Computational protocol: Westerdykella reniformis sp. nov., producing the antibiotic metabolites melinacidin IV and chetracin B

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Protocol publication

[…] The ITS, nLSU, and β-tubulin amplicons were sent to a commercial sequencing facility (Eurofins MWG Biotech) and sequenced on a 3730xl DNA Analyzer coupled with BigDye Terminator v. 3.1 Cycle Sequencing reagents, Applied Biosystems (ABI). The generated sequences were compared with other fungal DNA sequences from NCBI’s GenBank sequence database using a Blastn search algorithm. Phylogenetic analysis of the ITS rDNA gene were performed using the software Molecular Evolutionary Genetics Analysis v. 5 (MEGA5) (). Sequence data generated in this study were aligned with additional sequences of representative Westerdykella spp. as well as several isolates belonging to Sporormiaceae and other Pleosporales available in GenBank (). In total, 34 sequences were aligned using the ClustalW algorithm, with a DNA Gap Open Penalty = 15.0, DNA Gap Extension Penalty = 6.66 and a delay divergent cutoff of 30 %. For result optimization, the alignments were refined by manual correction when needed. The evolutionary history was inferred using the neighbor-joining method employing the maximum composite likelihood model using pairwise deletion and the clade stability was evaluated using the bootstrap method (n = 2000 bootstrap replications). Novel sequences were accessioned in GenBank under accession numbers JX235699–JX235707.A multigene phylogeny was constructed using 23 isolates (). Relevant sequence data were downloaded from GenBank and used to construct aligned and trimmed ITS, nLSU and β-tubulin data matrices in MEGA5. A Bayesian analysis was performed using MrBayes 3.2 () with the following settings: nst = 6, therefore using GTR (General Time Reversible) model; rates = invgama, setting across-site rate variation for gamma distribution with a proportion of invariant sites; MCMC heated chain set with nchains = 4 and temp = 0.2, ngen = 500 000, samplefreq = 100, sumt burnin = 1250; the analysis was continued for 500 000 generations in order obtain an average standard deviation of split frequencies below 0.01. The first 25 % of sampled trees were discarded as burn-in. Resulting trees were viewed in FigTree v. 1.3.1. Sequence alignments and trees presented were deposited in TreeBASE (accession number 13676). […]

Pipeline specifications

Software tools BLASTN, MEGA, Clustal W, MrBayes, FigTree
Databases TreeBASE
Application Phylogenetics
Organisms Oryza sativa, Staphylococcus warneri, Watanabea reniformis
Diseases Staphylococcal Infections, Proteus Syndrome
Chemicals Vancomycin