Computational protocol: Mitochondrial Genome Rearrangements in the Scleractinia/Corallimorpharia Complex: Implications for Coral Phylogeny

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Protocol publication

[…] Sequences were verified and assembled using SeqManII (DNAstar v5.0) or Sequencher v4.8 (Gene Codes Corporation) and then analyzed in Vector NTI v9.0 (InforMax). Open-reading frames (ORFs) of length more than 50 (amino acids) were translated using National Center for Biotechnology Information translation table 4 and compared with the databases using BlastX (). No novel ORFs were identified on this basis. MEGA v5.0 () with a weighted matrix of Clustal W () was used to align the identical putative ORFs and rRNA genes with previously published data. The 5′- and 3′-ends of the rRNA genes were predicted using the program SINA on the Silva ribosomal RNA database site (www.arb-silva.de/, last accessed February 1, 2014) using the default settings (). tRNAs were predicted using tRNAscan-SE search server v1.21 (). rRNA loci were identified on the basis of sequence similarity. Finally, Vector NTI v9.0 was used to generate maps of the mt genomes based on the assembled sequence data. [...] The double cut and join (DCJ) distance metric (), implemented in GRAPPA (; ), was used to calculate the pairwise DCJ and breakpoint distances (BPDs) from the gene order data and to generate pairwise distance matrixes. Gene order phylogenies (DCJ and BPD) were estimated with FastME ().Because gene order is a single character with multiple states (), bootstrapping is not applicable, hence the reliability of each branch was estimated by applying a jackknife resampling technique that in each iteration randomly removed 25% of the initial orthologous gene sets. Note that, because the data set consisted of only 13 protein-coding genes, higher removal rates (e.g., 50%) are unable to resolve the tree branching order. Jackknifing was used to generate 1,000 matrices, which were imported into FastME and used to obtain 1,000 DCJ- and BPD-based trees. Finally, the CONSENSE program in the PHYLIP software package () was used to calculate majority-rule consensus trees with percent values at each node. Each value represents the percentage of trees supporting a clade defined by a node. […]

Pipeline specifications

Software tools Sequencher, BLASTX, MEGA-V, Clustal W, tRNAscan-SE, FastME
Applications Genome annotation, Phylogenetics, GWAS
Organisms Corallimorphus profundus