title: Ultradeep pyrosequencing of hepatitis C virus hypervariable region 1 in quasispecies analysis.
abstract: Genetic variability of hepatitis C virus (HCV) determines pathogenesis of infection, including viral persistence and resistance to treatment. The aim of the present study was to characterize HCV genetic heterogeneity within a hypervariable region 1 (HVR1) of a chronically infected patient by ultradeep 454 sequencing strategy. Three independent sequencing error correction methods were applied. First correction method (Method I) implemented cut-off for genetic variants present in less than 1%. In the second method (Method II), a condition to call a variant was bidirectional coverage of sequencing reads. Third method (Method III) used Short Read Assembly into Haplotypes (ShoRAH) program. After the application of these three different algorithms, HVR1 population consisted of 8, 40, and 186 genetic haplotypes. The most sensitive method was ShoRAH, allowing to reconstruct haplotypes constituting as little as 0.013% of the population. The most abundant genetic variant constituted only 10.5%. Seventeen haplotypes were present in a frequency above 1%, and there was wide dispersion of the population into very sparse haplotypes. Our results indicate that HCV HVR1 heterogeneity and quasispecies population structure may be reconstructed by ultradeep sequencing. However, credible analysis requires proper reconstruction methods, which would distinguish sequencing error from real variability in vivo.
authors.affiliation: Department of Immunopathology of Infectious and Parasitic Diseases, Medical University of Warsaw, 3c Pawińskiego Street, 02-106 Warsaw, Poland. kcaraballo@wum.edu.pl
authors.forename: Kamila
authors.initials: K
authors.lastname: Caraballo Cortés
authors.forename: Osvaldo
authors.initials: O
authors.lastname: Zagordi
authors.forename: Tomasz
authors.initials: T
authors.lastname: Laskus
authors.forename: Rafał
authors.initials: R
authors.lastname: Płoski
authors.forename: Iwona
authors.initials: I
authors.lastname: Bukowska-Ośko
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Pawełczyk
authors.forename: Hanna
authors.initials: H
authors.lastname: Berak
authors.forename: Marek
authors.initials: M
authors.lastname: Radkowski
mesh_terms: D014644:Genetic Variation; D016174:Hepacivirus / Q000235:genetics* / Q000472:pathogenicity; D006526:Hepatitis C / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D013045:Species Specificity; D014759:Viral Envelope Proteins / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics*
chemical_list: C400562:HVR1 protein, Hepatitis C virus; D014759:Viral Envelope Proteins; D014764:Viral Proteins