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A method for near full-length amplification and sequencing for six hepatitis C virus genotypes
BACKGROUND: Hepatitis C virus (HCV) is a rapidly evolving RNA virus that has been classified into seven genotypes. All HCV genotypes cause chronic hepatitis, which ultimately leads to liver diseases such as cirrhosis. The genotypes are unevenly distributed across the globe, with genotypes 1 and 3 be...
Autores principales: | , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4797172/ https://www.ncbi.nlm.nih.gov/pubmed/26988550 http://dx.doi.org/10.1186/s12864-016-2575-8 |
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author | Bull, Rowena A. Eltahla, Auda A. Rodrigo, Chaturaka Koekkoek, Sylvie M. Walker, Melanie Pirozyan, Mehdi R. Betz-Stablein, Brigid Toepfer, Armin Laird, Melissa Oh, Steve Heiner, Cheryl Maher, Lisa Schinkel, Janke Lloyd, Andrew R. Luciani, Fabio |
author_facet | Bull, Rowena A. Eltahla, Auda A. Rodrigo, Chaturaka Koekkoek, Sylvie M. Walker, Melanie Pirozyan, Mehdi R. Betz-Stablein, Brigid Toepfer, Armin Laird, Melissa Oh, Steve Heiner, Cheryl Maher, Lisa Schinkel, Janke Lloyd, Andrew R. Luciani, Fabio |
author_sort | Bull, Rowena A. |
collection | PubMed |
description | BACKGROUND: Hepatitis C virus (HCV) is a rapidly evolving RNA virus that has been classified into seven genotypes. All HCV genotypes cause chronic hepatitis, which ultimately leads to liver diseases such as cirrhosis. The genotypes are unevenly distributed across the globe, with genotypes 1 and 3 being the most prevalent. Until recently, molecular epidemiological studies of HCV evolution within the host and at the population level have been limited to the analyses of partial viral genome segments, as it has been technically challenging to amplify and sequence the full-length of the 9.6 kb HCV genome. Although recent improvements have been made in full genome sequencing methodologies, these protocols are still either limited to a specific genotype or cost-inefficient. RESULTS: In this study we describe a genotype-specific protocol for the amplification and sequencing of the near-full length genome of all six major HCV genotypes. We applied this protocol to 122 HCV positive clinical samples, and had a successful genome amplification rate of 90 %, when the viral load was greater than 15,000 IU/ml. The assay was shown to have a detection limit of 1–3 cDNA copies per reaction. The method was tested with both Illumina and PacBio single molecule, real-time (SMRT) sequencing technologies. Illumina sequencing resulted in deep coverage and allowed detection of rare variants as well as HCV co-infection with multiple genotypes. The application of the method with PacBio RS resulted in sequence reads greater than 9 kb that covered the near full-length HCV amplicon in a single read and enabled analysis of the near full-length quasispecies. CONCLUSIONS: The protocol described herein can be utilised for rapid amplification and sequencing of the near-full length HCV genome in a cost efficient manner suitable for a wide range of applications. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-016-2575-8) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-4797172 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-47971722016-03-18 A method for near full-length amplification and sequencing for six hepatitis C virus genotypes Bull, Rowena A. Eltahla, Auda A. Rodrigo, Chaturaka Koekkoek, Sylvie M. Walker, Melanie Pirozyan, Mehdi R. Betz-Stablein, Brigid Toepfer, Armin Laird, Melissa Oh, Steve Heiner, Cheryl Maher, Lisa Schinkel, Janke Lloyd, Andrew R. Luciani, Fabio BMC Genomics Methodology Article BACKGROUND: Hepatitis C virus (HCV) is a rapidly evolving RNA virus that has been classified into seven genotypes. All HCV genotypes cause chronic hepatitis, which ultimately leads to liver diseases such as cirrhosis. The genotypes are unevenly distributed across the globe, with genotypes 1 and 3 being the most prevalent. Until recently, molecular epidemiological studies of HCV evolution within the host and at the population level have been limited to the analyses of partial viral genome segments, as it has been technically challenging to amplify and sequence the full-length of the 9.6 kb HCV genome. Although recent improvements have been made in full genome sequencing methodologies, these protocols are still either limited to a specific genotype or cost-inefficient. RESULTS: In this study we describe a genotype-specific protocol for the amplification and sequencing of the near-full length genome of all six major HCV genotypes. We applied this protocol to 122 HCV positive clinical samples, and had a successful genome amplification rate of 90 %, when the viral load was greater than 15,000 IU/ml. The assay was shown to have a detection limit of 1–3 cDNA copies per reaction. The method was tested with both Illumina and PacBio single molecule, real-time (SMRT) sequencing technologies. Illumina sequencing resulted in deep coverage and allowed detection of rare variants as well as HCV co-infection with multiple genotypes. The application of the method with PacBio RS resulted in sequence reads greater than 9 kb that covered the near full-length HCV amplicon in a single read and enabled analysis of the near full-length quasispecies. CONCLUSIONS: The protocol described herein can be utilised for rapid amplification and sequencing of the near-full length HCV genome in a cost efficient manner suitable for a wide range of applications. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-016-2575-8) contains supplementary material, which is available to authorized users. BioMed Central 2016-03-17 /pmc/articles/PMC4797172/ /pubmed/26988550 http://dx.doi.org/10.1186/s12864-016-2575-8 Text en © Bull et al. 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Methodology Article Bull, Rowena A. Eltahla, Auda A. Rodrigo, Chaturaka Koekkoek, Sylvie M. Walker, Melanie Pirozyan, Mehdi R. Betz-Stablein, Brigid Toepfer, Armin Laird, Melissa Oh, Steve Heiner, Cheryl Maher, Lisa Schinkel, Janke Lloyd, Andrew R. Luciani, Fabio A method for near full-length amplification and sequencing for six hepatitis C virus genotypes |
title | A method for near full-length amplification and sequencing for six hepatitis C virus genotypes |
title_full | A method for near full-length amplification and sequencing for six hepatitis C virus genotypes |
title_fullStr | A method for near full-length amplification and sequencing for six hepatitis C virus genotypes |
title_full_unstemmed | A method for near full-length amplification and sequencing for six hepatitis C virus genotypes |
title_short | A method for near full-length amplification and sequencing for six hepatitis C virus genotypes |
title_sort | method for near full-length amplification and sequencing for six hepatitis c virus genotypes |
topic | Methodology Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4797172/ https://www.ncbi.nlm.nih.gov/pubmed/26988550 http://dx.doi.org/10.1186/s12864-016-2575-8 |
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