A near full-length open reading frame next generation sequencing assay for genotyping and identification of resistance-associated variants in hepatitis C virus

• Published in: Journal of clinical virology 2018-08, Vol.105, p.49-56
• Main Authors: Pedersen, M.S., Fahnøe, U., Hansen, T.A., Pedersen, A.G., Jenssen, H., Bukh, J., Schønning, K.
• Format: Article
• Language: English
• Subjects: Drug Resistance, Viral - genetics; Genetic Variation; Genotype; Genotyping; Genotyping Techniques; Hepacivirus - genetics; Hepatitis C - blood; High-Throughput Nucleotide Sequencing - methods; Humans; NGS; Open Reading Frames; Phylogeny; RAS; Replicon; RT-PCR; Sequence Analysis, DNA; Subgenome; Viral Nonstructural Proteins - genetics
• ISSN: 1386-6532; 1873-5967
• DOI: 10.1016/j.jcv.2018.05.012

•A near full-length HCV amplicon was generated using a single set of primers.•HCV genotype 1a, 1b, 2b, 3a, 3b, 3h, 4a, 4d, 4o, 4r samples were sequenced.•Samples with HCV RNA down to 4 Log IU/mL were sequenced.•Method generated variation was estimated below 1%.•The method identified dual infections and the presence of subgenomic replicons. The current treatment options for hepatitis C virus (HCV), based on direct acting antivirals (DAA), are dependent on virus genotype and previous treatment experience. Treatment failures have been associated with detection of resistance-associated substitutions (RASs) in the DAA targets of HCV, the NS3, NS5A and NS5 B proteins. To develop a next generation sequencing based method that provides genotype and detection of HCV NS3, NS5A, and NS5 B RASs without prior knowledge of sample genotype. In total, 101 residual plasma samples from patients with HCV covering 10 different viral subtypes across 4 genotypes with viral loads of 3.84-7.61 Log IU/mL were included. All samples were de-identified and consequently prior treatment status for patients was unknown. Almost full open reading frame amplicons (∼ 9 kb) were generated using RT-PCR with a single primer set. The resulting amplicons were sequenced with high throughput sequencing and analysed using an in-house developed script for detecting RASs. The method successfully amplified and sequenced 94% (95/101) of samples with an average coverage of 14,035; four of six failed samples were genotype 4a. Samples analysed twice yielded reproducible nucleotide frequencies across all sites. RASs were detected in 21/95 (22%) samples at a 15% threshold. The method identified one patient infected with two genotype 2b variants, and the presence of subgenomic deletion variants in 8 (8.4%) of 95 successfully sequenced samples. The presented method may provide identification of HCV genotype, RASs detection, and detect multiple HCV infection without prior knowledge of sample genotype.