Computational protocol: Rasamsonia, a new genus comprising thermotolerant and thermophilic Talaromyces and Geosmithia species

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Protocol publication

[…] Strains were grown on MEA for 7–14 days prior to DNA extraction. Genomic DNA was extracted using the UltracleanTM Microbial DNA isolation Kit (MoBio, Solana Beach, U.S.A.) according to the manufacturer’s instructions. For the phylogenetic analysis of the Eurotialean taxa, parts of the RPB2, Tsr1 and Cct8 genes were sequenced. The latter two genes were chosen, since they were among the best-performing genes for recovering robust phylogenies in the study by Aguileta et al. (). The partial RPB2, Tsr1 and Cct8 sequences consists of exon data only and the alignment of the data set was performed using the Muscle software on the translated amino acid data. The ITS (incl. 5.8S rDNA), β-tubulin and calmodulin loci were sequenced in order to study the phylogenetic relationship among T. emersonii, T. byssochlamydoides, T. eburneus, G. argillacea and G. cylindrospora. The obtained sequences were aligned using the software Muscle on the EMBL-EBI website and subsequently combined. Primers used for amplification and sequencing and annealing temperatures are shown in Table . Maximum likelihood analysis was performed on the two data sets using RAxML version 7.2.8. Each locus was treated as a separate partition. The number of bootstrap replicates was set on 1,000 replicates (Stamatakis et al. ). A second measure of branch support was conducted using a Bayesian tree inference (BI) analysis. Prior to analysis, the best nucleotide substitution models were determined for each partition with MrModeltest v2.2 (Nylander ). A BI analysis was performed with MrBayes v3.1.2 (Huelsenbeck and Ronquist ). One tree was saved per 100 generations, and the run was automatically ended when the standard deviation of split frequencies was below 0.01. To avoid suboptimal trees being taken into account for the consensus tree, a burn-in of 25% of the saved trees was used. Coccidioides immitis sequences were downloaded from the full genome data set (Sharpton et al. ) and used as an outgroup in the study of the phylogeny of selected Trichocomaceae. Trichocoma paradoxa CBS 247.57 and CBS 103.73 were used as an outgroup in the phylogenetic analysis. […]

Pipeline specifications

Software tools MUSCLE, RAxML, MrModelTest, MrBayes
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Rasamsonia emersonii