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 Mancini; Ewan Birney; Mark Akeson; Francesco Nicassio; David A Matthews; Tommaso  Leonardi

doi:10.1093/nar/gkac144

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

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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 language	English
Article number	gkac144
Pages (from-to)	3475-3489
Number of pages	15
Journal	Nucleic Acids Research
Volume	50
Issue number	6
Early online date	4 Mar 2022
DOIs	https://doi.org/10.1093/nar/gkac144
Publication status	E-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.

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© 2022 The Author(s).

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Ugolini, C., Mulroney, L., Leger, A., Castelli, M., Criscuolo, E., Kavanagh Williamson, M., Davidson, A. D., Almuqrin, A. M., Giambruno, R., Jain, M., Frige, G., Olsen, H., Tzertzinis, G., Schildkraut, I., Wulf, M. G., Corrêa Jr, I. R., Ettwiller, L., Clementi, N., Clementi, M., ... Leonardi, T. (2022). Nanopore ReCappable sequencing maps SARS-CoV-2 5′ capping sites and provides new insights into the structure of sgRNAs. Nucleic Acids Research, 50(6), 3475-3489. Article gkac144. Advance online publication. https://doi.org/10.1093/nar/gkac144

@article{9605b9812e5342c49a6895ecb12200ba,

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

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.",

author = "Camilla Ugolini and Logan Mulroney and Adrien Leger and Matteo Castelli and Elena Criscuolo and {Kavanagh Williamson}, Maia and Davidson, {Andrew D} and Almuqrin, {Abdulaziz M} and Roberto Giambruno and Miten Jain and Gianmaria Frige and Hugh Olsen and George Tzertzinis and Ira Schildkraut and Wulf, {Madalee G.} and {Corr{\^e}a Jr}, {Ivan R.} and Laurence Ettwiller and Nicola Clementi and Massimo Clementi and Nicasio Mancini and Ewan Birney and Mark Akeson and Francesco Nicassio and Matthews, {David A} and Tommaso Leonardi",

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: {\textcopyright} 2022 The Author(s).",

year = "2022",

month = mar,

day = "4",

doi = "10.1093/nar/gkac144",

language = "English",

volume = "50",

pages = "3475--3489",

journal = "Nucleic Acids Research",

issn = "0305-1048",

publisher = "Oxford University Press",

number = "6",

}

Ugolini, C, Mulroney, L, Leger, A, Castelli, M, Criscuolo, E, Kavanagh Williamson, M, Davidson, AD, Almuqrin, AM, Giambruno, R, Jain, M, Frige, G, Olsen, H, Tzertzinis, G, Schildkraut, I, Wulf, MG, Corrêa Jr, IR, Ettwiller, L, Clementi, N, Clementi, M, Mancini, N, Birney, E, Akeson, M, Nicassio, F, Matthews, DA & Leonardi, T 2022, 'Nanopore ReCappable sequencing maps SARS-CoV-2 5′ capping sites and provides new insights into the structure of sgRNAs', Nucleic Acids Research, vol. 50, no. 6, gkac144, pp. 3475-3489. https://doi.org/10.1093/nar/gkac144

TY - JOUR

T1 - Nanopore ReCappable sequencing maps SARS-CoV-2 5′ capping sites and provides new insights into the structure of sgRNAs

AU - Ugolini, Camilla

AU - Mulroney, Logan

AU - Leger, Adrien

AU - Castelli, Matteo

AU - Criscuolo, Elena

AU - Kavanagh Williamson, Maia

AU - Davidson, Andrew D

AU - Almuqrin, Abdulaziz M

AU - Giambruno, Roberto

AU - Jain, Miten

AU - Frige, Gianmaria

AU - Olsen, Hugh

AU - Tzertzinis, George

AU - Schildkraut, Ira

AU - Wulf, Madalee G.

AU - Corrêa Jr, Ivan R.

AU - Ettwiller, Laurence

AU - Clementi, Nicola

AU - Clementi, Massimo

AU - Mancini, Nicasio

AU - Birney, Ewan

AU - Akeson, Mark

AU - Nicassio, Francesco

AU - Matthews, David A

AU - Leonardi, Tommaso

N1 - 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).

PY - 2022/3/4

Y1 - 2022/3/4

N2 - 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.

AB - 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.

U2 - 10.1093/nar/gkac144

DO - 10.1093/nar/gkac144

M3 - Article (Academic Journal)

C2 - 35244721

SN - 0305-1048

VL - 50

SP - 3475

EP - 3489

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 6

M1 - gkac144

ER -

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

Abstract

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