title: A method for near full-length amplification and sequencing for six hepatitis C virus genotypes.
abstract: BACKGROUND
  Hepatitis C virus (HCV) is a rapidly evolving RNA virus that has been classified into seven genotypes. All HCV genotypes cause chronic hepatitis, which ultimately leads to liver diseases such as cirrhosis. The genotypes are unevenly distributed across the globe, with genotypes 1 and 3 being the most prevalent. Until recently, molecular epidemiological studies of HCV evolution within the host and at the population level have been limited to the analyses of partial viral genome segments, as it has been technically challenging to amplify and sequence the full-length of the 9.6 kb HCV genome. Although recent improvements have been made in full genome sequencing methodologies, these protocols are still either limited to a specific genotype or cost-inefficient.
  
  
  RESULTS
  In this study we describe a genotype-specific protocol for the amplification and sequencing of the near-full length genome of all six major HCV genotypes. We applied this protocol to 122 HCV positive clinical samples, and had a successful genome amplification rate of 90%, when the viral load was greater than 15,000 IU/ml. The assay was shown to have a detection limit of 1-3 cDNA copies per reaction. The method was tested with both Illumina and PacBio single molecule, real-time (SMRT) sequencing technologies. Illumina sequencing resulted in deep coverage and allowed detection of rare variants as well as HCV co-infection with multiple genotypes. The application of the method with PacBio RS resulted in sequence reads greater than 9 kb that covered the near full-length HCV amplicon in a single read and enabled analysis of the near full-length quasispecies.
  
  
  CONCLUSIONS
  The protocol described herein can be utilised for rapid amplification and sequencing of the near-full length HCV genome in a cost efficient manner suitable for a wide range of applications.
authors.affiliation: School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, 2052, Australia. r.bull@unsw.edu.au.
authors.forename: Rowena A
authors.initials: RA
authors.lastname: Bull
authors.affiliation: School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, 2052, Australia.
authors.forename: Auda A
authors.initials: AA
authors.lastname: Eltahla
authors.affiliation: School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, 2052, Australia.
authors.forename: Chaturaka
authors.initials: C
authors.lastname: Rodrigo
authors.affiliation: Department of Medical Microbiology, Section of Clinical Virology, Academic Medical Center, Public Health Service, Amsterdam, The Netherlands.
authors.forename: Sylvie M
authors.initials: SM
authors.lastname: Koekkoek
authors.affiliation: School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, 2052, Australia.
authors.forename: Melanie
authors.initials: M
authors.lastname: Walker
authors.affiliation: School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, 2052, Australia.
authors.forename: Mehdi R
authors.initials: MR
authors.lastname: Pirozyan
authors.affiliation: School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, 2052, Australia.
authors.forename: Brigid
authors.initials: B
authors.lastname: Betz-Stablein
authors.affiliation: Pacific Biosciences, Menlo Park, CA, USA.
authors.forename: Armin
authors.initials: A
authors.lastname: Toepfer
authors.affiliation: Pacific Biosciences, Menlo Park, CA, USA.
authors.forename: Melissa
authors.initials: M
authors.lastname: Laird
authors.affiliation: Pacific Biosciences, Menlo Park, CA, USA.
authors.forename: Steve
authors.initials: S
authors.lastname: Oh
authors.affiliation: Pacific Biosciences, Menlo Park, CA, USA.
authors.forename: Cheryl
authors.initials: C
authors.lastname: Heiner
authors.affiliation: The Kirby Institute, University of New South Wales, Sydney, Australia.
authors.forename: Lisa
authors.initials: L
authors.lastname: Maher
authors.affiliation: Department of Medical Microbiology, Section of Clinical Virology, Academic Medical Center, Public Health Service, Amsterdam, The Netherlands.
authors.forename: Janke
authors.initials: J
authors.lastname: Schinkel
authors.affiliation: School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, 2052, Australia.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Lloyd
authors.affiliation: School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, 2052, Australia.
authors.forename: Fabio
authors.initials: F
authors.lastname: Luciani
mesh_terms: D060085:Coinfection / Q000175:diagnosis; D016679:Genome, Viral; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000175:diagnosis; D006801:Humans; D057230:Limit of Detection; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010802:Phylogeny; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA / Q000379:methods*; D019562:Viral Load
chemical_list: D012367:RNA, Viral