title: The Influence of Hepatitis C Virus Genetic Region on Phylogenetic Clustering Analysis.
abstract: Sequencing is important for understanding the molecular epidemiology and viral evolution of hepatitis C virus (HCV) infection. To date, there is little standardisation among sequencing protocols, in-part due to the high genetic diversity that is observed within HCV. This study aimed to develop a novel, practical sequencing protocol that covered both conserved and variable regions of the viral genome and assess the influence of each subregion, sequence concatenation and unrelated reference sequences on phylogenetic clustering analysis. The Core to the hypervariable region 1 (HVR1) of envelope-2 (E2) and non-structural-5B (NS5B) regions of the HCV genome were amplified and sequenced from participants from the Australian Trial in Acute Hepatitis C (ATAHC), a prospective study of the natural history and treatment of recent HCV infection. Phylogenetic trees were constructed using a general time-reversible substitution model and sensitivity analyses were completed for every subregion. Pairwise distance, genetic distance and bootstrap support were computed to assess the impact of HCV region on clustering results as measured by the identification and percentage of participants falling within all clusters, cluster size, average patristic distance, and bootstrap value. The Robinson-Foulds metrics was also used to compare phylogenetic trees among the different HCV regions. Our results demonstrated that the genomic region of HCV analysed influenced phylogenetic tree topology and clustering results. The HCV Core region alone was not suitable for clustering analysis; NS5B concatenation, the inclusion of reference sequences and removal of HVR1 all influenced clustering outcome. The Core-E2 region, which represented the highest genetic diversity and longest sequence length in this study, provides an ideal method for clustering analysis to address a range of molecular epidemiological questions.
authors.affiliation: The Kirby Institute, University of New South Wales Australia, Sydney, Australia.
authors.forename: François M J
authors.initials: FM
authors.lastname: Lamoury
authors.affiliation: The Kirby Institute, University of New South Wales Australia, Sydney, Australia.
authors.forename: Brendan
authors.initials: B
authors.lastname: Jacka
authors.affiliation: The Kirby Institute, University of New South Wales Australia, Sydney, Australia.
authors.forename: Sofia
authors.initials: S
authors.lastname: Bartlett
authors.affiliation: Inflammation and Infection Research Centre, School of Medical Sciences, University of New South Wales Australia, Sydney, Australia.
authors.forename: Rowena A
authors.initials: RA
authors.lastname: Bull
authors.affiliation: The Kirby Institute, University of New South Wales Australia, Sydney, Australia.
authors.forename: Arthur
authors.initials: A
authors.lastname: Wong
authors.affiliation: The Kirby Institute, University of New South Wales Australia, Sydney, Australia.
authors.forename: Janaki
authors.initials: J
authors.lastname: Amin
authors.affiliation: Academic Medical Centre, Department of Medical Microbiology, Section of Clinical Virology, Amsterdam, The Netherlands.
authors.forename: Janke
authors.initials: J
authors.lastname: Schinkel
authors.affiliation: BC Centre for Excellence in HIV/AIDS, Vancouver, Canada; Department of Medicine, University of British Columbia, Vancouver, Canada.
authors.forename: Art F
authors.initials: AF
authors.lastname: Poon
authors.affiliation: The Kirby Institute, University of New South Wales Australia, Sydney, Australia.
authors.forename: Gail V
authors.initials: GV
authors.lastname: Matthews
authors.affiliation: The Kirby Institute, University of New South Wales Australia, Sydney, Australia.
authors.forename: Jason
authors.initials: J
authors.lastname: Grebely
authors.affiliation: The Kirby Institute, University of New South Wales Australia, Sydney, Australia; HIV/Immunology/Infectious Diseases Clinical Services Unit, St Vincent's Hospital, Sydney, Australia.
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Dore
authors.affiliation: The Kirby Institute, University of New South Wales Australia, Sydney, Australia.
authors.forename: Tanya L
authors.initials: TL
authors.lastname: Applegate
mesh_terms: D016000:Cluster Analysis; D019143:Evolution, Molecular; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D010802:Phylogeny; D012367:RNA, Viral / Q000235:genetics; D017423:Sequence Analysis, RNA
chemical_list: D012367:RNA, Viral