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Metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance

Nature Microbiology volume 5, pages 443–454 (2020)Cite this article

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An Author Correction to this article was published on 21 January 2020

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Abstract

Metagenomic next-generation sequencing (mNGS), the shotgun sequencing of RNA and DNA from clinical samples, has proved useful for broad-spectrum pathogen detection and the genomic surveillance of viral outbreaks. An additional target enrichment step is generally needed for high-sensitivity pathogen identification in low-titre infections, yet available methods using PCR or capture probes can be limited by high cost, narrow scope of detection, lengthy protocols and/or cross-contamination. Here, we developed metagenomic sequencing with spiked primer enrichment (MSSPE), a method for enriching targeted RNA viral sequences while simultaneously retaining metagenomic sensitivity for other pathogens. We evaluated MSSPE for 14 different viruses, yielding a median tenfold enrichment and mean 47% (±16%) increase in the breadth of genome coverage over mNGS alone. Virus detection using MSSPE arboviral or haemorrhagic fever viral panels was comparable in sensitivity to specific PCR, demonstrating 95% accuracy for the detection of Zika, Ebola, dengue, chikungunya and yellow fever viruses in plasma samples from infected patients. Notably, sequences from re-emerging and/or co-infecting viruses that have not been specifically targeted a priori, including Powassan and Usutu, were successfully enriched using MSSPE. MSSPE is simple, low cost, fast and deployable on either benchtop or portable nanopore sequencers, making this method directly applicable for diagnostic laboratory and field use.

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Fig. 1: MSSPE viral primer design and metagenomic sequencing workflow.
Fig. 2: Spiked primer enrichment of viral sequences using MSSPE.
Fig. 3: Improvements in viral genome coverage using MSSPE.

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Data availability

Sequence data were deposited in the NCBI Sequence Read Archive after removal of human genomic reads (NCBI BioProject accession no. PRJNA578816, umbrella BioProject accession no. PRJNA171119). The data that support the findings of the study are available from the corresponding author on reasonable request. MSSPE primer sequences tested in this study are provided in Supplementary Table 21. Source data for Fig. 2 are presented with this paper.

Code availability

SURPI+, SURPIrt and MSSPE-design have been deposited on Github and are available for download for research use only at https://github.com/chiulab/SURPI-plus-dist, https://github.com/chiulab/SURPIrt-dist and https://github.com/chiulab/MSSPE-design, respectively.

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Acknowledgements

We thank N. Loman and J. Quick at the University of Birmingham for providing ZIKV tiling multiplex PCR primers. The following viral RNA extracts were obtained through Biodefense and Emerging Infections Resources, the National Institute of Allergy and Infectious Diseases (NIAID) and the National Institutes of Health (NIH): CCHFV, IbAr10200, NR-37382; LASV, Josiah, NR-31821; Rift Valley Fever Virus, ZH501, NR-37379; MeV, Edmonston strain, NR-44104. This work was also funded in part by Abbott Laboratories (C.Y.C.), NIH grant no. R33-AI129455 (C.Y.C.) from the NIAID, NIH grant no. R01-HL105704 (C.Y.C.) from the National Heart, Lung, and Blood Institute, the California Initiative to Advance Precision Medicine (C.Y.C.), the Charles and Helen Schwab Foundation (C.Y.C.), the Steven and Alexandra Cohen Foundation (C.Y.C.), United States Department of Defense award W81XWH-17-1-0681 (C.Y.C.), the Wellcome Trust and Royal Society/Sir Henry Dale Fellowship grant no. 204311/Z/16/Z (N.R.F.), the Global Challenges Research Fund grant no. 005073 (N.R.F.), the Oxford John Fell Research Fund grant no. 005166 (N.R.F.) and Africa Oxford grant no. AfiOx-48 (N.R.F.).

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Authors and Affiliations

  1. Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA

    Xianding Deng, Asmeeta Achari, Scot Federman, Guixia Yu, Sneha Somasekar, Shaun Arevalo, Steve Miller & Charles Y. Chiu

  2. UCSF–Abbott Viral Diagnostics and Discovery Center, San Francisco, CA, USA

    Xianding Deng, Asmeeta Achari, Scot Federman, Guixia Yu, Sneha Somasekar & Charles Y. Chiu

  3. Research Institute for Medicines, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal

    Inês Bártolo & Nuno Taveira

  4. Viral and Rickettsial Disease Laboratory, California Department of Public Health, Richmond, CA, USA

    Shigeo Yagi, Kristina Hsieh, Sharon Messenger & Debra A. Wadford

  5. Institut National de Recherche Biomédicale, Kinshasa, Democratic Republic of the Congo

    Placide Mbala-Kingebeni, Jimmy Kapetshi, Steve Ahuka-Mundeke & Jean-Jacques Muyembe-Tamfum

  6. Boston Children’s Hospital, Boston, MA, USA

    Asim A. Ahmed

  7. Harvard Medical School, Boston, MA, USA

    Asim A. Ahmed

  8. Massachussetts General Hospital, Boston, MA, USA

    Vijay Ganesh

  9. Department of Virology and Immunology, Texas Biomedical Research Institute, San Antonio, TX, USA

    Manasi Tamhankar & Jean L. Patterson

  10. Institute for Human Virology Nigeria, Abuja, Nigeria

    Nicaise Ndembi

  11. Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD, USA

    Nicaise Ndembi

  12. Universite de Yaoundé I, Yaoundé, Cameroon

    Dora Mbanya

  13. University of Bamenda, Bamenda, Cameroon

    Dora Mbanya

  14. Université des Montagnes, Bangangté, Cameroon

    Lazare Kaptue

  15. University of Missouri-Kansas City, Kansas City, MO, USA

    Carole McArthur

  16. Instituto Mexicano del Seguro Social, Mexico City, Mexico

    José E. Muñoz-Medina & Cesar R. Gonzalez-Bonilla

  17. Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mexico

    Susana López & Carlos F. Arias

  18. Blood Systems Research Institute, San Francisco, CA, USA

    Mars Stone & Michael Busch

  19. Abbott Laboratories, Abbott Park, IL, USA

    Mary Rodgers, Gavin Cloherty & John R. Hackett Jr.

  20. Department of Zoology, University of Oxford, Oxford, UK

    Nuno R. Faria, Julien Thézé & Oliver G. Pybus

  21. Angolan National Institute of Health Research, Luanda, Angola

    Zoraima Neto & Joana Morais

  22. Instituto Universitário Egas Moniz (IUEM), Monte de Caparica, Portugal

    Nuno Taveira

  23. Department of Medicine, Division of Infectious Diseases, University of California San Francisco, San Francisco, CA, USA

    Charles Y. Chiu

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  1. Xianding Deng
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Contributions

C.Y.C. conceived, designed, and supervised the study, developed MSSPE-design software and SURPIrt pathogen identification software for nanopore sequencing and analysed data. X.D. coordinated the study, performed experiments and analysed data. A.A., G.Y. and S.S. performed experiments. S.F. and J.T. performed the bioinformatics analysis of sequence data. I.B., N.R.F., O.G.P., Z.N., J.M. and N.T. collected YFV samples from patients and extracted the viral RNA. S.Y., K.H., S. Me. and D.A.W. collected CHIKV and DENV samples from febrile travellers returning to the United States and extracted the viral RNA. P.M.-K., J.K., S.A.-M. and J.-J.M.-T. collected Ebola samples from patients and extracted the viral RNA. A.A.A. collected a clinical CSF sample from a patient with POWV meningoencephalitis. V.G. collected a clinical CSF sample from a patient with JCV meningoencephalitis. M.T. and J.L.P. cultured the Ebola Kikwit strain for use in MSSPE experiments. N.N., D.M., L.K., C.M., M.R., G.C. and J.R.H.Jr. collected clinical HIV samples from patients in Cameroon, genotyped the strains and performed qRT–PCR for viral titre estimates. J.E.M.-M., C.R.G.-B., S.L. and C.F.A. collected clinical ZIKV samples from patients in Mexico. S.A. and S. Mi. provided clinical HCV samples from patients in California, USA. M.S. and M.B. collected ZIKV and DENV samples from infected blood donors. C.Y.C. and X.D. wrote the manuscript. C.Y.C., X.D., M.R. and G.C. edited the manuscript. All authors read the manuscript and agreed to its contents.

Corresponding author

Correspondence to Charles Y. Chiu.

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Competing interests

C.Y.C. is the director of the UCSF–Abbott Viral Diagnostics and Discovery Center and receives research support funding from Abbott Laboratories, Inc. X.D. and C.Y.C. are inventors on a patent application titled ‘Spiked Primer Design for Targeted Enrichment of Metagenomic Libraries’ (US application no. 62/667,344, filed 4 May 2018 by the University of California San Francisco) that includes a description of the methods and primer sets presented in this paper. A.A.A. is an employee of Karius, Inc.

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Supplementary Figs. 1 and 2 and Supplementary Tables 1–18.

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Deng, X., Achari, A., Federman, S. et al. Metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance. Nat Microbiol 5, 443–454 (2020). https://doi.org/10.1038/s41564-019-0637-9

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