where i is the index of position on the PWM. This distance was used to derive phylogenetic trees for the 50 and 30ss separately, using the program Phylip (41). Scoring a putative splice site Given a PWM of a given splice-site type, a log-odds scoring matrix was constructed to score the splice sites. A pseudo- count of 0.0001 was added to avoid logarithms of zero. The small pseudocount ensures a strong penalty for mismatches at the exon–intron junctions. The scores obtained by each LOD score matrix were rescaled to fall in the 0–100 range. All positive scores, from 0 to the maximum, were rescaled to lie between 50 and 100, and all negative scores, from the minimum score to 0, were rescaled to lie between 0 and 50. A score close to 50 means the background model is favored, a score close to 100 favors the splice-site motif in question, and a score close to 0 implies that the splice site is different from both the background and the splice-site distribution. Finally, strongly conserved splice sites have a bigger spread of scores, so a single mismatch can drop the score dramatically, whereas less con- served splice sites are more tolerant of mismatches compared to the highest scoring sequences. Classification of introns PWMs were ﬁrst generated for each splice-site type and BPS using human curation. The U12-type BPS consists of an 8 nt pyrimidine-rich sequence (approximately TTTTCCTT), fol- lowed by two positions enriched in As and two positions enriched in pyrimidines (C or T). The U12-type BPS requires an A, either at positions 9 or 10 (42). In order to take this into account, we constructed two U12-type BPS PWMs, one with a highly conserved A at position 9 (U12-BPS-A9), and another with a highly conserved A at position 10 (U12- BPS-A10). A sequence was considered a good BPS if it had a score greater than 65 for either one of the two BPS PWMs and if it was located between 8 and 30 nt upstream of the 30ss. The short fragments around the exon–intron boundaries were scored using the LOD matrices, and each intron received a score for 50ss, 30ss and BPS for U12-type introns. The best examples of each type, those with high 50ss scores and consis- tent dinucleotide boundaries, were selected. For U12-type GT–AG introns, an additional criterion for the presence of a BPS between 8 and 38 nt upstream of the 30 end was used to eliminate possible contaminants. The best set of introns for each subtype was used to generate new species and splice- site type speciﬁc PWMs, which were then used to rescore the splice sites and reclassify them. Intron classiﬁcation was unambiguous in almost all cases, either due to their distinctive boundaries or their 50ss scores, with the exception of a few introns with similar scores for U2- and U12-type GT–AG 50ss matrices. The introns that remained unclassiﬁed were subsequently classiﬁed using criteria described below. Previous classiﬁcation schemes (6,8,25) assumed that U12- dependent splicing required a good BPS within 10–20 nt from the 30 end of the intron (22), but recent data has shown this is not always necessary (7,25,43). Thus, we used the presence of an optimal U12-type BPS only to discriminate between Nucleic Acids Research, 2006, Vol. 34, No. 14 3957 introns that could not be unambiguously classiﬁed by their 50ss scores alone. If the U12-type 50ss score was greater than the U2-type 50ss score by more than 25 then it was assigned the U12-type GT–AG. If the U12-type 50ss score was greater than the U2-type 50ss score by more than 10 and the intron had a good U12-type BPS, then again it was assigned the U12-type GT–AG. In all other cases it was assigned the U2-type GT–AG. The thresholds of 25 and 10 are arbitrary, but were opti- mized by human curators to minimize the error rate, which was estimated at <1% by manual scrutiny. The borderline cases could be classiﬁed in either group, and their unambigu- ous assignment will require experimental veriﬁcation. Finally, introns with a minimum 50ss score less than 50 are kept in the database, and can be accessed from the SpliceRack website, but are not used in our analysis. Introns with non-canonical boundaries A few of the introns with dinucleotide boundaries that do not ﬁt into the canonical categories are real, and we delineate the strategy to identify them. In general, current datasets are biased against non-canonical splice sites, but it is possible to gather some information on them from the RefSeq sequences, as we show below. To identify non-canonical U12-type introns from this unclassiﬁed set, we chose those with good matches to the U12-type 50ss, with a non-canonical dinucleotide at the intron’s 30 end, and a signiﬁcant number of these introns were found (Table 1). Many of these introns have a U12- BPS. Analogous to the scheme outlined above, those introns without a match to the U12-type BPS were only classiﬁed as U12-type when the score for the U12-type 50ss was >25 score units higher than that for the U2-type 50ss. These introns were included in the U12-type RT–RN category (where R is a purine, and N is any nucleotide). Some GC–AG introns revealed a strong match to the PWM for U12-type GT–AG 50ss, except for the mismatch at posi- tion +2. We searched for GC–AG introns with the GCATCC motif from positions +1 to +6 of the 50ss, and found that most of these introns had a match to the BPS at an optimal distance from the 30ss. These introns were named U12-type GC–AG. U2-type AT–AC introns (or AT–AC type II introns) were identiﬁed by searching for unclassiﬁed AT–AC introns that bear a good match to the U2-type 50ss, except for the intron’s terminal dinucleotides. A PWM for U2-type AT–AC 50ss was generated from the PWM for U2-type GT–AG 50 ss, by changing the full conservation from G to A at position +1. Only those introns with a minimal 50ss score of 75 were reclassiﬁed as U2-type AT–AC. Putative examples of other classes of non-canonical U2- type introns were identiﬁed by searching for all exon–intron boundaries with good 50 GT–AG U2-type splice sites (score greater than 70), with a non-canonical dinucleotide boundary at the 30 terminus of the intron and a good PPT near the 30 terminus in the intron. Introns that could not be classiﬁed into any of the seven categories either lack a canonical exon–intron boundary dinu- cleotide, and/or have all 50ss scores below 40. These can be accessed through SpliceRack. Most of these unknown splice sites are probably due to sequencing or annotation errors in