Confirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencing

Abstract

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. WES has a mutation limit of detection (LoD) at variant allele frequencies (VAF) of 5%. Putative mutations called at ≤ 5% VAF are frequently due to sequencing errors, therefore reporting these subclonal mutations incurs risk of significant false positives. 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. Each variant was then tested using NuProbe NGSure, to confirm the original WES calls. NGSure utilizes Blocker Displacement Amplification to first enrich the allelic fraction of the mutation and then uses Sanger sequencing to determine mutation identity. 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. In the 66 cancer-related variants, the disconfirmed rate was 82% (54/66). This data demonstrates Blocker Displacement Amplification allelic enrichment coupled with Sanger sequencing can be used to confirm putative mutations ≤ 5% VAF. By implementing this method, next-generation sequencing can reliably report low-level variants at a high sensitivity, without the cost of high sequencing depth.

KRAS mutation allele frequency threshold alters prognosis in right‐sided resected pancreatic cancer

“…Patients were categorized as high mKRAS or low mKRAS if they had KRAS mutant allele frequencies greater than or less than 20%, respectively. Traditionally, allele frequency cut‐offs are set at 5% 39–41 . Previous studies looking at KRAS‐mutant tumor heterogeneity found that average KRAS mutation frequencies were between 10% and 20% 42 .…”

“…Traditionally, allele frequency cut-offs are set at 5%. [39][40][41] Previous studies looking at KRAS-mutant tumor heterogeneity found that average KRAS mutation frequencies were between 10% and 20%. 42 Twenty percent (20%) was chosen as our allele frequency cutoff as the distribution of the KRAS allele frequencies in our sample were more dispersed near this cut-off of 10% and 20%.…”

RNA-SSNV: A Reliable Somatic Single Nucleotide Variant Identification Framework for Bulk RNA-Seq Data

“…Interestingly, RNA VAF was significantly higher than DNA VAF within expressed mutations of RNA–DNA overlap part ( Figure 5C right comparison, p < 1 e-5), suggesting an expression tendency for the mutant allele. The common cancer WES study has a mutation limit of detection (LoD) at 5% VAF, and reporting these subclonal mutations incurs the risk of sequencing error–induced false positives ( Yan et al, 2021 ). For these low-VAF (<0.05) DNA somatic mutations, their RNA VAFs were much higher, with median values of 0.374 in LUSC, 0.342 in BLCA, and 0.241 in GBM.…”

PEAC: An Ultrasensitive and Cost-Effective MRD Detection System in Non-small Cell Lung Cancer Using Plasma Specimen

“…The competitive hybridization reaction enables PCR not only to sensitively recognize and selectively amplify SNVs, but also to do so across a broad temperature window spanning 8 • C, thus conveniently accomplishing multiplexing purposes. Several BDA-derived methods showed high sensitivity to detect low-abundant ctDNA mutations with cost-effective experimental procedures (49)(50)(51)(52). Therefore, we believe that these kind of technologies are quite appropriate to implement a highly sensitive, multiplexingenabled, and cost-effective MRD detection system for NSCLC.…”