Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing Approach

Hepatitis A virus (HAV) is one of the most common causes of acute viral hepatitis in humans. Although HAV has a relatively small genome, there are several factors limiting whole genome sequencing such as PCR amplification artefacts and ambiguities in de novo assembly. The recently developed Oxford N...

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Autores: Batista, Frederico M., Stapleton, Tina, Lowther, James A., Fonseca, Vera G., Shaw, Rebecca, Pond, Christopher, Walker, David I., van Aerle, Ronny|||0000-0002-2605-6518, Martínez-Urtaza, Jaime|||0000-0001-6219-0418
Tipo de recurso: artículo
Fecha de publicación:2020
País:España
Institución:Universitat Autònoma de Barcelona
Repositorio:Dipòsit Digital de Documents de la UAB
Idioma:inglés
OAI Identifier:oai:ddd.uab.cat:252663
Acceso en línea:https://ddd.uab.cat/record/252663
https://dx.doi.org/urn:doi:10.3389/fmicb.2020.00874
Access Level:acceso abierto
Palabra clave:Hepatitis A virus
Picornaviridae
Genome
Nanopore sequencing
Metagenomics
SNV
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spelling Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing ApproachBatista, Frederico M.Stapleton, TinaLowther, James A.Fonseca, Vera G.Shaw, RebeccaPond, ChristopherWalker, David I.van Aerle, Ronny|||0000-0002-2605-6518Martínez-Urtaza, Jaime|||0000-0001-6219-0418Hepatitis A virusPicornaviridaeGenomeNanopore sequencingMetagenomicsSNVHepatitis A virus (HAV) is one of the most common causes of acute viral hepatitis in humans. Although HAV has a relatively small genome, there are several factors limiting whole genome sequencing such as PCR amplification artefacts and ambiguities in de novo assembly. The recently developed Oxford Nanopore technologies (ONT) allows single-molecule sequencing of long-size fragments of DNA or RNA using PCR-free strategies. We have sequenced the whole genome of HAV using a PCR-free approach by direct reverse-transcribed sequencing. We were able to sequence HAV cDNA and obtain reads over 7 kilobases in length containing almost the whole genome of the virus. The comparison of these raw long nanopore reads with the HAV reference wild type revealed a nucleotide sequence identity between 81.1 and 96.6%. By de novo assembly of all HAV reads we obtained a consensus sequence of 7362 bases, with a nucleotide sequence identity of 99.0% with the genome of the HAV strain pHM175/18f. When the assembly was performed using as reference the HAV strain pHM175/18f a consensus with a sequence similarity of 99.8 % was obtained. We have also used an ONT amplicon-based assay to sequence two fragments of the VP3 and VP1 regions which showed a sequence similarity of 100% with matching regions of the consensus sequence obtained using the direct cDNA sequencing approach. This study showed the applicability of ONT sequencing technologies to obtain the whole genome of HAV by direct cDNA nanopore sequencing, highlighting the utility of this PCR-free approach for HAV characterization and potentially other viruses of the Picornaviridae family. 22020-01-0120202020-01-01Articlehttp://purl.org/coar/resource_type/c_6501VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfhttps://ddd.uab.cat/record/252663https://dx.doi.org/urn:doi:10.3389/fmicb.2020.00874reponame:Dipòsit Digital de Documents de la UABinstname:Universitat Autònoma de BarcelonaInglésengopen accesshttp://purl.org/coar/access_right/c_abf2Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.https://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:ddd.uab.cat:2526632026-06-06T12:50:31Z
dc.title.none.fl_str_mv Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing Approach
title Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing Approach
spellingShingle Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing Approach
Batista, Frederico M.
Hepatitis A virus
Picornaviridae
Genome
Nanopore sequencing
Metagenomics
SNV
title_short Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing Approach
title_full Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing Approach
title_fullStr Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing Approach
title_full_unstemmed Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing Approach
title_sort Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing Approach
dc.creator.none.fl_str_mv Batista, Frederico M.
Stapleton, Tina
Lowther, James A.
Fonseca, Vera G.
Shaw, Rebecca
Pond, Christopher
Walker, David I.
van Aerle, Ronny|||0000-0002-2605-6518
Martínez-Urtaza, Jaime|||0000-0001-6219-0418
author Batista, Frederico M.
author_facet Batista, Frederico M.
Stapleton, Tina
Lowther, James A.
Fonseca, Vera G.
Shaw, Rebecca
Pond, Christopher
Walker, David I.
van Aerle, Ronny|||0000-0002-2605-6518
Martínez-Urtaza, Jaime|||0000-0001-6219-0418
author_role author
author2 Stapleton, Tina
Lowther, James A.
Fonseca, Vera G.
Shaw, Rebecca
Pond, Christopher
Walker, David I.
van Aerle, Ronny|||0000-0002-2605-6518
Martínez-Urtaza, Jaime|||0000-0001-6219-0418
author2_role author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Hepatitis A virus
Picornaviridae
Genome
Nanopore sequencing
Metagenomics
SNV
topic Hepatitis A virus
Picornaviridae
Genome
Nanopore sequencing
Metagenomics
SNV
description Hepatitis A virus (HAV) is one of the most common causes of acute viral hepatitis in humans. Although HAV has a relatively small genome, there are several factors limiting whole genome sequencing such as PCR amplification artefacts and ambiguities in de novo assembly. The recently developed Oxford Nanopore technologies (ONT) allows single-molecule sequencing of long-size fragments of DNA or RNA using PCR-free strategies. We have sequenced the whole genome of HAV using a PCR-free approach by direct reverse-transcribed sequencing. We were able to sequence HAV cDNA and obtain reads over 7 kilobases in length containing almost the whole genome of the virus. The comparison of these raw long nanopore reads with the HAV reference wild type revealed a nucleotide sequence identity between 81.1 and 96.6%. By de novo assembly of all HAV reads we obtained a consensus sequence of 7362 bases, with a nucleotide sequence identity of 99.0% with the genome of the HAV strain pHM175/18f. When the assembly was performed using as reference the HAV strain pHM175/18f a consensus with a sequence similarity of 99.8 % was obtained. We have also used an ONT amplicon-based assay to sequence two fragments of the VP3 and VP1 regions which showed a sequence similarity of 100% with matching regions of the consensus sequence obtained using the direct cDNA sequencing approach. This study showed the applicability of ONT sequencing technologies to obtain the whole genome of HAV by direct cDNA nanopore sequencing, highlighting the utility of this PCR-free approach for HAV characterization and potentially other viruses of the Picornaviridae family.
publishDate 2020
dc.date.none.fl_str_mv 2
2020-01-01
2020
2020-01-01
dc.type.none.fl_str_mv Article
http://purl.org/coar/resource_type/c_6501
VoR
http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv https://ddd.uab.cat/record/252663
https://dx.doi.org/urn:doi:10.3389/fmicb.2020.00874
url https://ddd.uab.cat/record/252663
https://dx.doi.org/urn:doi:10.3389/fmicb.2020.00874
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
https://creativecommons.org/licenses/by/4.0/
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
https://creativecommons.org/licenses/by/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv reponame:Dipòsit Digital de Documents de la UAB
instname:Universitat Autònoma de Barcelona
instname_str Universitat Autònoma de Barcelona
reponame_str Dipòsit Digital de Documents de la UAB
collection Dipòsit Digital de Documents de la UAB
repository.name.fl_str_mv
repository.mail.fl_str_mv
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