Implementation of Next-Generation Sequencing for Hepatitis B Virus Resistance Testing and Genotyping in a Clinical Microbiology Laboratory

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 limitation...

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Bibliographic Details
Published in:Journal of clinical microbiology 2016-01, Vol.54 (1), p.127-133
Main Authors: Lowe, Christopher F, Merrick, Linda, Harrigan, P Richard, Mazzulli, Tony, Sherlock, Christopher H, Ritchie, Gordon
Format: Article
Language:English
Subjects:
Costs and Cost Analysis
DNA, Viral - chemistry
DNA, Viral - genetics
Genotyping Techniques - methods
Hepatitis B virus
Hepatitis B virus - drug effects
Hepatitis B virus - genetics
Hepatitis B virus - isolation & purification
Hepatitis B, Chronic - virology
High-Throughput Nucleotide Sequencing - methods
Humans
Microbial Sensitivity Tests - methods
Mutation
Polymerase Chain Reaction
Reproducibility of Results
Virology
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Summary: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
ISSN:0095-1137
1098-660X
DOI:10.1128/jcm.02229-15