TY - JOUR T1 - Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples JF - bioRxiv DO - 10.1101/098913 SP - 098913 AU - Josh Quick AU - Nathan D Grubaugh AU - Steven T Pullan AU - Ingra M Claro AU - Andrew D Smith AU - Karthik Gangavarapu AU - Glenn Oliveira AU - Refugio Robles-Sikisaka AU - Thomas F Rogers AU - Nathan A Beutler AU - Dennis R Burton AU - Lia Laura Lewis-Ximenez AU - Jaqueline Goes de Jesus AU - Marta Giovanetti AU - Sarah Hill AU - Allison Black AU - Trevor Bedford AU - Miles W Carroll AU - Marcio Nunes AU - Luiz Carlos Alcantara, Jr. AU - Ester C Sabino AU - Sally A Baylis AU - Nuno Faria AU - Matthew Loose AU - Jared T Simpson AU - Oliver G Pybus AU - Kristian G Andersen AU - Nicholas J Loman Y1 - 2017/01/01 UR - http://biorxiv.org/content/early/2017/01/09/098913.abstract N2 - 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. ER -