BMC Genomics 2006, 7:273 http://www.biomedcentral.com/1471-2164/7/273 SeFcxigournersedi1stribution of ancestral alternative exons and "other" Score distribution of ancestral alternative exons and "other" exons. X axis: the SVM 'alternativity score'. Y Axis: probabil- ity density. 'ancestral alt', score distribution of ancestral alternative exons in the training set; 'other', score distribu- tion of 4655 constitutive exons plus 182 human specific alter- native exons in the training set. (A) Model trained with 221 ancestral alternative exons from Yeo et al. [13]. (B) Model trained with 215 ancestral alternative exons from Sorek et al. [15]. be used to obtain another estimator that is to a large extent independent from the former. We trained a second model on the basis of this second set of exons versus the set of 4837 other exons (Figure 1B), and employed the resulting model on the entire set of 120,964 conserved exons. After decomposing the score distribution, the resulting fraction of ancestral alternative exons was 3.9% (± 0.5%), very similar to the result obtained using the first training set. Combining these two results, we conclude that 3.75% (± 0.65%) of the exons in our set of 120,964 known human-mouse conserved exons are ancestral alter- native cassette exons (Figure 2). FDmieogcuuosremecp2onsisteiorvneodf esxconres distribution of 120,964 human- Decomposition of score distribution of 120,964 human- mouse conserved exons. The blue distribution represents the scores of all exons. Its bigger right tail represents the contribution of the ancestral alternative exons. This distribu- tion was decomposed (see text) into "ancestral alternative" (transparent) and 'other' (superimposed in red) distributions. The area under the "ancestral alt" curve represents the per- centage of ancestral alternative exons out of the entire exons set. The hidden conserved alternative exons Our method enables, for each known exon, to assess its probability of being alternatively spliced. However, some conserved internal alternatively spliced exons are not rep- resented in EST data due to sampling limitations of ESTs. How many such exons still await discovery? To assess their number we used the output of the Exoniphy program [16]. This program, designed by Siepel and Haussler, identifies evolutionarily conserved protein-coding exons using a phylogenetic hidden Markov model, which simul- taneously describes exon structure and exon evolution [16]. Using human mouse and rat genome alignment, Exoniphy predicted 177,040 putative exons on the UCSC hg16 genome version (Methods). As these predictions do not depend on existence of ESTs, they can be used to assess the number of conserved coding exons that are still undetected. Out of our set of 120,964 conserved exons, 105,740 (87%) are accurately predicted by Exoniphy. This indi- cates the small amount of false negative predictions, namely, most true conserved exons are predicted by Exoniphy. To search for possible new alternatively spliced exons in the remaining 71,300 Exoniphy predictions, we took all such predictions that satisfy the following criteria: (i) Found within an intron of a known RefSeq gene (ii) Do not overlap with any EST/RNA, (iii) Do not overlap Page 3 of 8 (page number not for citation purposes)