Computational protocol: An expanded phylogeny of social amoebas (Dictyostelia) shows increasing diversity and new morphological patterns

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Protocol publication

[…] A ~2000 base pair (bp) fragment of SSU rDNA was amplified using the same primers as in []. The PCR program consisted of 5 minutes at 95°C, followed by 30 cycles of 30 seconds at 95°C, 1 minute at 56°C, and 2 minutes at 72°C, with a final elongation step of 10 minutes at 72°C. Following amplification, PCR products were separated on 1% agarose gels and DNA purified with MultiScreenHTS Vacuum Manifold and MultiScreen-PCR96 Filter Plate from Millipore (http://www.millipore.com). The extracted DNA was then directly sequenced using the same primers used for amplification by Macrogen (Korea) on an ABI 373 sequencer. Sequences were edited with the program Sequencher version 3.0 (Gene Codes Corporation Inc.). Edited sequences were manually added to an existing alignment []. Only unambiguously aligned regions were used for phylogenetic analysis. [...] Phylogenetic analyses were carried out using Bayesian inference with MrBayes version 3.1.2 [] and maximum likelihood using RaxML version 7.0.4 [,]. MrBayes analyses utilized the MC3 search algorithm and the GTR+I+G model [], with parameters determined through the course of the run. Searches were conducted with two independent sets of four chains for 1-10 million generations, with results saved every 10 generations. At the end of the run (split frequency less than 0.01), a 20% burnin was discarded before determining the optimal topology and posterior probabilities (biPP) of clades. Maximum likelihood analyses consisted of 1000 bootstrap replicates (mlBP) using the GTR+I+G model with parameters determined from a BioNJ starting tree. […]

Pipeline specifications

Software tools Sequencher, MrBayes, RAxML
Application Phylogenetics
Organisms Dictyostelium discoideum