Computational protocol: Identification and Characterization of a Rare Fungus, Quambalaria cyanescens, Isolated from the Peritoneal Fluid of a Patient after Nocturnal Intermittent Peritoneal Dialysis

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Protocol publication

[…] The internal transcribed spacer region (ITS), the small subunit of the ribosomal RNA gene (SSU) and the D1/D2 domain of the 26S rRNA gene were used as targets for the molecular identification of the fungal isolate. Total DNA extraction, PCR amplification, and sequencing were performed as described previously [,]. The primers used are listed in . The annealing temperatures used to amplify the ITS, SSU and D1/D2 domain of the 26S rDNA genes were 58°C, 52°C and 56°C, respectively. The sequenced data (ITS, SSU, and D1/D2 sequences) were subjected to a BLASTn search against the non-redundant (nr) NCBI-nucleotide database for species identification. Unique ITS and D1/D2 nucleotide sequences from the isolate, together with an additional 14 sequences for the ITS region and D1/D2 domain, respectively, of the Quambalaria species were compiled for multilocus phylogenetic analysis (). Microstroma juglandis was used as an outgroup strain in the phylogenetic analysis. Multiple sequence alignments of collected ITS and D1/D2 nucleotide sequences were generated using M-Coffee []. Individual alignments were concatenated for Bayesian Markov Chain Monte Carlo (MCMC) analysis partitioned by the gene. Bayesian tree analyses were performed using MrBayes v3.2.2 with reversible jump MCMC averaging over the entire general time reversible (GTR) rates and gamma-distributed rate heterogeneity for all subsets of the partitioned scheme. A total of 1,000,000 generations were run with a sampling frequency of 100, and diagnostics were calculated for every 1,000 generations. The first 2,500 trees were discarded with a burn-in setting of 25%. Convergence was assessed with a standard deviation of split frequencies below 0.01, no noticeable trend for the plot of the generation versus the log probability of the data, and a potential scale reduction factor (PSRF) close to 1.0 for all parameters. […]

Pipeline specifications

Software tools BLASTN, M-Coffee, MrBayes
Applications Phylogenetics, Protein structure analysis
Organisms Bacteria, Homo sapiens
Diseases Peritonitis
Chemicals Echinocandins