title: Deep sequencing is an appropriate tool for the selection of unique Hepatitis C virus (HCV) variants after single genomic amplification.
abstract: Hepatitis C virus (HCV) evolves rapidly in a single host and circulates as a quasispecies wich is a complex mixture of genetically distinct virus's but closely related namely variants. To identify intra-individual diversity and investigate their functional properties in vitro, it is necessary to define their quasispecies composition and isolate the HCV variants. This is possible using single genome amplification (SGA). This technique, based on serially diluted cDNA to amplify a single cDNA molecule (clonal amplicon), has already been used to determine individual HCV diversity. In these studies, positive PCR reactions from SGA were directly sequenced using Sanger technology. The detection of non-clonal amplicons is necessary for excluding them to facilitate further functional analysis. Here, we compared Next Generation Sequencing (NGS) with De Novo assembly and Sanger sequencing for their ability to distinguish clonal and non-clonal amplicons after SGA on one plasma specimen. All amplicons (n = 42) classified as clonal by NGS were also classified as clonal by Sanger sequencing. No double peaks were seen on electropherograms for non-clonal amplicons with position-specific nucleotide variation below 15% by NGS. Altogether, NGS circumvented many of the difficulties encountered when using Sanger sequencing after SGA and is an appropriate tool to reliability select clonal amplicons for further functional studies.
authors.affiliation: INSERM U966, Université François Rabelais and CHRU de Tours, Tours, France.
authors.forename: Thibault
authors.initials: T
authors.lastname: Guinoiseau
authors.affiliation: INSERM U966, Université François Rabelais and CHRU de Tours, Tours, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Moreau
authors.affiliation: INSERM U966, Université François Rabelais and CHRU de Tours, Tours, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Hohnadel
authors.affiliation: Institut de Recherche pour le Développement (IRD) UMI 174 PHPT-Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.
authors.forename: Nicole
authors.initials: N
authors.lastname: Ngo-Giang-Huong
authors.affiliation: UMR INSERM U930, Université François Rabelais, Tours, France.
authors.forename: Celine
authors.initials: C
authors.lastname: Brulard
authors.affiliation: UMR INSERM U930, Université François Rabelais, Tours, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Vourc'h
authors.affiliation: INSERM U966, Université François Rabelais and CHRU de Tours, Tours, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Goudeau
authors.affiliation: INSERM U966, Université François Rabelais and CHRU de Tours, Tours, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Gaudy-Graffin
mesh_terms: D005260:Female; D014644:Genetic Variation / Q000235:genetics; D016679:Genome, Viral / Q000235:genetics; D023281:Genomics / Q000379:methods; D006023:Glycoproteins / Q000235:genetics; D016174:Hepacivirus / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D006023:Glycoproteins