ArticleScientific reports2021
Confirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencing.
Article in Scientific reports, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 34 papers.
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34 citing papers in PubMed, 46 citations in OpenAlex.
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- Low Allele Frequency Variants Identified on Germline Multi-Gene Panel Testing for Cancer Predisposition Can Suggest the Presence of Constitutional Mosaicism.Clinical cancer research : an official journal of the American Association for Cancer Research · 2025Article
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- Genomic Insights into Idiopathic Granulomatous Mastitis through Whole-Exome Sequencing: A Case Report of Eight Patients.International journal of molecular sciences · 2024Article
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Authors and funding
7 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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.
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