Alternative splicing (AS), a process which enables the production of multiple mRNA transcripts by the same gene via the variable inclusion of parts of the primary transcript, is very widespread in eukaryotes - almost all multi-exonic human genes are believed to undergo AS [1, 2]. Thus, AS is a major contributor to the complexity and diversity of eukaryotic transcriptomes and proteomes. The splice variants produced can either exhibit different properties (e.g. half-life, translational efficiency), be translated into different protein isoforms with potentially different functions, or can be degraded via pathways such as the nonsense-mediated decay (NMD) . AS can often be specific to a tissue type or developmental stage, and the majority of human AS events are believed to be regulated in this sense . The regulation of AS has been shown to play an important role in several developmental processes in various organisms, and defects in AS can lead to diseases .
Subtle AS, involving splice sites separated by a distance of 2-12 nt, is an important, evolutionarily widespread subclass of AS . Such AS is called subtle because the resulting mRNA isoforms differ by only a few nucleotides. While alternative acceptors (AA) and alternative donors (AD) together constitute about a third of all AS events in humans, subtle AS events comprise about a third of AA and AD events - for example, subtle events constitute 1,586 (38%) out of 4,179 AA events and 774 (28%) out of 2,728 AD events in the "alt events" track of the UCSC genome browser  for a combined total of 34% (2,360/6,907). Another reason for treating these events separately is that the mechanisms behind such events are likely different from those involving splice sites separated by larger distances - for example, the emergence of a second polypyrimidine tract can be observed for alternative acceptors separated by 8 or more nucleotides, and events which result in a frame-preserving difference of transcript length are seen to be more common than frame-shifting ones, once we move beyond a difference of 12 nt [7, 8]. In the following, we shall use the notation Δx to denote a subtle splice event involving sites separated by x nucleotides, so for example, the class Δ3 shall be used to mean all GYNGYN and NAGNAG AS events (Y stands for C or T; N for A, C, G, or T), and so on.
It is a matter of debate as to what fraction of subtle AS events are truly functional, as opposed to being a result of a noisy process in which the spliceosome stochastically selects between nearby competing alternatives [5, 9–11]. Consistent with estimations that a fraction of those subtle AS events is under purifying selection , there are several known cases where they result in functionally different protein isoforms or affect the translational efficiency when located in the untranslated regions (UTR) . Moreover, subtle AS can also have a decidedly unsubtle effect in cases where a premature stop codon can be created, which is especially likely in cases where the splice sites are separated by a distance which is not a multiple of 3. Mutations that create frame-preserving tandem splice sites affecting the coding region are selected against , and in the case of ABCA4 (Δ3 acceptor, ) and WT1 (Δ9 donor, ) are associated with human disease. In the human EDA gene, AS at a conserved Δ6 donor leads to isoforms with distinct receptor binding specificity . For more examples and further details regarding the functional consequences of subtle AS, the reader is referred to .
TassDB1 (TAndem Splice Site DataBase, version 1), the first database devoted to subtle AS, provides large collections of Δ3 donors and acceptors in eight species . We have extended TassDB1 considerably, to create TassDB2, which provides a comprehensive collection of all human and mouse donors and acceptors in the Δ2-Δ12 range. We note that while TassDB provided data on 8 species, TassDB2 only includes 2 species, human and mouse. This is because the transcriptome coverage by ESTs/mRNAs in the remaining species was insufficient for detection of a non-negligible number of AS events involving the larger distances in the Δ2-Δ12 range. TassDB2 includes data on the conservation of the tandem motifs in five vertebrates (human, mouse, dog, chicken and zebrafish). Thus, TassDB2 provides comprehensive information on 22 event types, compared to 2 (NAGNAG and GYNGYN) in TassDB1. Thus TassDB2 is effectively a new database rather than just a simple extension. A user-friendly search interface features both a "quick search" mode, in which a user can search using gene symbol, as well as an "advanced search" mode, in which several different criteria can be specified by the user, and the possibility to download result datasets.