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Nat Protoc. 2017 Jun;12(6):1261-1276. doi: 10.1038/nprot.2017.066. Epub 2017 May 24.

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

Author information

1
Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, UK.
2
The Scripps Research Institute, La Jolla, California, USA.
3
Public Health England, National Infection Service, Porton Down, Salisbury, UK.
4
Department of Infectious Disease and Institute of Tropical Medicine, University of Saõ Paulo, Saõ Paulo, Brazil.
5
Scripps Translational Science Institute, La Jolla, California, USA.
6
Massachusetts General Hospital, Boston, Massachusetts, USA.
7
Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.
8
Fundação Oswaldo Cruz (FIOCRUZ), Salvador, Brazil.
9
University of Rome, Tor Vergata, Italy.
10
Department of Zoology, University of Oxford, Oxford, UK.
11
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
12
Department of Epidemiology, University of Washington, Seattle, Washington, USA.
13
University of Southampton, South General Hospital, Southampton, UK.
14
Instituto Evandro Chagas, Belem, Brazil.
15
Paul-Ehrlich-Institut, Langen, Germany.
16
DeepSeq, School of Life Sciences, University of Nottingham, Nottingham, UK.
17
OICR, Toronto, Canada.

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.

PMID:
28538739
PMCID:
PMC5902022
DOI:
10.1038/nprot.2017.066
[Indexed for MEDLINE]
Free PMC Article

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