Targeted gene sequencing with Nanopore enables rapid and accurate confirmatory diagnostic of Tilapia lake virus

Abstract

Infectious diseases represent one of the major challenges to sustainable aquaculture production. Rapid and accurate diagnosis of emerging pathogens during early-suspected disease cases is critical to facilitate timely response to deploy adequate control measures and prevent or reduce spread. Currently, most laboratories use PCR to amplify partial pathogen genomic regions combined with sequencing of PCR amplicon(s) using conventional Sanger sequencing services for confirmatory diagnosis. The main limitation of this approach is the uncertain turnaround time. Here, we report an innovative approach using Oxford Nanopore Technologies (ONT)-based amplicon sequencing. Using clinical samples from infected fish, we applied this approach for the rapid confirmation (in less than 12 h) of tilapia lake virus (TiLV), a disease-causing virus affecting global tilapia aquaculture. Despite the known high error rate of Nanopore sequencing at the single read level, the obtained consensus sequences after polishing exhibit strikingly high identity to their respective Illumina and Sanger-verified references (99.83-100 % identity). This study suggests that ONT-based amplicon sequencing is a promising platform to deploy in regional aquatic animal health diagnostic laboratories in low and medium income countries, for fast and accurate confirmation and phylogenetic/genotyping of emerging infectious pathogens from field samples within a single day.