A new method for next-generation sequencing of the full hepatitis b virus genome from a clinical specimen: Impact for virus genotyping
Autor(a) principal: | |
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Data de Publicação: | 2020 |
Outros Autores: | , , , , , |
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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Repositório Institucional da UNESP |
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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 |
|
_version_ |
1808129120891043840 |