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

Josh Quick, Nathan D Grubaugh, Steven T Pullan, Ingra M Claro, Andrew D Smith, 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 Hill, Allison Black, Trevor Bedford, Miles W Carroll, Marcio Nunes, Luiz Carlos Alcantara Jr., Ester C Sabino, Sally A Baylis, Nuno Faria, Matthew Loose, Jared T Simpson, Oliver G Pybus, Kristian G Andersen, View ORCID ProfileNicholas J Loman
doi: https://doi.org/10.1101/098913
Josh Quick
1Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, UK.
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Nathan D Grubaugh
2The Scripps Research Institute, La Jolla, CA, USA.
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Steven T Pullan
3Public Health England, National infection Service, Porton Down, Salisbury, UK.
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Ingra M Claro
4Department of Infectious Disease and Institute of Tropical Medicine, University of Saõ Paulo, Saõ Paulo, Brazil.
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Andrew D Smith
1Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, UK.
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Karthik Gangavarapu
2The Scripps Research Institute, La Jolla, CA, USA.
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Glenn Oliveira
5Scripps Translational Science Institute, La Jolla, CA, USA.
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Refugio Robles-Sikisaka
2The Scripps Research Institute, La Jolla, CA, USA.
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Thomas F Rogers
2The Scripps Research Institute, La Jolla, CA, USA.
6Massachusetts General Hospital, Boston, USA.
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Nathan A Beutler
2The Scripps Research Institute, La Jolla, CA, USA.
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Dennis R Burton
2The Scripps Research Institute, La Jolla, CA, USA.
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Lia Laura Lewis-Ximenez
7Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro
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Jaqueline Goes de Jesus
8Fiocruz Bahia, Salvador, Brazil.
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Marta Giovanetti
8Fiocruz Bahia, Salvador, Brazil.
9University of Rome, Tor Vergata.
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Sarah Hill
10Department of Zoology, University of Oxford, Oxford, UK.
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Allison Black
11Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, USA.
12Department of Epidemiology, University of Washington, Seattle, USA.
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Trevor Bedford
11Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, USA.
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Miles W Carroll
3Public Health England, National infection Service, Porton Down, Salisbury, UK.
13University of Southampton, South General Hospital, Southampton, UK.
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Marcio Nunes
14Instituto Evandro Chagas, Belem, Brazil.
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Luiz Carlos Alcantara Jr.
7Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro
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Ester C Sabino
4Department of Infectious Disease and Institute of Tropical Medicine, University of Saõ Paulo, Saõ Paulo, Brazil.
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Sally A Baylis
15Paul-Ehrlich-Institut, Langen, Germany.
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Nuno Faria
9University of Rome, Tor Vergata.
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Matthew Loose
16University of Nottingham, Nottingham, UK.
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Jared T Simpson
17OICR, Toronto, Canada.
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Oliver G Pybus
10Department of Zoology, University of Oxford, Oxford, UK.
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Kristian G Andersen
2The Scripps Research Institute, La Jolla, CA, USA.
5Scripps Translational Science Institute, La Jolla, CA, USA.
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Nicholas J Loman
1Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, UK.
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  • ORCID record for Nicholas J Loman
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Abstract

Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples without isolation remains challenging for viruses such as Zika, where 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, optimised 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 starting with clinical samples in 1-2 days 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.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted January 09, 2017.
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Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples
Josh Quick, Nathan D Grubaugh, Steven T Pullan, Ingra M Claro, Andrew D Smith, 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 Hill, Allison Black, Trevor Bedford, Miles W Carroll, Marcio Nunes, Luiz Carlos Alcantara Jr., Ester C Sabino, Sally A Baylis, Nuno Faria, Matthew Loose, Jared T Simpson, Oliver G Pybus, Kristian G Andersen, Nicholas J Loman
bioRxiv 098913; doi: https://doi.org/10.1101/098913
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Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples
Josh Quick, Nathan D Grubaugh, Steven T Pullan, Ingra M Claro, Andrew D Smith, 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 Hill, Allison Black, Trevor Bedford, Miles W Carroll, Marcio Nunes, Luiz Carlos Alcantara Jr., Ester C Sabino, Sally A Baylis, Nuno Faria, Matthew Loose, Jared T Simpson, Oliver G Pybus, Kristian G Andersen, Nicholas J Loman
bioRxiv 098913; doi: https://doi.org/10.1101/098913

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