Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching

Abstract High-throughput single-stranded DNA sequencing (ssDNA-seq) of cell-free DNA from plasma and other bodily fluids is a powerful method for non-invasive prenatal testing, and diagnosis of cancers and other diseases. Here, we developed a facile ssDNA-seq method, which exploits a novel template-...

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Autores principales:	Douglas C. Wu, Alan M. Lambowitz
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f6d262c943124d31bc0c968b144146f6
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spelling	oai:doaj.org-article:f6d262c943124d31bc0c968b144146f62021-12-02T11:53:08ZFacile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching10.1038/s41598-017-09064-w2045-2322https://doaj.org/article/f6d262c943124d31bc0c968b144146f62017-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-09064-whttps://doaj.org/toc/2045-2322Abstract High-throughput single-stranded DNA sequencing (ssDNA-seq) of cell-free DNA from plasma and other bodily fluids is a powerful method for non-invasive prenatal testing, and diagnosis of cancers and other diseases. Here, we developed a facile ssDNA-seq method, which exploits a novel template-switching activity of thermostable group II intron reverse transcriptases (TGIRTs) for DNA-seq library construction. This activity enables TGIRT enzymes to initiate DNA synthesis directly at the 3′ end of a DNA strand while simultaneously attaching a DNA-seq adapter without end repair, tailing, or ligation. Initial experiments using this method to sequence E. coli genomic DNA showed that the TGIRT enzyme has surprisingly robust DNA polymerase activity. Further experiments showed that TGIRT-seq of plasma DNA from a healthy individual enables analysis of nucleosome positioning, transcription factor-binding sites, DNA methylation sites, and tissues-of-origin comparably to established methods, but with a simpler workflow that captures precise DNA ends.Douglas C. WuAlan M. LambowitzNature 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 Douglas C. Wu Alan M. Lambowitz Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching
description	Abstract High-throughput single-stranded DNA sequencing (ssDNA-seq) of cell-free DNA from plasma and other bodily fluids is a powerful method for non-invasive prenatal testing, and diagnosis of cancers and other diseases. Here, we developed a facile ssDNA-seq method, which exploits a novel template-switching activity of thermostable group II intron reverse transcriptases (TGIRTs) for DNA-seq library construction. This activity enables TGIRT enzymes to initiate DNA synthesis directly at the 3′ end of a DNA strand while simultaneously attaching a DNA-seq adapter without end repair, tailing, or ligation. Initial experiments using this method to sequence E. coli genomic DNA showed that the TGIRT enzyme has surprisingly robust DNA polymerase activity. Further experiments showed that TGIRT-seq of plasma DNA from a healthy individual enables analysis of nucleosome positioning, transcription factor-binding sites, DNA methylation sites, and tissues-of-origin comparably to established methods, but with a simpler workflow that captures precise DNA ends.
format	article
author	Douglas C. Wu Alan M. Lambowitz
author_facet	Douglas C. Wu Alan M. Lambowitz
author_sort	Douglas C. Wu
title	Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching
title_short	Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching
title_full	Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching
title_fullStr	Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching
title_full_unstemmed	Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching
title_sort	facile single-stranded dna sequencing of human plasma dna via thermostable group ii intron reverse transcriptase template switching
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/f6d262c943124d31bc0c968b144146f6
work_keys_str_mv	AT douglascwu facilesinglestrandeddnasequencingofhumanplasmadnaviathermostablegroupiiintronreversetranscriptasetemplateswitching AT alanmlambowitz facilesinglestrandeddnasequencingofhumanplasmadnaviathermostablegroupiiintronreversetranscriptasetemplateswitching
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Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching

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