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Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5\u2032 cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs.<\/jats:p>","DOI":"10.1093\/nar\/gkac144","type":"journal-article","created":{"date-parts":[[2022,2,16]],"date-time":"2022-02-16T07:34:39Z","timestamp":1644996879000},"page":"3475-3489","source":"Crossref","is-referenced-by-count":31,"title":["Nanopore ReCappable sequencing maps SARS-CoV-2 5\u2032 capping sites and provides new insights into the structure of sgRNAs"],"prefix":"10.1093","volume":"50","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2269-2071","authenticated-orcid":false,"given":"Camilla","family":"Ugolini","sequence":"first","affiliation":[{"name":"Center for Genomic Science of IIT@SEMM, Fondazione Istituto Italiano di Tecnologia, 20139 Milano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0534-0165","authenticated-orcid":false,"given":"Logan","family":"Mulroney","sequence":"additional","affiliation":[{"name":"Center for Genomic Science of IIT@SEMM, Fondazione Istituto Italiano di Tecnologia, 20139 Milano, Italy"},{"name":"European Molecular Biology Laboratory, European Bioinformatics Institute, Hinxton, UK"},{"name":"Biomolecular Engineering Department, UC Santa Cruz, CA 95064, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8772-6776","authenticated-orcid":false,"given":"Adrien","family":"Leger","sequence":"additional","affiliation":[{"name":"European Molecular Biology Laboratory, European Bioinformatics Institute, Hinxton, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0295-7941","authenticated-orcid":false,"given":"Matteo","family":"Castelli","sequence":"additional","affiliation":[{"name":"Laboratory of Microbiology and Virology, Vita-Salute San Raffaele University; via Olgettina 58, 20132 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0185-3147","authenticated-orcid":false,"given":"Elena","family":"Criscuolo","sequence":"additional","affiliation":[{"name":"Laboratory of Microbiology and Virology, Vita-Salute San Raffaele University; via Olgettina 58, 20132 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4659-8613","authenticated-orcid":false,"given":"Maia Kavanagh","family":"Williamson","sequence":"additional","affiliation":[{"name":"School of Cellular and Molecular Medicine, Faculty of Life Sciences, University Walk, University of Bristol, Bristol\u00a0BS8 1TD, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1136-4008","authenticated-orcid":false,"given":"Andrew D","family":"Davidson","sequence":"additional","affiliation":[{"name":"School of Cellular and Molecular Medicine, Faculty of Life Sciences, University Walk, University of Bristol, Bristol\u00a0BS8 1TD, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abdulaziz","family":"Almuqrin","sequence":"additional","affiliation":[{"name":"School of Cellular and Molecular Medicine, Faculty of Life Sciences, University Walk, University of Bristol, Bristol\u00a0BS8 1TD, UK"},{"name":"Department of Clinical Laboratory Sciences, King Saud University, Riyadh, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4566-261X","authenticated-orcid":false,"given":"Roberto","family":"Giambruno","sequence":"additional","affiliation":[{"name":"Center for Genomic Science of IIT@SEMM, Fondazione Istituto Italiano di Tecnologia, 20139 Milano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4571-3982","authenticated-orcid":false,"given":"Miten","family":"Jain","sequence":"additional","affiliation":[{"name":"Biomolecular Engineering Department, UC Santa Cruz, CA 95064, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3253-7091","authenticated-orcid":false,"given":"Gianmaria","family":"Frig\u00e8","sequence":"additional","affiliation":[{"name":"Department of Experimental Oncology, IEO European Institute of Oncology IRCCS, 20139 Milano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7293-8853","authenticated-orcid":false,"given":"Hugh","family":"Olsen","sequence":"additional","affiliation":[{"name":"Biomolecular Engineering Department, UC Santa Cruz, CA 95064, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8821-8060","authenticated-orcid":false,"given":"George","family":"Tzertzinis","sequence":"additional","affiliation":[{"name":"New England Biolabs, Ipswich, MA 01938 3, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1143-6630","authenticated-orcid":false,"given":"Ira","family":"Schildkraut","sequence":"additional","affiliation":[{"name":"New England Biolabs, Ipswich, MA 01938 3, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7601-413X","authenticated-orcid":false,"given":"Madalee G","family":"Wulf","sequence":"additional","affiliation":[{"name":"New England Biolabs, Ipswich, MA 01938 3, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3169-6878","authenticated-orcid":false,"suffix":"Jr","given":"Ivan R","family":"Corr\u00eaa","sequence":"additional","affiliation":[{"name":"New England Biolabs, Ipswich, MA 01938 3, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3957-6539","authenticated-orcid":false,"given":"Laurence","family":"Ettwiller","sequence":"additional","affiliation":[{"name":"New England Biolabs, Ipswich, MA 01938 3, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicola","family":"Clementi","sequence":"additional","affiliation":[{"name":"Laboratory of Microbiology and Virology, Vita-Salute San Raffaele University; 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