Computational protocol: A New Multilocus Sequence Typing Scheme and Its Application for the Characterization of Photobacterium damselae subsp. damselae Associated with Mortality in Cetaceans

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[…] A MLST scheme was developed choosing six PDD housekeeping genes: gyrB (DNA gyrase, subunit B) and toxR (positive transcriptional regulator-ToxR protein), both already proposed by Takahashi et al. (), glp (Glucose-6-phosphate isomerase), metG (Methionyl-tRNA synthetase), pnt (Transhydrogenase alpha subunit), and pyrC (Dihydroorotase). These latter were selected as they had been previously used for the Vibrio vulnificus MLST scheme (Bisharat et al., ; All chosen genes belonged to the PDD chromosome I, except for pnt, belonging to the chromosome II.Following DNA extraction, PCR reactions were performed using the following thermal conditions: 10′ at 95⋅C, followed by 35 amplification cycles with conditions as reported in the Supplementary Table . Every mix reaction contained 0.2 mM of dNTP's, 0.5 μM of each primer, 1.5 mM of Mg2Cl and 2U of taqPolymerase Gold (Applied Biosystem, Foster City, CA, USA). Primers were designed with the CLC DNA workbench® software version 5.7.1 (CLC Bio-Qiagen, Aarhus, Denmark) using the PDD (ADBS01000000) as reference genome. Amplicons were Sanger sequenced by BigDye Terminator chemistry (Applied Biosystems) using the same primers. Sequence data analysis and classification was performed using the CLC DNA workbench® software version 5.7.1 (CLC Bio-Qiagen).A different number was assigned to each allele of the six genes studied. In the same way, a different sequence type (ST) was assigned to each combination of alleles. The ratio between non-synonymous substitutions and synonymous substitutions (dN/dS) (Nei and Gojobori, ) and the index of association (Smith et al., ) was calculated using START2 (Jolley et al., ). The Tajima's D neutrality test (Tajima, ), the Fu and Li's F neutrality test (Fu and Li, ), the number of variable sites, the number of point and InDel mutations and the average of guanine plus cytosine (G+C) for the proposed genes were calculated using DnaSP v.5.0 (Librado and Rozas, ). Clonal complex (CC) assignment was performed using eBURSTv.3 software (; Feil and Enright, ), and confirmed with the graphical representation of the Minimum Spanning Tree (MST). The MST was constructed with the Bionumerics® 7 software (Applied Maths, Sint-Martens-Latem, Belgium), using the allele combinations as categorical values. The ClonalFrame software, with default parameters, was used to assess the amount of recombination among the isolates under study and among the obtained STs, by calculating the r/m ratio (ratio of rates at which nucleotides become substituted as a result of recombination and mutation; Didelot and Falush, ). This ratio is usually categorized as low (< 1), intermediate (1–2), or high (>2) (Vos and Didelot, ). [...] The 78 sequences of each gene were aligned using Clustal IW with MEGA6 (Tamura et al., ), translated to aminoacids and back-translated to nucleotides. ToxR was previously aligned using clustal Omega ( in order to mark off the InDel gaps. For each gene (glp, gyrB, metG, pnt, pyrC and toxR) the evolutionary history (MEGA6) was inferred using the Maximum Likelihood (ML) method (Tamura-Nei model; Tamura and Nei, ) and rooted with the PDP sequence of each gene. Initial trees for the heuristic search were automatically obtained by applying Neighbor-Joining (NJ) and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. All positions containing gaps and missing data were eliminated. In the final dataset a total of 480 (glp), 537 (gyrB), 429 (metG), 396 (pnt), 507 (pyrC), and 372 (toxR) positions were present, respectively.The sequences obtained from the 78 isolates were concatenated for each isolate in the following order using START2 (Jolley et al., ): glp, gyrB, metG, pnt, pyrC, and toxR. For comparison purposes, the phylogenetic tree was built using: (a) the Neighbor-joining method based on a multiple alignment (with Kimura corrections), and with the Jukes and Cantor correction, using the Bionumerics® 7 software. None position was discarded, for a total of 2739 bases in the final dataset. The tree topology was tested using 500 bootstrap replicates; (b) the Minimum Evolution method (Rzhetsky and Nei, ), using MEGA6 (Tamura et al., ), with the Neighbor-joining algorithm (Saitou and Nei, ) used to generate the initial tree; (c) a Bayesian approximation using Markov chain Monte Carlo (MCMC) algorithms with the BEAST software v1.8 (Drummond et al., ). […]

Pipeline specifications

Software tools DnaSP, BURST, BioNumerics, ClonalFrame, MEGA, Clustal Omega, BEAST
Databases PubMLST
Applications Phylogenetics, WGS analysis
Organisms Photobacterium damselae subsp. damselae, Homo sapiens, Stenella coeruleoalba, Prunus mume