{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T02:21:34Z","timestamp":1760235694798,"version":"build-2065373602"},"reference-count":41,"publisher":"MDPI AG","issue":"18","license":[{"start":{"date-parts":[[2021,9,17]],"date-time":"2021-09-17T00:00:00Z","timestamp":1631836800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["31970153, 3191101787"],"award-info":[{"award-number":["31970153, 3191101787"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Influenza viruses still pose a serious threat to humans, and we have not yet been able to effectively predict future pandemic strains and prepare vaccines in advance. One of the main reasons is the high genetic diversity of influenza viruses. We do not know the individual clonotypes of a virus population because some are the majority and others make up only a small fraction of the population. First-generation (FGS) and next-generation sequencing (NGS) technologies have inherent limitations that are unable to resolve a minority clonotype\u2019s information in the virus population. Third-generation sequencing (TGS) technologies with ultra-long reads have the potential to solve this problem but have a high error rate. Here, we evaluated emerging direct RNA sequencing and cDNA sequencing with the MinION platform and established a novel approach that combines the high accuracy of Illumina sequencing technology and long reads of nanopore sequencing technology to resolve both variants and clonotypes of influenza virus. Furthermore, a new program was written to eliminate the effect of nanopore sequencing errors for the analysis of the results. By using this pipeline, we identified 47 clonotypes in our experiment. We conclude that this approach can quickly discriminate the clonotypes of virus genes, allowing researchers to understand virus adaptation and evolution at the population level.<\/jats:p>","DOI":"10.3390\/ijms221810069","type":"journal-article","created":{"date-parts":[[2021,9,17]],"date-time":"2021-09-17T21:23:29Z","timestamp":1631913809000},"page":"10069","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Discriminating Clonotypes of Influenza A Virus Genes by Nanopore Sequencing"],"prefix":"10.3390","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4289-452X","authenticated-orcid":false,"given":"Ying","family":"Cao","sequence":"first","affiliation":[{"name":"National Virus Resource Center, Chinese Academy of Sciences, Wuhan 430071, China"},{"name":"CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China"},{"name":"State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources & Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning 530004, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4727-088X","authenticated-orcid":false,"given":"Haizhou","family":"Liu","sequence":"additional","affiliation":[{"name":"National Virus Resource Center, Chinese Academy of Sciences, Wuhan 430071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8195-3628","authenticated-orcid":false,"given":"Yi","family":"Yan","sequence":"additional","affiliation":[{"name":"National Virus Resource Center, Chinese Academy of Sciences, Wuhan 430071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wenjun","family":"Liu","sequence":"additional","affiliation":[{"name":"CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China"},{"name":"State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources & Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning 530004, China"},{"name":"Savaid Medical School, University of Chinese Academy of Sciences, Beijing 100039, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Di","family":"Liu","sequence":"additional","affiliation":[{"name":"National Virus Resource Center, Chinese Academy of Sciences, Wuhan 430071, China"},{"name":"Savaid Medical School, University of Chinese Academy of Sciences, Beijing 100039, China"},{"name":"CAS Key Laboratory of Special Pathogens, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan 430071, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9588-5291","authenticated-orcid":false,"given":"Jing","family":"Li","sequence":"additional","affiliation":[{"name":"CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China"},{"name":"Savaid Medical School, University of Chinese Academy of Sciences, Beijing 100039, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,9,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1457","DOI":"10.1093\/oxfordjournals.molbev.a026057","article-title":"Positive selection on the H3 hemagglutinin gene of human influenza virus A","volume":"16","author":"Bush","year":"1999","journal-title":"Mol. Biol. Evol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"4171","DOI":"10.1073\/pnas.90.9.4171","article-title":"Rates of spontaneous mutation among RNA viruses","volume":"90","author":"Drake","year":"1993","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"13910","DOI":"10.1073\/pnas.96.24.13910","article-title":"Mutation rates among RNA viruses","volume":"96","author":"Drake","year":"1999","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"443","DOI":"10.1126\/science.287.5452.443","article-title":"Emerging infectious diseases of wildlife\u2014Threats to biodiversity and human health","volume":"287","author":"Daszak","year":"2000","journal-title":"Science"},{"key":"ref_5","unstructured":"H\u00e4rtl, G. (2017, December 13). Up to 650,000 People Die of Respiratory Diseases Linked to Seasonal Flu each Year. Available online: https:\/\/www.who.int\/mediacentre\/news\/releases\/2017\/seasonal-flu\/zh\/."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1126\/science.1254664","article-title":"Influenza and the Live Poultry Trade","volume":"344","author":"Gao","year":"2014","journal-title":"Science"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1455","DOI":"10.1128\/JVI.02692-15","article-title":"Genetics, Receptor Binding, Replication, and Mammalian Transmission of H4 Avian Influenza Viruses Isolated from Live Poultry Markets in China","volume":"90","author":"Liang","year":"2016","journal-title":"J. Virol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"30331","DOI":"10.2807\/1560-7917.ES.2016.21.35.30331","article-title":"Isolation of H5N6, H7N9 and H9N2 avian influenza A viruses from air sampled at live poultry markets in China, 2014 and 2015","volume":"21","author":"Zhou","year":"2016","journal-title":"Eurosurveillance"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"5909","DOI":"10.1038\/s41467-020-19671-3","article-title":"Dominant subtype switch in avian influenza viruses during 2016\u20132019 in China","volume":"11","author":"Bi","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3142","DOI":"10.1038\/ncomms4142","article-title":"Dynamic reassortments and genetic heterogeneity of the human-infecting influenza A (H7N9) virus","volume":"5","author":"Cui","year":"2014","journal-title":"Nat. Commun."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Nowak, M.A. (2006). Evolutionary Dynamics, Harvard University Press.","DOI":"10.2307\/j.ctvjghw98"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1888","DOI":"10.1056\/NEJMoa1304459","article-title":"Human Infection with a Novel Avian-Origin Influenza A (H7N9) Virus","volume":"368","author":"Gao","year":"2013","journal-title":"N. Engl. J. Med."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1926","DOI":"10.1016\/S0140-6736(13)60938-1","article-title":"Origin and diversity of novel avian influenza A H7N9 viruses causing human infection: Phylogenetic, structural, and coalescent analyses","volume":"381","author":"Liu","year":"2013","journal-title":"Lancet"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"16151","DOI":"10.1038\/nmicrobiol.2016.151","article-title":"Intra-host dynamics of Ebola virus during 2014","volume":"1","author":"Ni","year":"2016","journal-title":"Nat. Microbiol."},{"key":"ref_15","first-page":"401","article-title":"Genomic epidemiology reveals multiple introductions of Zika virus into the United States","volume":"546","author":"Grubaugh","year":"2017","journal-title":"Nat. Cell Biol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"411","DOI":"10.1038\/nature22402","article-title":"Zika virus evolution and spread in the Americas","volume":"546","author":"Metsky","year":"2017","journal-title":"Nature"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1038\/nrg2626","article-title":"Sequencing technologies\u2014The next generation","volume":"11","author":"Soverini","year":"2010","journal-title":"Nat. Rev. Genet."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1038\/nature07517","article-title":"Accurate whole human genome sequencing using reversible terminator chemistry","volume":"456","author":"Bentley","year":"2008","journal-title":"Nature"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1038\/s41437-018-0070-5","article-title":"A new hybrid approach for MHC genotyping: High-throughput NGS and long read MinION nanopore sequencing, with application to the non-model vertebrate Alpine chamois (Rupicapra rupicapra)","volume":"121","author":"Fuselli","year":"2018","journal-title":"Heredity"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41598-018-31518-y","article-title":"Draft genome sequence of first monocot-halophytic species Oryza coarctata reveals stress-specific genes","volume":"8","author":"Mondal","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41598-018-23634-6","article-title":"Elucidation of the bacterial communities associated with the harmful microalgae Alexandrium tamarense and Cochlodinium polykrikoides using nanopore sequencing","volume":"8","author":"Shin","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"14408","DOI":"10.1038\/s41598-018-32615-8","article-title":"Direct RNA Sequencing of the Coding Complete Influenza A Virus Genome","volume":"8","author":"Keller","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_23","first-page":"228","article-title":"Real-time, portable genome sequencing for Ebola surveillance","volume":"530","author":"Quick","year":"2016","journal-title":"Nat. Cell Biol."},{"key":"ref_24","first-page":"766","article-title":"MinION nanopore sequencing of an influenza genome","volume":"6","author":"Wang","year":"2015","journal-title":"Front. Microbiol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"406","DOI":"10.1038\/nature22401","article-title":"Establishment and cryptic transmission of Zika virus in Brazil and the Americas","volume":"546","author":"Faria","year":"2017","journal-title":"Nature"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1186\/s13742-015-0051-z","article-title":"Bacterial and viral identification and differentiation by amplicon sequencing on the MinION nanopore sequencer","volume":"4","author":"Kilianski","year":"2015","journal-title":"GigaScience"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Bo\u017ea, V., Brejova, B., and Vina\u0159, T. (2017). DeepNano: Deep recurrent neural networks for base calling in MinION nanopore reads. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0178751"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1038\/nmeth.4577","article-title":"Highly parallel direct RNA sequencing on an array of nanopores","volume":"15","author":"Garalde","year":"2018","journal-title":"Nat. Methods"},{"key":"ref_29","unstructured":"Viehweger, A., Krautwurst, S., Lamkiewicz, K., Madhugiri, R., Ziebuhr, J., H\u00f6lzer, M., and Marz, M. (2018). Nanopore direct RNA sequencing reveals modification in full-length coronavirus genomes. bioRxiv."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1186\/2047-217X-1-18","article-title":"SOAPdenovo2: An empirically improved memory-efficient short-read de novo assembler","volume":"1","author":"Luo","year":"2012","journal-title":"GigaScience"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"802","DOI":"10.1101\/gr.072033.107","article-title":"De novo bacterial genome sequencing: Millions of very short reads assembled on a desktop computer","volume":"18","author":"Hernandez","year":"2008","journal-title":"Genome Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1754","DOI":"10.1093\/bioinformatics\/btp324","article-title":"Fast and accurate short read alignment with Burrows-Wheeler transform","volume":"25","author":"Li","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"2078","DOI":"10.1093\/bioinformatics\/btp352","article-title":"The Sequence Alignment\/Map format and SAMtools","volume":"25","author":"Li","year":"2009","journal-title":"Bioinformatics"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"810","DOI":"10.1016\/j.chom.2016.10.022","article-title":"Genesis, Evolution and Prevalence of H5N6 Avian Influenza Viruses in China","volume":"20","author":"Bi","year":"2016","journal-title":"Cell Host Microbe"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"11307","DOI":"10.1038\/ncomms11307","article-title":"Fast and sensitive mapping of nanopore sequencing reads with GraphMap","volume":"7","author":"Wilm","year":"2016","journal-title":"Nat. Commun."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"596","DOI":"10.1128\/JVI.02005-07","article-title":"The Influenza Virus Resource at the National Center for Biotechnology Information","volume":"82","author":"Bao","year":"2008","journal-title":"J. Virol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1188","DOI":"10.1101\/gr.849004","article-title":"WebLogo: A Sequence Logo Generator","volume":"14","author":"Crooks","year":"2004","journal-title":"Genome Res."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1870","DOI":"10.1093\/molbev\/msw054","article-title":"MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets","volume":"33","author":"Kumar","year":"2016","journal-title":"Mol. Biol. Evol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"680","DOI":"10.1093\/bioinformatics\/btq003","article-title":"CD-HIT Suite: A web server for clustering and comparing biological sequences","volume":"26","author":"Huang","year":"2010","journal-title":"Bioinform."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1038\/msb.2011.75","article-title":"Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega","volume":"7","author":"Sievers","year":"2011","journal-title":"Mol. Syst. Biol."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Miller, M.A., Pfeiffer, W., and Schwartz, T. (2010, January 14). Creating the CIPRES Science Gateway for inference of large phylogenetic trees. Proceedings of the 2010 Gateway Computing Environments Workshop (GCE), New Orleans, LA, USA.","DOI":"10.1109\/GCE.2010.5676129"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/18\/10069\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:01:29Z","timestamp":1760166089000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/18\/10069"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,9,17]]},"references-count":41,"journal-issue":{"issue":"18","published-online":{"date-parts":[[2021,9]]}},"alternative-id":["ijms221810069"],"URL":"https:\/\/doi.org\/10.3390\/ijms221810069","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2021,9,17]]}}}