Bréhélin et al. BMC Genomics 2010, 11:35 http://www.biomedcentral.com/1471-2164/11/35 that associates with each D. melanogaster gene its best BLAST hit in S. cerevisiae, using different e-value cutoff to control the number and accuracy of functional trans- fers. Here again we observe for the BP ontology that using expression context greatly improves the accuracy achieved using only sequence similarity. Next, the same experiments were conducted on P. fal- ciparum using S. cerevisiae as reference species (tran- scriptomic studies Le Roch vs. Gash and Bozdech vs. Spellman) and with all available GO annotations (IEA included). In each experiment, the HMM was learned using the P. falciparum-yeast orthologs selected with the RBH procedure, and was then used to assess the functional annotation transfers of all gene pairs with BLAST sequence similarity below 0.1. Results were com- pared with the accuracy of the method that associates with each gene P. falciparum of its best BLAST hit in S. cerevisiae, using different e-value cutoffs (see Figure 3). Like for D. melanogaster, taking coexpression context into account outperforms the accuracy achieved when using only sequence information. Table 1 is an estimate of the method potential on genes that have no, or only non-curated GO annotations. This table shows that, depending on the threshold chosen, several dozen genes with no BP annotation can be associated with a S. cere- visiae gene annotated in this ontology. Moreover, even more genes that only possess non-curated annotations (mainly derived from sequence similarity) can be asso- ciated with a S. cerevisiae gene with curated annotations. This reveals the potential of our method for improving annotations based on sequence similarity only, by using information derived from gene expression. Appraisal of the proposed annotation transfers We next investigated the potential of co-coexpression analysis to improve simple homology-based annotation transfers in a more careful and systematic way. The method was applied to the P. falciparum vs. S. cerevisiae and P. falciparum vs. D. melanogaster studies LR-GA, LR-SP, BO-GA, BO-SP, and BO-PI. For each analysis, a model was learned using gene pairs selected by RBH, and was then used to assess functional transfers for gene pairs with BLAST sequence similarity below 0.1. Page 6 of 14 For each experiment, our method outputs the gene pairs authorizing a functional transfer in an organized way, by presenting in cluster pairs the coexpressed genes whose coexpression has been conserved. Outputs of all above analyses along with useful links to appropriate databases and BLAST alignments can be browsed in the Supple- mentary Data files (see Additional Files 1, 2, 3, 4 and 5). All pairs were analyzed in all clusters to validate (or invalidate) the identified pairs and, in addition, we checked that P. falciparum genes were associated with the same yeast genes independently of the data sets con- sidered (for example in LR-GA versus BO-GA or BO- GA versus BO-SP). In cases where P. falciparum genes were paired both with yeast and Drosophila genes, we checked that the two latter genes had concordant anno- tations, further supporting the inter-species annotation transfer. When both genes of the output pairs had a previously inferred function, we observed nearly no annotation conflict. All discrepancies could be attributed to an obvious error, or incomplete analysis of the Plas- modium gene. We did not detect any Plasmodium gene with known function, paired with hypothetical Saccharo- myces or Drosophila genes. However, on several occa- sions, hypothetical Plasmodium genes were paired with hypothetical genes in the reference species—mostly Dro- sophila, which is not yet as extensively annotated as yeast. Several hypothetical P. falciparum genes were paired with annotated S. cerevisiae/D. melanogaster genes. In some of these pairs, the P. falciparum gene is only par- tially annotated, as for example in the LR-GA co-coex- pression analysis, the gene pairs (PFE1240w; YPL207W) and (PF11_0090; YGR103W) in the first cluster pair (cluster pair #0), or the gene pair (PF14_0661; YOR145C) in cluster pair #1 (see Additional File 1). In other pairs, the P. falciparum gene has absolutely no GO data. See, for example, the pairs (PF11_0471; YCR072C), (PF07_0121; YHR170W), or (PF10_0200; YNL132W) in the cluster pair #0 of the LR-GA analysis. The comparative analysis therefore allows correction of incomplete annotations and proposes annotations for completely unannotated P. falciparum genes. Several Table 1 Number of uncharacterized or poorly characterized genes of P.falciparum that can be annotated by the LR-GA or BO-SP comparisons with different ratio thresholds. ξ ratio threshold LR-GA BO-SP # unchar. genes # IEA char. genes # unchar. genes # IEA char. genes 40 4 74 1 77 20 47 90 31 106 10 80 127 92 115 5 142 163 129 176 2 183 214 167 230 Lines “# unchar. genes” show the number of genes without any annotation in the BP ontology that can be associated with an S. cerevisiae gene annotated in this ontology. Lines “# IEA char. genes” show the number of genes with only electronic non-curated annotations (GO Evidence Code IEA) in the BP ontology that can be associated with an S. cerevisiae gene with a curated annotation in this ontology.