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

Martin Schou Pedersen, Ulrik Fahnøe, Thomas Arn Hansen, Anders Gorm Pedersen, Håvard Jenssen, Jens Bukh, Kristian Schønning

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

BACKGROUND:
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.

OBJECTIVE:
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.

STUDY DESIGN:
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.

RESULTS:
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.

CONCLUSIONS:
The presented method may provide identification of HCV genotype, RASs detection, and detect multiple HCV infection without prior knowledge of sample genotype.
Original languageEnglish
JournalJournal of Clinical Virology
Volume105
Pages (from-to)49-56
ISSN1386-6532
DOIs
Publication statusPublished - 2018

Keywords

  • Genotyping
  • NGS
  • RAS
  • RT-PCR
  • Replicon
  • Subgenome

Cite this