BMC Genomics 2006, 7:273 http://www.biomedcentral.com/1471-2164/7/273 spliced might be significantly higher than the 7.2% we cal- culated. However, many lines of evidence suggest that the majority of apparently species-specific alternative splicing events observed in dbEST represent non-functional alter- native splicing [9,13]. As there are currently no computa- tional methods to distinguish functional from non- functional species-specific splicing, at this stage it is impossible to determine the contribution of species-spe- cific alternative splicing to the fraction of functional alter- native exons. Based on the calculated fraction of 7.2% (± 1.1%), we esti- mate that 7,500–10,000 exons in the set of ~121,700 con- served human exons (including the putative hidden exons) are ancestral alternatively spliced. This is a surpris- ingly small number; however, we note that our set of con- served exons is not the complete exon repertoire in the human genome, which was estimated to contain ~200,000 exons [22]. Presumably, fast-evolving exons (for example, in immune- or reproduction-related genes [22]) escape our set due to low human-mouse conserva- tion. Nevertheless, even if we assume that the same rate of alter- native splicing that we found holds for the complete set of ~200,000 exons in human (distributed within ~25,000 genes), our results predict that the number of ancestral alternative exons in human is somewhere between 12,000–16,500. At first glance this number seems to con- tradict the current paradigm of alternative splicing abun- dance in human genes (the higher estimates are that 74% of all human genes are alternatively spliced [5]). Indeed, even if each alternatively spliced gene contains only one ancestral alternative exon (which is frequently untrue), the fraction of such genes would still be 48%-66%, less than 74% of all human genes. However, all high estimates of alternatively spliced genes were derived from analyses that took into account also species-specific splicing events, which, as mentioned above, are much more abun- dant than ancestral alternative splicing (and their func- tionality is yet to be proven). Our estimate solely refers to ancestral, non-species specific alternative exons. Our estimation of the fraction of alternative exons is com- posed of three elements: estimation for the number of known exons that undergo conserved exon-skipping (3.75%), extrapolation using the fraction of the 'exon- skipping' type out of all types of alternative splicing (yielding a factor of 1.75), and additional estimation for the number of ancestral alternative exons still hidden in the human genome (0.6%). Whereas our results show that the basic estimate of 3.75% (± 0.65%) is robust, the two subsequent extrapolations are less reliable (due to the difficulty in assigning proper error bounds). These extrap- olations should, therefore, be viewed with caution. Several factors might have influenced our results. First, it is still possible that our training sets of exons are some- how biased and do not represent the actual qualities of alternative and constitutive exons in the genome. Although we are not aware of any reason for such bias, if it exists it could influence the resulting numbers. The fact that we obtained very close estimates using two different training sets, compiled in two separate studies by different methods, makes such bias less likely. Second, the number of 'hidden' alternative exons might be higher than what we detected, due to under-representation of very short and highly conserved exons in the Exoniphy predictions. Finally, some exons can be alternatively spliced in several ways, e.g., they can be both skipped and have an alterna- tive 3' splice site. Our analysis, which assumed that ~40% of all alternative exons are of the exon-skipping type, did not take into account such complex cases. It is noteworthy that our method does not classify each specific exon as ancestral alternative or 'other'. Instead, it assigns the exon a score that could be used to calculate the probability that it is ancestral alternative. Calculated over the entire set of conserved exons in the human genome, these probabilities enabled us to accurately estimate the number of ancestral alternative exons, but not to accu- rately pinpoint each one of them. For the same reason, the method cannot uncover the full complexity of the tran- scriptome, as it does not directly provide the number of alternative transcripts of each gene. We could not find a set of parameters that produce SVM- based classifier that would distinguish between species- specific alternatively spliced exons and constitutive exons. This could stem from several factors. First, for the species- specific alternative exons we, naturally, did not have the comparative-genomics-based parameters (such as exon- conservation and flanking-intron conservation). As these are strong classifying parameters in our SVM classifier [11] their absence in this case limits its ability to distinguish between the two populations. Second, the other character- istics of species-specific alternative exons (such as their size distribution and divisibility by 3) resemble those of the constitutive exons [9]. Conclusion To our knowledge, this report contains the first estimation for the number of ancestral alternatively spliced exons in the human genome. Most or all of these exons represent functional and highly important splicing events (other- wise they would not have been conserved in evolution over millions of years). In the quest to reveal the full com- plexity of the transcriptome, the central parameter will be the total number of human functional transcripts. While this number still remains unknown, our estimate is an important signpost towards revealing it. Page 5 of 8 (page number not for citation purposes)