A set of 43 337 splice junction pairs was extracted from mammalian GenBank 
annotated genes. Expressed sequence tag (EST) sequences support 22 489 of them. 
Of these, 98.71% contain canonical dinucleotides GT and AG for donor and 
acceptor sites, respectively; 0.56% hold non-canonical GC-AG splice site pairs; 
and the remaining 0.73% occurs in a lot of small groups (with a maximum size of 
0.05%). Studying these groups we observe that many of them contain splicing 
dinucleotides shifted from the annotated splice junction by one position. After 
close examination of such cases we present a new classification consisting of 
only eight observed types of splice site pairs (out of 256 a priori possible 
combinations). EST alignments allow us to verify the exonic part of the splice 
sites, but many non-canonical cases may be due to intron sequencing errors. This 
idea is given substantial support when we compare the sequences of human genes 
having non-canonical splice sites deposited in GenBank by high throughput genome 
sequencing projects (HTG). A high proportion (156 out of 171) of the human 
non-canonical and EST-supported splice site sequences had a clear match in the 
human HTG. They can be classified after corrections as: 79 GC-AG pairs (of which 
one was an error that corrected to GC-AG), 61 errors that were corrected to 
GT-AG canonical pairs, six AT-AC pairs (of which two were errors that corrected 
to AT-AC), one case was produced from non-existent intron, seven cases were 
found in HTG that were deposited to GenBank and finally there were only two 
cases left of supported non-canonical splice sites. If we assume that 
approximately the same situation is true for the whole set of annotated 
mammalian non-canonical splice sites, then the 99.24% of splice site pairs 
should be GT-AG, 0.69% GC-AG, 0.05% AT-AC and finally only 0.02% could consist 
of other types of non-canonical splice sites. We analyze several characteristics 
of EST-verified splice sites and build weight matrices for the major groups, 
which can be incorporated into gene prediction programs. We also present a set 
of EST-verified canonical splice sites larger by two orders of magnitude than 
the current one (22 199 entries versus approximately 600) and finally, a set of 
290 EST-supported non-canonical splice sites. Both sets should be significant 
for future investigations of the splicing mechanism.