title: Implementation of Next-Generation Sequencing for Hepatitis B Virus Resistance Testing and Genotyping in a Clinical Microbiology Laboratory.
abstract: Sanger sequencing or DNA hybridization have been the primary modalities for hepatitis B (HBV) resistance testing and genotyping; however, there are limitations, such as low sensitivity and the inability to detect novel mutations. Next-generation sequencing (NGS) for HBV can overcome these limitations, but there is limited guidance for clinical microbiology laboratories to validate this novel technology. In this study, we describe an approach to implementing deep pyrosequencing for HBV resistance testing and genotyping in a clinical virology laboratory. A nested PCR targeting the pol region of HBV (codons 143 to 281) was developed, and the PCR product was sequenced by the 454 Junior (Roche). Interpretation was performed by ABL TherapyEdge based on European Association for the Study of the Liver (EASL) guidelines. Previously characterized HBV samples by INNO-LiPA (LiPA) were compared to NGS with discordant results arbitrated by Sanger sequencing. Genotyping of 105 distinct samples revealed a concordance of 95.2% (100/105), with Sanger sequencing confirming the NGS result. Resistance testing by NGS was concordant with LiPA in 85% (68/80) of previously characterized samples. Additional mutations were found in 8 samples, which related to the identification of low-level mutant subpopulations present at <10% (6/8). To balance the costs of testing for the validation study, reproducibility of the NGS was investigated through an analysis of sequence variants at loci not associated with resistance in a single patient sample. Our validation approach attempts to balance costs with efficient data acquisition.
authors.affiliation: Division of Microbiology and Virology, Providence Health Care, Vancouver, British Columbia, Canada Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada clowe@providencehealth.bc.ca.
authors.forename: Christopher F
authors.initials: CF
authors.lastname: Lowe
authors.affiliation: Division of Microbiology and Virology, Providence Health Care, Vancouver, British Columbia, Canada.
authors.forename: Linda
authors.initials: L
authors.lastname: Merrick
authors.affiliation: BC Centre for Excellence in HIV/AIDS, Vancouver, British Columbia, Canada.
authors.forename: P Richard
authors.initials: PR
authors.lastname: Harrigan
authors.affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Department of Microbiology, Mount Sinai Hospital/University Health Network, Toronto, Ontario, Canada.
authors.forename: Tony
authors.initials: T
authors.lastname: Mazzulli
authors.affiliation: Division of Microbiology and Virology, Providence Health Care, Vancouver, British Columbia, Canada Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Christopher H
authors.initials: CH
authors.lastname: Sherlock
authors.affiliation: Division of Microbiology and Virology, Providence Health Care, Vancouver, British Columbia, Canada Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Gordon
authors.initials: G
authors.lastname: Ritchie
mesh_terms: D003365:Costs and Cost Analysis; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D060005:Genotyping Techniques / Q000379:methods*; D006515:Hepatitis B virus / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D019694:Hepatitis B, Chronic / Q000821:virology*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D009154:Mutation; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results
chemical_list: D004279:DNA, Viral