Computational protocol: The acute transcriptional response of the coral Acropora millepora to immune challenge: expression of GiMAP/IAN genes links the innate immune responses of corals with those of mammals and plants

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Protocol publication

[…] mRNA was isolated as previously described []. RNA-seq libraries were prepared and sequenced by GeneWorks Pty (Australia) on an Illumina Genome Analyzer I. For each condition (control, MDP and pIC), four libraries of single end 35 bp sequences were obtained, yielding an average of 2.8 million reads per library. These samples are biological replicates, coming from different coral colonies. The sequencing data have been deposited in the GEO database with accession ID GSE46389. The reads were mapped onto the latest A. millepora transcriptome assembly [] using the Bowtie mapping software v0.12.7 []. Differential gene expression was inferred based on these counts using the edgeR package with common dispersion estimates [], comparing each treatment (MDP and pIC) to the controls. P-values for differential gene expression were corrected for multiple testing using the Benjamini and Hochberg method [], and an adjusted p-value threshold of 0.05 was used.GO annotations were as previously used [], and GO-enrichment analyses were carried out with the Goseq package []. [...] GiMAPs were identified with BlastP and HMMER (hmmer.org) searches, using the AIG1 domain (pfam04548), focusing on species for which sequenced well- annotated whole genome data are available. In equivocal cases, assignments were made on the basis of a BLAST e-value difference of at least 1E-4 between similarity to the AIG1 domain and alternatives. Sequences were aligned with MAFFT 6.717b [] using the accurate L-INS-I method. Positions containing over 95% gaps were removed from the alignment. The dataset and the alignment used for phylogenetic analyses are provided as Additional files and . Maximum likelihood trees were inferred with PhyML 3.0 [] using the LG amino acid substitution model [], with four substitution rate categories approximating a gamma distribution whose rate was estimated, and an invariant category. The starting trees were computed using BioNJ and the topologies were optimised by nearest neighbour interchange and sub-tree pruning and regrafting. The branch support was estimated using approximate likelihood tests [] and with the bootstrap procedure, using 100 replicates. For Bayesian Inference, Mr Bayes 3.2-cvs was used as described in Forêt et al. []. […]

Pipeline specifications

Software tools Bowtie, edgeR, GOseq, BLASTP, HMMER, MAFFT, PhyML
Databases Pfam
Applications Phylogenetics, RNA-seq analysis
Organisms Drosophila melanogaster, Homo sapiens