RT Journal Article
SR Electronic
T1 Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 098913
DO 10.1101/098913
A1 Josh Quick
A1 Nathan D Grubaugh
A1 Steven T Pullan
A1 Ingra M Claro
A1 Andrew D Smith
A1 Karthik Gangavarapu
A1 Glenn Oliveira
A1 Refugio Robles-Sikisaka
A1 Thomas F Rogers
A1 Nathan A Beutler
A1 Dennis R Burton
A1 Lia Laura Lewis-Ximenez
A1 Jaqueline Goes de Jesus
A1 Marta Giovanetti
A1 Sarah Hill
A1 Allison Black
A1 Trevor Bedford
A1 Miles W Carroll
A1 Marcio Nunes
A1 Luiz Carlos Alcantara, Jr.
A1 Ester C Sabino
A1 Sally A Baylis
A1 Nuno Faria
A1 Matthew Loose
A1 Jared T Simpson
A1 Oliver G Pybus
A1 Kristian G Andersen
A1 Nicholas J Loman
YR 2017
UL http://biorxiv.org/content/early/2017/01/09/098913.abstract
AB 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.