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We here present NOseq, a method to detect m6A residues in defined amplicons by virtue of their resistance to chemical deamination, effected by nitrous acid. Partial deamination in NOseq affects all exocyclic amino groups present in nucleobases and thus also changes sequence information. The method uses a mapping algorithm specifically adapted to the sequence degeneration caused by deamination events. Thus, m6A sites with partial modification levels of \u223c50% were detected in defined amplicons, and this threshold can be lowered to \u223c10% by combination with m6A immunoprecipitation. NOseq faithfully detected known m6A sites in human rRNA, and the long non-coding RNA MALAT1, and positively validated several m6A candidate sites, drawn from miCLIP data with an m6A antibody, in the transcriptome of Drosophila melanogaster. Conceptually related to bisulfite sequencing, NOseq presents a novel amplicon-based sequencing approach for the validation of m6A sites in defined sequences.<\/jats:p>","DOI":"10.1093\/nar\/gkaa1173","type":"journal-article","created":{"date-parts":[[2020,11,20]],"date-time":"2020-11-20T20:19:23Z","timestamp":1605903563000},"page":"e23-e23","source":"Crossref","is-referenced-by-count":45,"title":["NOseq: amplicon sequencing evaluation method for RNA m6A sites after chemical deamination"],"prefix":"10.1093","volume":"49","author":[{"given":"Stephan","family":"Werner","sequence":"first","affiliation":[{"name":"Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University Mainz, Staudingerweg 5, 55128 Mainz, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aurellia","family":"Galliot","sequence":"additional","affiliation":[{"name":"Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University Mainz, Staudingerweg 5, 55128 Mainz, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Florian","family":"Pichot","sequence":"additional","affiliation":[{"name":"Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University Mainz, Staudingerweg 5, 55128 Mainz, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Thomas","family":"Kemmer","sequence":"additional","affiliation":[{"name":"Institute of Computer Science, Johannes Gutenberg-University Mainz, Staudingerweg 9, 55128 Mainz, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Virginie","family":"Marchand","sequence":"additional","affiliation":[{"name":"Universit\u00e9 de Lorraine, CNRS, INSERM, Epitranscriptomics and Sequencing (EpiRNA-Seq) Core Facility, UMS2008\/US40 IBSLor, Biop\u00f4le UL, F-54000 Nancy, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maksim V","family":"Sednev","sequence":"additional","affiliation":[{"name":"Institute of Organic Chemistry, Julius Maximilian University W\u00fcrzburg, Am Hubland, 97074 W\u00fcrzburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tina","family":"Lence","sequence":"additional","affiliation":[{"name":"Institute of Molecular Biology, Ackermannweg 4, 55128 Mainz, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jean-Yves","family":"Roignant","sequence":"additional","affiliation":[{"name":"Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University Mainz, Staudingerweg 5, 55128 Mainz, Germany"},{"name":"Institute of Molecular Biology, Ackermannweg 4, 55128 Mainz, Germany"},{"name":"G\u00e9nopode - Center for Integrative Genomics, Universit\u00e9 de Lausanne, 1015 Lausanne, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Julian","family":"K\u00f6nig","sequence":"additional","affiliation":[{"name":"Institute of Molecular Biology, Ackermannweg 4, 55128 Mainz, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4548-2299","authenticated-orcid":false,"given":"Claudia","family":"H\u00f6bartner","sequence":"additional","affiliation":[{"name":"Institute of Organic Chemistry, Julius Maximilian University W\u00fcrzburg, Am Hubland, 97074 W\u00fcrzburg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8018-334X","authenticated-orcid":false,"given":"Yuri","family":"Motorin","sequence":"additional","affiliation":[{"name":"Universit\u00e9 de Lorraine, CNRS, UMR7365 IMoPA, Biop\u00f4le UL, F-54000 Nancy, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Andreas","family":"Hildebrandt","sequence":"additional","affiliation":[{"name":"Institute of Computer Science, Johannes Gutenberg-University Mainz, Staudingerweg 9, 55128 Mainz, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0154-0928","authenticated-orcid":false,"given":"Mark","family":"Helm","sequence":"additional","affiliation":[{"name":"Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University Mainz, Staudingerweg 5, 55128 Mainz, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"286","published-online":{"date-parts":[[2020,12,11]]},"reference":[{"key":"2021022716422522200_B1","doi-asserted-by":"crossref","first-page":"349","DOI":"10.1146\/annurev-genet-120417-031522","article-title":"Chemical modifications in the life of an mRNA transcript","volume":"52","author":"Nachtergaele","year":"2018","journal-title":"Annu. 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