In total, GENOA aligned about 85,900 human cDNAs and about 890,300 ESTs to the human genome. The relatively low fraction of aligned ESTs (about 18%), and average aligned length of about 550 bases (the average lengths were not significantly different between different tissues, see Additional data file 6), reflect the stringent alignment-quality criteria that were imposed so as to be as confident as possible in the inferred splicing patterns. The aligned sequences yielded about 17,800 gene regions with more than one transcript aligned that exhibited a multi-exon structure. Of these, about 60% exhibited evidence of alternative splicing of internal exons. Our analysis did not examine differences in 3'-terminal and 5'-terminal exons, inclusion of which is frequently dictated by alternative polyadenylation or alternative transcription start sites and therefore does not represent 'pure' AS [73,74]. The EST alignments were then used to categorize all internal exons as: constitutive exons; A3Es, A5Es, skipped exons, multiply alternatively spliced exons (for example, exons that exhibited both skipping and alternative 5' splice site usage), and exons that contained retained introns. An internal exon present in at
