Bréhélin et al. BMC Genomics 2010, 11:35 http://www.biomedcentral.com/1471-2164/11/35 use the initial K-MEANS clusterings to set parameters Θ0. Namely, means and covariance matrices of the Gaussian distributions are estimated from the profiles of the genes associated with each prototype. Prior probabil- ities of the prototypes are estimated by the proportion of genes associated with, and probability transitions between prototypes are estimated by the proportion of genes whose coexpression has been conserved. Testing for coexpression conservation Let k and k’ be two gene clusters for which we want to know if they reveal conserved coexpression. For this purpose, we designed a statistical test that uses the number nkk’ of gene pairs of L between the two clusters. More precisely, we compute the probability of observing nkk’ or more gene pairs of L between cluster k and a randomly composed cluster of size #k’. This p-value can be written as P(r  nkk | , k, # k)  min(# k,# k)  unkk P(r  u | , k, # k)(,2) with P(r = u|L, k, #k’) being the probability of obser- ving u gene pairs of L between cluster k and a ran- domly composed cluster of size #k’. Let G’ be the number of genes of the reference species. We can mark the genes of the reference species that are paired with a gene of k in L. The probability P(r = u|L, k, #k’ ) is actually the probability of finding u marked genes when picking #k’ genes among the G’ genes, and thus follows an hypergeometric distribution. In our experiments, all cluster pairs that get a p-value below 10-3, as computed by Expression (2), are considered to reveal conserved coexpression, and a direct transition is added in the HMM between the associated prototypes. Accuracy estimate on the Gene Ontology Each ontology (MF, BP or CC) is a pseudo-hierarchy describing generalization relationships between hun- dreds of terms. The most general term is at the top of the ontology, while the terms at the bottom are the most specific ones. The specificity of a term can be assessed by its depth in the ontology, i.e. the number of levels that separate this term from the top. In the fol- lowing, we consider a term as “specific” if it is deeper than the 4th level of the ontology. Note that a gene may be annotated with several GO terms of the same ontol- ogy. Moreover, due to the generalization relationship, when a gene is annotated with a term t, it is also anno- tated with all the upper level terms that generalize t (a principle known as the “true path rule” in GO context). Let g be a gene of the query species already annotated in a given ontology (for example BP), and let A be the “specific” annotations associated with g in this ontology. Now, let A’ be the set of specific annotations transferred Page 12 of 14 to g by one of the methods assessed in the experiments. Four different methods are tested: the RBH procedure, the best BLAST procedure, the co-coexpression approach, and OPI. For the first three approaches, A’ is the set of specific annotations associated with the refer- ence gene used for the transfer; for the OPI approach, A’ is the set of specific annotations (plus the specific annotations of the upper levels) that have been pre- dicted for g with a FDR below the cutoff. The transfer accuracy is estimated by the proportion of annotations of A’ that are in A, i.e. |A|A A’| ’| , with |·| denoting the cardinal number of a set. Transfers involving the same specific annotations have accuracy 1, while those invol- ving completely different specific annotations have accu- racy 0. The global accuracy of a method is then estimated by averaging the accuracy of all transfers involving a query gene already annotated in the ontol- ogy. We used annotations available on the GO website and provided by GeneDB http://www.genedb.org/, the Saccharomyces Genome Database http://www.yeastgen- ome.org/, and FlyBase http://flybase.bio.indiana.edu/ for P. falciparum, S. cerevisiae, and D. melanogaster, respectively. Additional file 1: Le Roch - Gasch analysis. This file presents the cluster pairs identified as revealing a conservation of coexpression when comparing the Le Roch and Gasch data. This file also provide additional information on the available functional annotations, as well as links to the BLAST alignments and the different databases (click on the ‘?’s to access PlasmoDB, SGD, and Amigo databases). Gene functional annotations are as follows. The short description immediately following each P. falciparum gene comes from PlasmoDB (red = functional gene, blue = putative gene, black = hypothetical gene). Other annotations are Gene Ontology annotations (red = Molecular Function, green = Biological Process, blue = Cellular Component). Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 35-S1.HTML ] Additional file 2: Le Roch - Spellman analysis. This file presents the cluster pairs identified as revealing a conservation of coexpression when comparing the Le Roch and Spellman data. This file also provide additional information on the available functional annotations, as well as links to the BLAST alignments and the different databases (click on the ‘?’s to access PlasmoDB, SGD, and Amigo databases). Gene functional annotations are as follows. The short description immediately following each P. falciparum gene comes from PlasmoDB (red = functional gene, blue = putative gene, black = hypothetical gene). Other annotations are Gene Ontology annotations (red = Molecular Function, green = Biological Process, blue = Cellular Component). Click here for file [ http://www.biomedcentral.com/content/supplementary/1471-2164-11- 35-S2.HTML ] Additional file 3: Bozdech - Gasch analysis. This file presents the cluster pairs identified as revealing a conservation of coexpression when comparing the Bozdech and Gasch data. This file also provide additional information on the available functional annotations, as well as links to the BLAST alignments and the different databases (click on the ‘?’s to access PlasmoDB, SGD, and Amigo databases). Gene functional annotations are as follows. The short description immediately following each P. falciparum gene comes from PlasmoDB (red = functional gene, blue = putative gene, black = hypothetical gene). Other annotations are