title: Comparison of Next-Generation Sequencing Technologies for Comprehensive Assessment of Full-Length Hepatitis C Viral Genomes.
abstract: Affordable next-generation sequencing (NGS) technologies for hepatitis C virus (HCV) may potentially identify both viral genotype and resistance genetic motifs in the era of directly acting antiviral (DAA) therapies. This study compared the ability of high-throughput NGS methods to generate full-length, deep, HCV sequence data sets and evaluated their utility for diagnostics and clinical assessment. NGS methods using (i) unselected HCV RNA (metagenomics), (ii) preenrichment of HCV RNA by probe capture, and (iii) HCV preamplification by PCR implemented in four United Kingdom centers were compared. Metrics of sequence coverage and depth, quasispecies diversity, and detection of DAA resistance-associated variants (RAVs), mixed HCV genotypes, and other coinfections were compared using a panel of samples with different viral loads, genotypes, and mixed HCV genotypes/subtypes [geno(sub)types]. Each NGS method generated near-complete genome sequences from more than 90% of samples. Enrichment methods and PCR preamplification generated greater sequence depth and were more effective for samples with low viral loads. All NGS methodologies accurately identified mixed HCV genotype infections. Consensus sequences generated by different NGS methods were generally concordant, and majority RAVs were consistently detected. However, methods differed in their ability to detect minor populations of RAVs. Metagenomic methods identified human pegivirus coinfections. NGS provided a rapid, inexpensive method for generating whole HCV genomes to define infecting genotypes, RAVs, comprehensive viral strain analysis, and quasispecies diversity. Enrichment methods are particularly suited for high-throughput analysis while providing the genotype and information on potential DAA resistance.
authors.affiliation: MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom.
authors.forename: Emma
authors.initials: E
authors.lastname: Thomson
authors.affiliation: Oxford Genomics Centre, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.
authors.forename: Camilla L C
authors.initials: CL
authors.lastname: Ip
authors.affiliation: Virus Reference Department, Public Health England, London, United Kingdom.
authors.forename: Anjna
authors.initials: A
authors.lastname: Badhan
authors.affiliation: University College London (UCL), Division of Infection and Immunity, London, United Kingdom.
authors.forename: Mette T
authors.initials: MT
authors.lastname: Christiansen
authors.affiliation: MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom.
authors.forename: Walt
authors.initials: W
authors.lastname: Adamson
authors.affiliation: Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: M Azim
authors.initials: MA
authors.lastname: Ansari
authors.affiliation: Virus Reference Department, Public Health England, London, United Kingdom.
authors.forename: David
authors.identifier: 0000-0002-8458-9662
authors.initials: D
authors.lastname: Bibby
authors.affiliation: University College London (UCL), Division of Infection and Immunity, London, United Kingdom.
authors.forename: Judith
authors.initials: J
authors.lastname: Breuer
authors.affiliation: Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: Anthony
authors.initials: A
authors.lastname: Brown
authors.affiliation: Oxford Genomics Centre, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.
authors.forename: Rory
authors.initials: R
authors.lastname: Bowden
authors.affiliation: University College London (UCL), Division of Infection and Immunity, London, United Kingdom.
authors.forename: Josie
authors.identifier: 0000-0002-0149-5768
authors.initials: J
authors.lastname: Bryant
authors.affiliation: Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Bonsall
authors.affiliation: MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom.
authors.forename: Ana
authors.initials: A
authors.lastname: Da Silva Filipe
authors.affiliation: MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom.
authors.forename: Chris
authors.initials: C
authors.lastname: Hinds
authors.affiliation: Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: Emma
authors.initials: E
authors.lastname: Hudson
authors.affiliation: Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: Paul
authors.initials: P
authors.lastname: Klenerman
authors.affiliation: Virus Reference Department, Public Health England, London, United Kingdom.
authors.forename: Kieren
authors.initials: K
authors.lastname: Lythgow
authors.affiliation: Virus Reference Department, Public Health England, London, United Kingdom.
authors.forename: Jean L
authors.initials: JL
authors.lastname: Mbisa
authors.affiliation: MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom.
authors.forename: John
authors.initials: J
authors.lastname: McLauchlan
authors.affiliation: Virus Reference Department, Public Health England, London, United Kingdom.
authors.forename: Richard
authors.initials: R
authors.lastname: Myers
authors.affiliation: Oxford Genomics Centre, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.
authors.forename: Paolo
authors.initials: P
authors.lastname: Piazza
authors.affiliation: University College London (UCL), Division of Infection and Immunity, London, United Kingdom.
authors.forename: Sunando
authors.initials: S
authors.lastname: Roy
authors.affiliation: Oxford Genomics Centre, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom.
authors.forename: Amy
authors.initials: A
authors.lastname: Trebes
authors.affiliation: MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom.
authors.forename: Vattipally B
authors.initials: VB
authors.lastname: Sreenu
authors.affiliation: Roslin Institute, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Witteveldt
authors.affiliation: Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom ellie.barnes@ndm.ox.ac.uk.
authors.forename: Eleanor
authors.initials: E
authors.lastname: Barnes
authors.affiliation: Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom Roslin Institute, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Peter
authors.initials: P
authors.lastname: Simmonds
mesh_terms: D024882:Drug Resistance, Viral; D016679:Genome, Viral; D005838:Genotype; D016174:Hepacivirus / Q000145:classification / Q000235:genetics*; D006526:Hepatitis C / Q000175:diagnosis* / Q000821:virology*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D017422:Sequence Analysis, DNA / Q000379:methods*; D006113:United Kingdom