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

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dc.citation.articleNumber11640en_US
dc.citation.journalTitleScientific Reportsen_US
dc.citation.volumeNumber11en_US
dc.contributor.authorYan, Yan Helenen_US
dc.contributor.authorChen, Sherry X.en_US
dc.contributor.authorCheng, Lauren Y.en_US
dc.contributor.authorRodriguez, Alyssa Y.en_US
dc.contributor.authorTang, Ruien_US
dc.contributor.authorCabrera, Karinaen_US
dc.contributor.authorZhang, David Yuen_US
dc.contributor.orgSystems, Synthetic, and Physical Biologyen_US
dc.date.accessioned2021-06-17T19:01:35Zen_US
dc.date.available2021-06-17T19:01:35Zen_US
dc.date.issued2021en_US
dc.description.abstractWhole 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.en_US
dc.identifier.citationYan, Yan Helen, Chen, Sherry X., Cheng, Lauren Y., et al.. "Confirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencing." <i>Scientific Reports,</i> 11, (2021) Springer Nature: https://doi.org/10.1038/s41598-021-91142-1.en_US
dc.identifier.digitals41598-021-91142-1en_US
dc.identifier.doihttps://doi.org/10.1038/s41598-021-91142-1en_US
dc.identifier.urihttps://hdl.handle.net/1911/110747en_US
dc.language.isoengen_US
dc.publisherSpringer Natureen_US
dc.rightsThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.en_US
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/en_US
dc.titleConfirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencingen_US
dc.typeJournal articleen_US
dc.type.dcmiTexten_US
dc.type.publicationpublisher versionen_US
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