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

Research output: Contribution to journalArticle


  • Nathan D Grubaugh
  • Steven T Pullan
  • Ingra M Claro
  • Karthik Gangavarapu
  • Glenn Oliveira
  • Refugio Robles-Sikisaka
  • Thomas F Rogers
  • Nathan A Beutler
  • Dennis R Burton
  • Lia Laura Lewis-Ximenez
  • Jaqueline Goes de Jesus
  • Marta Giovanetti
  • Sarah C Hill
  • Allison Black
  • Trevor Bedford
  • Miles W Carroll
  • Marcio Nunes
  • Luiz Carlos Alcantara
  • Ester C Sabino
  • Sally A Baylis
  • Nuno R Faria
  • Matthew Loose
  • Jared T Simpson
  • Oliver G Pybus
  • Kristian G Andersen

Colleges, School and Institutes

External organisations

  • Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom.
  • The Scripps Research Institute, Department of Immunology and Microbial Science, La Jolla, California 92037, USA.
  • Public Health England, National Infection Service, Porton Down, Salisbury, UK.
  • Department of Infectious Disease and Institute of Tropical Medicine, University of Saõ Paulo, Saõ Paulo, Brazil.
  • Scripps Translational Science Institute, La Jolla, California, USA.
  • Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.
  • Fundação Oswaldo Cruz (FIOCRUZ), Salvador, Brazil.
  • Department of Zoology, University of Oxford
  • Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
  • Instituto Evandro Chagas, Belem, Brazil.
  • Paul-Ehrlich-Institut, Langen, Germany.
  • DeepSeq, School of Life Sciences, University of Nottingham, Nottingham, UK.
  • OICR, Toronto, Canada.


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.


Original languageEnglish
Pages (from-to)1261-1276
Number of pages16
JournalNature protocols
Issue number6
Early online date24 May 2017
Publication statusPublished - Jun 2017


  • Genomic analysis, Next-generation sequencing, Viral epidemiology, Viral genetics, Virology