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PMID: 28538739 Published · ppublish English Journal Article

Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples.

Nature protocols ·Vol. 12 ·No. 6 ·2017-06-00 ·Pages 1261-1276

Quick J, Grubaugh ND, Pullan ST, Claro IM, Smith AD, Gangavarapu K, Oliveira G, Robles-Sikisaka R, Rogers TF, Beutler NA, Burton DR, Lewis-Ximenez LL, de Jesus JG, Giovanetti M, Hill SC, Black A, Bedford T, Carroll MW, Nunes M, Alcantara LC, Sabino EC, Baylis SA, Faria NR, Loose M, Simpson JT, Pybus OG, Andersen KG, Loman NJ

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.

MeSH Terms
DNA Viruses/genetics Genome, Viral Molecular Epidemiology/methods Multiplex Polymerase Chain Reaction/methods RNA Viruses/genetics Sequence Analysis, DNA/methods
Authors & Affiliations
28 authors, click to expand affiliations / ORCID
Quick Joshua
Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, UK.
Grubaugh Nathan D ORCID
The Scripps Research Institute, La Jolla, California, USA.
Pullan Steven T
Public Health England, National Infection Service, Porton Down, Salisbury, UK.
Claro Ingra M
Department of Infectious Disease and Institute of Tropical Medicine, University of Saõ Paulo, Saõ Paulo, Brazil.
Smith Andrew D
Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, UK.
Gangavarapu Karthik
The Scripps Research Institute, La Jolla, California, USA.
Oliveira Glenn
Scripps Translational Science Institute, La Jolla, California, USA.
Robles-Sikisaka Refugio
The Scripps Research Institute, La Jolla, California, USA.
Rogers Thomas F
The Scripps Research Institute, La Jolla, California, USA. | Massachusetts General Hospital, Boston, Massachusetts, USA.
Beutler Nathan A
The Scripps Research Institute, La Jolla, California, USA.
Burton Dennis R
The Scripps Research Institute, La Jolla, California, USA.
Lewis-Ximenez Lia Laura
Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.
de Jesus Jaqueline Goes
Fundação Oswaldo Cruz (FIOCRUZ), Salvador, Brazil.
Giovanetti Marta
Fundação Oswaldo Cruz (FIOCRUZ), Salvador, Brazil. | University of Rome, Tor Vergata, Italy.
Hill Sarah C
Department of Zoology, University of Oxford, Oxford, UK.
Black Allison ORCID
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. | Department of Epidemiology, University of Washington, Seattle, Washington, USA.
Bedford Trevor
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
Carroll Miles W
Public Health England, National Infection Service, Porton Down, Salisbury, UK. | University of Southampton, South General Hospital, Southampton, UK.
Nunes Marcio
Instituto Evandro Chagas, Belem, Brazil.
Alcantara Luiz Carlos ORCID
Fundação Oswaldo Cruz (FIOCRUZ), Salvador, Brazil.
Sabino Ester C
Department of Infectious Disease and Institute of Tropical Medicine, University of Saõ Paulo, Saõ Paulo, Brazil.
Baylis Sally A
Paul-Ehrlich-Institut, Langen, Germany.
Faria Nuno R
Department of Zoology, University of Oxford, Oxford, UK.
Loose Matthew
DeepSeq, School of Life Sciences, University of Nottingham, Nottingham, UK.
Simpson Jared T
OICR, Toronto, Canada.
Pybus Oliver G
Department of Zoology, University of Oxford, Oxford, UK.
Andersen Kristian G
The Scripps Research Institute, La Jolla, California, USA. | Scripps Translational Science Institute, La Jolla, California, USA.
Loman Nicholas J
Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, UK.
References (42)
42 references, click to expand
  1. Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West Africa.
    Nature. 2015 Aug 6;524(7563):97-101 PMID: 26083749
  2. Primer3 on the WWW for general users and for biologist programmers.
    Methods Mol Biol. 2000;132:365-86 PMID: 10547847
  3. Genomic surveillance elucidates Ebola virus origin and transmission during the 2014 outbreak.
    Science. 2014 Sep 12;345(6202):1369-72 PMID: 25214632
  4. Tablet--next generation sequence assembly visualization.
    Bioinformatics. 2010 Feb 1;26(3):401-2 PMID: 19965881
  5. Performance comparison of benchtop high-throughput sequencing platforms.
    Nat Biotechnol. 2012 May;30(5):434-9 PMID: 22522955
  6. Isolation of a Novel Insect-Specific Flavivirus from Culiseta melanura in the Northeastern United States.
    Vector Borne Zoonotic Dis. 2016 Mar;16(3):181-90 PMID: 26807512
  7. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing.
    J Comput Biol. 2012 May;19(5):455-77 PMID: 22506599
  8. Actionable diagnosis of neuroleptospirosis by next-generation sequencing.
    N Engl J Med. 2014 Jun 19;370(25):2408-17 PMID: 24896819
  9. Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies.
    Nucleic Acids Res. 1990 Feb 25;18(4):999-1005 PMID: 2179874
  10. Genetic and phenotypic variation of foot-and-mouth disease virus during serial passages in a natural host.
    J Virol. 2007 Oct;81(20):11341-51 PMID: 17686868
  11. A guide to the design and use of mismatched and degenerate primers.
    PCR Methods Appl. 1994 Feb;3(4):S39-47 PMID: 8173508
  12. PriSM: a primer selection and matching tool for amplification and sequencing of viral genomes.
    Bioinformatics. 2011 Jan 15;27(2):266-7 PMID: 21068001
  13. CLIMB (the Cloud Infrastructure for Microbial Bioinformatics): an online resource for the medical microbiology community.
    Microb Genom. 2016 Sep 20;2(9):e000086 PMID: 28785418
  14. Rapid outbreak sequencing of Ebola virus in Sierra Leone identifies transmission chains linked to sporadic cases.
    Virus Evol. 2016 Jun 22;2(1):vew016 PMID: 28694998
  15. Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples.
    Genome Biol. 2014;15(11):519 PMID: 25403361
  16. Sequencing technologies - the next generation.
    Nat Rev Genet. 2010 Jan;11(1):31-46 PMID: 19997069
  17. The evolution of Ebola virus: Insights from the 2013-2016 epidemic.
    Nature. 2016 Oct 12;538(7624):193-200 PMID: 27734858
  18. Comparison of Next-Generation Sequencing Technologies for Comprehensive Assessment of Full-Length Hepatitis C Viral Genomes.
    J Clin Microbiol. 2016 Oct;54(10):2470-84 PMID: 27385709
  19. Trimmomatic: a flexible trimmer for Illumina sequence data.
    Bioinformatics. 2014 Aug 1;30(15):2114-20 PMID: 24695404
  20. 1970s and 'Patient 0' HIV-1 genomes illuminate early HIV/AIDS history in North America.
    Nature. 2016 Nov 3;539(7627):98-101 PMID: 27783600
  21. Snakemake--a scalable bioinformatics workflow engine.
    Bioinformatics. 2012 Oct 1;28(19):2520-2 PMID: 22908215
  22. Unusual distribution of mutations associated with serial bottleneck passages of human immunodeficiency virus type 1.
    J Virol. 2000 Oct;74(20):9546-52 PMID: 11000225
  23. Norovirus Whole-Genome Sequencing by SureSelect Target Enrichment: a Robust and Sensitive Method.
    J Clin Microbiol. 2016 Oct;54(10):2530-7 PMID: 27487952
  24. Complete Genome Sequence of Zika Virus Isolated from Semen.
    Genome Announc. 2016 Oct 13;4(5): PMID: 27738033
  25. Virus genomes reveal factors that spread and sustained the Ebola epidemic.
    Nature. 2017 Apr 20;544(7650):309-315 PMID: 28405027
  26. Outbreak of Zika Virus Infection, Chiapas State, Mexico, 2015, and First Confirmed Transmission by Aedes aegypti Mosquitoes in the Americas.
    J Infect Dis. 2016 Nov 1;214(9):1349-1356 PMID: 27436433
  27. Mobile real-time surveillance of Zika virus in Brazil.
    Genome Med. 2016 Sep 29;8(1):97 PMID: 27683027
  28. Real-time digital pathogen surveillance - the time is now.
    Genome Biol. 2015 Jul 30;16(1):155 PMID: 27391693
  29. Automated degenerate PCR primer design for high-throughput sequencing improves efficiency of viral sequencing.
    Virol J. 2012 Nov 06;9:261 PMID: 23131097
  30. Genome analysis linking recent European and African influenza (H5N1) viruses.
    Emerg Infect Dis. 2007 May;13(5):713-8 PMID: 17553249
  31. A modified RNA-Seq approach for whole genome sequencing of RNA viruses from faecal and blood samples.
    PLoS One. 2013 Jun 10;8(6):e66129 PMID: 23762474
  32. Coming of age: ten years of next-generation sequencing technologies.
    Nat Rev Genet. 2016 May 17;17(6):333-51 PMID: 27184599
  33. Direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased high-throughput sequencing approach.
    PLoS One. 2009;4(1):e4219 PMID: 19156205
  34. Real-time, portable genome sequencing for Ebola surveillance.
    Nature. 2016 Feb 11;530(7589):228-232 PMID: 26840485
  35. Integrative genomics viewer.
    Nat Biotechnol. 2011 Jan;29(1):24-6 PMID: 21221095
  36. Genome Sequence of a Candidate World Health Organization Reference Strain of Zika Virus for Nucleic Acid Testing.
    Genome Announc. 2016 Sep 01;4(5): PMID: 27587826
  37. The Sequence Alignment/Map format and SAMtools.
    Bioinformatics. 2009 Aug 15;25(16):2078-9 PMID: 19505943
  38. A new arenavirus in a cluster of fatal transplant-associated diseases.
    N Engl J Med. 2008 Mar 6;358(10):991-8 PMID: 18256387
  39. Skip the alignment: degenerate, multiplex primer and probe design using K-mer matching instead of alignments.
    PLoS One. 2012;7(4):e34560 PMID: 22485178
  40. Clinical Sequencing Uncovers Origins and Evolution of Lassa Virus.
    Cell. 2015 Aug 13;162(4):738-50 PMID: 26276630
  41. Distinct Zika Virus Lineage in Salvador, Bahia, Brazil.
    Emerg Infect Dis. 2016 Oct;22(10):1788-92 PMID: 27448188
  42. Multiplex degenerate primer design for targeted whole genome amplification of many viral genomes.
    Adv Bioinformatics. 2014;2014:101894 PMID: 25157264
Article Info
Journal
Nature protocols
Abbr.
Nat Protoc
ISSN
1750-2799
DOI
10.1038/nprot.2017.066
Published
2017-06-00
Epub
2017-00-24
Pages
1261-1276
Language
English
Region
England
NLM ID
101284307
PMCID
PMC5902022
Subset
IM
Grants
Medical Research Council · MR/L015080/1 · United Kingdom
NIGMS NIH HHS · R35 GM119774 · United States
Medical Research Council · MR/M501621/1 · United Kingdom
NIAID NIH HHS · T32 AI007244 · United States
NIGMS NIH HHS · U54 GM111274 · United States
Medical Research Council · MC_PC_15100 · United Kingdom
External Links
PubMed source DOI: 10.1038/nprot.2017.066 PMC Full Text View journal details
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