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

Joshua 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 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; Nicholas J Loman

doi:10.1038/nprot.2017.066

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

Joshua 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 C Hill, Allison Black, Trevor Bedford, Miles W Carroll, Marcio Nunes, Luiz Carlos AlcantaraEster C Sabino, Sally A Baylis, Nuno R Faria, Matthew Loose, Jared T Simpson, Oliver G Pybus, Kristian G Andersen, 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 (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 language	English
Pages (from-to)	1261-1276
Number of pages	16
Journal	Nature protocols
Volume	12
Issue number	6
DOIs	https://doi.org/10.1038/nprot.2017.066
Publication status	Published - 24 May 2017

Keywords

Genomic analysis
Next-generation sequencing
Viral epidemiology
Viral genetics
Virology

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1038/nprot.2017.066Licence: None: All rights reserved

Quick_et_al_Multiplex_PCR_method_Nature_Protocols
Final Version of Record published as above. Checked 30/6/2017
Accepted author manuscript, 674 KBLicence: None: All rights reserved

https://doi.org/10.1038/nprot.2017.066Licence: None: All rights reserved

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Quick, J., Grubaugh, N. D., Pullan, S. T., Claro, I. M., Smith, A. D., Gangavarapu, K., Oliveira, G., Robles-Sikisaka, R., Rogers, T. F., Beutler, N. A., Burton, D. R., Lewis-Ximenez, L. L., de Jesus, J. G., Giovanetti, M., Hill, S. C., Black, A., Bedford, T., Carroll, M. W., Nunes, M., ... Loman, N. J. (2017). Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nature protocols, 12(6), 1261-1276. https://doi.org/10.1038/nprot.2017.066

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

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 = "Genomic analysis, Next-generation sequencing, Viral epidemiology, Viral genetics, Virology",

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

year = "2017",

month = may,

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doi = "10.1038/nprot.2017.066",

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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 2017, 'Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples', Nature protocols, vol. 12, no. 6, pp. 1261-1276. https://doi.org/10.1038/nprot.2017.066

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T1 - Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples

AU - Quick, Joshua

AU - Grubaugh, Nathan D

AU - Pullan, Steven T

AU - Claro, Ingra M

AU - Smith, Andrew D

AU - Gangavarapu, Karthik

AU - Oliveira, Glenn

AU - Robles-Sikisaka, Refugio

AU - Rogers, Thomas F

AU - Beutler, Nathan A

AU - Burton, Dennis R

AU - Lewis-Ximenez, Lia Laura

AU - de Jesus, Jaqueline Goes

AU - Giovanetti, Marta

AU - Hill, Sarah C

AU - Black, Allison

AU - Bedford, Trevor

AU - Carroll, Miles W

AU - Nunes, Marcio

AU - Alcantara, Luiz Carlos

AU - Sabino, Ester C

AU - Baylis, Sally A

AU - Faria, Nuno R

AU - Loose, Matthew

AU - Simpson, Jared T

AU - Pybus, Oliver G

AU - Andersen, Kristian G

AU - Loman, Nicholas J

PY - 2017/5/24

Y1 - 2017/5/24

N2 - 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.

AB - 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.

KW - Genomic analysis

KW - Next-generation sequencing

KW - Viral epidemiology

KW - Viral genetics

KW - Virology

U2 - 10.1038/nprot.2017.066

DO - 10.1038/nprot.2017.066

M3 - Article

C2 - 28538739

SN - 1754-2189

VL - 12

SP - 1261

EP - 1276

JO - Nature protocols

JF - Nature protocols

IS - 6

ER -

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

Abstract

Keywords

UN SDGs

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