Abstract
Genotyping of hepatitis C virus (HCV) plays an important role in the treatment of HCV. As new genotype-specific treatment options become available, it has become increasingly important to have accurate HCV genotype and subtype information to ensure that the most appropriate treatment regimen is selected. Most current genotyping methods are unable to detect mixed genotypes from two or more HCV infections. Next generation sequencing (NGS) allows for rapid and low cost mass sequencing of viral genomes and provides an opportunity to probe the viral population from a single host. In this paper, the possibility of using short NGS reads for direct HCV genotyping without genome assembly was evaluated. We surveyed the publicly-available genetic content of three HCV drug target regions (NS3, NS5A, NS5B) in terms of whether these genes contained genotype-specific regions that could predict genotype. Six genotypes and 38 subtypes were included in this study. An automated phylogenetic analysis based HCV genotyping method was implemented and used to assess different HCV target gene regions. Candidate regions of 250-bp each were found for all three genes that have enough genetic information to predict HCV genotypes/subtypes. Validation using public datasets shows 100% genotyping accuracy. To test whether these 250-bp regions were sufficient to identify mixed genotypes, we developed a random primer-based method to sequence HCV plasma samples containing mixtures of two HCV genotypes in different ratios. We were able to determine the genotypes without ambiguity and to quantify the ratio of the abundances of the mixed genotypes in the samples. These data provide a proof-of-concept that this random primed, NGS-based short-read genotyping approach does not need prior information about the viral population and is capable of detecting mixed viral infection.
Highlights
About 130–170 million people are infected with hepatitis C virus (HCV), with a global prevalence of infection estimated at 2%-3% [1,2]
More than 20000 HCV NS3, NS5A and NS5B sequences were collected from the May 2014 version of Los Alamos HCV database. 75 total HCV subtypes were collected
Only subtypes with at least 3 sequences were included in the analysis. 37 subtypes had less than 3 sequences and were excluded from the analysis and the HCV reference tree construction for NS3 and NS5A. 39 subtypes were excluded for NS5B according to the same criteria
Summary
About 130–170 million people are infected with HCV, with a global prevalence of infection estimated at 2%-3% [1,2]. Six major genotypes (HCV 1–6) have been described, each containing multiple subtypes [4,5,6]. In 2013 the US FDA approved the Abbott m2000 RealTime HCV Genotype II assay (Abbott Molecular Inc., Des Plaines, IL, USA), which is a Polymerase Chain Reaction (PCR)-based assay targeting specific regions of the 50NCR and the NS5B genes, and reports the genotypes as 1, 1a, 1b, 2, 3, 4, 5, and 6 [14]. It is known that the m2000 assay does not resolve all HCV genotypes [15] in which cases ‘indeterminate’, ‘mixed’, ‘genotype X reactivity with Y’, or only the major genotype were reported. To fully resolve the genotype, additional testing is required in 9–10% of cases
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