Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples.
Joshua QuickNathan D GrubaughSteven T PullanIngra M ClaroAndrew D SmithKarthik GangavarapuGlenn OliveiraRefugio Robles-SikisakaThomas F RogersNathan A BeutlerDennis R BurtonLia Laura Lewis-XimenezJaqueline Goes de JesusMarta GiovanettiSarah C HillAllison BlackTrevor BedfordMiles W CarrollMarcio NunesLuiz Carlos Junior AlcantaraEster C SabinoSally A BaylisNuno R FariaMatthew LooseJared T SimpsonOliver G PybusKristian G AndersenNicholas J LomanPublished in: Nature protocols (2017)
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.
Keyphrases
- zika virus
- single cell
- real time pcr
- sars cov
- dengue virus
- randomized controlled trial
- infectious diseases
- aedes aegypti
- high throughput
- genome wide
- cancer therapy
- genetic diversity
- clinical practice
- functional connectivity
- electronic health record
- white matter
- gene expression
- high resolution
- cell free
- patient reported outcomes
- resting state
- molecularly imprinted
- solid state