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Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing

Enteroviruses are small RNA viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. In routine diagnostic laboratories, enteroviruses are identified by PCR-based methods, often combined with partial sequencing for genotyping. In t...

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Autores principales: Grädel, Carole, Terrazos Miani, Miguel A., Baumann, Christian, Barbani, Maria Teresa, Neuenschwander, Stefan, Leib, Stephen L., Suter-Riniker, Franziska, Ramette, Alban
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472277/
https://www.ncbi.nlm.nih.gov/pubmed/32752120
http://dx.doi.org/10.3390/v12080841
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author Grädel, Carole
Terrazos Miani, Miguel A.
Baumann, Christian
Barbani, Maria Teresa
Neuenschwander, Stefan
Leib, Stephen L.
Suter-Riniker, Franziska
Ramette, Alban
author_facet Grädel, Carole
Terrazos Miani, Miguel A.
Baumann, Christian
Barbani, Maria Teresa
Neuenschwander, Stefan
Leib, Stephen L.
Suter-Riniker, Franziska
Ramette, Alban
author_sort Grädel, Carole
collection PubMed
description Enteroviruses are small RNA viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. In routine diagnostic laboratories, enteroviruses are identified by PCR-based methods, often combined with partial sequencing for genotyping. In this proof-of-principle study, we assessed direct RNA sequencing (DRS) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. The approach was complemented by sequencing the corresponding viral cDNA via Illumina MiSeq sequencing. DRS of total RNA extracted from three different enterovirus-positive stool samples produced long RNA fragments, covering between 59% and 99.6% of the most similar reference genome sequences. The identification of the enterovirus sequences in the samples was confirmed by short-read cDNA sequencing. Sequence identity between DRS and Illumina MiSeq enterovirus consensus sequences ranged between 94% and 97%. Here, we show that nanopore DRS can be used to correctly identify enterovirus genotypes from patient stool samples with high viral load and that the approach also provides rich metatranscriptomic information on sample composition for all life domains.
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spelling pubmed-74722772020-09-04 Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing Grädel, Carole Terrazos Miani, Miguel A. Baumann, Christian Barbani, Maria Teresa Neuenschwander, Stefan Leib, Stephen L. Suter-Riniker, Franziska Ramette, Alban Viruses Article Enteroviruses are small RNA viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. In routine diagnostic laboratories, enteroviruses are identified by PCR-based methods, often combined with partial sequencing for genotyping. In this proof-of-principle study, we assessed direct RNA sequencing (DRS) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. The approach was complemented by sequencing the corresponding viral cDNA via Illumina MiSeq sequencing. DRS of total RNA extracted from three different enterovirus-positive stool samples produced long RNA fragments, covering between 59% and 99.6% of the most similar reference genome sequences. The identification of the enterovirus sequences in the samples was confirmed by short-read cDNA sequencing. Sequence identity between DRS and Illumina MiSeq enterovirus consensus sequences ranged between 94% and 97%. Here, we show that nanopore DRS can be used to correctly identify enterovirus genotypes from patient stool samples with high viral load and that the approach also provides rich metatranscriptomic information on sample composition for all life domains. MDPI 2020-07-31 /pmc/articles/PMC7472277/ /pubmed/32752120 http://dx.doi.org/10.3390/v12080841 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Grädel, Carole
Terrazos Miani, Miguel A.
Baumann, Christian
Barbani, Maria Teresa
Neuenschwander, Stefan
Leib, Stephen L.
Suter-Riniker, Franziska
Ramette, Alban
Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing
title Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing
title_full Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing
title_fullStr Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing
title_full_unstemmed Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing
title_short Whole-Genome Sequencing of Human Enteroviruses from Clinical Samples by Nanopore Direct RNA Sequencing
title_sort whole-genome sequencing of human enteroviruses from clinical samples by nanopore direct rna sequencing
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472277/
https://www.ncbi.nlm.nih.gov/pubmed/32752120
http://dx.doi.org/10.3390/v12080841
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