Nanopore long reads enable the first complete genome assembly of a Malaysian Vibrio parahaemolyticus isolate bearing the pVa plasmid associated with acute hepatopancreatic necrosis disease

Abstract

BackgroundVibrio parahaemolyticus MVP1 was isolated from a Malaysian aquaculture farm affected with shrimp acute hepatopancreatic necrosis disease (AHPND). Its genome was previously sequenced on the Illumina MiSeq platform and assembled de novo producing a relatively fragmented assembly. Despite identifying the binary toxin genes in the MVP1 draft genome that were linked to AHPND, the toxin genes were localized on a very small contig precluding proper analysis of gene neighbourhood. MethodsThe genome of Vibrio parahaemolyticus MVP1 was sequenced on the Nanopore MinION device to obtain long reads that can span longer repeats and improve genome contiguity. De novo genome assembly was subsequently performed using long-read only assembler (Flye) followed by genome polishing as well as hybrid assembler (Unicycler). ResultsLong-read only assembly produced three complete circular MVP1 contigs consisting of chromosome 1, chromosome 2 and the pVa plasmid that pirABvp binary toxin genes. Polishing of the long read assembly with Illumina short reads was necessary to remove indel errors. The complete assembly of the pVa plasmid could not be achieved using Illumina reads due to the presence of identical repetitive elements flanking the binary toxin genes leading to multiple contigs. Whereas these regions were fully spanned by the Nanopore long reads resulting in a single contig. In addition, alignment of Illumina reads to the complete genome assembly indicated there is sequencing bias as read depth was lowest in low-GC genomic regions. Comparative genomic analysis revealed the presence of a gene cluster coding for additional insecticidal toxins in chromosome 2 of MVP1 that may further contribute to host pathogenesis pending functional validation. Scanning of all publicly available V. parahaemolyticus genomes revealed the presence of a single AinS-family quorum-sensing system in this species that can be targeted for future microbial management. ConclusionsWe generated the first chromosome-scale genome assembly of a Malaysian pirABVp-bearing V. parahaemolyticus isolate. Structural variations identified from comparative genomic analysis provide new insights into the genomic features of V. parahaemolyticus MVP1 that may be associated with host colonization and pathogenicity.