Computational protocol: Towards a phylogenetic reappraisal of Parmulariaceae and Asterinaceae (Dothideomycetes)

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Protocol publication

[…] PCR amplicons of the regions targeted in this study served as templates for DNA sequencing reactions with the BigDye® Terminator Cycle Sequencing Kit v. 3.1 (Applied Biosystems Life Technologies, Carlsbad, CA, USA) following the protocol of the manufacturer. DNA sequencing reactions used the same primers as those for the PCR reactions. DNA sequencing amplicons were purified through Sephadex® G-50 Superfine columns (Sigma Aldrich, St. Louis, MO) in MultiScreen HV plates (Millipore, Billerica, MA). Purified sequence reactions were run on an ABI Prism 3730xl DNA Sequencer (Life Technologies, Carlsbad, CA, USA).DNA sequence data were analysed in MEGA (Molecular Evolutionary Genetics Analysis) v. 6.0 (). Consensus sequences were generated and imported into MEGA for initial alignment and the construction of sequence datasets. Sequences obtained from , TreeBASE study S10245, and from GenBank (www.ncbi.nlm.nih.gov) and the novel sequences generated on this study were aligned using MAFFT v. 7 (http://mafft.cbrc.jp/alignment/server/index.html; ) and manually improved in MEGA as indicated. [...] Appropriate gene models were selected using MrModeltest v. 2.3 () and applied to the gene partition. Based on the results of MrModeltest, a Bayesian phylogenetic analysis was performed with MrBayes v. 3.1.2 applying a general time-reversible (GTR+I+G) substitution model with inverse gamma rates and dirichlet base frequencies and a heating parameter set at 0.01. Saccharomyces cerevisiae DAOM 216365 (JN938921) served as outgroup for the phylogenetic analyses. Posterior probabilities were determined by Markov Chain Monte Carlo sampling (MCMC) in MrBayes v. 3.2.1 (). Six simultaneous Markov chains were run for 10 000 000 generations and trees were sampled every 100th generation and 10 000 trees were obtained. The first 2 000 trees, representing the burn-in phase were discarded, while the remaining 8 000 trees were used for calculating posterior probabilities. Bayesian posterior probabilities are presented on the left of each node (). Sequences derived in this study were lodged in GenBank (http://www.ncbi.nlm.nih.gov/genbank) (), the alignment and tree in TreeBASE (www.MycoBank.org) (study number 17355) and taxonomic novelties in MycoBank (www.MycoBank.org; ). […]

Pipeline specifications

Software tools MEGA, MAFFT, MrModelTest, MrBayes
Databases TreeBASE
Application Phylogenetics