Comparison of next generation sequencing technologies for the comprehensive assessment of full-length hepatitis C viral genomes

Thomson, E. et al. (2016) Comparison of next generation sequencing technologies for the comprehensive assessment of full-length hepatitis C viral genomes. Journal of Clinical Microbiology, 54(10), pp. 2470-2484. (doi:10.1128/JCM.00330-16) (PMID:27385709) (PMCID:PMC5035407)

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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 anti-viral (DAA) therapies. This study compared the ability of high-throughput NGS methods to generate full-length, deep, HCV viral sequence datasets and evaluated their utility for diagnostics and clinical assessment.NGS methods using (1) unselected HCV RNA (metagenomic); (2) pre-enrichment of HCV RNA by probe capture and (3) HCV pre-amplification by PCR implemented in four UK centres were compared. Metrics of sequence coverage and depth, quasispecies diversity and detection of DAA-resistance associated variants (RAVs), mixed HCV genotype and other co-infections were compared using a panel of samples of varying viral load, genotype and mixed HCV geno(sub)types. Each NGS method generated near complete genome sequences from over 90% of samples. Enrichment methods and PCR pre-amplification generated greater sequence depth and were more effective for low viral load samples. 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 co-infections. 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 genotype and information on potential DAA resistance.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Hinds, Mr Christopher and Adamson, Dr Walt and Thomson, Dr Emma and Vattipally, Dr Sreenu and Da Silva Filipe, Dr Ana and McLauchlan, Professor John
Authors: Thomson, E., Ip, C. L.C., Badhan, A., Christiansen, M. T., Adamson, W., Ansari, M. A., Bibby, D., Breuer, J., Brown, A., Bowden, R., Bryant, J., Bonsall, D., Da Silva Filipe, A., Hinds, C., Hudson, E., Klenerman, P., Lythgow, K., Mbisa, J. L., McLauchlan, J., Myers, R., Piazza, P., Roy, S., Trebes, A., Sreenu, V. B., Witteveldt, J., Barnes, E., and Simmonds, P.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:Journal of Clinical Microbiology
Publisher:American Society for Microbiology
ISSN:0095-1137
ISSN (Online):1098-660X
Published Online:06 July 2016
Copyright Holders:Copyright © 2016 Thomson et al.
First Published:First published in Journal of Clinical Microbiology 2016
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
644641Establishment of a Resource for Long-Term Study of Hepatitis C Virus Infection in the UKJohn McLauchlanMedical Research Foundation (MED RES)C0365MVLS III - CENTRE FOR VIRUS RESEARCH