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Oxford Nanopore Technologies allows for real-time viral sequencing on a local laptop system for point-of-care testing. Seoul orthohantavirus (Seoul virus, SEOV), harbored by Rattus norvegicus and R. rattus, causes mild hemorrhagic fever with renal syndrome and poses an important threat to public health worldwide. We evaluated the deployable MinION system to obtain high-fidelity entire-length sequences of SEOV for the genome identification of accurate infectious sources and their genetic diversity. One-step amplicon-based nanopore sequencing was performed from SEOV 80\u201339 specimens with different viral copy numbers and SEOV-positive wild rats. The KU-ONT-SEOV-consensus module was developed to analyze SEOV genomic sequences generated from the nanopore system. Using amplicon-based nanopore sequencing and the KU-ONT-consensus pipeline, we demonstrated novel molecular diagnostics for acquiring full-length SEOV genome sequences, with sufficient read depth in less than 6 h. The consensus sequence accuracy of the SEOV small, medium, and large genomes showed 99.75\u2013100% (for SEOV 80\u201339 isolate) and 99.62\u201399.89% (for SEOV-positive rats) identities. This study provides useful insights into on-site diagnostics based on nanopore technology and the genome epidemiology of orthohantaviruses for a quicker response to hantaviral outbreaks.<\/jats:p>","DOI":"10.3390\/v15071542","type":"journal-article","created":{"date-parts":[[2023,7,13]],"date-time":"2023-07-13T02:14:48Z","timestamp":1689214488000},"page":"1542","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["A Development of Rapid Whole-Genome Sequencing of Seoul orthohantavirus Using a Portable One-Step Amplicon-Based High Accuracy Nanopore System"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2132-0674","authenticated-orcid":false,"given":"Kyungmin","family":"Park","sequence":"first","affiliation":[{"name":"Department of Microbiology, College of Medicine, Korea University, Seoul 02841, Republic of Korea"},{"name":"BK21 Graduate Program, Department of Biomedical Sciences, Korea University College of Medicine, Seoul 02841, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juyoung","family":"Noh","sequence":"additional","affiliation":[{"name":"Department of Microbiology, College of Medicine, Korea University, Seoul 02841, Republic of Korea"},{"name":"BK21 Graduate Program, Department of Biomedical Sciences, Korea University College of Medicine, Seoul 02841, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kijin","family":"Kim","sequence":"additional","affiliation":[{"name":"Centre for Infectious Disease Genomics and One Health, Faculty of Health Sciences, Simon Fraser University, Burnaby, BC V5A 1S6, Canada"},{"name":"Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3543-9713","authenticated-orcid":false,"given":"Jongwoo","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Microbiology, College of Medicine, Korea University, Seoul 02841, Republic of Korea"},{"name":"BK21 Graduate Program, Department of Biomedical Sciences, Korea University College of Medicine, Seoul 02841, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hee-Kyung","family":"Cho","sequence":"additional","affiliation":[{"name":"Department of Microbiology, College of Medicine, Korea University, Seoul 02841, Republic of Korea"},{"name":"BK21 Graduate Program, Department of Biomedical Sciences, Korea University College of Medicine, Seoul 02841, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seong-Gyu","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Microbiology, College of Medicine, Korea University, Seoul 02841, Republic of Korea"},{"name":"BK21 Graduate Program, Department of Biomedical Sciences, Korea University College of Medicine, Seoul 02841, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eunyoung","family":"Yang","sequence":"additional","affiliation":[{"name":"Department of Microbiology, College of Medicine, Korea University, Seoul 02841, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3178-8569","authenticated-orcid":false,"given":"Won-Keun","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Microbiology, College of Medicine, Hallym University, Chuncheon 24252, Republic of Korea"},{"name":"Institute of Medical Research, College of Medicine, Hallym University, Chuncheon 24252, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0796-8332","authenticated-orcid":false,"given":"Jin-Won","family":"Song","sequence":"additional","affiliation":[{"name":"Department of Microbiology, College of Medicine, Korea University, Seoul 02841, Republic of Korea"},{"name":"BK21 Graduate Program, Department of Biomedical Sciences, Korea University College of Medicine, Seoul 02841, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,7,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1002\/rmv.1722","article-title":"Hantavirus infections in Europe and their impact on public health","volume":"23","author":"Vaheri","year":"2013","journal-title":"Rev. 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