title: Development and Validation of a Template-Independent Next-Generation Sequencing Assay for Detecting Low-Level Resistance-Associated Variants of Hepatitis C Virus.
abstract: To develop hepatitis C virus (HCV) direct-acting antiviral (DAA) drugs that can treat most HCV genotypes and offer higher barriers for treatment-resistant mutations, it is important to study resistance-associated variants (RAVs). Current commercially available RAV detection assays rely on genotype- or subtype-specific template-dependent PCR amplification. These assays are limited to genotypes and subtypes that are often prevalent in developed countries because of availability of public sequence databases. To support global clinical trials of DAAs, we developed and validated a template-independent (TI) next-generation sequencing (NGS) assay for HCV whole genome sequencing that can perform HCV subtyping, detect HCV mixed genotype or subtype infection, and identify low-level RAVs at a 5% fraction of the viral population with sensitivity and positive predictive value ≥ 0.9. We compared TI-NGS with commercial genotype- or subtype-specific Sanger sequencing assays, and found that TI-NGS both confirmed most of variants called by Sanger sequencing and avoided biases likely caused by PCR primers used in Sanger sequencing. To confirm TI-NGS assay's variant calls at the discrepant positions with Sanger sequencing, we custom designed template-dependent NGS assays and obtained 100% concordance with the TI-NGS assay. The ability to reliably detect low-level RAVs in HCV samples of any subtype without PCR primer-related bias makes this TI-NGS assay an important tool in studying HCV DAA drug resistance.
authors.affiliation: Section of Translational Molecular Biomarkers, Merck & Co, Inc., Rahway, New Jersey. Electronic address: bo_wei@merck.com.
authors.forename: Bo
authors.initials: B
authors.lastname: Wei
authors.affiliation: Section of EDS-Biometrics Research, Merck & Co, Inc., Rahway, New Jersey.
authors.forename: John
authors.initials: J
authors.lastname: Kang
authors.affiliation: Section of Translational Molecular Biomarkers, Merck & Co, Inc., Rahway, New Jersey.
authors.forename: Miho
authors.initials: M
authors.lastname: Kibukawa
authors.affiliation: Section of Translational Molecular Biomarkers, Merck & Co, Inc., Rahway, New Jersey.
authors.forename: Lei
authors.initials: L
authors.lastname: Chen
authors.affiliation: Section of Translational Molecular Biomarkers, Merck & Co, Inc., Rahway, New Jersey.
authors.forename: Ping
authors.initials: P
authors.lastname: Qiu
authors.affiliation: Section of Biology Discovery, Merck & Co, Inc., Rahway, New Jersey.
authors.forename: Fred
authors.initials: F
authors.lastname: Lahser
authors.affiliation: Section of Companion Diagnostics, Merck & Co, Inc., Rahway, New Jersey.
authors.forename: Matthew
authors.initials: M
authors.lastname: Marton
authors.affiliation: Section of Translational Molecular Biomarkers, Merck & Co, Inc., Rahway, New Jersey.
authors.forename: Diane
authors.initials: D
authors.lastname: Levitan
mesh_terms: D000483:Alleles; D000998:Antiviral Agents / Q000494:pharmacology* / Q000627:therapeutic use; D019295:Computational Biology / Q000379:methods; D024882:Drug Resistance, Viral; D005787:Gene Frequency; D014644:Genetic Variation; D016679:Genome, Viral; D005838:Genotype; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics*; D006526:Hepatitis C / Q000175:diagnosis / Q000188:drug therapy / Q000821:virology*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods* / Q000592:standards; D006801:Humans; D008826:Microbial Sensitivity Tests; D058889:Molecular Typing; D012367:RNA, Viral; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017211:Treatment Failure; D016896:Treatment Outcome; D019562:Viral Load
chemical_list: D000998:Antiviral Agents; D012367:RNA, Viral