Early MinION™ nanopore single-molecule sequencing technology enables the characterization of hepatitis B virus genetic complexity in clinical samples

Until recently, the method of choice to characterize viral diversity consisted in cloning PCR amplicons of full-length viral genomes and Sanger-sequencing of multiple clones. However, this is extremely laborious, time-consuming, and low-throughput. Next generation short-read sequencing appears also...

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Vydáno v:	PloS one Ročník 13; číslo 3; s. e0194366
Hlavní autoři:	Sauvage, Virginie, Boizeau, Laure, Candotti, Daniel, Vandenbogaert, Mathias, Servant-Delmas, Annabelle, Caro, Valérie, Laperche, Syria
Médium:	Journal Article
Jazyk:	angličtina
Vydáno:	United States Public Library of Science 22.03.2018 Public Library of Science (PLoS)
Témata:	Biology and Life Sciences Cloning Consortia Deoxyribonucleic acid DNA Ebola virus Ebolavirus Gene expression Gene sequencing Genetic diversity Genome, Viral - genetics Genomes Hepatitis Hepatitis B Hepatitis B - blood Hepatitis B - diagnosis Hepatitis B - virology Hepatitis B virus Hepatitis B virus - genetics High-Throughput Nucleotide Sequencing - methods Humans Infections Laboratories Life Sciences Medicine and health sciences Methods Microbiology and Parasitology Mutation Nanopores New technology Pathogens Polymerase Chain Reaction Polymorphism Polymorphism, Single Nucleotide - genetics Population Porosity Recombination, Genetic - genetics Research and Analysis Methods Sequence Analysis, DNA - methods Technology Technology assessment Viral infections Viral Proteins - genetics Virology Viruses France
ISSN:	1932-6203, 1932-6203
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Abstract	Until recently, the method of choice to characterize viral diversity consisted in cloning PCR amplicons of full-length viral genomes and Sanger-sequencing of multiple clones. However, this is extremely laborious, time-consuming, and low-throughput. Next generation short-read sequencing appears also limited by its inability to directly sequence full-length viral genomes. The MinION™ device recently developed by Oxford Nanopore Technologies can be a promising alternative by applying long-read single-molecule sequencing directly to the overall amplified products generated in a PCR reaction. This new technology was evaluated by using hepatitis B virus (HBV) as a model. Several previously characterized HBV-infected clinical samples were investigated including recombinant virus, variants that harbored deletions and mixed population. Original MinION device was able to generate individual complete 3,200-nt HBV genome sequences and to identify recombinant variants. MinION was particularly efficient in detecting HBV genomes with multiple large in-frame deletions and spliced variants concomitantly with non-deleted parental genomes. However, an average-12% sequencing error rate per individual reads associated to a low throughput challenged single-nucleotide resolution, polymorphism calling and phasing mutations directly from the sequencing reads. Despite this high error rate, the pairwise identity of MinION HBV consensus genome was consistent with Sanger sequencing method. MinION being under continuous development, further studies are needed to evaluate its potential use for viral infection characterization.
AbstractList	Until recently, the method of choice to characterize viral diversity consisted in cloning PCR amplicons of full-length viral genomes and Sanger-sequencing of multiple clones. However, this is extremely laborious, time-consuming, and low-throughput. Next generation short-read sequencing appears also limited by its inability to directly sequence full-length viral genomes. The MinION™ device recently developed by Oxford Nanopore Technologies can be a promising alternative by applying long-read single-molecule sequencing directly to the overall amplified products generated in a PCR reaction. This new technology was evaluated by using hepatitis B virus (HBV) as a model. Several previously characterized HBV-infected clinical samples were investigated including recombinant virus, variants that harbored deletions and mixed population. Original MinION device was able to generate individual complete 3,200-nt HBV genome sequences and to identify recombinant variants. MinION was particularly efficient in detecting HBV genomes with multiple large in-frame deletions and spliced variants concomitantly with non-deleted parental genomes. However, an average-12% sequencing error rate per individual reads associated to a low throughput challenged single-nucleotide resolution, polymorphism calling and phasing mutations directly from the sequencing reads. Despite this high error rate, the pairwise identity of MinION HBV consensus genome was consistent with Sanger sequencing method. MinION being under continuous development, further studies are needed to evaluate its potential use for viral infection characterization. Until recently, the method of choice to characterize viral diversity consisted in cloning PCR amplicons of full-length viral genomes and Sanger-sequencing of multiple clones. However, this is extremely laborious, time-consuming, and low-throughput. Next generation short-read sequencing appears also limited by its inability to directly sequence full-length viral genomes. The MinION™ device recently developed by Oxford Nanopore Technologies can be a promising alternative by applying long-read single-molecule sequencing directly to the overall amplified products generated in a PCR reaction. This new technology was evaluated by using hepatitis B virus (HBV) as a model. Several previously characterized HBV-infected clinical samples were investigated including recombinant virus, variants that harbored deletions and mixed population. Original MinION device was able to generate individual complete 3,200-nt HBV genome sequences and to identify recombinant variants. MinION was particularly efficient in detecting HBV genomes with multiple large in-frame deletions and spliced variants concomitantly with non-deleted parental genomes. However, an average-12% sequencing error rate per individual reads associated to a low throughput challenged single-nucleotide resolution, polymorphism calling and phasing mutations directly from the sequencing reads. Despite this high error rate, the pairwise identity of MinION HBV consensus genome was consistent with Sanger sequencing method. MinION being under continuous development, further studies are needed to evaluate its potential use for viral infection characterization.Until recently, the method of choice to characterize viral diversity consisted in cloning PCR amplicons of full-length viral genomes and Sanger-sequencing of multiple clones. However, this is extremely laborious, time-consuming, and low-throughput. Next generation short-read sequencing appears also limited by its inability to directly sequence full-length viral genomes. The MinION™ device recently developed by Oxford Nanopore Technologies can be a promising alternative by applying long-read single-molecule sequencing directly to the overall amplified products generated in a PCR reaction. This new technology was evaluated by using hepatitis B virus (HBV) as a model. Several previously characterized HBV-infected clinical samples were investigated including recombinant virus, variants that harbored deletions and mixed population. Original MinION device was able to generate individual complete 3,200-nt HBV genome sequences and to identify recombinant variants. MinION was particularly efficient in detecting HBV genomes with multiple large in-frame deletions and spliced variants concomitantly with non-deleted parental genomes. However, an average-12% sequencing error rate per individual reads associated to a low throughput challenged single-nucleotide resolution, polymorphism calling and phasing mutations directly from the sequencing reads. Despite this high error rate, the pairwise identity of MinION HBV consensus genome was consistent with Sanger sequencing method. MinION being under continuous development, further studies are needed to evaluate its potential use for viral infection characterization. Until recently, the method of choice to characterize viral diversity consisted in cloning PCR amplicons of full-length viral genomes and Sanger-sequencing of multiple clones. However, this is extremely laborious, time-consuming, and low-throughput. Next generation short-read sequencing appears also limited by its inability to directly sequence full-length viral genomes. The MinION ™ device recently developed by Oxford Nanopore Technologies can be a promising alternative by applying long-read single-molecule sequencing directly to the overall amplified products generated in a PCR reaction. This new technology was evaluated by using hepatitis B virus (HBV) as a model. Several previously characterized HBV-infected clinical samples were investigated including recombinant virus, variants that harbored deletions and mixed population. Original MinION device was able to generate individual complete 3,200-nt HBV genome sequences and to identify recombinant variants. MinION was particularly efficient in detecting HBV genomes with multiple large in-frame deletions and spliced variants concomitantly with non-deleted parental genomes. However, an average-12% sequencing error rate per individual reads associated to a low throughput challenged single-nucleotide resolution, polymorphism calling and phasing mutations directly from the sequencing reads. Despite this high error rate, the pairwise identity of MinION HBV consensus genome was consistent with Sanger sequencing method. MinION being under continuous development, further studies are needed to evaluate its potential use for viral infection characterization.
Author	Vandenbogaert, Mathias Servant-Delmas, Annabelle Sauvage, Virginie Boizeau, Laure Laperche, Syria Caro, Valérie Candotti, Daniel
AuthorAffiliation	Oklahoma State University, UNITED STATES 1 Institut National de la Transfusion Sanguine (INTS), Département D’études des Agents Transmissibles par le Sang, Centre National de Référence Risques Infectieux Transfusionnels, Paris, France 2 Institut Pasteur, Genotyping of Pathogens Pole, Laboratory for Urgent Response to Biological Threats, Environment and Infectious Risks, Paris, France
AuthorAffiliation_xml	– name: 1 Institut National de la Transfusion Sanguine (INTS), Département D’études des Agents Transmissibles par le Sang, Centre National de Référence Risques Infectieux Transfusionnels, Paris, France – name: 2 Institut Pasteur, Genotyping of Pathogens Pole, Laboratory for Urgent Response to Biological Threats, Environment and Infectious Risks, Paris, France – name: Oklahoma State University, UNITED STATES
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Copyright	2018 Sauvage et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License. Distributed under a Creative Commons Attribution 4.0 International License 2018 Sauvage et al 2018 Sauvage et al
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SubjectTerms	Biology and Life Sciences Cloning Consortia Deoxyribonucleic acid DNA Ebola virus Ebolavirus Gene expression Gene sequencing Genetic diversity Genome, Viral - genetics Genomes Hepatitis Hepatitis B Hepatitis B - blood Hepatitis B - diagnosis Hepatitis B - virology Hepatitis B virus Hepatitis B virus - genetics High-Throughput Nucleotide Sequencing - methods Humans Infections Laboratories Life Sciences Medicine and health sciences Methods Microbiology and Parasitology Mutation Nanopores New technology Pathogens Polymerase Chain Reaction Polymorphism Polymorphism, Single Nucleotide - genetics Population Porosity Recombination, Genetic - genetics Research and Analysis Methods Sequence Analysis, DNA - methods Technology Technology assessment Viral infections Viral Proteins - genetics Virology Viruses
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Title	Early MinION™ nanopore single-molecule sequencing technology enables the characterization of hepatitis B virus genetic complexity in clinical samples
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