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MULTIPLEX PCR METHOD FOR MINION AND ILLUMINA SEQUENCING OF ZIKA AND OTHER VIRUS GENOMES DIRECTLY FROM CLINICAL SAMPLES
Quick, Joshua et al. | Date Issued: 2017
Author
Quick, Joshua
Grubaugh, Nathan D.
Pullan, Steven T.
Claro, Ingra M.
Smith, Andrew D.
Gangavarapu, Karthik
Oliveira, Glenn
Robles-Sikisaka, Refugio
Rogers, Thomas F.
Beutler, Nathan A.
Burton, Dennis R.
Lewis-Ximenez, Lia Laura
Jesus, Jaqueline Goes de
Giovanetti, Marta
Hill, Sarah C.
Black, Allison
Bedford, Trevor
Carroll, Miles W.
Nunes, Marcio
Alcantara, Luiz Carlos
Sabino, Ester C.
Baylis, Sally A.
Faria, Nuno R.
Loose, Matthew
Simpson, Jared T.
Pybus, Oliver G.
Andersen, Kristian G.
Loman, Nicholas J.
Affilliation
University of Birmingham. School of Biosciences. Institute of Microbiology and Infection. Birmingham, UK.
The Scripps Research Institute. La Jolla, California, USA.
Public Health England. National Infection Service., Porton Down, Salisbury, UK.
Universidade de São Paulo.Instituto de Medicina Tropical. Departamento de Doenças Infecciosas. São Paulo, SP., Brasil.
University of Birmingham. School of Biosciences. Institute of Microbiology and Infection. Birmingham, UK.
The Scripps Research Institute. La Jolla, California, USA.
Scripps Translational Science Institute. La Jolla, California, USA.
The Scripps Research Institute. La Jolla, California, USA.
The Scripps Research Institute. La Jolla, California, USA / Massachusetts General Hospital. Boston, Massachusetts, USA..
The Scripps Research Institute. La Jolla, California, USA.
The Scripps Research Institute. La Jolla, California, USA.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ. Brasil.
Fundação Oswaldo Cruz. Instituto Gonçalo Muniz. Salvador, BA, Brasil.
Fundação Oswaldo Cruz. Instituto Gonçalo Muniz. Salvador, BA, Brasil / University of Rome. Tor Vergata, Italy.
University of Oxford. Department of Zoology. Oxford, UK.
Fred Hutchinson Cancer Research Center. Vaccine and Infectious Disease Division. Seattle, Washington, USA / University of Washington. Department of Epidemiology. Seattle, Washington, USA.
Fred Hutchinson Cancer Research Center. Vaccine and Infectious Disease Division. Seattle, Washington, USA.
Public Health England. National Infection Service. Porton Down, Salisbury, UK / University of Southampton. South General Hospital. Southampton, UK.
Instituto Evandro Chagas. Belém, PA, Brazil.
Fundação Oswaldo Cruz. Instituto Gonçalo Muniz. Salvador, BA, Brasil.
Universidade de São Paulo.Instituto de Medicina Tropical. Departamento de Doenças Infecciosas. São Paulo, SP, Brasil.
Paul-Ehrlich-Institut. Langen, Germany.
University of Oxford. Department of Zoology. Oxford, UK.
University of Nottingham. School of Life Sciences. DeepSeq. Nottingham, UK.
OICR. Toronto, Canada.
University of Oxford. Department of Zoology. Oxford, UK.
The Scripps Research Institute. La Jolla, California, USA / Scripps Translational Science Institute, La Jolla. California, USA..
University of Birmingham. School of Biosciences. Institute of Microbiology and Infection. Birmingham, UK.
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.
Keywords in Portuguese
Zika virus
Sequenciação do genoma
Amostras clínicas
Reação em Cadeia da Polimerase Multiplex
Genomas de vírus
 
Keywords
Zika virus
Genome sequencing
Multiplex PCR method
Clinical samples
Virus genomes
 
Publisher
Nature Publishing Group
Citation
QUICK, Joshua et al. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nature Protocols, v. 12, n. 6, p. 1261-1276, Jun. 2017.
DOI
10.1038/nprot.2017.066
ISSN
1754-2189