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

Background: The genome of Vibrio parahaemolyticus MVP1, isolated from a Malaysian aquaculture farm with shrimp acute hepatopancreatic necrosis disease (AHPND), was previously sequenced using Illumina MiSeq 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. Methods: The genome of MVP1 was sequenced on Nanopore MinION to obtain long reads to improve genome contiguity. De novo genome assembly was performed using long-read only assembler followed by genome polishing and hybrid assembler. Results: Long-read assembly produced three complete circular MVP1 contigs: chromosome 1, chromosome 2 and the pVa plasmid encoding pirABvp binary toxin genes. Polishing of the long-read assembly with Illumina short reads was necessary to remove indel errors. Complete assembly of the pVa plasmid could not be achieved using Illumina reads due to identical repetitive elements flanking the binary toxin genes leading to multiple contigs. These regions were fully spanned by the Nanopore long-reads resulting in a single contig. 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 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 publicly available V. parahaemolyticus genomes revealed the presence of a single AinS-family quorum-sensing system that can be targeted for future microbial management. Conclusions: We 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.