Mary Wilson
Well-known member
Posted January 20, 2024.
doi: https://doi.org/10.1101/2024.01.19.24301519
Derek Tshiabuila1*#, Wonderful Choga1#, San E. James1,2#, Tongai Maponga3#, Wolfgang Preiser3, Gert van Zyl3, Monika Moir1, Stephanie van Wyk4, Jennifer Giandhari2, Sureshnee Pillay2, Ugochukwu J. Anyaneji2, Richard J. Lessells2, Yeshnee Naidoo1, Tomasz Janusz Sanko1, Eduan Wilkinson1, Houriiyah Tegally1, Cheryl Baxter1, Darren P. Martin5, Tulio de Oliveira1,2
Abstract
Chronic hepatitis B virus (HBV) infection remains a significant public health concern, particularly in Africa, where there is a substantial substantial. HBV is an enveloped virus, with isolates being classified into ten phylogenetically distinct genotypes (A-J) determined based on full-genome sequence data or reverse hybridization-based diagnostic tests. In practice, limitations are noted in that diagnostic sequencing, generally using Sanger sequencing, tends to focus only on the S-gene, yielding little or no information on intra-patient HBV genetic diversity with very low-frequency variants and reverse hybridization detects only known genotype-specific mutations. To resolve these limitations, we developed an Oxford Nanopore Technology (ONT)-based HBV genotyping protocol suitable for clinical virology, yielding complete HBV genome sequences and extensive data on intra-patient HBV diversity. Specifically, the protocol involves tiling-based PCR amplification of HBV sequences, library preparation using the ONT Rapid Barcoding Kit, ONT GridION sequencing, genotyping using Genome Detective software, recombination analysis using jpHMM and RDP5 software, and drug resistance profiling using Geno2pheno software. We prove the utility of our protocol by efficiently generating and characterizing high-quality near full-length HBV genomes from 148 left-over diagnostic Hepatitis B patient samples obtained in the Western Cape province of South Africa, providing valuable insights into the genetic diversity and epidemiology of HBV in this region of the world.
(Download PDF)
https://www.medrxiv.org/content/10.1101/2024.01.19.24301519v1
doi: https://doi.org/10.1101/2024.01.19.24301519
Derek Tshiabuila1*#, Wonderful Choga1#, San E. James1,2#, Tongai Maponga3#, Wolfgang Preiser3, Gert van Zyl3, Monika Moir1, Stephanie van Wyk4, Jennifer Giandhari2, Sureshnee Pillay2, Ugochukwu J. Anyaneji2, Richard J. Lessells2, Yeshnee Naidoo1, Tomasz Janusz Sanko1, Eduan Wilkinson1, Houriiyah Tegally1, Cheryl Baxter1, Darren P. Martin5, Tulio de Oliveira1,2
Abstract
Chronic hepatitis B virus (HBV) infection remains a significant public health concern, particularly in Africa, where there is a substantial substantial. HBV is an enveloped virus, with isolates being classified into ten phylogenetically distinct genotypes (A-J) determined based on full-genome sequence data or reverse hybridization-based diagnostic tests. In practice, limitations are noted in that diagnostic sequencing, generally using Sanger sequencing, tends to focus only on the S-gene, yielding little or no information on intra-patient HBV genetic diversity with very low-frequency variants and reverse hybridization detects only known genotype-specific mutations. To resolve these limitations, we developed an Oxford Nanopore Technology (ONT)-based HBV genotyping protocol suitable for clinical virology, yielding complete HBV genome sequences and extensive data on intra-patient HBV diversity. Specifically, the protocol involves tiling-based PCR amplification of HBV sequences, library preparation using the ONT Rapid Barcoding Kit, ONT GridION sequencing, genotyping using Genome Detective software, recombination analysis using jpHMM and RDP5 software, and drug resistance profiling using Geno2pheno software. We prove the utility of our protocol by efficiently generating and characterizing high-quality near full-length HBV genomes from 148 left-over diagnostic Hepatitis B patient samples obtained in the Western Cape province of South Africa, providing valuable insights into the genetic diversity and epidemiology of HBV in this region of the world.
(Download PDF)
https://www.medrxiv.org/content/10.1101/2024.01.19.24301519v1