For technical reasons (lack of CPU because after each step we have to apply several prediction methods), we repeat these two moves N/10 times (N number of residues in the protein).
In order to investigate the effect of the threshold relaxation method, we repeated the whole experiment by using natural threshold <img src="http://journals.plos.org/plosone/article/asset?id=info?doi/10.1371/journal.pone.0012382.e069.PNG" class= inline-graphic"/> for all methods.
To demonstrate that our findings are not dependent on the background correction method, we repeated the analysis with non-background corrected gene expression data.
As a positive control for the method, we repeated the same analysis for frames captured at the moment animals expressed their judgment by inserting their snout at one of the two choice ports.
To assess the FDR for our method, we repeated all steps using randomized human and mouse UTR sequences maintaining overall dimer frequencies, and calculated the number of selected entries at each step (Additional file 3).
To check that the density decrease between seed sites and flanks was not an artefact of the method, we repeated our SNP density analysis 1000 times by sampling 2043 random motifs of seven nucleotides and computing SNP distributions.
Finally, to illustrate empirically the benefit of this distribution-based MI method, we repeated the analyses above with the observations < LOD excluded completely or replaced by log(LOD)/2 or log(LOD).
In addition, to exclude the possibility that our results were influenced by the normalization method, we repeated all the analyses with a different method, i.e. the median normalization method in which each sample was centred based on the median value of all the 147 expressed miRNAs.
In order to test whether the presence of this module in Wnt5 genes is due to sensitivity of our method, we repeated exactly the same analysis (same parameters and for the same species) for the constitutively expressed gene of b-actin, finding absence of the module, with exception that of zebrafish.
To confirm reproducibility of the applied method, we repeated for 20 samples the leukocyte telomere length measurement by using an additional reference gene, acidic ribosomal phosphoprotein PO (36B4), 300 nM forward primer (5′-CAGCAAGTGGGAAGGTGTAATCC-3′), 500 nM reverse primer (5′-CCCATTCTATCATCAACGGGTACAA-3′).
