H.sapiens M.musculus D.melanogaster C.elegans A.thaliana 11 763 11 756 40 637 185 3 40 825 11 6 1 18 40 843 0.453 0.007 0.044 19 278 19 269 93 699 351 0 94 072 0* 0 0 0 94 072 0.373 0 0 22 434 22 354 111 351 903 0 112 254 162 27 2 191 112 445 0.804 0 0.170 Total 99 087 98 656 604 036 4480 24 608 562 1086* 353 66 1505* 610 067 0.736 0.003 0.247 3958 Nucleic Acids Research, 2006, Vol. 34, No. 14 Table 1. A synopsis of the collection of splice sites in SpliceRack Number of accessionsa Number of genesa U2-type GT–AG U2-type GC–AG U2-type II (AT–AC) Total U2-type U12-type GT–AG U12-type AT–AC Other U12-typeb Total U12-type Total number % U2-type GC–AG % U2-type AT–AC % U12-type 23 788 23 505 183 678 1602 15 185 295 469 169 33 671 185 966 0.865 0.008 0.361 21 824 21 772 174 671 1439 6 176 116 444 151 30 625 176 741 0.817 0.003 0.354 This table shows the total number of introns in SpliceRack for each subtype. The * indicates that there are 22 U12-type-like GT–AG introns in C.elegans, which were not included in the counts of U12-type introns (see text for details). aThe number of accessions (RefSeqs) includes alternatively spliced isoforms. bOther U12-type introns refers to those U12-type introns lacking the canonical U12-type terminal dinucleotide pairs, which were sorted in the RT–RN and GC–AG categories (see text for details). the genome (44), as shown in previous examinations of non- canonical splice sites (45–47). The unclassiﬁed introns in our dataset represent a small fraction (<1.5%) of the total number of introns. RESULTS AND DISCUSSION SpliceRack We have created the largest compilations of splice-site sequences to-date. The splice-site sequences for the ﬁve spe- cies were extracted from the RefSeq collection of NCBI (35). Introns were classiﬁed into various splice-site subtypes, based on scoring methods derived from PWMs, as explained in the Materials and Methods section (Table 1). Our data is publicly available through the SpliceRack website (see http://katahdin. cshl.edu:9331/SpliceRack/). Tools to explore SpliceRack SpliceRack contains sequences ﬂanking the 50ss (38 nt long, 8 nt within the exon and 30 nt within the intron), and the 30ss (43 nt long, 35 nt within the intron and 8 nt within the exon). We have developed several useful tools, available on the SpliceRack website, that allow exploration of the splice sites and their ﬂanking sequences. Each tool results in the selection of a subset of splice sites. The genes containing these splice sites can be analysed for functional and other biases by using GObar (48), which is provided as a built-in tool. Retrieve splice sites by type. This tool allows users to retrieve splice site ﬂanking sequences by specifying one or more splice-site subtypes, cut-off 50ss or 30ss scores, and species (Figure 2). A similar display is obtained in the ‘Search for genes’ tool, which allows visualization of all splice sites within a queried gene. Introns with weak splice-site bound- aries can be retrieved using a minimum 50ss score of 40 and a maximum of 50. Search for patterns. This tool can be used to locate potential protein-binding sites by searching for sequences that match user-speciﬁed patterns in the form of regular expressions. For example, IUPAC SNP codes as well as standard symbols, such as ‘+’ and ‘*’ can be used. The search can be restricted by species, splice-site type and start position, as explained on the website. The neural-speciﬁc splicing factor Fox-1 binds to the hex- amer UGCAUG. In order to ﬁnd introns with the hexamer in the vicinity downstream of the 50ss, the database was queried for U2-type GT–AG 50ss with the pattern N{13,31}TGCATG. The N allows match to any nucleotide, the 13 and 31 ensure that the TGCATG motif will start anywhere from position 13 on the ﬂanking sequence, which is after the U1 binding site, to position 31. This query results in 1221 introns. By using the GObar function, we conﬁrmed that the genes containing these motifs are enriched for function in neural tissues (49). This analysis will not ﬁnd genes that have this motif deep inside the intron. Search for motifs. This tool is similar to the search for patterns tool, except the patterns are now of ﬁxed-length, which speeds up the search. For example, to ﬁnd the fre- quency of potential stop codons (TAG,TTA,TAA) at the last 3 nt of an exon, the search is carried out for human U2-type GT–AG 50 ss with a motif start position of 5. A query with the sequence TAG returns 4591 introns, while TRA (R is a purine), representing the other two stop codons, returns an additional 1439 introns. Search for matrices. This tools ﬁnds matches to a user- supplied PWM in the sequences in SpliceRack. Users can also supply cut-off scores, and the tool calculates the frequency of the motif’s occurrence, starting from a user- speciﬁed position relative to the exon–intron boundary. It also displays the rank (based on counts) of a searched motif relative to the frequencies for all the motifs that are of the length of the PWM and start at the user-speciﬁed start site. Results can be limited by species, 50ss or 30ss and splice- site type. This tool can be used to locate sequences in the neighborhood of splice sites that can serve as protein-binding sites.