Nanopore ReCappable sequencing maps SARS-CoV-2 5′ capping sites and provides new insights into the structure of sgRNAs

Camilla Ugolini, Logan Mulroney, Adrien Leger, Matteo Castelli, Elena Criscuolo, Maia Kavanagh Williamson, Andrew D Davidson, Abdulaziz M Almuqrin, Roberto Giambruno, Miten Jain, Gianmaria Frige, Hugh Olsen, George Tzertzinis, Ira Schildkraut, Madalee G. Wulf, Ivan R. Corrêa Jr, Laurence Ettwiller, Nicola Clementi, Massimo Clementi, Nicasio ManciniEwan Birney, Mark Akeson, Francesco Nicassio, David A Matthews*, Tommaso Leonardi*

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

9 Citations (Scopus)
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Abstract

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested subgenomic RNAsused to express structural and accessory proteins. 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′ 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.
Original languageEnglish
Article numbergkac144
Pages (from-to)3475-3489
Number of pages15
JournalNucleic Acids Research
Volume50
Issue number6
Early online date4 Mar 2022
DOIs
Publication statusE-pub ahead of print - 4 Mar 2022

Bibliographical note

Funding Information:
D.A.M. and A.D.D. were supported by the United States Food and Drug Administration [HHSF223201510104C] 'Ebola Virus Disease: correlates of protection, determinants of outcome and clinical management' amended to incorporate urgent COVID-19 studies; M.K.W. was supported by a Medical Research Council, UK [MR/R020566/1 to A.D.D.]; the UCSC Nanopore Group was supported by NIH [HG010053]; Oxford Nanopore Technologies [SC20130149]; Associazione Italiana per la Ricerca sul Cancro [IG22851 to F.N.]. Funding for open access charge: Medical Research Council [MR/R020566/1]; Associazione Italiana per la Ricerca sul Cancro [IG22851]; U.S. Food and Drug Administration [HHSF223201510104C]; Oxford Nanopore Technologies [SC20130149]; National Institutes of Health [HG010053]. Conflict of interest statement. A.L., L.M., M.A., M.J. and T.L. have received financial support fromOxfordNanopore Technologies (ONT) for travel and accommodations to attend and present at ONT events. T.L. is a paid consultant to STORM therapeutics limited. E.B. and M.A. are paid consultants to ONT. E.B. and M.A. are shareholders of ONT. M.A. is an inventor on 11 UC patents licensed to ONT (6 267 872, 6 465 193, 6 746 594, 6 936 433, 7 060 50, 8 500 982, 8 679 747, 9 481 908, 9 797 013, 10 059 988, 10 081 835); M.A. received research funding from ONT; A.L. is currently an employee of ONT;G.T., I.S.,M.G.W., I.R.C.J., L.E. are employees of New England Biolabs Inc. New England Biolabs commercialises reagents for molecular biology applications.

Publisher Copyright:
© 2022 The Author(s).

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