BMC Genomics 2006, 7:273 http://www.biomedcentral.com/1471-2164/7/273 with any processed pseudogene, and (iv) Are flanked by canonical splice sites both in the human and the mouse genomes. The first two criteria screened the vast majority of the remaining predictions, as most of the predicted exons were either overlapped (fully or partially) by an expressed sequence, or not contained within any known intron. We collected 783 putative novel exons conforming to the above criteria, and applied our classifiers on each of these. Figure 3 presents the 'alternativity scores' distribution of the 783 putative exons. Indeed, it is apparent that this population of sequences is highly enriched in true alterna- tive exons. Using the criterion described above to decom- pose the distribution, we calculated that 93.1% (± 3.7%) of these exons (~730 exons) represent true cassette exons. These results are in agreement with recent estimates that few hundred ancestral alternative exons are still undetec- ted in the human genome [17]. Our analysis indicates that hidden, novel exons contrib- ute at least 0.6% (out of ~122,000 conserved exons) to the overall fraction of ancestral alternative exons. Combined with our result from the non-hidden conserved exons, we estimate that 4.3% (± 0.65%) of all conserved exons in human are ancestral alternatively spliced cassette exons. We note that Exoniphy is expected to fail in the identifica- tion of very short and highly conserved exons (A. Siepel, personal communications). As alternative exons tend to be smaller and more conserved than constitutive ones [10] the actual numbers of 'hidden' alternative exons might be higher than that we calculate. Additional types of alternative splicing Both our training sets are composed solely of ancestral alternatively spliced exons of the "cassette" type, i.e., such that undergo exon-skipping. Exon skipping is the most abundant type in human, composing ~40% of all alterna- tive splicing events in the genome [18]. Events such as mutually exclusive exons, intron-retention, alternative 5' and 3' splice sites, etc., comprise the remaining 60% of the events. Due to the composition of the training sets, we expected our classifiers to be biased towards identifying mainly cassette (skipped) exons. Indeed, manually inspecting EST support for 50 of the conserved exons that had the highest alternativity scores indicates that 70% of them are alternatively spliced in an exon-skipping man- ner, while only 30% show other patterns of alternative splicing. A similar 70:30 ratio was observed in the results obtained by using another classification rule, and also in the experimentally verified predictions of that rule [10]. Hence, exon-skipping cases are over-represented in our predictions, and consist a fraction of 0.7 of our set instead of the expected 0.4 [18]. SFcigoureredi3stribution for the exons predicted by 'Exoniphy' Score distribution for the exons predicted by 'Exoniphy'. 'ancestral alt', score distribution of ancestral alternative exons in the training set; 'other', score distribution of 4655 constitutive exons plus 182 human specific alternative exons in the training set; 'exoniphy', scores of 783 predicted Exoni- phy exons. The distribution of the Exoniphy exons is clearly biased toward the 'ancestral alt' distribution, indicating that many of the Exoniphy exons are truly ancestral alternative exons. To reach the total fraction of exons in the genome that undergo all types of alternative splicing, one must, there- fore, multiply our previous results by a factor of 0.7/0.4 = 1.75 (this applies only to the known, non-hidden exons, as all the hidden exons are, by definition, of the cassette exons type). Thus, our estimate for the fraction of exons that are ancestrally alternatively spliced is 3.75% * 1.75 + 0.6% = 7.2%. The error in this estimate is at least ± 1.1% (0.65*1.75) due to the original error estimate; additional unknown error in the 1.75 factor may increase the error bound. Discussion Our analysis indicates that 7.2% (± 1.1%) of the exons in the human genome are ancestrally alternatively spliced, i.e., undergo alternative splicing both in human and in mouse. As our positive training set was composed solely of ancestral alternative exons, the classifier cannot detect species-specific alternatively spliced exons. However, some alternative splicing events are species specific: For example, the primate-specific Alu-derived exon 8 in the RNA-editing enzyme ADAR2 is contained in 40% of all ADAR2 transcripts in human, thus changing the RNA- editing activity of the mature protein [19]. Moreover, EST analyses have shown that species-specific alternative splic- ing might be up to 3 or 4 times more abundant than con- served alternative splicing [10,20,21]. In this light, the total number of human exons that are alternatively Page 4 of 8 (page number not for citation purposes)