Abstract

Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples (i.e., without isolation and culture) remains challenging for viruses such as Zika, for which metagenomic sequencing methods may generate insufficient numbers of viral reads. Here we present a protocol for generating coding-sequence-complete genomes, comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimized library preparation methods for the portable MinION sequencer (Oxford Nanopore Technologies) and the Illumina range of instruments, and a bioinformatics pipeline for generating consensus sequences. The MinION protocol does not require an Internet connection for analysis, making it suitable for field applications with limited connectivity. Our method relies on multiplex PCR for targeted enrichment of viral genomes from samples containing as few as 50 genome copies per reaction. Viral consensus sequences can be achieved in 1–2 d by starting with clinical samples and following a simple laboratory workflow. This method has been successfully used by several groups studying Zika virus evolution and is facilitating an understanding of the spread of the virus in the Americas. The protocol can be used to sequence other viral genomes using the online Primal Scheme primer designer software. It is suitable for sequencing either RNA or DNA viruses in the field during outbreaks or as an inexpensive, convenient method for use in the lab.

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Acknowledgements

The authors thank the Brazilian Ministry of Health and the Latin American Community Engagement Networks (LACENs) of Natal, João Pessoa, Recife, Maceió and Salvador for their support. We thank T. Fredeking from Antibody Systems for providing the Zika virus samples from Colombia. We thank K. Brunker for testing the Primal Scheme software. The Zika in Brazil Real-time Analysis (ZiBRA) project (http://www.zibraproject.org) is supported by the Medical Research Council/Wellcome Trust/Newton Fund Zika Rapid Response Initiative (grant no, ZK/16-078), which also provides J.Q.'s salary. N.J.L. is supported by a Medical Research Council Bioinformatics Fellowship as part of the Cloud Infrastructure for Microbial Bioinformatics (CLIMB) project. Primal Scheme is hosted on the CLIMB platform, where pipeline development and MinION data analysis was performed59. N.D.G. is supported by National Institutes of Health (NIH) training grant 5T32AI007244-33. K.G.A. is a PEW Biomedical Scholar, and his work is supported by NIH National Center for Advancing Translational Studies Clinical and Translational Science Award UL1TR001114 and National Institute of Allergy and Infectious Diseases (NIAID) contract HHSN272201400048C. A.B. and T.B. were supported by NIH awards R35 GM119774 and U54 GM111274. T.B. is a Pew Biomedical Scholar. A.B. is supported by the National Science Foundation Graduate Research Fellowship Program under Grant no. DGE-1256082. N.R.F. was funded by a Sir Henry Dale Fellowship (Wellcome Trust/Royal Society grant 204311/Z/16/Z). Work at the Paul-Ehrlich-Institut was supported by a grant ('Sicherheit von Blut(produkten) und Geweben hinsichtlich der Abwesenheit von Zikaviren') from the German Ministry of Health. This study was supported by USAID Emerging Pandemic Threats Program-2 PREDICT-2 (cooperative agreement AID-OAA-A-14-00102). The contents of this article are the responsibility of the authors and do not necessarily reflect the views of USAID or the US government.

Author information

Affiliations

  1. Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, UK.

    • Joshua Quick
    • , Andrew D Smith
    •  & Nicholas J Loman
  2. The Scripps Research Institute, La Jolla, California, USA.

    • Nathan D Grubaugh
    • , Karthik Gangavarapu
    • , Refugio Robles-Sikisaka
    • , Thomas F Rogers
    • , Nathan A Beutler
    • , Dennis R Burton
    •  & Kristian G Andersen
  3. Public Health England, National Infection Service, Porton Down, Salisbury, UK.

    • Steven T Pullan
    •  & Miles W Carroll
  4. Department of Infectious Disease and Institute of Tropical Medicine, University of Saõ Paulo, Saõ Paulo, Brazil.

    • Ingra M Claro
    •  & Ester C Sabino
  5. Scripps Translational Science Institute, La Jolla, California, USA.

    • Glenn Oliveira
    •  & Kristian G Andersen
  6. Massachusetts General Hospital, Boston, Massachusetts, USA.

    • Thomas F Rogers
  7. Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.

    • Lia Laura Lewis-Ximenez
  8. Fundação Oswaldo Cruz (FIOCRUZ), Salvador, Brazil.

    • Jaqueline Goes de Jesus
    • , Marta Giovanetti
    •  & Luiz Carlos Alcantara Jr.
  9. University of Rome, Tor Vergata, Italy.

    • Marta Giovanetti
  10. Department of Zoology, University of Oxford, Oxford, UK.

    • Sarah C Hill
    • , Nuno R Faria
    •  & Oliver G Pybus
  11. Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

    • Allison Black
    •  & Trevor Bedford
  12. Department of Epidemiology, University of Washington, Seattle, Washington, USA.

    • Allison Black
  13. University of Southampton, South General Hospital, Southampton, UK.

    • Miles W Carroll
  14. Instituto Evandro Chagas, Belem, Brazil.

    • Marcio Nunes
  15. Paul-Ehrlich-Institut, Langen, Germany.

    • Sally A Baylis
  16. DeepSeq, School of Life Sciences, University of Nottingham, Nottingham, UK.

    • Matthew Loose
  17. OICR, Toronto, Canada.

    • Jared T Simpson

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Contributions

J.Q. and N.J.L. conceived the project. J.Q., N.D.G., K.G.A. and N.J.L. designed the experiments and wrote the manuscript. J.Q., A.D.S. and O.G.P. built the online primer design tool. J.T.S. modified nanopolish to support R9/R9.4 data and indels. M.L. wrote the demultiplexing software. N.J.L. designed and implemented the MinION bioinformatics pipeline. N.J.L., T.B. and K.G. built the Docker image. N.D.G., K.G., G.O., R.R.-S. and K.G.A. developed the Illumina sequencing protocol and bioinformatics pipeline. L.L.L.-X. collected the chikungunya sample, performed clinical diagnosis and received local approvals. S.A.B. performed molecular diagnostics and curated Zika and Chikungunya control material. N.D.G., S.T.P., I.M.C., K.G., G.O., R.R.-S., T.F.R., N.A.B., J.G.d.J., M.G., S.H. and A.B. performed the experiments. All other authors tested the protocol and provided feedback. All authors have read and approved the contents of the manuscript.

Competing interests

J.Q., N.J.L. and J.T.S. have received expenses and/or honoraria to speak at Oxford Nanopore Technologies and Illumina events. N.J.L., M.L. and J.T.S. have received free-of-charge reagents from Oxford Nanopore Technologies as members of the developer group. N.J.L. has received free-of-charge reagents from Oxford Nanopore Technologies in support of this project. N.J.L. and M.W.C. have received free-of-charge reagents from Oxford Nanopore Technologies to support previous work on Ebola virus. J.T.S. has received research funding from Oxford Nanopore Technologies.

Corresponding author

Correspondence to Nicholas J Loman.

Supplementary information

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    Supplementary Tables

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