title: Determination of hepatitis C virus genotype by Pyrosequencing.
abstract: A simple sequencing-based assay is described for genotyping of hepatitis C virus (HCV). RT-PCR was employed to amplify a 237-nucleotide-long fragment from the 5' untranslated region (UTR) of the genome using one biotinylated and one normal primer. Subsequent to capture of the PCR products on streptavidin-coated beads, single-stranded DNA separation, and hybridization of sequencing primer, Pyrosequencing was performed. The genotype of 98 samples out of which 77 samples were from American veterans and 21 samples were from Iran was determined. The samples from the American veterans contained six different subtypes, while five subtypes were found in Iranian samples. For rapid population-specific HCV subtyping, a multiplex assay was developed. This study demonstrates the suitability of this technology for low-cost, high throughput and accurate microbial genotyping.
authors.affiliation: Stanford Genome Technology Center, Stanford University, 855 California Avenue, Palo Alto, CA 94304, USA.
authors.forename: Elahe
authors.initials: E
authors.lastname: Elahi
authors.forename: Nader
authors.initials: N
authors.lastname: Pourmand
authors.forename: Ramsey
authors.initials: R
authors.lastname: Chaung
authors.forename: Ara
authors.initials: A
authors.lastname: Rofoogaran
authors.forename: Judie
authors.initials: J
authors.lastname: Boisver
authors.forename: Katayon
authors.initials: K
authors.lastname: Samimi-Rad
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Davis
authors.forename: Mostafa
authors.initials: M
authors.lastname: Ronaghi
mesh_terms: D020121:5' Untranslated Regions / Q000737:chemistry; D001483:Base Sequence; D004279:DNA, Viral / Q000737:chemistry*; D005838:Genotype; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D020121:5' Untranslated Regions; D004279:DNA, Viral