Bréhélin et al. BMC Genomics 2010, 11:35 http://www.biomedcentral.com/1471-2164/11/35 Page 7 of 14 Figure 3 Estimate of the method accuracy with GO: P. falciparum vs. S. cerevisiae. Accuracies achieved on the Le Roch-Gasch (orange curve) and Bozdech-Spellman (blue curve) comparisons, estimated on the MF (left) and BP (right) ontologies with different ξ thresholds—from left to right on each curve: 40, 20, 10, 5, 2, 1. The x-axis shows the number of gene pairs authorizing annotation transfers. The brown curves show the accuracy achieve when associating each gene of P. falciparum with its best BLAST hit in S. cerevisiae, for different e-value thresholds. pairs associating hypothetical P. falciparum genes have surprisingly good or even excellent BLAST matches, with e-values lower than 1.e-40. For the others, with moderate or poor BLAST matches, careful examination was crucial to assess the proposed annotation transfer. This led to the identification of several homologs with probably the same function, generally with amino acid compositional biases and the occurrence of low com- plexity inserts in the Plasmodium genes. See for exam- ple the pairs (PF10_0278; YKR081C), e-value = 2e-05, and (PF14_0072; YNR046W), e-value = 2e-09, in the cluster pair #0 of the LR-GA co-coexpression analysis. For some other pairs, however, the sequence alignments are clearly irrelevant, aligning repeated domains, low complexity segments, or very short proportions of one gene—e.g. in the LR-GA analysis, (PFI0635c; YML093W) and (PF13_0256; YCL037C) in the cluster pair #0. Thus, the co-coexpression analyses are very interesting indica- tors to identify hypothetical Plasmodium genes that have sequence and expression similarities with a yeast and/or Drosophila functional gene, but they cannot entirely replace an expert examination of the sequences to support or reject the possible annotation transfers they propose. Three tables summarize the most interesting annota- tion transfers proposed by the co-coexpression analysis: Additional File 6 shows the results obtained when focusing on the cytosolic and organellar (mitochon- drial and apicoplast) ribosomal proteins (GO:0003735) based on comparative analysis between P. falciparum and S. cerevisiae expression profiles (LR-GA; BO-GA and BO-SP) and between P. falciparum and D. melanogaster expression pro- files (BO-PI). For genes encoding ribosomal proteins of the cytosol, the co-coexpression analyses enable the recovery of 57 functional genes previously avail- able in the PlasmoDB v5.4 resource, the refinement of 8 incomplete or wrong annotations, an inference of a function for 2 hypothetical genes, and provide no information on 9 already known genes. For genes encoding mitochondrial ribosomal proteins, 6 could be annotated on the basis of the co-coexpression analysis (4 confirmed PlasmoDB v5.4 annotation, 1 refinement of an incomplete annotation and 1 func- tional inference of hypothetical genes). None of the 30 apicoplast ribosomal proteins could be annotated according to the co-coexpression analysis, which is consistent with the lack of plastid in yeast and Dro- sophila, and consequently provides a negative con- trol for this procedure. Additional File 7 shows the pairing of P. falci- parum genes with S. cerevisiae or D. melanogaster genes functionally annotated as factors of the ribo- some assembly and biogenesis (GO:0042254) in the nucleolus and/or nucleus or cytoplasm, factors involved in the rRNA metabolic process (GO:0016072) and tRNA processing (GO:0008033). In these 102 pairs, co-coexpression analysis con- firmed the functional inference of 7 Plasmodium genes, a proposed refinement or correction of 46 genes and inference of a function for 49 hypothetical genes. In this analysis, careful examination was criti- cal for reviewing gene pairs when BLAST matches were questionable—see, for example, (PFL2295w; YKL099C), e-value = 0.095 found in BO-GA and BO-SP and predicting a nucleolar rRNA processing protein associated with U3 snoRNA. A few unex- pected situations were discovered, such as PFA0330w known as the PfAARP2 protein [30] that