{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,17]],"date-time":"2026-01-17T16:46:21Z","timestamp":1768668381325,"version":"3.49.0"},"reference-count":52,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T00:00:00Z","timestamp":1768435200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Guangxi Natural Science Foundation","award":["2025GXNSFBA069111"],"award-info":[{"award-number":["2025GXNSFBA069111"]}]},{"name":"Guangxi Natural Science Foundation","award":["2025GXNSFBA069574"],"award-info":[{"award-number":["2025GXNSFBA069574"]}]},{"name":"Guangxi Municipal and County Scientific Research Project","award":["2024KY1238"],"award-info":[{"award-number":["2024KY1238"]}]},{"name":"Guangxi Municipal and County Scientific Research Project","award":["XKJ2401"],"award-info":[{"award-number":["XKJ2401"]}]},{"name":"First Batch of the Guangxi Medical Young Reserve Talents Training Program"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Background: The enhanced sensitivity of next-generation sequencing (NGS) for assessing hepatitis B virus (HBV) quasispecies heterogeneity over clone-based sequencing (CBS) is well documented. However, its comparative reliability for genotype determination remains an open question. Objective: This study aimed to directly compare the performance of NGS and CBS for genotyping HBV using the entire viral genome. Methods: We selected five challenging clinical samples that previously could not be subgenotyped or showed conflicting results when using direct sequencing of the S open reading frame (ORF). The full HBV genome from these subjects was amplified and then analyzed in parallel by both NGS and CBS. Phylogenetic analysis was subsequently used to assign genotypes. Results: Both methods identified a range of genotypes, including B, C, and I, as well as aberrant and recombinant forms. For three of the five subjects, genotyping results were identical between the two platforms. In the remaining two cases, however, CBS revealed greater complexity, identifying additional subgenotypes and recombinant\/aberrant strains not detected by NGS. Notably, for three individuals, the genotypes determined by both modern methods contradicted earlier results from 2011 based on direct S ORF sequencing. Furthermore, the specific mutations detected were incongruent between the platforms, with CBS identifying a higher number of variants than NGS. Conclusions: Our findings indicate that genotyping results from NGS and CBS can be discordant. Contrary to expectations, CBS may uncover more genetic diversity, including a greater number of subgenotypes and mutations, than NGS in certain contexts. The study also confirms that genotyping based solely on direct sequencing of the S ORF can be unreliable and lead to misclassification.<\/jats:p>","DOI":"10.3390\/v18010112","type":"journal-article","created":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T08:26:36Z","timestamp":1768465596000},"page":"112","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Full-Genome Hepatitis B Virus Genotyping: A Juxtaposition of Next-Generation and Clone-Based Sequencing Approaches\u2014Comparing Genotyping Methods of Hepatitis B Virus"],"prefix":"10.3390","volume":"18","author":[{"given":"Li-Ping","family":"Hu","sequence":"first","affiliation":[{"name":"Guangxi Zhuang Autonomous Region Center for Disease Prevention and Control, Guangxi Key Laboratory for the Prevention and Control of Viral Hepatitis, Nanning 530028, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qin-Yan","family":"Chen","sequence":"additional","affiliation":[{"name":"Guangxi Zhuang Autonomous Region Center for Disease Prevention and Control, Guangxi Key Laboratory for the Prevention and Control of Viral Hepatitis, Nanning 530028, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mei-Lin","family":"Huang","sequence":"additional","affiliation":[{"name":"Guangxi Zhuang Autonomous Region Center for Disease Prevention and Control, Guangxi Key Laboratory for the Prevention and Control of Viral Hepatitis, Nanning 530028, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wen-Jia","family":"Zhang","sequence":"additional","affiliation":[{"name":"Guangxi Zhuang Autonomous Region Center for Disease Prevention and Control, Guangxi Key Laboratory for the Prevention and Control of Viral Hepatitis, Nanning 530028, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiao-Qian","family":"Huang","sequence":"additional","affiliation":[{"name":"Guangxi Zhuang Autonomous Region Center for Disease Prevention and Control, Guangxi Key Laboratory for the Prevention and Control of Viral Hepatitis, Nanning 530028, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xian-Feng","family":"Yi","sequence":"additional","affiliation":[{"name":"The Animal Husbandry Research Institute of Guangxi Zhuang Autonomous Region, Nanning 530010, China"},{"name":"Drug and Food Vocational College, Guangxi Vocational University of Agriculture, Nanning 530007, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hui-Hua","family":"Jia","sequence":"additional","affiliation":[{"name":"The Animal Husbandry Research Institute of Guangxi Zhuang Autonomous Region, Nanning 530010, China"},{"name":"Drug and Food Vocational College, Guangxi Vocational University of Agriculture, Nanning 530007, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1159\/000360947","article-title":"Genotypes and genetic variability of hepatitis B virus","volume":"57","author":"Kramvis","year":"2014","journal-title":"Intervirology"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"854","DOI":"10.1007\/s12072-016-9745-2","article-title":"Hepatitis B virus genotypes: Epidemiological and clinical relevance in Asia","volume":"10","author":"Tian","year":"2016","journal-title":"Hepatol. Int."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1087","DOI":"10.1002\/jmv.25421","article-title":"Analysis of HBV X gene quasispecies characteristics by next-generation sequencing and cloning-based sequencing and its association with hepatocellular carcinoma progression","volume":"91","author":"Mei","year":"2019","journal-title":"J. Med. Virol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"947","DOI":"10.1111\/j.1469-0691.2010.03374.x","article-title":"HBV subgenotype misclassification expands quasi-subgenotype A3","volume":"17","author":"Pourkarim","year":"2011","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1007\/s11262-008-0205-5","article-title":"Complete genomic sequence and phylogenetic relatedness of hepatitis B virus isolates in Cambodia","volume":"36","author":"Huy","year":"2008","journal-title":"Virus Genes"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"972","DOI":"10.1099\/jgv.0.001147","article-title":"Genetic diversity of hepatitis B virus in Yunnan, China: Identification of novel subgenotype C17, an intergenotypic B\/I recombinant, and B\/C recombinants","volume":"101","author":"Feng","year":"2020","journal-title":"J. General. Virol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"104322","DOI":"10.1016\/j.meegid.2020.104322","article-title":"Characterization of a novel hepatitis B virus subgenotype B10 among chronic hepatitis B patients in Yunnan, China","volume":"83","author":"Liu","year":"2020","journal-title":"Infect. Genet. Evol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1016\/j.ijid.2020.01.048","article-title":"Novel hepatitis B virus subgenotype A8 and quasi-subgenotype D12 in African\u2013Belgian chronic carriers","volume":"93","author":"Thijssen","year":"2020","journal-title":"Int. J. Infect. Dis."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"a021436","DOI":"10.1101\/cshperspect.a021436","article-title":"Hepatitis B virus genotypes and variants","volume":"5","author":"Lin","year":"2015","journal-title":"Cold Spring Harb. Perspect. Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"347","DOI":"10.3138\/canlivj-2023-0003","article-title":"Hepatitis B virus genotypes influence clinical outcomes: A review","volume":"6","author":"Keeshan","year":"2023","journal-title":"Can. Liver J."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1016\/j.bpg.2017.04.010","article-title":"Natural history of acute and chronic hepatitis B: The role of HBV genotypes and mutants","volume":"31","author":"Lin","year":"2017","journal-title":"Best. Pract. Res. Clin. Gastroenterol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"e941","DOI":"10.1016\/j.ijid.2010.03.020","article-title":"Hepatitis B virus genotyping: Current methods and clinical implications","volume":"14","author":"Guirgis","year":"2010","journal-title":"Int. J. Infect. Dis."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1002\/rmv.400","article-title":"Hepatitis B virus genotypes: Comparison of genotyping methods","volume":"14","author":"Bartholomeusz","year":"2004","journal-title":"Rev. Med. Virol."},{"key":"ref_14","first-page":"79","article-title":"Comparison of PCR-based genotyping methods for hepatitis B virus","volume":"29","author":"Lim","year":"2007","journal-title":"Malays. J. Pathol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1038\/nrg.2016.49","article-title":"Coming of age: Ten years of next-generation sequencing technologies","volume":"17","author":"Goodwin","year":"2016","journal-title":"Nat. Rev. Genet."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"101421","DOI":"10.1016\/j.rineng.2023.101421","article-title":"Challenges in clinical interpretation of next-generation sequencing data: Advantages and Pitfalls","volume":"20","author":"Karakoyun","year":"2023","journal-title":"Results Eng."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"4087","DOI":"10.1128\/JCM.01723-13","article-title":"Comparison of next-generation sequencing and clone-based sequencing in analysis of hepatitis B virus reverse transcriptase quasispecies heterogeneity","volume":"51","author":"Gong","year":"2013","journal-title":"J. Clin. Microbiol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"3572","DOI":"10.1017\/S0950268815000242","article-title":"The prevalence of mutations in the major hydrophilic region of the surface antigen of hepatitis B virus varies with subgenotype","volume":"143","author":"Wang","year":"2015","journal-title":"Epidemiol. Infect."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1099\/vir.0.026666-0","article-title":"A complex hepatitis B virus (X\/C) recombinant is common in Long An county, Guangxi and may have originated in southern China","volume":"92","author":"Fang","year":"2011","journal-title":"J. General. Virol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"799","DOI":"10.1002\/jmv.23533","article-title":"Hepatitis B virus candidate subgenotype I1 varies in distribution throughout Guangxi, China and may have originated in Long An county, Guangxi","volume":"85","author":"Li","year":"2013","journal-title":"J. Med. Virol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"878","DOI":"10.1002\/jmv.23278","article-title":"Prevalence of hepatitis B virus infection in a highly endemic area of southern China after catch-up immunization","volume":"84","author":"Fang","year":"2012","journal-title":"J. Med. Virol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"5437","DOI":"10.1128\/jvi.69.9.5437-5444.1995","article-title":"A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients","volume":"69","author":"Li","year":"1995","journal-title":"J. Virol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"3068","DOI":"10.1128\/AAC.01318-07","article-title":"Rolling circle amplification, a powerful tool for genetic and functional studies of complete hepatitis B virus genomes from low-level infections and for directly probing covalently closed circular DNA","volume":"52","author":"Margeridon","year":"2008","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2719","DOI":"10.1016\/S0021-9258(18)53833-3","article-title":"Fidelity of phi 29 DNA polymerase. Comparison between protein-primed initiation and DNA polymerization","volume":"268","author":"Esteban","year":"1993","journal-title":"J. Biol. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"531","DOI":"10.1016\/j.tibtech.2003.09.010","article-title":"Whole genome amplification: Abundant supplies of DNA from precious samples or clinical specimens","volume":"21","author":"Lasken","year":"2003","journal-title":"Trends Biotechnol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"i884","DOI":"10.1093\/bioinformatics\/bty560","article-title":"fastp: An ultra-fast all-in-one FASTQ preprocessor","volume":"34","author":"Chen","year":"2018","journal-title":"Bioinformatics"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1038\/nmeth.1923","article-title":"Fast gapped-read alignment with Bowtie 2","volume":"9","author":"Langmead","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1754","DOI":"10.1093\/bioinformatics\/btp324","article-title":"Fast and accurate short read alignment with Burrows\u2013Wheeler transform","volume":"25","author":"Li","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e102","DOI":"10.1093\/nar\/gkab576","article-title":"Accurate assembly of minority viral haplotypes from next-generation sequencing through efficient noise reduction","volume":"49","author":"Knyazev","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2032","DOI":"10.1093\/bioinformatics\/btv098","article-title":"Sambamba: Fast processing of NGS alignment formats","volume":"31","author":"Tarasov","year":"2015","journal-title":"Bioinformatics"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1016\/B978-0-12-394585-3.00015-8","article-title":"Analysis of deoxyribonucleic acid (DNA) sequence data using BioEdit","volume":"Volume 15","author":"Elkins","year":"2013","journal-title":"Forensic DNA Biology: A Laboratory Manual"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1870","DOI":"10.1093\/molbev\/msw054","article-title":"Molecular evolutionary genetics analysis version 7.0 for bigger datasets","volume":"33","author":"Kumar","year":"2016","journal-title":"Mol. Biol. Evol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"381","DOI":"10.1080\/14737159.2021.1913055","article-title":"Next-generation sequencing for the diagnosis of hepatitis B: Current status and future prospects","volume":"21","author":"Cortese","year":"2021","journal-title":"Expert. Rev. Mol. Diagn."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1128\/JCM.02229-15","article-title":"Implementation of next-generation sequencing for hepatitis B virus resistance testing and genotyping in a clinical microbiology laboratory","volume":"54","author":"Lowe","year":"2016","journal-title":"J. Clin. Microbiol."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Hebeler-Barbosa, F., Wolf, I.R., Valente, G.T., Mello, F.C.d.A., Lampe, E., Pardini, M.I.d.M.C., and Grotto, R.M.T. (2020). A new method for next-generation sequencing of the full hepatitis B virus genome from a clinical specimen: Impact for virus genotyping. Microorganisms, 8.","DOI":"10.3390\/microorganisms8091391"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"618","DOI":"10.1038\/s41575-020-0296-6","article-title":"The evolution and clinical impact of hepatitis B virus genome diversity","volume":"17","author":"Revill","year":"2020","journal-title":"Nat. Rev. Gastroenterol. Hepatol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1281","DOI":"10.1016\/j.jhep.2017.07.011","article-title":"How viral genetic variants and genotypes influence disease and treatment outcome of chronic hepatitis B. Time for an individualised approach?","volume":"67","author":"Rajoriya","year":"2017","journal-title":"J. Hepatol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1093\/bib\/bbaa101","article-title":"Epidemiological data analysis of viral quasispecies in the next-generation sequencing era","volume":"22","author":"Knyazev","year":"2021","journal-title":"Brief. Bioinform."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"199317","DOI":"10.1016\/j.virusres.2024.199317","article-title":"The dynamic variation position and predominant quasispecies of hepatitis B virus: Novel predictors of early hepatocarcinoma","volume":"341","author":"Zhang","year":"2024","journal-title":"Virus Res."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"110197","DOI":"10.1016\/j.virol.2024.110197","article-title":"Evolution and diversity of the hepatitis B virus genome: Clinical implications","volume":"598","author":"Xie","year":"2024","journal-title":"Virology"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"105178","DOI":"10.1016\/j.antiviral.2021.105178","article-title":"Early dynamic changes of quasispecies in the reverse transcriptase region of hepatitis B virus in telbivudine treatment","volume":"195","author":"Tang","year":"2021","journal-title":"Antivir. Res."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"e28615","DOI":"10.1002\/jmv.28612","article-title":"Nucleos(t)ide analogues altered quasispecies composition of hepatitis B virus (HBV)-resistant mutations in serum HBV DNA and serum HBV RNA","volume":"95","author":"Liao","year":"2023","journal-title":"J. Med. Virol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"106291","DOI":"10.1016\/j.antiviral.2025.106291","article-title":"Pretreatment viral quasispecies characteristics and evolutionary phases correlate with HBsAg seroconversion in peginterferon-alfa-2a-treated children with HBeAg-positive chronic hepatitis B","volume":"244","author":"Yang","year":"2025","journal-title":"Antivir. Res."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"7152","DOI":"10.3748\/wjg.v20.i23.7152","article-title":"Molecular identification of hepatitis B virus genotypes\/subgenotypes: Revised classification hurdles and updated resolutions","volume":"20","author":"Pourkarim","year":"2014","journal-title":"World J. Gastroenterol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1016\/j.meegid.2013.03.021","article-title":"Hepatitis B virus subgenotyping: History, effects of recombination, misclassifications, and corrections","volume":"16","author":"Shi","year":"2013","journal-title":"Infect. Genet. Evol."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Fu, Y., Zeng, Y., Chen, T., Chen, H., Lin, N., Lin, J., Liu, X., Huang, E., Wu, S., and Wu, S. (2019). Characterization and clinical significance of natural variability in hepatitis B virus reverse transcriptase in treatment-naive Chinese patients by Sanger sequencing and next-generation sequencing. J. Clin. Microbiol., 57.","DOI":"10.1128\/JCM.00119-19"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1708","DOI":"10.3748\/wjg.v24.i16.1708","article-title":"Naturally occurring hepatitis B virus reverse transcriptase mutations related to potential antiviral drug resistance and liver disease progression","volume":"24","author":"Choi","year":"2018","journal-title":"World J. Gastroenterol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"4985","DOI":"10.3748\/wjg.v25.i33.4985","article-title":"Tenofovir is a more suitable treatment than entecavir for chronic hepatitis B patients carrying naturally occurring rtM204I mutations","volume":"25","author":"Choe","year":"2019","journal-title":"World J. Gastroenterol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"220","DOI":"10.1186\/s12985-025-02829-0","article-title":"Conserved recombination patterns across hepatitis B genotypes: A retrospective study","volume":"22","author":"Tshiabuila","year":"2025","journal-title":"Virol. J."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"e01477","DOI":"10.1016\/j.heliyon.2019.e01477","article-title":"A novel hepatitis B virus recombinant genotype D4\/E identified in a South African population","volume":"5","author":"Matlou","year":"2019","journal-title":"Heliyon"},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Locarnini, S.A., Littlejohn, M., and Yuen, L.K.W. (2021). Origins and Evolution of the Primate Hepatitis B Virus. Front. Microbiol., 12.","DOI":"10.3389\/fmicb.2021.653684"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"e22358","DOI":"10.1016\/j.heliyon.2023.e22358","article-title":"Characterization of Hepatitis B virus based complete genome analysis improves molecular surveillance and enables identification of a recombinant C\/D strain in the Netherlands","volume":"9","author":"Cremer","year":"2023","journal-title":"Heliyon"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/1\/112\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,17]],"date-time":"2026-01-17T05:25:46Z","timestamp":1768627546000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/18\/1\/112"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,15]]},"references-count":52,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["v18010112"],"URL":"https:\/\/doi.org\/10.3390\/v18010112","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,15]]}}}