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Nevertheless, when using different methods, the results do not always agree. The sampling scheme used in different studies may partially explain the differences that are found, but there is also the possibility that some of the identified positively selected amino acid sites are false positives. This is especially important in the context of very large-scale projects where hundreds of analyses have been performed for the same protein-coding gene. To account for these issues, in this work, we have identified positively selected amino acid sites in SARS-CoV-2 and 15 other coronavirus species, using both codeML and FUBAR, and compared the location of such sites in the different species. Moreover, we also compared our results to those that are available in the COV2Var database and the frequency of the 10 most frequent variants and predicted protein location to identify those sites that are supported by multiple lines of evidence. Amino acid changes observed at these sites should always be of concern. The information reported for SARS-CoV-2 can also be used to identify variants of concern in other coronaviruses.<\/jats:p>","DOI":"10.3390\/ijms25042428","type":"journal-article","created":{"date-parts":[[2024,2,19]],"date-time":"2024-02-19T10:40:15Z","timestamp":1708339215000},"page":"2428","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Multiple Lines of Evidence Support 199 SARS-CoV-2 Positively Selected Amino Acid Sites"],"prefix":"10.3390","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4657-6991","authenticated-orcid":false,"given":"Pedro","family":"Ferreira","sequence":"first","affiliation":[{"name":"Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade (i3S), Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal"},{"name":"Instituto de Biologia Molecular e Celular (IBMC), Rua Alfredo Allen 208, 4200-135 Porto, Portugal"},{"name":"School of Medicine and Biomedical Sciences (ICBAS), Porto University, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6332-7807","authenticated-orcid":false,"given":"Ricardo","family":"Soares","sequence":"additional","affiliation":[{"name":"Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade (i3S), Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal"},{"name":"Instituto de Biologia Molecular e Celular (IBMC), Rua Alfredo Allen 208, 4200-135 Porto, Portugal"},{"name":"School of Medicine and Biomedical Sciences (ICBAS), Porto University, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal"},{"name":"Faculdade de Ci\u00eancias da Universidade do Porto (FCUP), Rua do Campo Alegre s\/n, 4169-007 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6476-7206","authenticated-orcid":false,"given":"Hugo","family":"L\u00f3pez-Fern\u00e1ndez","sequence":"additional","affiliation":[{"name":"CINBIO, Department of Computer Science, ESEI\u2014Escuela Superior de Ingenier\u00eda Inform\u00e1tica, Universidade de Vigo, 32004 Ourense, Spain"},{"name":"CINBIO, SING Research Group, Galicia Sur Health Research Institute (IIS Galicia Sur), SERGAS-UVIGO, 36213 Vigo, Spain"}]},{"given":"No\u00e9","family":"Vazquez","sequence":"additional","affiliation":[{"name":"CINBIO, Department of Computer Science, ESEI\u2014Escuela Superior de Ingenier\u00eda Inform\u00e1tica, Universidade de Vigo, 32004 Ourense, Spain"},{"name":"CINBIO, SING Research Group, Galicia Sur Health Research Institute (IIS Galicia Sur), SERGAS-UVIGO, 36213 Vigo, Spain"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8749-2703","authenticated-orcid":false,"given":"Miguel","family":"Reboiro-Jato","sequence":"additional","affiliation":[{"name":"CINBIO, Department of Computer Science, ESEI\u2014Escuela Superior de Ingenier\u00eda Inform\u00e1tica, Universidade de Vigo, 32004 Ourense, Spain"},{"name":"CINBIO, SING Research Group, Galicia Sur Health Research Institute (IIS Galicia Sur), SERGAS-UVIGO, 36213 Vigo, Spain"}]},{"given":"Cristina P.","family":"Vieira","sequence":"additional","affiliation":[{"name":"Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade (i3S), Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal"},{"name":"Instituto de Biologia Molecular e Celular (IBMC), Rua Alfredo Allen 208, 4200-135 Porto, Portugal"}]},{"given":"Jorge","family":"Vieira","sequence":"additional","affiliation":[{"name":"Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade (i3S), Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal"},{"name":"Instituto de Biologia Molecular e Celular (IBMC), Rua Alfredo Allen 208, 4200-135 Porto, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2024,2,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1467","DOI":"10.1136\/bmj.1.5448.1467","article-title":"Cultivation of a novel type of common-cold virus in organ cultures","volume":"1","author":"Tyrrell","year":"1965","journal-title":"Br. 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