BAMClipper: removing primers from alignments to minimize false-negative mutations in amplicon next-generation sequencing

Abstract Amplicon-based next-generation sequencing (NGS) has been widely adopted for genetic variation detection in human and other organisms. Conventional data analysis paradigm includes primer trimming before read mapping. Here we introduce BAMClipper that removes primer sequences after mapping or...

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Autores principales:	Chun Hang Au, Dona N. Ho, Ava Kwong, Tsun Leung Chan, Edmond S. K. Ma
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/6e4c0ae171ba4d6baeb272e2a5123b23
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spelling	oai:doaj.org-article:6e4c0ae171ba4d6baeb272e2a5123b232021-12-02T11:41:00ZBAMClipper: removing primers from alignments to minimize false-negative mutations in amplicon next-generation sequencing10.1038/s41598-017-01703-62045-2322https://doaj.org/article/6e4c0ae171ba4d6baeb272e2a5123b232017-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-01703-6https://doaj.org/toc/2045-2322Abstract Amplicon-based next-generation sequencing (NGS) has been widely adopted for genetic variation detection in human and other organisms. Conventional data analysis paradigm includes primer trimming before read mapping. Here we introduce BAMClipper that removes primer sequences after mapping original sequencing reads by soft-clipping SAM/BAM alignments. Mutation detection accuracy was affected by the choice of primer handling approach based on real NGS datasets of 7 human peripheral blood or breast cancer tissue samples with known BRCA1/BRCA2 mutations and >130000 simulated NGS datasets with unique mutations. BAMClipper approach detected a BRCA1 deletion (c.1620_1636del) that was otherwise missed due to edge effect. Simulation showed high false-negative rate when primers were perfectly trimmed as in conventional practice. Among the other 6 samples, variant allele frequencies of 5 BRCA1/BRCA2 mutations (indel or single-nucleotide variants) were diluted by apparently wild-type primer sequences from an overlapping amplicon (17 to 82% under-estimation). BAMClipper was robust in both situations and all 7 mutations were detected. When compared with Cutadapt, BAMClipper was faster and maintained equally high primer removal effectiveness. BAMClipper is implemented in Perl and is available under an open source MIT license at https://github.com/tommyau/bamclipper.Chun Hang AuDona N. HoAva KwongTsun Leung ChanEdmond S. K. MaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-7 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Chun Hang Au Dona N. Ho Ava Kwong Tsun Leung Chan Edmond S. K. Ma BAMClipper: removing primers from alignments to minimize false-negative mutations in amplicon next-generation sequencing
description	Abstract Amplicon-based next-generation sequencing (NGS) has been widely adopted for genetic variation detection in human and other organisms. Conventional data analysis paradigm includes primer trimming before read mapping. Here we introduce BAMClipper that removes primer sequences after mapping original sequencing reads by soft-clipping SAM/BAM alignments. Mutation detection accuracy was affected by the choice of primer handling approach based on real NGS datasets of 7 human peripheral blood or breast cancer tissue samples with known BRCA1/BRCA2 mutations and >130000 simulated NGS datasets with unique mutations. BAMClipper approach detected a BRCA1 deletion (c.1620_1636del) that was otherwise missed due to edge effect. Simulation showed high false-negative rate when primers were perfectly trimmed as in conventional practice. Among the other 6 samples, variant allele frequencies of 5 BRCA1/BRCA2 mutations (indel or single-nucleotide variants) were diluted by apparently wild-type primer sequences from an overlapping amplicon (17 to 82% under-estimation). BAMClipper was robust in both situations and all 7 mutations were detected. When compared with Cutadapt, BAMClipper was faster and maintained equally high primer removal effectiveness. BAMClipper is implemented in Perl and is available under an open source MIT license at https://github.com/tommyau/bamclipper.
format	article
author	Chun Hang Au Dona N. Ho Ava Kwong Tsun Leung Chan Edmond S. K. Ma
author_facet	Chun Hang Au Dona N. Ho Ava Kwong Tsun Leung Chan Edmond S. K. Ma
author_sort	Chun Hang Au
title	BAMClipper: removing primers from alignments to minimize false-negative mutations in amplicon next-generation sequencing
title_short	BAMClipper: removing primers from alignments to minimize false-negative mutations in amplicon next-generation sequencing
title_full	BAMClipper: removing primers from alignments to minimize false-negative mutations in amplicon next-generation sequencing
title_fullStr	BAMClipper: removing primers from alignments to minimize false-negative mutations in amplicon next-generation sequencing
title_full_unstemmed	BAMClipper: removing primers from alignments to minimize false-negative mutations in amplicon next-generation sequencing
title_sort	bamclipper: removing primers from alignments to minimize false-negative mutations in amplicon next-generation sequencing
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/6e4c0ae171ba4d6baeb272e2a5123b23
work_keys_str_mv	AT chunhangau bamclipperremovingprimersfromalignmentstominimizefalsenegativemutationsinampliconnextgenerationsequencing AT donanho bamclipperremovingprimersfromalignmentstominimizefalsenegativemutationsinampliconnextgenerationsequencing AT avakwong bamclipperremovingprimersfromalignmentstominimizefalsenegativemutationsinampliconnextgenerationsequencing AT tsunleungchan bamclipperremovingprimersfromalignmentstominimizefalsenegativemutationsinampliconnextgenerationsequencing AT edmondskma bamclipperremovingprimersfromalignmentstominimizefalsenegativemutationsinampliconnextgenerationsequencing
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BAMClipper: removing primers from alignments to minimize false-negative mutations in amplicon next-generation sequencing

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