Computational protocol: Comparative Genomics of Large Mitochondria in Placozoans

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Protocol publication

[…] The DNA sequences of each strain were assembled separately using the Phred, Phrap, and Consed package, release 15.0 [–]. Potential genes were identified using the National Center for Biotechnology Information's ORF FINDER, using the Mold, Protozoan, and Coelenterate Mitochondria genetic code. For ORFs of unknown function, only those greater than 100 bp and not overlapping other known genes (i.e., respiratory chain subunits or structural RNAs) were annotated. tRNAs were inferred using the program tRNAscan-SE 1.21 (http://lowelab.ucsc.edu/tRNAscan-SE). Each of the 12 placozoan inferred gene sequences (atp6, cob, cox1–3, nad1–6, and nad4L) was aligned to its homologous sequence in sponges (Axinella corrugata, Geodia neptuni, and Tethya actinia), cnidarians (Metridium senile, Acropora tenuis, Aurelia aurita, Briareum asbestinum, and Ricordea florida), bilaterians (Artemia franciscana, Katharina tunicata, Saccoglossus kowalevskii, and Strongylocentratus purpuratus), the choanoflagellate Monosiga brevicollis, and the chytrid fungus Monoblepharella using the program CLUSTALW [] and edited manually, in order to predict translational start sites and intron-exon boundaries. These alignments were edited using Gblocks, version 0.91b [], so as to exclude gaps and large nonconserved regions, and the output alignments were concatenated for phylogenetic analyses. Two likelihood-based phylogenetic inference procedures were employed: ML and Bayesian, and both used the mtREV model of amino acid substitution. The program PHYML v2.4.4 [,] was used to run the ML analysis and to obtain bootstrap support values (4,000 replicates). The Bayesian analysis was run in MrBayes [,], and posterior probabilities were obtained after 500,000 generations with a burn-in of 25%. All parameter values were the same as in Dellaporta et al. []. Relative-rate tests were performed on each of the 12 respiratory chain genes individually using the program RRTree []. Two lineages were defined: one composed of only placozoan sequences and the other composed of sponge and cnidarian sequences. The outgroup contained Monoblepharella and Monosiga sequences. A guide tree topology obtained from MrBayes was used in RRTree. […]

Pipeline specifications

Software tools Consed, Open Reading Frame Finder, tRNAscan-SE, Clustal W, Gblocks, PhyML, MrBayes
Applications Genome annotation, Phylogenetics
Organisms Trichoplax adhaerens