Computational protocol: Complete Genome Sequence of Vibrio sp. Strain 2521-89, a Close Relative of Vibrio cholerae Isolated from Lake Water in New Mexico, USA

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Protocol publication

[…] The Vibrio genus contains over 140 different species, including Vibrio cholerae, the causative agent of cholera. This species alone harbors over 200 different serogroups based on the O antigen, several of which are associated with human infections (). Here, we describe a potentially novel Vibrio species, Vibrio sp. strain 2521-89, which is a close relative of Vibrio cholerae but is markedly divergent from it.Genomic DNA was extracted using an ArchivePure DNA cell/tissue kit (5 PRIME) according to the manufacturer’s instructions. Sequencing libraries were prepared using the RSII SMRTbell template preparation kit 1.0 (PacBio) and sequenced with the P6v2 single-molecule real-time (SMRT) sequencing platform (PacBio). The complete genome has a total size of 4,123,863 bp and consists of two chromosomes (3,017,468 bp and 1,106,395 bp). The DNA G+C content was determined to be 47.2 mol%. Genome annotation was done using RAST 2.0 () and the Prokaryotic Genome Annotation Pipeline (PGAP) (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/). PGAP annotations identified 3,814 coding sequences, 31 rRNA sequences (11 5S rRNA, 10 16S rRNA, and 10 23S rRNA sequences), and 104 tRNA sequences. Vibrio sp. 2521-89 contains genes encoding the transcriptional activator ToxR protein, one of the major regulators of virulence genes in V. cholerae (), but it lacks both genes encoding the cholera toxin and the toxin-coregulated pilus cluster.Species identity was determined by calculating average nucleotide identity (ANI) and in silico digital DNA-DNA hybridization (dDDH) between Vibrio sp. 2521-89 and V. cholerae N16961, Vibrio metoecus OP3H, or Vibrio mimicus CAIM 602 using JSpecies 1.2.1 () and GGDC 2.1 (), respectively. Vibrio sp. 2521-89 has an ANI value of 95.1% with V. cholerae N16961 and values ranging from 84.8% to 87.9% with other closely related Vibrio species. Its dDDH value with V. cholerae N16961 is 61.8% (58.9% to 64.6% range, with 95% confidence interval), compared to less than 32.6% with other Vibrio species. Despite the ANI value being borderline to the 95% species cutoff (), a dDDH value well below the 70% species cutoff () suggests that Vibrio sp. 2521-89 is potentially a novel species. Parsnp 1.2 () was used to perform core-genome single-nucleotide polymorphism (SNP)-based phylogenetic analysis of Vibrio sp. 2521-89 and closely related vibrios. This analysis showed the novel strain grouping strongly in a monophyletic cluster with V. cholerae isolates, albeit displaying an unusually long branch length, possibly representing a speciation event. […]

Pipeline specifications

Software tools RAST, PGAP, JSpeciesWS, GGDC, Parsnp
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Hemisus marmoratus, Vibrio cholerae