Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing

Ingra M. Claro; Mariana S. Ramundo; Thais M. Coletti; Camila A.M. da Silva; Ian N. Valenca; Darlan S. Candido; Flavia C.S. Sales; Erika R. Manuli; Jaqueline G. de Jesus; Anderson de Paula; Alvina Clara Felix; Pamela dos Santos Andrade; Mariana C. Pinho; William M. Souza; Mariene R. Amorim; José Luiz Proenca-Modena; Esper G. Kallas; José Eduardo Levi; Nuno Rodrigues Faria; Ester C. Sabino; Nicholas J. Loman; Joshua Quick

doi:10.12688/wellcomeopenres.17170.2

Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing

Ingra M. Claro, Mariana S. Ramundo, Thais M. Coletti, Camila A.M. da Silva, Ian N. Valenca, Darlan S. Candido, Flavia C.S. Sales, Erika R. Manuli, Jaqueline G. de Jesus, Anderson de Paula, Alvina Clara Felix, Pamela dos Santos Andrade, Mariana C. Pinho, William M. Souza, Mariene R. Amorim, José Luiz Proenca-Modena, Esper G. Kallas, José Eduardo Levi, Nuno Rodrigues Faria, Ester C. SabinoNicholas J. Loman^*, Joshua Quick^*

^*Corresponding author for this work

Biosciences

Research output: Contribution to journal › Article › peer-review

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Abstract

Emerging and re-emerging viruses are a global health concern. Genome sequencing as an approach for monitoring circulating viruses is currently hampered by complex and expensive methods. Untargeted, metagenomic nanopore sequencing can provide genomic information to identify pathogens, prepare for or even prevent outbreaks.

SMART (Switching Mechanism at the 5′ end of RNA Template) is a popular approach for RNA-Seq but most current methods rely on oligo-dT priming to target polyadenylated mRNA molecules. We have developed two random primed SMART-Seq approaches, a sequencing agnostic approach ‘SMART-9N’ and a version compatible rapid adapters available from Oxford Nanopore Technologies ‘Rapid SMART-9N’. The methods were developed using viral isolates, clinical samples, and compared to a gold-standard amplicon-based method. From a Zika virus isolate the SMART-9N approach recovered 10kb of the 10.8kb RNA genome in a single nanopore read. We also obtained full genome coverage at a high depth coverage using the Rapid SMART-9N, which takes only 10 minutes and costs up to 45% less than other methods. We found the limits of detection of these methods to be 6 focus forming units (FFU)/mL with 99.02% and 87.58% genome coverage for SMART-9N and Rapid SMART-9N respectively. Yellow fever virus plasma samples and SARS-CoV-2 nasopharyngeal samples previously confirmed by RT-qPCR with a broad range of Ct-values were selected for validation. Both methods produced greater genome coverage when compared to the multiplex PCR approach and we obtained the longest single read of this study (18.5 kb) with a SARS-CoV-2 clinical sample, 60% of the virus genome using the Rapid SMART-9N method.

This work demonstrates that SMART-9N and Rapid SMART-9N are sensitive, low input, and long-read compatible alternatives for RNA virus detection and genome sequencing and Rapid SMART-9N improves the cost, time, and complexity of laboratory work.

Original language	English
Article number	241
Number of pages	22
Journal	Wellcome Open Research
Volume	6
DOIs	https://doi.org/10.12688/wellcomeopenres.17170.2
Publication status	Published - 24 Apr 2023

Bibliographical note

Funding Information:
This work was supported by a Medical Research Council-São Paulo Research Foundation (FAPESP) CADDE partnership award (MR/S0195/1 and FAPESP 18/14389-0) (https://caddecentre.org); FAPESP/Newton Funds (FAPESP 2017/08012-9), Bill & Melinda Gates Foundation (INV-034540 and INV-034652), and I.M.C (FAPESP 2018/17176-8). M.S.R. is supported by the Faculdade de Medicina da Universidade de São Paulo (FMUSP). T.M.C. (2019/07544-2), C.A.M.S. (2019/21301-5), F.C.S.S. (2018/25468-9), J.G.J. (2018/17176-8, 2019/12000-1, 18/14389-0) and E.C.S (2018/14389-0) are supported by FAPESP. P.S.A. is supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (88887.596940/2021-00). W.M.S is supported by FAPESP (2017/13981-0, 2019/24251-9), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (408338/2018-0), and the National Institutes of Health (AI120942). D.S.C is supported by the Clarendon Fund and by the Department of Zoology, University of Oxford. M.R.A is supported by CAPES (88887.356527/2019-00). N.R.F. is supported by a Wellcome Trust Royal Society Sir Henry Dale Fellowship (204311). N.J.L. and J.Q. are supported by the Wellcome ARTIC network (206298).

Publisher Copyright:
Copyright: © 2023 Claro IM et al.

Keywords

diagnostic
genomic surveillance
metagenomic
nanopore sequencing
RNA virus
SARS-CoV-2
YFV
ZIKV

ASJC Scopus subject areas

Medicine (miscellaneous)
General Biochemistry,Genetics and Molecular Biology

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.12688/wellcomeopenres.17170.2Licence: Creative Commons: Attribution (CC BY)

ClaroI2023RapidFinal published version, 3.43 MBLicence: Creative Commons: Attribution (CC BY)

Putting genomic surveillance at the heart of viural epidemic response
Loman, N.
THE WELLCOME TRUST
5/09/17 → 4/09/25
Project: Research

Cite this

Claro, I. M., Ramundo, M. S., Coletti, T. M., da Silva, C. A. M., Valenca, I. N., Candido, D. S., Sales, F. C. S., Manuli, E. R., de Jesus, J. G., de Paula, A., Felix, A. C., Andrade, P. D. S., Pinho, M. C., Souza, W. M., Amorim, M. R., Proenca-Modena, J. L., Kallas, E. G., Levi, J. E., Faria, N. R., ... Quick, J. (2023). Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing. Wellcome Open Research, 6, Article 241. https://doi.org/10.12688/wellcomeopenres.17170.2

@article{0da3db8c2a214ee3961f7244a91cef7f,

title = "Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing",

abstract = "Emerging and re-emerging viruses are a global health concern. Genome sequencing as an approach for monitoring circulating viruses is currently hampered by complex and expensive methods. Untargeted, metagenomic nanopore sequencing can provide genomic information to identify pathogens, prepare for or even prevent outbreaks. SMART (Switching Mechanism at the 5′ end of RNA Template) is a popular approach for RNA-Seq but most current methods rely on oligo-dT priming to target polyadenylated mRNA molecules. We have developed two random primed SMART-Seq approaches, a sequencing agnostic approach {\textquoteleft}SMART-9N{\textquoteright} and a version compatible rapid adapters available from Oxford Nanopore Technologies {\textquoteleft}Rapid SMART-9N{\textquoteright}. The methods were developed using viral isolates, clinical samples, and compared to a gold-standard amplicon-based method. From a Zika virus isolate the SMART-9N approach recovered 10kb of the 10.8kb RNA genome in a single nanopore read. We also obtained full genome coverage at a high depth coverage using the Rapid SMART-9N, which takes only 10 minutes and costs up to 45% less than other methods. We found the limits of detection of these methods to be 6 focus forming units (FFU)/mL with 99.02% and 87.58% genome coverage for SMART-9N and Rapid SMART-9N respectively. Yellow fever virus plasma samples and SARS-CoV-2 nasopharyngeal samples previously confirmed by RT-qPCR with a broad range of Ct-values were selected for validation. Both methods produced greater genome coverage when compared to the multiplex PCR approach and we obtained the longest single read of this study (18.5 kb) with a SARS-CoV-2 clinical sample, 60% of the virus genome using the Rapid SMART-9N method. This work demonstrates that SMART-9N and Rapid SMART-9N are sensitive, low input, and long-read compatible alternatives for RNA virus detection and genome sequencing and Rapid SMART-9N improves the cost, time, and complexity of laboratory work.",

keywords = "diagnostic, genomic surveillance, metagenomic, nanopore sequencing, RNA virus, SARS-CoV-2, YFV, ZIKV",

author = "Claro, {Ingra M.} and Ramundo, {Mariana S.} and Coletti, {Thais M.} and {da Silva}, {Camila A.M.} and Valenca, {Ian N.} and Candido, {Darlan S.} and Sales, {Flavia C.S.} and Manuli, {Erika R.} and {de Jesus}, {Jaqueline G.} and {de Paula}, Anderson and Felix, {Alvina Clara} and Andrade, {Pamela dos Santos} and Pinho, {Mariana C.} and Souza, {William M.} and Amorim, {Mariene R.} and Proenca-Modena, {Jos{\'e} Luiz} and Kallas, {Esper G.} and Levi, {Jos{\'e} Eduardo} and Faria, {Nuno Rodrigues} and Sabino, {Ester C.} and Loman, {Nicholas J.} and Joshua Quick",

note = "Funding Information: This work was supported by a Medical Research Council-S{\~a}o Paulo Research Foundation (FAPESP) CADDE partnership award (MR/S0195/1 and FAPESP 18/14389-0) (https://caddecentre.org); FAPESP/Newton Funds (FAPESP 2017/08012-9), Bill & Melinda Gates Foundation (INV-034540 and INV-034652), and I.M.C (FAPESP 2018/17176-8). M.S.R. is supported by the Faculdade de Medicina da Universidade de S{\~a}o Paulo (FMUSP). T.M.C. (2019/07544-2), C.A.M.S. (2019/21301-5), F.C.S.S. (2018/25468-9), J.G.J. (2018/17176-8, 2019/12000-1, 18/14389-0) and E.C.S (2018/14389-0) are supported by FAPESP. P.S.A. is supported by Coordena{\c c}{\~a}o de Aperfei{\c c}oamento de Pessoal de N{\'i}vel Superior (CAPES) (88887.596940/2021-00). W.M.S is supported by FAPESP (2017/13981-0, 2019/24251-9), Conselho Nacional de Desenvolvimento Cient{\'i}fico e Tecnol{\'o}gico (CNPq) (408338/2018-0), and the National Institutes of Health (AI120942). D.S.C is supported by the Clarendon Fund and by the Department of Zoology, University of Oxford. M.R.A is supported by CAPES (88887.356527/2019-00). N.R.F. is supported by a Wellcome Trust Royal Society Sir Henry Dale Fellowship (204311). N.J.L. and J.Q. are supported by the Wellcome ARTIC network (206298). Publisher Copyright: Copyright: {\textcopyright} 2023 Claro IM et al.",

year = "2023",

month = apr,

day = "24",

doi = "10.12688/wellcomeopenres.17170.2",

language = "English",

volume = "6",

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Claro, IM, Ramundo, MS, Coletti, TM, da Silva, CAM, Valenca, IN, Candido, DS, Sales, FCS, Manuli, ER, de Jesus, JG, de Paula, A, Felix, AC, Andrade, PDS, Pinho, MC, Souza, WM, Amorim, MR, Proenca-Modena, JL, Kallas, EG, Levi, JE, Faria, NR, Sabino, EC, Loman, NJ & Quick, J 2023, 'Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing', Wellcome Open Research, vol. 6, 241. https://doi.org/10.12688/wellcomeopenres.17170.2

TY - JOUR

T1 - Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing

AU - Claro, Ingra M.

AU - Ramundo, Mariana S.

AU - Coletti, Thais M.

AU - da Silva, Camila A.M.

AU - Valenca, Ian N.

AU - Candido, Darlan S.

AU - Sales, Flavia C.S.

AU - Manuli, Erika R.

AU - de Jesus, Jaqueline G.

AU - de Paula, Anderson

AU - Felix, Alvina Clara

AU - Andrade, Pamela dos Santos

AU - Pinho, Mariana C.

AU - Souza, William M.

AU - Amorim, Mariene R.

AU - Proenca-Modena, José Luiz

AU - Kallas, Esper G.

AU - Levi, José Eduardo

AU - Faria, Nuno Rodrigues

AU - Sabino, Ester C.

AU - Loman, Nicholas J.

AU - Quick, Joshua

N1 - Funding Information: This work was supported by a Medical Research Council-São Paulo Research Foundation (FAPESP) CADDE partnership award (MR/S0195/1 and FAPESP 18/14389-0) (https://caddecentre.org); FAPESP/Newton Funds (FAPESP 2017/08012-9), Bill & Melinda Gates Foundation (INV-034540 and INV-034652), and I.M.C (FAPESP 2018/17176-8). M.S.R. is supported by the Faculdade de Medicina da Universidade de São Paulo (FMUSP). T.M.C. (2019/07544-2), C.A.M.S. (2019/21301-5), F.C.S.S. (2018/25468-9), J.G.J. (2018/17176-8, 2019/12000-1, 18/14389-0) and E.C.S (2018/14389-0) are supported by FAPESP. P.S.A. is supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (88887.596940/2021-00). W.M.S is supported by FAPESP (2017/13981-0, 2019/24251-9), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (408338/2018-0), and the National Institutes of Health (AI120942). D.S.C is supported by the Clarendon Fund and by the Department of Zoology, University of Oxford. M.R.A is supported by CAPES (88887.356527/2019-00). N.R.F. is supported by a Wellcome Trust Royal Society Sir Henry Dale Fellowship (204311). N.J.L. and J.Q. are supported by the Wellcome ARTIC network (206298). Publisher Copyright: Copyright: © 2023 Claro IM et al.

PY - 2023/4/24

Y1 - 2023/4/24

N2 - Emerging and re-emerging viruses are a global health concern. Genome sequencing as an approach for monitoring circulating viruses is currently hampered by complex and expensive methods. Untargeted, metagenomic nanopore sequencing can provide genomic information to identify pathogens, prepare for or even prevent outbreaks. SMART (Switching Mechanism at the 5′ end of RNA Template) is a popular approach for RNA-Seq but most current methods rely on oligo-dT priming to target polyadenylated mRNA molecules. We have developed two random primed SMART-Seq approaches, a sequencing agnostic approach ‘SMART-9N’ and a version compatible rapid adapters available from Oxford Nanopore Technologies ‘Rapid SMART-9N’. The methods were developed using viral isolates, clinical samples, and compared to a gold-standard amplicon-based method. From a Zika virus isolate the SMART-9N approach recovered 10kb of the 10.8kb RNA genome in a single nanopore read. We also obtained full genome coverage at a high depth coverage using the Rapid SMART-9N, which takes only 10 minutes and costs up to 45% less than other methods. We found the limits of detection of these methods to be 6 focus forming units (FFU)/mL with 99.02% and 87.58% genome coverage for SMART-9N and Rapid SMART-9N respectively. Yellow fever virus plasma samples and SARS-CoV-2 nasopharyngeal samples previously confirmed by RT-qPCR with a broad range of Ct-values were selected for validation. Both methods produced greater genome coverage when compared to the multiplex PCR approach and we obtained the longest single read of this study (18.5 kb) with a SARS-CoV-2 clinical sample, 60% of the virus genome using the Rapid SMART-9N method. This work demonstrates that SMART-9N and Rapid SMART-9N are sensitive, low input, and long-read compatible alternatives for RNA virus detection and genome sequencing and Rapid SMART-9N improves the cost, time, and complexity of laboratory work.

AB - Emerging and re-emerging viruses are a global health concern. Genome sequencing as an approach for monitoring circulating viruses is currently hampered by complex and expensive methods. Untargeted, metagenomic nanopore sequencing can provide genomic information to identify pathogens, prepare for or even prevent outbreaks. SMART (Switching Mechanism at the 5′ end of RNA Template) is a popular approach for RNA-Seq but most current methods rely on oligo-dT priming to target polyadenylated mRNA molecules. We have developed two random primed SMART-Seq approaches, a sequencing agnostic approach ‘SMART-9N’ and a version compatible rapid adapters available from Oxford Nanopore Technologies ‘Rapid SMART-9N’. The methods were developed using viral isolates, clinical samples, and compared to a gold-standard amplicon-based method. From a Zika virus isolate the SMART-9N approach recovered 10kb of the 10.8kb RNA genome in a single nanopore read. We also obtained full genome coverage at a high depth coverage using the Rapid SMART-9N, which takes only 10 minutes and costs up to 45% less than other methods. We found the limits of detection of these methods to be 6 focus forming units (FFU)/mL with 99.02% and 87.58% genome coverage for SMART-9N and Rapid SMART-9N respectively. Yellow fever virus plasma samples and SARS-CoV-2 nasopharyngeal samples previously confirmed by RT-qPCR with a broad range of Ct-values were selected for validation. Both methods produced greater genome coverage when compared to the multiplex PCR approach and we obtained the longest single read of this study (18.5 kb) with a SARS-CoV-2 clinical sample, 60% of the virus genome using the Rapid SMART-9N method. This work demonstrates that SMART-9N and Rapid SMART-9N are sensitive, low input, and long-read compatible alternatives for RNA virus detection and genome sequencing and Rapid SMART-9N improves the cost, time, and complexity of laboratory work.

KW - diagnostic

KW - genomic surveillance

KW - metagenomic

KW - nanopore sequencing

KW - RNA virus

KW - SARS-CoV-2

KW - YFV

KW - ZIKV

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U2 - 10.12688/wellcomeopenres.17170.2

DO - 10.12688/wellcomeopenres.17170.2

M3 - Article

AN - SCOPUS:85159683183

SN - 2398-502X

VL - 6

JO - Wellcome Open Research

JF - Wellcome Open Research

M1 - 241

ER -

Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

UN SDGs

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Putting genomic surveillance at the heart of viural epidemic response

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