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 mu...

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Autores principales:	Yan Helen Yan, Sherry X. Chen, Lauren Y. Cheng, Alyssa Y. Rodriguez, Rui Tang, Karina Cabrera, David Yu Zhang
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/27644cc07d57469489f2b52234db2f81
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spelling	oai:doaj.org-article:27644cc07d57469489f2b52234db2f812021-12-02T17:51:21ZConfirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencing10.1038/s41598-021-91142-12045-2322https://doaj.org/article/27644cc07d57469489f2b52234db2f812021-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-91142-1https://doaj.org/toc/2045-2322Abstract 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.Yan Helen YanSherry X. ChenLauren Y. ChengAlyssa Y. RodriguezRui TangKarina CabreraDavid Yu ZhangNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-9 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Yan Helen Yan Sherry X. Chen Lauren Y. Cheng Alyssa Y. Rodriguez Rui Tang Karina Cabrera David Yu Zhang Confirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencing
description	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.
format	article
author	Yan Helen Yan Sherry X. Chen Lauren Y. Cheng Alyssa Y. Rodriguez Rui Tang Karina Cabrera David Yu Zhang
author_facet	Yan Helen Yan Sherry X. Chen Lauren Y. Cheng Alyssa Y. Rodriguez Rui Tang Karina Cabrera David Yu Zhang
author_sort	Yan Helen Yan
title	Confirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencing
title_short	Confirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencing
title_full	Confirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencing
title_fullStr	Confirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencing
title_full_unstemmed	Confirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencing
title_sort	confirming putative variants at ≤ 5% allele frequency using allele enrichment and sanger sequencing
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/27644cc07d57469489f2b52234db2f81
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Confirming putative variants at ≤ 5% allele frequency using allele enrichment and Sanger sequencing

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