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A New Method for Next-Generation Sequencing of the Full Hepatitis B Virus Genome from A Clinical Specimen: Impact for Virus Genotyping

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 f...

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Autores principales: Hebeler-Barbosa, Flavia, Wolf, Ivan Rodrigo, Valente, Guilherme Targino, Mello, Francisco Campello do Amaral, Lampe, Elisabeth, Pardini, Maria Inês de Moura Campos, Grotto, Rejane Maria Tommasini
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564258/
https://www.ncbi.nlm.nih.gov/pubmed/32932752
http://dx.doi.org/10.3390/microorganisms8091391
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author Hebeler-Barbosa, Flavia
Wolf, Ivan Rodrigo
Valente, Guilherme Targino
Mello, Francisco Campello do Amaral
Lampe, Elisabeth
Pardini, Maria Inês de Moura Campos
Grotto, Rejane Maria Tommasini
author_facet Hebeler-Barbosa, Flavia
Wolf, Ivan Rodrigo
Valente, Guilherme Targino
Mello, Francisco Campello do Amaral
Lampe, Elisabeth
Pardini, Maria Inês de Moura Campos
Grotto, Rejane Maria Tommasini
author_sort Hebeler-Barbosa, Flavia
collection PubMed
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.
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spelling pubmed-75642582020-10-26 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 Wolf, Ivan Rodrigo Valente, Guilherme Targino Mello, Francisco Campello do Amaral Lampe, Elisabeth Pardini, Maria Inês de Moura Campos Grotto, Rejane Maria Tommasini Microorganisms Article 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. MDPI 2020-09-11 /pmc/articles/PMC7564258/ /pubmed/32932752 http://dx.doi.org/10.3390/microorganisms8091391 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
Hebeler-Barbosa, Flavia
Wolf, Ivan Rodrigo
Valente, Guilherme Targino
Mello, Francisco Campello do Amaral
Lampe, Elisabeth
Pardini, Maria Inês de Moura Campos
Grotto, Rejane Maria Tommasini
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
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_short A New Method for Next-Generation Sequencing of the Full Hepatitis B Virus Genome from A Clinical Specimen: Impact for Virus Genotyping
title_sort new method for next-generation sequencing of the full hepatitis b virus genome from a clinical specimen: impact for virus genotyping
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564258/
https://www.ncbi.nlm.nih.gov/pubmed/32932752
http://dx.doi.org/10.3390/microorganisms8091391
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