The results were compared with droplet digital PCR (ddPCR), and the obtained VAFs were similar. BDA was validated for use in WES confirmation of rare variants, providing an LoD of 0.1% VAF to determine if mutations found in child proband by WES were de novo or parental somatic mosaicism mutations 18. BDA technology is a novel PCR-based enrichment method for preferential amplification of low-level variants over the wildtype sequence 17. However, because Sanger sequencing has an LoD of 5–20% VAF, it cannot be used directly to confirm NGS variant findings with VAF < 5%.Īnother method used for low-level variant detection is Blocker Displacement Amplification technology (BDA). reported an extensive Sanger confirmation of 7845 variants called in a 14-gene NGS panel nearly all disconfirmed variants were concentrated at VAF < 20% 15. Sanger sequencing is considered the gold standard for pathology laboratories due to its robustness and well-established nature 15, 16. Sanger sequencing, also known as the “chain termination method”, is a fast, cost-effective sequencing method that has been used in this field for more than 40 years 14. Therefore, there is a strong need for orthogonal variant confirmation. reported that the proportion of false-positive variants was as high as 78% for single-nucleotide variants (SNVs) and 44% for indels in WES assessed by Sanger sequencing 13. This is due to limitation of sequencing depth, NGS intrinsic error, gene coverage nonuniformity, and errors from variant calling software 4, 5. But at a price of $50 k per sample 7, 11, 12, it is impractical for many laboratories to use ultra-deep NGS assays.Īdditionally, WES is susceptible to high false-positive rates caused by variant calling errors. To reliably make mutation calls at 0.1% VAF, WES at 35,000 × utilizing Unique Molecular Identifiers is needed (UMIs e.g., 70-gene Guardant 360 panel 8, 9 and 500-gene Illumina TruSight panel 10). For these applications, high clinical sensitivity can only be achieved when the NGS panel's mutation sensitivity reaches 0.1% to 0.5% VAF. However, higher mutation sensitivity is required to identify subclonal drug resistance mutations from tumor tissue or perform non-invasive tumor profiling with cell-free DNA from peripheral blood samples (“liquid biopsy”) 6, 7. For many tumor sequencing applications, 5% VAF mutation sensitivity is sufficient. In most laboratories, WES is performed at 100 × for somatic mutation discovery, which costs ~ $500 with an LoD of 5–10% VAF (Fig. WES has also been used to characterize mutational signatures across tumor types 3.įor NGS panels like WES, intrinsic sequencing error rate and sequencing depth are bottlenecks for detection of low-frequency variants. The exon region is roughly 1% of the human genome and responsible for tumor-causing mutations that lead to altered protein functions 2. One use of NGS is whole exome sequencing (WES), in which the entire exome is enriched and sequenced to detect disease-causing variants 1. Next-generation sequencing (NGS) quickly emerges as a primary clinical diagnostic platform. By implementing this method, next-generation sequencing can reliably report low-level variants at a high sensitivity, without the cost of high sequencing depth. This data demonstrates Blocker Displacement Amplification allelic enrichment coupled with Sanger sequencing can be used to confirm putative mutations ≤ 5% VAF. In the 66 cancer-related variants, the disconfirmed rate was 82% (54/66). Results showed that 52% of the 226 (117) putative variants were disconfirmed, among which 2% (5) putative variants were found to be misidentified in WES. NGSure utilizes Blocker Displacement Amplification to first enrich the allelic fraction of the mutation and then uses Sanger sequencing to determine mutation identity. Each variant was then tested using NuProbe NGSure, to confirm the original WES calls. Here we performed ~ 1000 × WES on fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue biopsy samples from a non-small cell lung cancer patient, and identified 226 putative mutations at between 0.5 and 5% VAF. Putative mutations called at ≤ 5% VAF are frequently due to sequencing errors, therefore reporting these subclonal mutations incurs risk of significant false positives. WES has a mutation limit of detection (LoD) at variant allele frequencies (VAF) of 5%. Whole exome sequencing (WES) is used to identify mutations in a patient’s tumor DNA that are predictive of tumor behavior, including the likelihood of response or resistance to cancer therapy.
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