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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| Format: | Article |
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MDPI AG
2020-09-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/8/9/1391 |
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| author | Flavia Hebeler-Barbosa Ivan Rodrigo Wolf Guilherme Targino Valente Francisco Campello do Amaral Mello Elisabeth Lampe Maria Inês de Moura Campos Pardini Rejane Maria Tommasini Grotto |
| author_facet | Flavia Hebeler-Barbosa Ivan Rodrigo Wolf Guilherme Targino Valente Francisco Campello do Amaral Mello Elisabeth Lampe Maria Inês de Moura Campos Pardini Rejane Maria Tommasini Grotto |
| author_sort | Flavia Hebeler-Barbosa |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-10T16:24:39Z |
| format | Article |
| id | doaj.art-a0180b71ba624f42972a24269282c247 |
| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-10T16:24:39Z |
| publishDate | 2020-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microorganisms |
| spelling | doaj.art-a0180b71ba624f42972a24269282c2472023-11-20T13:19:59ZengMDPI AGMicroorganisms2076-26072020-09-0189139110.3390/microorganisms8091391A New Method for Next-Generation Sequencing of the Full Hepatitis B Virus Genome from A Clinical Specimen: Impact for Virus GenotypingFlavia Hebeler-Barbosa0Ivan Rodrigo Wolf1Guilherme Targino Valente2Francisco Campello do Amaral Mello3Elisabeth Lampe4Maria Inês de Moura Campos Pardini5Rejane Maria Tommasini Grotto6Medical School, São Paulo State University (Unesp), Botucatu 18618-687, BrazilSchool of Agriculture, São Paulo State University (Unesp), Botucatu 18618-687, BrazilSchool of Agriculture, São Paulo State University (Unesp), Botucatu 18618-687, BrazilLaboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro 21040-900, BrazilLaboratory of Viral Hepatitis, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro 21040-900, BrazilMedical School, São Paulo State University (Unesp), Botucatu 18618-687, BrazilMedical School, São Paulo State University (Unesp), Botucatu 18618-687, BrazilHepatitis 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.https://www.mdpi.com/2076-2607/8/9/1391Hepatitis B virusNGSgenotypingphylogeny analysis |
| spellingShingle | Flavia Hebeler-Barbosa Ivan Rodrigo Wolf Guilherme Targino Valente Francisco Campello do Amaral Mello Elisabeth Lampe Maria Inês de Moura Campos Pardini Rejane Maria Tommasini Grotto A New Method for Next-Generation Sequencing of the Full Hepatitis B Virus Genome from A Clinical Specimen: Impact for Virus Genotyping Microorganisms Hepatitis B virus NGS genotyping phylogeny analysis |
| 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 | Hepatitis B virus NGS genotyping phylogeny analysis |
| url | https://www.mdpi.com/2076-2607/8/9/1391 |
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