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 less overlap in the specific ESTs chosen (and therefore in the specific AS events detected) than for random subsets of a smaller pool of ESTs, and increased numbers of ESTs give greater coverage of exons. However, there is no reason that the expected number of AS events detected per randomly sampled subset should depend on the size of the pool the subset was chosen from. While the error (standard deviation) of the measured AS frequency per gene should be lower when restricting to genes with larger minimum pools of ESTs, such a restriction would not change the expected value. Unfortunately, the reduction in error of the estimated AS frequency per gene is offset by an increase in the expected error of the tissue-level AS frequency resulting from the use of fewer genes. The inclusion of all genes with at least 20 tissue-derived ESTs represents a reasonable trade-off between these factors.
