A new method for next-generation sequencing of the full hepatitis b virus genome from a clinical specimen: Impact for virus genotyping

Detalhes bibliográficos
Autor(a) principal: Hebeler-Barbosa, Flavia [UNESP]
Data de Publicação: 2020
Outros Autores: Wolf, Ivan Rodrigo [UNESP], Valente, Guilherme Targino [UNESP], Mello, Francisco Campello Do Amaral, Lampe, Elisabeth, Pardini, Maria Inês de Moura Campos [UNESP], Grotto, Rejane Maria Tommasini [UNESP]
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Institucional da UNESP
Texto Completo: http://dx.doi.org/10.3390/microorganisms8091391
http://hdl.handle.net/11449/205202
Resumo: Hepatitis B virus (HBV) is an enveloped virus that induces chronic liver disease. HBV has been classified into eight genotypes (A–H) according to its genome sequence by using Sanger sequencing or reverse hybridization. Sanger sequencing is often restricted to analyzing the S gene and is inaccurate for detecting minority genetic variants, whereas reverse hybridization detects only known mutations. Next-generation sequencing (NGS) is a robust tool for clinical virology with different protocols available. The objective of this study was to develop a new method for the study of viral genetic polymorphisms or more accurate genotyping using genome amplification followed by NGS. Plasma obtained from five chronically infected HBV individuals was used for viral DNA isolation. HBV full-genome PCR amplification was the enrichment method for NGS. Primers were used to amplify all HBV genotypes in three overlapping amplicons, following a tagmentation step and Illumina NGS. For phylogenetic analysis, sequences were extracted from the HBVdb database. We were able to amplify a full HBV genome; further, NGS was shown to be a robust method and allowed better genotyping, mainly in patients carrying mixed genotypes, classified according to other techniques. This new method may be significant for whole genome analyses, including other viruses.
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spelling A new method for next-generation sequencing of the full hepatitis b virus genome from a clinical specimen: Impact for virus genotypingGenotypingHepatitis B virusNGSPhylogeny analysisHepatitis B virus (HBV) is an enveloped virus that induces chronic liver disease. HBV has been classified into eight genotypes (A–H) according to its genome sequence by using Sanger sequencing or reverse hybridization. Sanger sequencing is often restricted to analyzing the S gene and is inaccurate for detecting minority genetic variants, whereas reverse hybridization detects only known mutations. Next-generation sequencing (NGS) is a robust tool for clinical virology with different protocols available. The objective of this study was to develop a new method for the study of viral genetic polymorphisms or more accurate genotyping using genome amplification followed by NGS. Plasma obtained from five chronically infected HBV individuals was used for viral DNA isolation. HBV full-genome PCR amplification was the enrichment method for NGS. Primers were used to amplify all HBV genotypes in three overlapping amplicons, following a tagmentation step and Illumina NGS. For phylogenetic analysis, sequences were extracted from the HBVdb database. We were able to amplify a full HBV genome; further, NGS was shown to be a robust method and allowed better genotyping, mainly in patients carrying mixed genotypes, classified according to other techniques. This new method may be significant for whole genome analyses, including other viruses.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Medical School São Paulo State University (Unesp)Molecular Biology Laboratory of Clinical Hospital of Botucatu (HCFMB)School of Agriculture São Paulo State University (Unesp)Laboratory of Viral Hepatitis Oswaldo Cruz Institute FIOCRUZMedical School São Paulo State University (Unesp)School of Agriculture São Paulo State University (Unesp)FAPESP: 2017/07711-0Universidade Estadual Paulista (Unesp)Molecular Biology Laboratory of Clinical Hospital of Botucatu (HCFMB)FIOCRUZHebeler-Barbosa, Flavia [UNESP]Wolf, Ivan Rodrigo [UNESP]Valente, Guilherme Targino [UNESP]Mello, Francisco Campello Do AmaralLampe, ElisabethPardini, Maria Inês de Moura Campos [UNESP]Grotto, Rejane Maria Tommasini [UNESP]2021-06-25T10:11:30Z2021-06-25T10:11:30Z2020-09-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article1-12http://dx.doi.org/10.3390/microorganisms8091391Microorganisms, v. 8, n. 9, p. 1-12, 2020.2076-2607http://hdl.handle.net/11449/20520210.3390/microorganisms80913912-s2.0-85091354827Scopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengMicroorganismsinfo:eu-repo/semantics/openAccess2021-10-23T11:59:40Zoai:repositorio.unesp.br:11449/205202Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462024-08-05T19:48:47.109931Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false
dc.title.none.fl_str_mv A new method for next-generation sequencing of the full hepatitis b virus genome from a clinical specimen: Impact for virus genotyping
title A new method for next-generation sequencing of the full hepatitis b virus genome from a clinical specimen: Impact for virus genotyping
spellingShingle A new method for next-generation sequencing of the full hepatitis b virus genome from a clinical specimen: Impact for virus genotyping
Hebeler-Barbosa, Flavia [UNESP]
Genotyping
Hepatitis B virus
NGS
Phylogeny analysis
title_short A new method for next-generation sequencing of the full hepatitis b virus genome from a clinical specimen: Impact for virus genotyping
title_full A new method for next-generation sequencing of the full hepatitis b virus genome from a clinical specimen: Impact for virus genotyping
title_fullStr A new method for next-generation sequencing of the full hepatitis b virus genome from a clinical specimen: Impact for virus genotyping
title_full_unstemmed A new method for next-generation sequencing of the full hepatitis b virus genome from a clinical specimen: Impact for virus genotyping
title_sort A new method for next-generation sequencing of the full hepatitis b virus genome from a clinical specimen: Impact for virus genotyping
author Hebeler-Barbosa, Flavia [UNESP]
author_facet Hebeler-Barbosa, Flavia [UNESP]
Wolf, Ivan Rodrigo [UNESP]
Valente, Guilherme Targino [UNESP]
Mello, Francisco Campello Do Amaral
Lampe, Elisabeth
Pardini, Maria Inês de Moura Campos [UNESP]
Grotto, Rejane Maria Tommasini [UNESP]
author_role author
author2 Wolf, Ivan Rodrigo [UNESP]
Valente, Guilherme Targino [UNESP]
Mello, Francisco Campello Do Amaral
Lampe, Elisabeth
Pardini, Maria Inês de Moura Campos [UNESP]
Grotto, Rejane Maria Tommasini [UNESP]
author2_role author
author
author
author
author
author
dc.contributor.none.fl_str_mv Universidade Estadual Paulista (Unesp)
Molecular Biology Laboratory of Clinical Hospital of Botucatu (HCFMB)
FIOCRUZ
dc.contributor.author.fl_str_mv Hebeler-Barbosa, Flavia [UNESP]
Wolf, Ivan Rodrigo [UNESP]
Valente, Guilherme Targino [UNESP]
Mello, Francisco Campello Do Amaral
Lampe, Elisabeth
Pardini, Maria Inês de Moura Campos [UNESP]
Grotto, Rejane Maria Tommasini [UNESP]
dc.subject.por.fl_str_mv Genotyping
Hepatitis B virus
NGS
Phylogeny analysis
topic Genotyping
Hepatitis B virus
NGS
Phylogeny analysis
description Hepatitis B virus (HBV) is an enveloped virus that induces chronic liver disease. HBV has been classified into eight genotypes (A–H) according to its genome sequence by using Sanger sequencing or reverse hybridization. Sanger sequencing is often restricted to analyzing the S gene and is inaccurate for detecting minority genetic variants, whereas reverse hybridization detects only known mutations. Next-generation sequencing (NGS) is a robust tool for clinical virology with different protocols available. The objective of this study was to develop a new method for the study of viral genetic polymorphisms or more accurate genotyping using genome amplification followed by NGS. Plasma obtained from five chronically infected HBV individuals was used for viral DNA isolation. HBV full-genome PCR amplification was the enrichment method for NGS. Primers were used to amplify all HBV genotypes in three overlapping amplicons, following a tagmentation step and Illumina NGS. For phylogenetic analysis, sequences were extracted from the HBVdb database. We were able to amplify a full HBV genome; further, NGS was shown to be a robust method and allowed better genotyping, mainly in patients carrying mixed genotypes, classified according to other techniques. This new method may be significant for whole genome analyses, including other viruses.
publishDate 2020
dc.date.none.fl_str_mv 2020-09-01
2021-06-25T10:11:30Z
2021-06-25T10:11:30Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://dx.doi.org/10.3390/microorganisms8091391
Microorganisms, v. 8, n. 9, p. 1-12, 2020.
2076-2607
http://hdl.handle.net/11449/205202
10.3390/microorganisms8091391
2-s2.0-85091354827
url http://dx.doi.org/10.3390/microorganisms8091391
http://hdl.handle.net/11449/205202
identifier_str_mv Microorganisms, v. 8, n. 9, p. 1-12, 2020.
2076-2607
10.3390/microorganisms8091391
2-s2.0-85091354827
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv Microorganisms
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv 1-12
dc.source.none.fl_str_mv Scopus
reponame:Repositório Institucional da UNESP
instname:Universidade Estadual Paulista (UNESP)
instacron:UNESP
instname_str Universidade Estadual Paulista (UNESP)
instacron_str UNESP
institution UNESP
reponame_str Repositório Institucional da UNESP
collection Repositório Institucional da UNESP
repository.name.fl_str_mv Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)
repository.mail.fl_str_mv
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