{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"medRxiv"}],"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T12:52:27Z","timestamp":1768567947521,"version":"3.49.0"},"posted":{"date-parts":[[2024,1,20]]},"group-title":"Genetic and Genomic Medicine","reference-count":55,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2024,1,25]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Chronic hepatitis B virus (HBV) infection remains a significant public health concern, particularly in Africa, where there is a substantial burden. HBV is an enveloped virus, with isolates being classified into ten phylogenetically distinct genotypes (A \u2013 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.<\/jats:p>","DOI":"10.1101\/2024.01.19.24301519","type":"posted-content","created":{"date-parts":[[2024,1,20]],"date-time":"2024-01-20T11:35:14Z","timestamp":1705750514000},"source":"Crossref","is-referenced-by-count":1,"title":["An Oxford Nanopore Technology-Based Hepatitis B Virus Sequencing Protocol Suitable For Genomic Surveillance Within Clinical Diagnostic Settings"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5221-2126","authenticated-orcid":false,"given":"Derek","family":"Tshiabuila","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7606-0569","authenticated-orcid":false,"given":"Wonderful","family":"Choga","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5736-664X","authenticated-orcid":false,"given":"San E.","family":"James","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6876-3712","authenticated-orcid":false,"given":"Tongai","family":"Maponga","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0254-7910","authenticated-orcid":false,"given":"Wolfgang","family":"Preiser","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3021-5101","authenticated-orcid":false,"given":"Gert","family":"van Zyl","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1095-1910","authenticated-orcid":false,"given":"Monika","family":"Moir","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2655-8518","authenticated-orcid":false,"given":"Stephanie","family":"van Wyk","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2944-4583","authenticated-orcid":false,"given":"Jennifer","family":"Giandhari","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9288-7996","authenticated-orcid":false,"given":"Sureshnee","family":"Pillay","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ugochukwu J.","family":"Anyaneji","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0926-710X","authenticated-orcid":false,"given":"Richard J.","family":"Lessells","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9040-963X","authenticated-orcid":false,"given":"Yeshnee","family":"Naidoo","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4154-7067","authenticated-orcid":false,"given":"Tomasz Janusz","family":"Sanko","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2503-9441","authenticated-orcid":false,"given":"Eduan","family":"Wilkinson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Houriiyah","family":"Tegally","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6033-7655","authenticated-orcid":false,"given":"Cheryl","family":"Baxter","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8785-0870","authenticated-orcid":false,"given":"Darren P.","family":"Martin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3027-5254","authenticated-orcid":false,"given":"Tulio","family":"de Oliveira","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"issue":"10","key":"2024012902100480000_2024.01.19.24301519v2.1","doi-asserted-by":"crossref","first-page":"879","DOI":"10.1016\/S2468-1253(23)00197-8","article-title":"Global prevalence, cascade of care, and prophylaxis coverage of hepatitis B in 2022: a modelling study","volume":"8","year":"2023","journal-title":"Lancet Gastroenterol Hepatol"},{"key":"2024012902100480000_2024.01.19.24301519v2.2","doi-asserted-by":"publisher","DOI":"10.1136\/bmjopen-2019-030211"},{"issue":"9","key":"2024012902100480000_2024.01.19.24301519v2.3","doi-asserted-by":"crossref","first-page":"796","DOI":"10.1016\/S2468-1253(22)00124-8","article-title":"Global, regional, and national burden of hepatitis B, 1990\u20132019: a systematic analysis for the Global Burden of Disease Study 2019","volume":"7","year":"2022","journal-title":"The Lancet Gastroenterology & Hepatology"},{"key":"2024012902100480000_2024.01.19.24301519v2.4","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2893.1997.tb00155.x"},{"key":"2024012902100480000_2024.01.19.24301519v2.5","doi-asserted-by":"crossref","unstructured":"Inoue J , Nakamura T , Masamune A . Roles of Hepatitis B Virus Mutations in the Viral Reactivation after Immunosuppression Therapies. Viruses. 2019;11(5).","DOI":"10.3390\/v11050457"},{"key":"2024012902100480000_2024.01.19.24301519v2.6","doi-asserted-by":"publisher","DOI":"10.1111\/j.1872-034X.2009.00601.x"},{"issue":"10","key":"2024012902100480000_2024.01.19.24301519v2.7","doi-asserted-by":"crossref","first-page":"495","DOI":"10.3390\/genes9100495","article-title":"The global hepatitis B virus genotype distribution approximated from available genotyping data","volume":"9","year":"2018","journal-title":"Genes"},{"key":"2024012902100480000_2024.01.19.24301519v2.8","first-page":"17","article-title":"Molecular characteristics and clinical relevance of African genotypes and subgenotypes of hepatitis B virus","volume":"108","year":"2018","journal-title":"SAMJ: South African Medical Journal"},{"issue":"9","key":"2024012902100480000_2024.01.19.24301519v2.9","doi-asserted-by":"crossref","first-page":"1939","DOI":"10.3390\/v14091939","article-title":"Hepatitis B Virus Research in South Africa","volume":"14","year":"2022","journal-title":"Viruses"},{"key":"2024012902100480000_2024.01.19.24301519v2.10","doi-asserted-by":"crossref","unstructured":"Abe H , Ushijima Y , Bikangui R , Ondo GN , Pemba CM , Zadeh VR , et al. Genetic Diversity of Hepatitis B and C Viruses Revealed by Continuous Surveillance from 2015 to 2021 in Gabon, Central Africa. Microorganisms. 2023;11(8).","DOI":"10.3390\/microorganisms11082046"},{"issue":"10","key":"2024012902100480000_2024.01.19.24301519v2.11","doi-asserted-by":"crossref","first-page":"e0240375","DOI":"10.1371\/journal.pone.0240375","article-title":"Global and regional dispersal patterns of hepatitis B virus genotype E from and in Africa: A full-genome molecular analysis","volume":"15","year":"2020","journal-title":"PLOS ONE"},{"key":"2024012902100480000_2024.01.19.24301519v2.12","unstructured":"Jennifer G , Sureshnee P , Eduan W , Houriiyah T , Ilya S , Maria S , et al. Early transmission of SARS-CoV-2 in South Africa: An epidemiological and phylogenetic report. medRxiv. 2020:2020.05.29.20116376."},{"issue":"11","key":"2024012902100480000_2024.01.19.24301519v2.13","doi-asserted-by":"crossref","first-page":"1403","DOI":"10.25122\/jml-2022-0051","article-title":"Comparison of different rapid screening tests and ELISA for HBV, HCV, and HIV among healthy blood donors and recipients at Jibla University Hospital Yemen","volume":"15","year":"2022","journal-title":"J Med Life"},{"key":"2024012902100480000_2024.01.19.24301519v2.14","doi-asserted-by":"publisher","DOI":"10.1128\/cmr.00064-18"},{"key":"2024012902100480000_2024.01.19.24301519v2.15","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.02011-08"},{"key":"2024012902100480000_2024.01.19.24301519v2.16","doi-asserted-by":"publisher","DOI":"10.1016\/j.jcv.2014.06.013"},{"issue":"12","key":"2024012902100480000_2024.01.19.24301519v2.17","doi-asserted-by":"crossref","first-page":"e0144816","DOI":"10.1371\/journal.pone.0144816","article-title":"Complex genotype mixtures analyzed by deep sequencing in two different regions of hepatitis B virus","volume":"10","year":"2015","journal-title":"PLoS One"},{"key":"2024012902100480000_2024.01.19.24301519v2.18","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.02229-15"},{"issue":"10","key":"2024012902100480000_2024.01.19.24301519v2.19","doi-asserted-by":"crossref","first-page":"2885","DOI":"10.1007\/s00705-016-2959-8","article-title":"Hepatitis B virus resistance substitutions: long-term analysis by next-generation sequencing","volume":"161","year":"2016","journal-title":"Archives of virology"},{"issue":"2","key":"2024012902100480000_2024.01.19.24301519v2.20","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1016\/j.jviromet.2013.06.015","article-title":"Analysis of hepatitis B virus genotyping and drug resistance gene mutations based on massively parallel sequencing","volume":"193","year":"2013","journal-title":"Journal of virological methods"},{"key":"2024012902100480000_2024.01.19.24301519v2.21","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1007\/s12072-015-9645-x","article-title":"Aligning to the sample-specific reference sequence to optimize the accuracy of next-generation sequencing analysis for hepatitis B virus","volume":"10","year":"2016","journal-title":"Hepatology International"},{"issue":"9","key":"2024012902100480000_2024.01.19.24301519v2.22","doi-asserted-by":"crossref","first-page":"e46345","DOI":"10.1371\/journal.pone.0046345","article-title":"Genotyping and Molecular Characterization of Hepatitis B Virus from Human Immunodeficiency Virus-Infected Individuals in Southern Africa","volume":"7","year":"2012","journal-title":"PLOS ONE"},{"issue":"1","key":"2024012902100480000_2024.01.19.24301519v2.23","doi-asserted-by":"crossref","first-page":"7081","DOI":"10.1038\/s41598-019-43524-9","article-title":"Illumina and Nanopore methods for whole genome sequencing of hepatitis B virus (HBV)","volume":"9","year":"2019","journal-title":"Scientific Reports"},{"issue":"4","key":"2024012902100480000_2024.01.19.24301519v2.24","doi-asserted-by":"crossref","first-page":"lqaa074","DOI":"10.1093\/nargab\/lqaa074","article-title":"Nanopore sequencing of native adeno-associated virus (AAV) single-stranded DNA using a transposase-based rapid protocol","volume":"2","year":"2020","journal-title":"NAR Genomics and Bioinformatics"},{"issue":"4","key":"2024012902100480000_2024.01.19.24301519v2.25","doi-asserted-by":"crossref","first-page":"e0265220","DOI":"10.1371\/journal.pone.0265220","article-title":"Comparison of different sequencing techniques for identification of SARS-CoV-2 variants of concern with multiplex real-time PCR","volume":"17","year":"2022","journal-title":"PLOS ONE"},{"key":"2024012902100480000_2024.01.19.24301519v2.26","doi-asserted-by":"publisher","DOI":"10.1101\/gr.092759.109"},{"issue":"1","key":"2024012902100480000_2024.01.19.24301519v2.27","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1186\/s13027-019-0253-6","article-title":"A meta-analysis on genetic variability of RT\/HBsAg overlapping region of hepatitis B virus (HBV) isolates of Bangladesh","volume":"14","year":"2019","journal-title":"Infectious Agents and Cancer"},{"key":"2024012902100480000_2024.01.19.24301519v2.28","doi-asserted-by":"crossref","first-page":"1040","DOI":"10.3389\/fpls.2020.01040","article-title":"Comparison Between Core Set Selection Methods Using Different Illumina Marker Platforms: A Case Study of Assessment of Diversity in Wheat","volume":"11","year":"2020","journal-title":"Front Plant Sci"},{"issue":"1","key":"2024012902100480000_2024.01.19.24301519v2.29","first-page":"1340","article-title":"Will long-read sequencing technologies replace short-read sequencing technologies in the next 10 years?","volume":"9","year":"2020","journal-title":"Afr J Lab Med"},{"key":"2024012902100480000_2024.01.19.24301519v2.30","doi-asserted-by":"crossref","unstructured":"Gionda PO , Gomes-Gouvea M , Malta FdM , Sebe P , Salles APM , Francisco RdS , et al. Analysis of the complete genome of HBV genotypes F and H found in Brazil and Mexico using the next generation sequencing method. Annals of Hepatology. 2022;27.","DOI":"10.1016\/j.aohep.2021.100569"},{"key":"2024012902100480000_2024.01.19.24301519v2.31","unstructured":"Stuart A , Marcia Maria Da Costa Nunes S , Emmanuel P , Barnabas K , Ana Carolina Gomes J , Jacqueline Farinha S , et al. Extraction-free direct PCR from dried serum spots permits HBV genotyping and RAS identification by Sanger and minION sequencing. bioRxiv. 2019:552539."},{"issue":"3","key":"2024012902100480000_2024.01.19.24301519v2.32","doi-asserted-by":"crossref","first-page":"e0194366","DOI":"10.1371\/journal.pone.0194366","article-title":"Early MinION\u2122 nanopore single-molecule sequencing technology enables the characterization of hepatitis B virus genetic complexity in clinical samples","volume":"13","year":"2018","journal-title":"PLoS One"},{"issue":"8","key":"2024012902100480000_2024.01.19.24301519v2.33","doi-asserted-by":"crossref","first-page":"453","DOI":"10.1038\/nrgastro.2009.107","article-title":"Hepatitis B virus variants","volume":"6","year":"2009","journal-title":"Nature Reviews Gastroenterology & Hepatology"},{"key":"2024012902100480000_2024.01.19.24301519v2.34","doi-asserted-by":"publisher","DOI":"10.1099\/vir.0.19749-0"},{"issue":"1","key":"2024012902100480000_2024.01.19.24301519v2.35","doi-asserted-by":"crossref","first-page":"5723","DOI":"10.1038\/s41598-023-32865-1","article-title":"Mapping hepatitis B virus genotypes on the African continent from 1997 to 2021: a systematic review with meta-analysis","volume":"13","year":"2023","journal-title":"Scientific Reports"},{"issue":"6","key":"2024012902100480000_2024.01.19.24301519v2.36","doi-asserted-by":"crossref","first-page":"e0018223","DOI":"10.1128\/mra.00182-23","article-title":"Hepatitis B Virus Genotype E\/A Recombinants from Blood Donors in Beira","volume":"12","year":"2023","journal-title":"Mozambique. Microbiol Resour Announc"},{"key":"2024012902100480000_2024.01.19.24301519v2.37","doi-asserted-by":"publisher","DOI":"10.1099\/vir.0.046524-0"},{"issue":"3-4","key":"2024012902100480000_2024.01.19.24301519v2.38","doi-asserted-by":"crossref","first-page":"232","DOI":"10.1159\/000361076","article-title":"Hepatitis B virus drug resistance tools: one sequence, two predictions","volume":"57","year":"2014","journal-title":"Intervirology"},{"key":"2024012902100480000_2024.01.19.24301519v2.39","doi-asserted-by":"publisher","DOI":"10.1099\/vir.0.043042-0"},{"issue":"8","key":"2024012902100480000_2024.01.19.24301519v2.40","doi-asserted-by":"crossref","first-page":"e0006629","DOI":"10.1371\/journal.pntd.0006629","article-title":"A systematic review of hepatitis B virus (HBV) drug and vaccine escape mutations in Africa: A call for urgent action","volume":"12","year":"2018","journal-title":"PLOS Neglected Tropical Diseases"},{"issue":"5","key":"2024012902100480000_2024.01.19.24301519v2.41","first-page":"337","article-title":"South African guideline for the management of chronic hepatitis B: 2013","volume":"103","year":"2013","journal-title":"South African Medical Journal"},{"key":"2024012902100480000_2024.01.19.24301519v2.42","doi-asserted-by":"publisher","DOI":"10.1016\/j.jcv.2014.03.018"},{"issue":"7854","key":"2024012902100480000_2024.01.19.24301519v2.43","doi-asserted-by":"crossref","first-page":"438","DOI":"10.1038\/s41586-021-03402-9","article-title":"Detection of a SARS-CoV-2 variant of concern in South Africa","volume":"592","year":"2021","journal-title":"Nature"},{"key":"2024012902100480000_2024.01.19.24301519v2.44","doi-asserted-by":"publisher","DOI":"10.1093\/molbev\/mst010"},{"issue":"1","key":"2024012902100480000_2024.01.19.24301519v2.45","doi-asserted-by":"crossref","first-page":"veaa087","DOI":"10.1093\/ve\/veaa087","article-title":"RDP5: a computer program for analyzing recombination in, and removing signals of recombination from, nucleotide sequence datasets","volume":"7","year":"2021","journal-title":"Virus Evol"},{"key":"2024012902100480000_2024.01.19.24301519v2.46","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/16.6.562"},{"key":"2024012902100480000_2024.01.19.24301519v2.47","doi-asserted-by":"publisher","DOI":"10.1006\/viro.1999.0056"},{"key":"2024012902100480000_2024.01.19.24301519v2.48","doi-asserted-by":"publisher","DOI":"10.1007\/BF00182389"},{"key":"2024012902100480000_2024.01.19.24301519v2.49","doi-asserted-by":"publisher","DOI":"10.1089\/aid.2005.21.98"},{"key":"2024012902100480000_2024.01.19.24301519v2.50","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.241370698"},{"key":"2024012902100480000_2024.01.19.24301519v2.51","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/16.7.573"},{"key":"2024012902100480000_2024.01.19.24301519v2.52","doi-asserted-by":"publisher","DOI":"10.1093\/molbev\/msx263"},{"key":"2024012902100480000_2024.01.19.24301519v2.53","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0003239"},{"key":"2024012902100480000_2024.01.19.24301519v2.54","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btq467"},{"issue":"8","key":"2024012902100480000_2024.01.19.24301519v2.55","doi-asserted-by":"crossref","first-page":"e71131","DOI":"10.1371\/journal.pone.0071131","article-title":"Frequent intra-subtype recombination among HIV-1 circulating in Tanzania","volume":"8","year":"2013","journal-title":"PLoS One"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2024.01.19.24301519","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T19:49:07Z","timestamp":1768506547000},"score":1,"resource":{"primary":{"URL":"http:\/\/medrxiv.org\/lookup\/doi\/10.1101\/2024.01.19.24301519"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,1,20]]},"references-count":55,"URL":"https:\/\/doi.org\/10.1101\/2024.01.19.24301519","relation":{"is-preprint-of":[{"id-type":"doi","id":"10.3390\/ijms252111702","asserted-by":"subject"}]},"subject":[],"published":{"date-parts":[[2024,1,20]]},"subtype":"preprint"}}