Targeted sequencing of both DNA strands barcoded and captured individually by RNA probes to identify genome-wide ultra-rare mutations

Abstract Next Generation Sequencing (NGS) has been widely implemented in biological research and has made a profound impact on patient care. One of the essential NGS applications is to identify disease-causing sequence variants, where high coverage and accuracy are needed. Here, we reported a novel...

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Autores principales:	Qing Wang, Xu Wang, Pheobe S. Tang, Grace M. O’leary, Ming Zhang
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/aca9a23e34c041d3a63565c43d988b71
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spelling	oai:doaj.org-article:aca9a23e34c041d3a63565c43d988b712021-12-02T16:07:56ZTargeted sequencing of both DNA strands barcoded and captured individually by RNA probes to identify genome-wide ultra-rare mutations10.1038/s41598-017-03448-82045-2322https://doaj.org/article/aca9a23e34c041d3a63565c43d988b712017-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-03448-8https://doaj.org/toc/2045-2322Abstract Next Generation Sequencing (NGS) has been widely implemented in biological research and has made a profound impact on patient care. One of the essential NGS applications is to identify disease-causing sequence variants, where high coverage and accuracy are needed. Here, we reported a novel NGS pipeline, termed a Sequencing System of Digitalized Barcode Encrypted Single-stranded Library from Extremely Low (quality and quantity) DNA Input with Probe-based DNA Enrichment by RNA probes targeting DNA duplex (DEEPER-Seq). This method combines an ultra-sensitive single-stranded library construction with barcoding error correction, termed DEEPER-Library; and a DNA capture approach using RNA probes targeting both DNA strands, termed DEEPER-Capture. DEEPER-Seq can create NGS libraries from as little as 20 pg DNA with PCR error correcting capabilities, and capture target sequences at an average ratio of 29.2% by targeting both DNA strands simultaneously with an over 98.6% coverage. Our method tags and sequences each of the two strands of a DNA duplex independently and only scores mutations that are found at the same position in both strands, which allows us to identify mutations with allelic fractions down to 0.03% in a whole exome sequencing (WES) study with a background error rate of one artificial error per 4.8 × 109 nucleotides.Qing WangXu WangPheobe S. TangGrace M. O’learyMing ZhangNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-14 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Qing Wang Xu Wang Pheobe S. Tang Grace M. O’leary Ming Zhang Targeted sequencing of both DNA strands barcoded and captured individually by RNA probes to identify genome-wide ultra-rare mutations
description	Abstract Next Generation Sequencing (NGS) has been widely implemented in biological research and has made a profound impact on patient care. One of the essential NGS applications is to identify disease-causing sequence variants, where high coverage and accuracy are needed. Here, we reported a novel NGS pipeline, termed a Sequencing System of Digitalized Barcode Encrypted Single-stranded Library from Extremely Low (quality and quantity) DNA Input with Probe-based DNA Enrichment by RNA probes targeting DNA duplex (DEEPER-Seq). This method combines an ultra-sensitive single-stranded library construction with barcoding error correction, termed DEEPER-Library; and a DNA capture approach using RNA probes targeting both DNA strands, termed DEEPER-Capture. DEEPER-Seq can create NGS libraries from as little as 20 pg DNA with PCR error correcting capabilities, and capture target sequences at an average ratio of 29.2% by targeting both DNA strands simultaneously with an over 98.6% coverage. Our method tags and sequences each of the two strands of a DNA duplex independently and only scores mutations that are found at the same position in both strands, which allows us to identify mutations with allelic fractions down to 0.03% in a whole exome sequencing (WES) study with a background error rate of one artificial error per 4.8 × 109 nucleotides.
format	article
author	Qing Wang Xu Wang Pheobe S. Tang Grace M. O’leary Ming Zhang
author_facet	Qing Wang Xu Wang Pheobe S. Tang Grace M. O’leary Ming Zhang
author_sort	Qing Wang
title	Targeted sequencing of both DNA strands barcoded and captured individually by RNA probes to identify genome-wide ultra-rare mutations
title_short	Targeted sequencing of both DNA strands barcoded and captured individually by RNA probes to identify genome-wide ultra-rare mutations
title_full	Targeted sequencing of both DNA strands barcoded and captured individually by RNA probes to identify genome-wide ultra-rare mutations
title_fullStr	Targeted sequencing of both DNA strands barcoded and captured individually by RNA probes to identify genome-wide ultra-rare mutations
title_full_unstemmed	Targeted sequencing of both DNA strands barcoded and captured individually by RNA probes to identify genome-wide ultra-rare mutations
title_sort	targeted sequencing of both dna strands barcoded and captured individually by rna probes to identify genome-wide ultra-rare mutations
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/aca9a23e34c041d3a63565c43d988b71
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Targeted sequencing of both DNA strands barcoded and captured individually by RNA probes to identify genome-wide ultra-rare mutations

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