title: A comparison of 454 sequencing and clonal sequencing for the characterization of hepatitis C virus NS3 variants.
abstract: We compared 454 amplicon sequencing with clonal sequencing for the characterization of intra-host hepatitis C virus (HCV) NS3 variants. Clonal and 454 sequences were obtained from 12 patients enrolled in a clinical phase I study for telaprevir, an NS3-4a protease inhibitor. Thirty-nine datasets were used to compare the consensus sequence, average pairwise distance, normalized Shannon entropy, phylogenetic tree topology and the number and frequency of variants derived from both sequencing techniques. In general, a good concordance was observed between both techniques for the majority of datasets. Discordant results were observed for 5 out of 39 clonal and 454 datasets, which could be attributed to primer-related selective amplification used for clonal sequencing. Both 454 and clonal datasets consisted of a few major variants and a large number of low-frequency variants. Telaprevir resistance-associated variants were observed in low frequencies and were detected more often by 454. We conclude that performance of 454 and clonal sequencing is comparable for the characterization of intra-host virus populations. Not surprisingly, 454 is superior for the detection of low frequency resistance-associated variants. However, despite the greater coverage, 454 failed to detect some low frequency variants detected by clonal sequencing.
authors.affiliation: Department of Medical Microbiology, Academic Medical Center, Amsterdam 1105 AZ, The Netherlands. Electronic address: k.y.ho@amc.uva.nl.
authors.forename: Cynthia K Y
authors.initials: CKY
authors.lastname: Ho
authors.affiliation: Department of Medical Microbiology, Academic Medical Center, Amsterdam 1105 AZ, The Netherlands. Electronic address: m.r.a.welkers@amc.uva.nl.
authors.forename: Matthijs R A
authors.initials: MRA
authors.lastname: Welkers
authors.affiliation: Department of Medical Microbiology, Academic Medical Center, Amsterdam 1105 AZ, The Netherlands. Electronic address: xvthomas86@gmail.com.
authors.forename: Xiomara V
authors.initials: XV
authors.lastname: Thomas
authors.affiliation: Department of Infectious Diseases, Vertex Pharmaceuticals Incorporated, Cambridge, MA 02139, USA. Electronic address: james_sullivan@vrtx.com.
authors.forename: James C
authors.initials: JC
authors.lastname: Sullivan
authors.affiliation: Department of Infectious Diseases, Vertex Pharmaceuticals Incorporated, Cambridge, MA 02139, USA. Electronic address: tara_kieffer@vrtx.com.
authors.forename: Tara L
authors.initials: TL
authors.lastname: Kieffer
authors.affiliation: Department of Gastroenterology and Hepatology, Academic Medical Center, Amsterdam 1104 AZ, The Netherlands. Electronic address: h.w.reesink@amc.uva.nl.
authors.forename: Henk W
authors.initials: HW
authors.lastname: Reesink
authors.affiliation: Department of Medical Microbiology, Academic Medical Center, Amsterdam 1105 AZ, The Netherlands. Electronic address: s.rebers@amc.uva.nl.
authors.forename: Sjoerd P H
authors.initials: SPH
authors.lastname: Rebers
authors.affiliation: Department of Medical Microbiology, Academic Medical Center, Amsterdam 1105 AZ, The Netherlands. Electronic address: m.d.dejong@amc.uva.nl.
authors.forename: Menno D
authors.initials: MD
authors.lastname: de Jong
authors.affiliation: Department of Medical Microbiology, Academic Medical Center, Amsterdam 1105 AZ, The Netherlands. Electronic address: j.schinkel@amc.uva.nl.
authors.forename: Janke
authors.initials: J
authors.lastname: Schinkel
authors.affiliation: Department of Medical Microbiology, Academic Medical Center, Amsterdam 1105 AZ, The Netherlands. Electronic address: r.molenkamp@amc.uva.nl.
authors.forename: Richard
authors.initials: R
authors.lastname: Molenkamp
mesh_terms: D016000:Cluster Analysis; D024882:Drug Resistance, Viral; D019143:Evolution, Molecular; D014644:Genetic Variation; D016174:Hepacivirus / Q000145:classification / Q000187:drug effects / Q000235:genetics*; D006526:Hepatitis C / Q000188:drug therapy / Q000821:virology; D006801:Humans; D010802:Phylogeny; D017422:Sequence Analysis, DNA / Q000379:methods; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
keywords: 454 sequencing; Clonal sequencing; Hepatitis C virus; NS3; Next-generation sequencing; Telaprevir; Viral variants