Our objective was to control for differences in EST abundance across tissues while retaining sufficient power to detect a reasonable fraction of AS events. For each tissue we considered genes that had at least 20 aligned EST sequences derived from human cDNA libraries specific to that tissue ('tissue-derived' ESTs). For each such gene, a random sample of 20 of these ESTs was chosen (without replacement) to represent the splicing of the given gene in the given human tissue. For the gene and tissue combinations included in this analysis, the median number of EST sequences per gene was not dramatically different between tissues, ranging from 25 to 35 (see Additional data file 1). The sampled ESTs for each gene were then compared to each other to identify AS events occurring within the given tissue (see Materials and methods). The random sampling was repeated 20 times and the mean fraction of AS genes observed in these 20 trials was used to assess the fraction of AS genes for each tissue (Figure 1a). Different random subsets of a relatively large pool will have
