Letter | Published:

Real-time, portable genome sequencing for Ebola surveillance

Nature volume 530, pages 228232 (11 February 2016) | Download Citation

Abstract

The Ebola virus disease epidemic in West Africa is the largest on record, responsible for over 28,599 cases and more than 11,299 deaths1. Genome sequencing in viral outbreaks is desirable to characterize the infectious agent and determine its evolutionary rate. Genome sequencing also allows the identification of signatures of host adaptation, identification and monitoring of diagnostic targets, and characterization of responses to vaccines and treatments. The Ebola virus (EBOV) genome substitution rate in the Makona strain has been estimated at between 0.87 × 10−3 and 1.42 × 10−3 mutations per site per year. This is equivalent to 16–27 mutations in each genome, meaning that sequences diverge rapidly enough to identify distinct sub-lineages during a prolonged epidemic2,3,4,5,6,7. Genome sequencing provides a high-resolution view of pathogen evolution and is increasingly sought after for outbreak surveillance. Sequence data may be used to guide control measures, but only if the results are generated quickly enough to inform interventions8. Genomic surveillance during the epidemic has been sporadic owing to a lack of local sequencing capacity coupled with practical difficulties transporting samples to remote sequencing facilities9. To address this problem, here we devise a genomic surveillance system that utilizes a novel nanopore DNA sequencing instrument. In April 2015 this system was transported in standard airline luggage to Guinea and used for real-time genomic surveillance of the ongoing epidemic. We present sequence data and analysis of 142 EBOV samples collected during the period March to October 2015. We were able to generate results less than 24 h after receiving an Ebola-positive sample, with the sequencing process taking as little as 15–60 min. We show that real-time genomic surveillance is possible in resource-limited settings and can be established rapidly to monitor outbreaks.

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European Nucleotide Archive

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Acknowledgements

The EMLab is a technical partner in the WHO Emerging and Dangerous Pathogens Laboratory Network (EDPLN), and the Global Outbreak Alert and Response Network (GOARN) and the deployments in West Africa have been coordinated and supported by the GOARN Operational Support Team at WHO/HQ and the African Union. This work was carried out in the context of the project EVIDENT (Ebola virus disease: correlates of protection, determinants of outcome, and clinical management) that received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 666100 and in the context of service contract IFS/2011/272-372 funded by Directorate-General for International Cooperation and Development. J.Q. is funded by the NIHR Surgical Reconstruction and Microbiology Research Centre (SRMRC). N.J.L. is funded by a Medical Research Council Special Training Fellowship in Biomedical Informatics (to September 2015) and a Medical Research Council Bioinformatics Fellowship. J.T.S. is supported by the Ontario Institute for Cancer Research through funding provided by the Government of Ontario. Dstl support was funded by the UK Ministry of Defence (MOD). Dstl authors thank S. Lonsdale, C. Lonsdale and C. Mayers for supply of RNA, previous assistance, and review of the manuscript. The views expressed in this paper are not necessarily endorsed by the UK MOD. A.R. was supported by EU Seventh Framework Programme [FP7/2007-2013] under Grant Agreement no. 278433-PREDEMICS and ERC Grant agreement no. 260864. We are grateful for the generous support of University of Birmingham alumni for donations in support of the pilot work. The MRC Cloud Infrastructure for Microbial Bioinformatics (CLIMB) cyberinfrastructure was used to conduct bioinformatics analysis. The authors would like to thank B. Oppenheim and C. Wardius for help with logistics and the staff of Alta Biosciences, University of Birmingham and Sigma-Aldrich for generating PCR primers especially rapidly for this project. The authors would like to thank scientists deployed from the Special Pathogens Program from the National Microbiology Laboratory, Public Health Agency of Canada, who worked on EBOV diagnostics in Guinea. We are grateful to I. Goodfellow, M. Cotten and P. Kellam for permission to include sequences from Sierra Leone in this analysis. We thank R. Vipond for assistance with validation experiments. We thank H. Eno and B. Myers for help with proofreading. We are thankful for the generous support of reagents and technical support from Oxford Nanopore. We thank the staff at Oxford Nanopore for technical and logistical support during this project with special thanks to S. Brooking, O. Hartwell, R. Pettett, C. Brown, G. Sanghera and R. Ronan. We thank T. Bedford and R. Neher for developing the Nextstrain website.

Author information

Author notes

    • Joshua Quick
    • , Nicholas J. Loman
    • , Sophie Duraffour
    • , Jared T. Simpson
    • , Ettore Severi
    •  & Lauren Cowley

    These authors contributed equally to this work.

Affiliations

  1. Institute of Microbiology and Infection, University of Birmingham, Birmingham B15 2TT, UK

    • Joshua Quick
    •  & Nicholas J. Loman
  2. The European Mobile Laboratory Consortium, Bernhard-Nocht-Institute for Tropical Medicine, D-20359 Hamburg, Germany

    • Sophie Duraffour
    • , Joseph Akoi Bore
    • , Raymond Koundouno
    • , Babak Afrough
    • , Amadou Bah
    • , Jonathan H. J. Baum
    • , Beate Becker-Ziaja
    • , Jan Peter Boettcher
    • , Mar Cabeza-Cabrerizo
    • , Álvaro Camino-Sánchez
    • , Lisa L. Carter
    • , Juliane Doerrbecker
    • , Theresa Enkirch
    • , Isabel García- Dorival
    • , Nicole Hetzelt
    • , Julia Hinzmann
    • , Tobias Holm
    • , Liana Eleni Kafetzopoulou
    • , Michel Koropogui
    • , Abigael Kosgey
    • , Eeva Kuisma
    • , Christopher H. Logue
    • , Antonio Mazzarelli
    • , Sarah Meisel
    • , Marc Mertens
    • , Janine Michel
    • , Didier Ngabo
    • , Katja Nitzsche
    • , Elisa Pallasch
    • , Livia Victoria Patrono
    • , Jasmine Portmann
    • , Johanna Gabriella Repits
    • , Natasha Y. Rickett
    • , Andreas Sachse
    • , Katrin Singethan
    • , Inês Vitoriano
    • , Rahel L. Yemanaberhan
    • , Elsa G. Zekeng
    • , Antonino Di Caro
    • , Roman Wölfel
    • , Kilian Stoecker
    • , Erna Fleischmann
    • , Martin Gabriel
    • , Stephan Günther
    •  & Miles W. Carroll
  3. Bernhard-Nocht-Institute for Tropical Medicine, D-20359 Hamburg, Germany

    • Sophie Duraffour
    • , Jonathan H. J. Baum
    • , Beate Becker-Ziaja
    • , Mar Cabeza-Cabrerizo
    • , Juliane Doerrbecker
    • , Tobias Holm
    • , Sarah Meisel
    • , Katja Nitzsche
    • , Elisa Pallasch
    • , Livia Victoria Patrono
    • , Rahel L. Yemanaberhan
    • , Martin Gabriel
    •  & Stephan Günther
  4. Ontario Institute for Cancer Research, Toronto M5G 0A3, Canada

    • Jared T. Simpson
  5. Department of Computer Science, University of Toronto, Toronto M5S 3G4, Canada

    • Jared T. Simpson
  6. European Centre for Disease Prevention and Control (ECDC), 171 65 Solna, Sweden

    • Ettore Severi
    • , Natacha Milhano
    •  & Christopher J. Williams
  7. National Infection Service, Public Health England, London NW9 5EQ, UK

    • Lauren Cowley
    •  & Amy Mikhail
  8. Institute of Evolutionary Biology, University of Edinburgh, Edinburgh EH9 2FL, UK

    • Gytis Dudas
    •  & Andrew Rambaut
  9. Postgraduate Training for Applied Epidemiology (PAE, German FETP), Robert Koch Institute, D-13302 Berlin, Germany

    • Nobila Ouédraogo
  10. National Infection Service, Public Health England, Porton Down, Wiltshire SP4 0JG, UK

    • Babak Afrough
    • , Eeva Kuisma
    • , Christopher H. Logue
    • , Didier Ngabo
    • , Inês Vitoriano
    • , Kuiama Lewandowski
    •  & Miles W. Carroll
  11. Swiss Tropical and Public Health Institute, 4002 Basel, Switzerland

    • Amadou Bah
  12. Robert Koch Institute, D-13302 Berlin, Germany

    • Jan Peter Boettcher
    • , Nicole Hetzelt
    • , Julia Hinzmann
    • , Janine Michel
    •  & Andreas Sachse
  13. University College London, London WC1E 6BT, UK

    • Lisa L. Carter
  14. Paul-Ehrlich-Institut, Division of Veterinary Medicine, D-63225 Langen, Germany

    • Theresa Enkirch
  15. Institute of Infection and Global Health, University of Liverpool, Liverpool L69 7BE, UK

    • Isabel García- Dorival
    • , Natasha Y. Rickett
    • , Elsa G. Zekeng
    • , Georgios Pollakis
    •  & Julian A. Hiscox
  16. Laboratory for Clinical and Epidemiological Virology, Department of Microbiology and Immunology, KU Leuven, Leuven B-3000, Belgium

    • Liana Eleni Kafetzopoulou
  17. Ministry of Health Guinea, Conakry BP 585, Guinea

    • Michel Koropogui
    • , Facinet Yattara
    •  & Sakoba Keïta
  18. Kenya Medical Research Institute, Nairobi P.O. BOX 54840 - 00200, Kenya

    • Abigael Kosgey
  19. National Institute for Infectious Diseases L. Spallanzani, 00149 Rome, Italy

    • Antonio Mazzarelli
    •  & Antonino Di Caro
  20. Friedrich-Loeffler-Institute, D-17493 Greifswald, Germany

    • Marc Mertens
  21. Federal Office for Civil Protection, Spiez Laboratory, 3700 Spiez, Switzerland

    • Jasmine Portmann
  22. Janssen-Cilag, Stockholm, Box 7073 – 19207, Sweden

    • Johanna Gabriella Repits
  23. NIHR Health Protection Research Unit in Emerging and Zoonotic Infections, University of Liverpool, Liverpool L69 7BE, UK

    • Natasha Y. Rickett
    • , Elsa G. Zekeng
    • , Georgios Pollakis
    •  & Julian A. Hiscox
  24. Institute of Virology, Technische Universität München, D-81675 Munich, Germany

    • Katrin Singethan
  25. Public Health Agency of Canada, Winnipeg, Manitoba R3E 3R2, Canada

    • Trina Racine
    •  & Alexander Bello
  26. Institut Pasteur Dakar, Dakar, DP 220 Senegal

    • Amadou Alpha Sall
    • , Ousmane Faye
    •  & Oumar Faye
  27. Laboratoire de Fièvres Hémorragiques de Guinée, Conakry BP 5680, Guinea

    • N’Faly Magassouba
  28. Sandia National Laboratories, PO Box 5800 MS1363, Albuquerque, New Mexico 87185-1363, USA

    • Cecelia V. Williams
    • , Victoria Amburgey
    •  & Linda Winona
  29. Ratoma Ebola Diagnostic Center, Conakry, Guinea

    • Cecelia V. Williams
    • , Victoria Amburgey
    • , Linda Winona
    • , Emily Davis
    • , Jon Gerlach
    •  & Frank Washington
  30. MRIGlobal, Kansas City, Missouri 64110-2241, USA

    • Emily Davis
    • , Jon Gerlach
    •  & Frank Washington
  31. Expertise France, Laboratoire K-plan de Forecariah en Guinée, 75006 Paris, France

    • Vanessa Monteil
    • , Marine Jourdain
    • , Marion Bererd
    • , Alimou Camara
    • , Hermann Somlare
    • , Abdoulaye Camara
    • , Marianne Gerard
    • , Guillaume Bado
    •  & Bernard Baillet
  32. Fédération des Laboratoires - HIA Bégin, 94163 Saint-Mandé cedex, France

    • Déborah Delaune
  33. Laboratoire de Biologie - Centre de Traitement des Soignants, Conakry, Guinea

    • Déborah Delaune
  34. World Health Organization, Conakry BP 817, Guinea

    • Koumpingnin Yacouba Nebie
    • , Abdoulaye Diarra
    • , Yacouba Savane
    • , Raymond Bernard Pallawo
    • , Isabelle Roger
    • , Boubacar Diallo
    •  & Pierre Formenty
  35. London School of Hygiene and Tropical Medicine, London EC1E 7HT, UK

    • Giovanna Jaramillo Gutierrez
  36. Norwegian Institute of Public Health, PO Box 4404 Nydalen, 0403 Oslo, Norway

    • Natacha Milhano
  37. Public Health Wales, Cardiff CF11 9LJ, UK

    • Christopher J. Williams
  38. Defence Science and Technology Laboratory (Dstl) Porton Down, Salisbury SP4 0JQ, UK

    • James Taylor
    • , Phillip Rachwal
    •  & Simon A. Weller
  39. Oxford Nanopore Technologies, Oxford OX4 4GA, UK

    • Daniel J. Turner
  40. Department of Cellular and Molecular Medicine, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK

    • David A. Matthews
  41. Academic Department of Military Medicine, Royal Centre for Defence Medicine, Birmingham B15 2TH, UK

    • Matthew K. O’ Shea
    • , Andrew McD. Johnston
    •  & Duncan Wilson
  42. Centre of Defence Pathology, Royal Centre for Defence Medicine, Birmingham B15 2TH, UK

    • Emma Hutley
  43. Queen Elizabeth Hospital, Birmingham B12 2TH, UK

    • Erasmus Smit
  44. Bundeswehr Institute of Microbiology, D-80937 Munich, Germany

    • Roman Wölfel
    • , Kilian Stoecker
    •  & Erna Fleischmann
  45. Institut National de Santé Publique, Conakry BP 1147, Guinea

    • Lamine Koivogui
  46. Fogarty International Center, National Institutes of Health, Bethesda, MD 20892-2220, USA

    • Andrew Rambaut
  47. Centre for Immunology, Infection and Evolution, University of Edinburgh, Edinburgh EH9 2FL, UK

    • Andrew Rambaut
  48. University of Southampton, South General Hospital, Southampton SO16 6YD, UK

    • Miles W. Carroll
  49. NIHR Health Protection Research Unit in Emerging and Zoonotic Infections, PHE Porton Down, UK

    • Miles W. Carroll

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Contributions

N.J.L., J.Q., M.K.O’S., D.W., S.G., M.W.C. conceived the study. N.J.L., J.Q., M.K.O’S., S.A.W., J.T., P.R., D.T. designed the lab in a suitcase and laboratory protocol and initial validation. J.Q., S.D., L.C., J.A.B., R.K., L.E.K., and A.Ma. performed MinION sequencing. N.J.L., J.Q. and J.T.S. performed bioinformatics analysis and wrote software. J.T.S. added variant calling support to the nanopolish software. N.J.L., J.Q., S.D., E.S., P.F., L.C., A.Mi., N.M. and I.R. analysed the data. G.D., A.R., N.J.L., J.Q. and G.P. performed phylogenetic analysis. J.A.H., D.A.M., G.P., K.L., B.A. assisted further validation experiments. M.W.C., M.Ga., S.G., A.D.C., K.S., E.F. and R.W. coordinated activities for the European Mobile Laboratories. N.J.L., J.Q., S.D., M.W.C., S.G., M.K.O’S., A.R., E.S., P.F., I.R., A.Mi., and L.C. wrote the manuscript. All other authors were involved either in sample collection, and/or logistical support and strategic oversight for the work.

Competing interests

J.Q., N.J.L. and J.T.S. have all received travel expenses and accommodation from Oxford Nanopore to speak at organised symposia. J.Q. and N.J.L. have received an honorarium payment to speak at an Oxford Nanopore meeting. N.J.L. is a member of the Oxford Nanopore MinION Access Programme and has received reagents free of charge as part of the MinION Access Programme and in support of this project but does not receive other financial compensation or hold shares. D.T. is an employee of Oxford Nanopore.

Corresponding author

Correspondence to Nicholas J. Loman.

MinION and Illumina raw sequence files have been deposited into the European Nucleotide Archive under project code PRJEB10571.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains a Field Guide to Nanopore Sequencing - a detailed discussion of logistical issues that arose during this project and Supplementary Tables 1-4.

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DOI

https://doi.org/10.1038/nature16996

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