A modified RNA-Seq approach for whole genome sequencing of RNA viruses from faecal and blood samples.

To date, very large scale sequencing of many clinically important RNA viruses has been complicated by their high population molecular variation, which creates challenges for polymerase chain reaction and sequencing primer design. Many RNA viruses are also difficult or currently not possible to cultu...

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Autores principales: Elizabeth M Batty, T H Nicholas Wong, Amy Trebes, Karène Argoud, Moustafa Attar, David Buck, Camilla L C Ip, Tanya Golubchik, Madeleine Cule, Rory Bowden, Charis Manganis, Paul Klenerman, Eleanor Barnes, A Sarah Walker, David H Wyllie, Daniel J Wilson, Kate E Dingle, Tim E A Peto, Derrick W Crook, Paolo Piazza
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Publicado: Public Library of Science (PLoS) 2013
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Acceso en línea:https://doaj.org/article/a2407922c58b47e5ad9a468cfd3fdb1b
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spelling oai:doaj.org-article:a2407922c58b47e5ad9a468cfd3fdb1b2021-11-18T07:42:26ZA modified RNA-Seq approach for whole genome sequencing of RNA viruses from faecal and blood samples.1932-620310.1371/journal.pone.0066129https://doaj.org/article/a2407922c58b47e5ad9a468cfd3fdb1b2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23762474/?tool=EBIhttps://doaj.org/toc/1932-6203To date, very large scale sequencing of many clinically important RNA viruses has been complicated by their high population molecular variation, which creates challenges for polymerase chain reaction and sequencing primer design. Many RNA viruses are also difficult or currently not possible to culture, severely limiting the amount and purity of available starting material. Here, we describe a simple, novel, high-throughput approach to Norovirus and Hepatitis C virus whole genome sequence determination based on RNA shotgun sequencing (also known as RNA-Seq). We demonstrate the effectiveness of this method by sequencing three Norovirus samples from faeces and two Hepatitis C virus samples from blood, on an Illumina MiSeq benchtop sequencer. More than 97% of reference genomes were recovered. Compared with Sanger sequencing, our method had no nucleotide differences in 14,019 nucleotides (nt) for Noroviruses (from a total of 2 Norovirus genomes obtained with Sanger sequencing), and 8 variants in 9,542 nt for Hepatitis C virus (1 variant per 1,193 nt). The three Norovirus samples had 2, 3, and 2 distinct positions called as heterozygous, while the two Hepatitis C virus samples had 117 and 131 positions called as heterozygous. To confirm that our sample and library preparation could be scaled to true high-throughput, we prepared and sequenced an additional 77 Norovirus samples in a single batch on an Illumina HiSeq 2000 sequencer, recovering >90% of the reference genome in all but one sample. No discrepancies were observed across 118,757 nt compared between Sanger and our custom RNA-Seq method in 16 samples. By generating viral genomic sequences that are not biased by primer-specific amplification or enrichment, this method offers the prospect of large-scale, affordable studies of RNA viruses which could be adapted to routine diagnostic laboratory workflows in the near future, with the potential to directly characterize within-host viral diversity.Elizabeth M BattyT H Nicholas WongAmy TrebesKarène ArgoudMoustafa AttarDavid BuckCamilla L C IpTanya GolubchikMadeleine CuleRory BowdenCharis ManganisPaul KlenermanEleanor BarnesA Sarah WalkerDavid H WyllieDaniel J WilsonKate E DingleTim E A PetoDerrick W CrookPaolo PiazzaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 6, p e66129 (2013)
institution DOAJ
collection DOAJ
language EN
topic Medicine
R
Science
Q
spellingShingle Medicine
R
Science
Q
Elizabeth M Batty
T H Nicholas Wong
Amy Trebes
Karène Argoud
Moustafa Attar
David Buck
Camilla L C Ip
Tanya Golubchik
Madeleine Cule
Rory Bowden
Charis Manganis
Paul Klenerman
Eleanor Barnes
A Sarah Walker
David H Wyllie
Daniel J Wilson
Kate E Dingle
Tim E A Peto
Derrick W Crook
Paolo Piazza
A modified RNA-Seq approach for whole genome sequencing of RNA viruses from faecal and blood samples.
description To date, very large scale sequencing of many clinically important RNA viruses has been complicated by their high population molecular variation, which creates challenges for polymerase chain reaction and sequencing primer design. Many RNA viruses are also difficult or currently not possible to culture, severely limiting the amount and purity of available starting material. Here, we describe a simple, novel, high-throughput approach to Norovirus and Hepatitis C virus whole genome sequence determination based on RNA shotgun sequencing (also known as RNA-Seq). We demonstrate the effectiveness of this method by sequencing three Norovirus samples from faeces and two Hepatitis C virus samples from blood, on an Illumina MiSeq benchtop sequencer. More than 97% of reference genomes were recovered. Compared with Sanger sequencing, our method had no nucleotide differences in 14,019 nucleotides (nt) for Noroviruses (from a total of 2 Norovirus genomes obtained with Sanger sequencing), and 8 variants in 9,542 nt for Hepatitis C virus (1 variant per 1,193 nt). The three Norovirus samples had 2, 3, and 2 distinct positions called as heterozygous, while the two Hepatitis C virus samples had 117 and 131 positions called as heterozygous. To confirm that our sample and library preparation could be scaled to true high-throughput, we prepared and sequenced an additional 77 Norovirus samples in a single batch on an Illumina HiSeq 2000 sequencer, recovering >90% of the reference genome in all but one sample. No discrepancies were observed across 118,757 nt compared between Sanger and our custom RNA-Seq method in 16 samples. By generating viral genomic sequences that are not biased by primer-specific amplification or enrichment, this method offers the prospect of large-scale, affordable studies of RNA viruses which could be adapted to routine diagnostic laboratory workflows in the near future, with the potential to directly characterize within-host viral diversity.
format article
author Elizabeth M Batty
T H Nicholas Wong
Amy Trebes
Karène Argoud
Moustafa Attar
David Buck
Camilla L C Ip
Tanya Golubchik
Madeleine Cule
Rory Bowden
Charis Manganis
Paul Klenerman
Eleanor Barnes
A Sarah Walker
David H Wyllie
Daniel J Wilson
Kate E Dingle
Tim E A Peto
Derrick W Crook
Paolo Piazza
author_facet Elizabeth M Batty
T H Nicholas Wong
Amy Trebes
Karène Argoud
Moustafa Attar
David Buck
Camilla L C Ip
Tanya Golubchik
Madeleine Cule
Rory Bowden
Charis Manganis
Paul Klenerman
Eleanor Barnes
A Sarah Walker
David H Wyllie
Daniel J Wilson
Kate E Dingle
Tim E A Peto
Derrick W Crook
Paolo Piazza
author_sort Elizabeth M Batty
title A modified RNA-Seq approach for whole genome sequencing of RNA viruses from faecal and blood samples.
title_short A modified RNA-Seq approach for whole genome sequencing of RNA viruses from faecal and blood samples.
title_full A modified RNA-Seq approach for whole genome sequencing of RNA viruses from faecal and blood samples.
title_fullStr A modified RNA-Seq approach for whole genome sequencing of RNA viruses from faecal and blood samples.
title_full_unstemmed A modified RNA-Seq approach for whole genome sequencing of RNA viruses from faecal and blood samples.
title_sort modified rna-seq approach for whole genome sequencing of rna viruses from faecal and blood samples.
publisher Public Library of Science (PLoS)
publishDate 2013
url https://doaj.org/article/a2407922c58b47e5ad9a468cfd3fdb1b
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