Computational protocol: Virulence Profiles of Vibrio vulnificus in German Coastal Waters, a Comparison of North Sea and Baltic Sea Isolates

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Protocol publication

[…] MLST analysis was performed based on ten housekeeping genes (glp, gyrB, mdh, metG, purM, dtdS, lysA, pntA, pyrC, tnaA) as described by Bisharat et al. [,] using primers and protocols published on the V. vulnificus Multilocus Sequence Typing website []. Electropherograms were assembled and trimmed using SeqMan Pro (v12; DNASTAR Lasergene, Madison, WI, USA) and Accelrys Gene (v2.5, Accelrys Inc., San Diego, CA, USA). Allele sequences at each locus and corresponding allelic profiles were compared to the PubMLST database. New allele sequences and allelic profiles (sequence types, STs) were submitted to the PubMLST database. A list of all strains with corresponding allelic profiles is provided in . To elucidate clonal relationships, allelic profiles were analyzed with the PHYLOViZ software using the goeBURST algorithm []. Related strains with allelic profiles differing in one of ten loci (single locus variants, SLV) were assigned to a distinct clonal complex. The PHYLOViZ software was used to generate a full Minimum Spanning Tree (MST) displaying the relationships between STs of the whole dataset. Allele sequences were concatenated in the order of loci used to define the allelic profile to generate a 4326 bp concatemer for each strain. A phylogenetic tree was constructed in MEGA ver. 6 [] based on the alignment of concatenated allele sequences using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) []. Distances were computed using the Maximum Composite Likelihood method [] and the reliability of the tree was assessed by bootstrap analysis with 1000 replicates. Sequences from clinical and environmental biotype 1 isolates from the Baltic Sea region from our previous study [] were included in the analysis for comparison. The same procedure was performed on concatemers of 1299 bp obtained by concatenating the sequences of three housekeeping genes in the order gyrB, dtdS and pyrC. […]

Pipeline specifications

Software tools DNASTAR Molecular Biology Suite, DNASTAR Lasergene, PHYLOViZ, BURST, MEGA
Databases PubMLST
Applications Phylogenetics, WGS analysis, Sanger sequencing
Organisms Vibrio vulnificus, Homo sapiens
Diseases Infection, Wound Infection