A novel standardized deep sequencing-based assay for hepatitis C virus genotype determination

Abstract

Hepatitis C virus (HCV) genotype and subtype (1a/1b) identification is needed to tailor anti-HCV therapy. Currently available methods accurately identify the genotype and differentiate subtypes 1a from 1b. However, these assays have not been designed to identify other HCV subtypes, nor to recognize mixed genotype/subtype infections, emphasizing the need for a high-resolution system based on phylogenetic analysis of reads obtained by deep sequencing of a relevant genome region. The aim of this study was to evaluate the performance of the Sentosa SQ HCV Genotyping Assay, a novel deep sequencing-based assay targeting the HCV nonstructural 5B (NS5B) region, in clinical samples from patients with an indication for anti-HCV therapy. A high concordance rate with Sanger sequencing of the NS5B region, the reference method, was found for genotype 1 to 6 determination, 1a/1b subtype identification, and genotype 4, 5 and 6 subtyping. Discrepancies were seen essentially for HCV genotype 2 subtyping. Overall, the performance of the deep sequencing-based assay in generating the genotypes/subtype information needed to tailor anti-HCV treatment was adequate in this study. Further improvements, such as a longer NS5B fragment analyzed and enriching the database of reference prototype strains used for subtype assignment would make it a method of choice for HCV genotyping and subtyping for future clinical practice and research.

Figure 1

Phylogenetic analysis of HCV NS5B sequences from the 99 patients included in the study. The neighbor-joining tree was based on alignment of nucleotide sequences from a portion of the NS5B gene spanning nucleotide positions 8325 to 9610. Branch lengths were calculated using a Kimura 2-parameter distance matrix with a Ts/Tv ratio of 2.0. Numbers at nodes correspond to the percentage of 1,000 replicates supporting the distal cluster. All reference strains are represented as subtype_isolate ID. In the center of the tree, a pie chart shows the proportions of the different genotypes among the 99 isolates, represented by different colors: red for genotype 1 (46.5%), blue for genotype 3 (14.1%), orange for genotype 4 (17.2%), green for genotype 2 (11.1%), gray for genotype 5 (2.0%), pink for genotype 6 (5.1%) and 2k/1b recombinant strains in hatched red (4.0%).