.…”