Nucleic Acids Research, 2005, Vol. 33, No. 17 5579 Figure 4. Percentage of symmetrical and non-symmetrical exons along the CDS. (A–D) Percentage of exons in different locations along the CDS, in increments of 0.1 (exon locations in CDS are normalized between 0 and 1, see Supplementary Data). A and B show the distribution of alternative exons, C and D show that of constitutive exons. all constitutive exons do not show any bias for speciﬁc loca- tions within the CDS: average location of 0.49 and 0.51 for symmetrical alternative and constitutive exons, respectively (Figures 4A, C and D). The difference between the average normalized location of symmetrical and non-symmetrical alternative exons is statistically signiﬁcant: P < 2.7 · 10 0.5 (t-test). There is also a slight drop in the percentage of sym- metrical alternative exons and all constitutive exons near the ends of the CDS, which may result from a tendency of the start and stop codons not to lie near exon–exon junctions (as it is in exons with average normalized location which is between 0 and 0.1 or 0.9 and 1). We also checked whether the 43% of non-symmetrical alternative exons, which have mRNA evidence for their skipping, differ from the rest of the non- symmetrical alternative exons with respect to a preference to reside in a speciﬁc location along the CDS, but found no such difference (data not shown). Analysis of the effect on the reading frame of alternative splicing events of non-symmetrical exons Skipping or inserting of cassette exons can be accompanied by other alternative splicing events (splicing of other exons, use of alternative initiation or termination, etc.). Careful analysis of all the differences between the exon-including isoforms and the exon-skipping ones may require a very sophisticated computational algorithm. Therefore, we decided to perform such an analysis manually on a random sample of alternative exons. For that, we randomly sampled a group of 30 symmet- rical alternative exons and 30 non-symmetrical ones of the 101 and 475 symmetrical and non-symmetrical exons, respect- ively, that are CDS-internal and have mRNA evidence in GenBank for both their inclusion and skipping. For each of the 60 sampled exons, we used the mRNA/genomic alignments in the UCSC genome browser (see Materials and Methods) to compare one of their exon-skipping isoforms with one of their exon-including isoforms, with regard to preservation of the reading frame between the two isoforms and generation of a PTC. Stop codons were considered as PTCs if they were located more than 55 nt upstream from the 30-most exon–exon junction (38). For the 30 non-symmetrical exons, we detected three cat- egories of differences between the including and skipping isoforms (Figure 5). For 53% (16/30) of these exons (category A), the reading frame downstream of the alternative exon is different in the skipping and including isoforms. Among these 16 exons, the skipping isoform contains a PTC in four cases and the including isoform contains a PTC in two cases. In category B, which contains 37% (11/30) of the exons, an alternative start codon ensures the use of the same reading frame downstream from the alternative exon (but introduces a different reading frame upstream to the alternat- ive exons). Among these 11 exons, the skipping isoform is PTC+ in one case. Category C contains three exons for which the including and skipping isoforms have a different reading frame (both upstream and downstream from the alternative exon). Among these three exons, the skipping isoform contains a PTC in one case, and, in another case, the including isoform is PTC+. To summarize, in 20% (6/30) of pairs the skipping isoform was PTC+; in 10% (3/30), the including isoform was PTC+; and, in 70%, none of the isoforms harbored a PTC. For comparison, none of the skipping or including isoforms of the 30 symmetrical alternative exons meets the deﬁnition of a PTC+ mRNA. We also examined the location in CDS of the alternative non-symmetrical exons in categories A and B, for which neither inclusion nor skipping introduces a PTC. For the 10 exons in category A, where both isoforms share the same reading frame upstream from the alternative exon, the average normalized location is 0.68 (median 0.76). For the 10 exons in