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.

Figure 1

Workflow for tiling amplicon sequencing.

Figure 2

Overview of multiplex primer design using primal online primer design tool. (a) Submission box for online primer design tool. (b) Primer table of results. (c) Schematic showing expected amplicon products for each pool in genomic context for the ZikaAsian and ChikAsianECSA schemes.

Figure 3

Overview of multiplex tiling PCR and pooling. (a) Schematic showing how the primers in pool ‘1’ and ‘2’ overlap between but not within reactions. (b) Amplicons generated by pool 1 and pool 2 primers from one sample are (c) pooled together, and (d) ligated with the same barcode during library preparation. (e) Samples are given unique barcodes so that (f) amplicons from many samples can be combined before platform specific sequencing.

Figure 4

Coverage plots for ZikaAsian scheme sequenced on MinION before (top panel) and after primer trimming and coverage normalisation (bottom panel). During the preprocessing step reads are trimmed using a BED file containing primer positions and read coverage is normalised. The coverage plot was produced using the Tablet genome viewer .