title: Genotype- and Subtype-Independent Full-Genome Sequencing Assay for Hepatitis C Virus.
abstract: Hepatitis C virus (HCV) exhibits a high genetic diversity and is classified into 6 genotypes, which are further divided into 66 subtypes. Current sequencing strategies require prior knowledge of the HCV genotype and subtype for efficient amplification, making it difficult to sequence samples with a rare or unknown genotype and/or subtype. Here, we describe a subtype-independent full-genome sequencing assay based on a random amplification strategy coupled with next-generation sequencing. HCV genomes from 17 patient samples with both common subtypes (1a, 1b, 2a, 2b, and 3a) and rare subtypes (2c, 2j, 3i, 4a, 4d, 5a, 6a, 6e, and 6j) were successfully sequenced. On average, 3.7 million reads were generated per sample, with 15% showing HCV specificity. The assembled consensus sequences covered 99.3% to 100% of the HCV coding region, and the average coverage was 6,070 reads/position. The accuracy of the generated consensus sequence was estimated to be >99% based on results from in vitro HCV replicon amplification, with the same extrapolated amount of input RNA molecules as that for the patient samples. Taken together, the HCV genomes from 17 patient samples were successfully sequenced, including samples with subtypes that have limited sequence information. This method has the potential to sequence any HCV patient sample, independent of genotype or subtype. It may be especially useful in confounding cases, like those with rare subtypes, intergenotypic recombination, or multiple genotype infections, and may allow greater insight into HCV evolution, its genetic diversity, and drug resistance development.
authors: Gilead Sciences, Inc., Foster City, California, USA.
authors: Charlotte
authors: C
authors: Hedskog
authors: Gilead Sciences, Inc., Foster City, California, USA.
authors: Krishna
authors: K
authors: Chodavarapu
authors: Gilead Sciences, Inc., Foster City, California, USA.
authors: Karin S
authors: KS
authors: Ku
authors: Gilead Sciences, Inc., Foster City, California, USA.
authors: Simin
authors: S
authors: Xu
authors: Gilead Sciences, Inc., Foster City, California, USA.
authors: Ross
authors: R
authors: Martin
authors: Gilead Sciences, Inc., Foster City, California, USA.
authors: Michael D
authors: MD
authors: Miller
authors: Gilead Sciences, Inc., Foster City, California, USA.
authors: Hongmei
authors: H
authors: Mo
authors: Gilead Sciences, Inc., Foster City, California, USA evguenia.svarovskaia@gilead.com.
authors: Evguenia
authors: E
authors: Svarovskaia
mesh_terms: D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics* / Q000302:isolation & purification; D006526:Hepatitis C / Q000821:virology; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D012367:RNA, Viral / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D012367:RNA, Viral