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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.
Highlights
Hepatitis C virus (HCV) is an enveloped positive sense single stranded RNA virus of 9600 bases, which infects 130–150 million people worldwide
Forty-two positive PCR products issued from single genome amplification (SGA) fulfilled the criteria of PCR selection process. were purified for sequencing
We first sequenced the HCV E1E2 amplicons derived from SGA by Next Generation Sequencing (NGS)
Summary
Hepatitis C virus (HCV) is an enveloped positive sense single stranded RNA virus of 9600 bases, which infects 130–150 million people worldwide. Most (70%) HCV infections become chronic and progress toward liver diseases such as cirrhosis and hepatocellular carcinoma[1]. 500 000 people die each year from hepatitis C-related liver diseases[2]. The error rate of HCV polymerase has been estimated in vitro to be 10−3 nucleotide substitutions per site per year[4]. This high mutation rate combined with a short generation time (1012 virions produced per day[5]) is at the origin of the quasispecies dynamics of RNA viruses
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