Nucleic Acids Research, 2006, Vol. 34, Web Server issue W525 us to identify candidate gene regulatory sequences with predicted biological activity, conﬁrmed using transgenic mouse assays (9). The newly updated version of the TFBScluster web server has many new features that make it a widely applicable resource for studying mammalian gene regulatory networks. For example, we have now implemented mouse and human versions to streamline analysis for researchers using murine or human systems as their principal experimental setting. Moreover, integration within TFBScluster of genomic sequence searches with gene expression proﬁling datasets provides the ability to datamine across diverse yet comple- mentary datasets, in order to increase the likely accuracy of computational predictions. We have implemented a much wider range of TFBSs, including IPUAC consensus sequences curated from the literature, published datasets and a large set of conserved positional weight matrices. Our emphasis has moved from the use of multiple alignments to the initial use of pair-wise alignments, such as human–mouse. Conserved TFBSs can now be further ﬁltered to retain only those sites that are also conserved between human/mouse and other species including dog and opossum. TFBScluster signiﬁcantly differs from other tools developed for the analysis of lower invertebrate species (3,10–12), tools that use limited datasets (13–16) or are restricted to sequence ﬂanking predicted transcription start- sites (17–24). TFBScluster shares the concept of identifying binding site clusters with the SynoR program (25). However, TFBScluster utilizes three datasets of IUPAC consensus sequences in addition to TRANSFAC matrices, and provides links to mainstream genome browsers thereby providing a variety of external information. METHODOLOGY OF TFBScluster TFBScluster utilizes three principle methods to help distinguish functional binding sites (true positives) from the background noise of non-functional sites (false positives): (i) queries focus upon TFBSs that form part of binding site clusters, (ii) TFBScluster only considers sites that are conserved between two or more species and (iii) searches can be restricted to areas of the genome thought to be involved in regulating gene expression. TFBScluster can subject candidate binding site clusters to a series of ﬁlters based upon associated gene expression, and by their location in areas of regulatory potential or active promoters. Motif datasets Genome-wide positions for three sets of IUPAC code deﬁned TFBS consensus sequences and one set of positional weight matrices have been determined. The ﬁrst IUPAC code set consists of 41 consensus sequences curated from the literature and the databases TRANSFAC (26) and JASPAR (27). Back- ground information and references for each IUPAC consensus sequence can be found at (http://hscl.cimr.cam.ac.uk/ TFBScluster_genome_35_ﬁlters_background.html). Five dataﬁles containing genome-wide collections of matching conserved sites with increasing levels of sequence con- servation have been generated for all 41 IUPAC consensus sequences. The ﬁrst dataﬁle contains ‘non-exact’ matches to the core sequence; both sequences match the IUPAC consensus, but degenerate IUPAC codes are allowed to differ between the two sequences. The second dataﬁle contains ‘exact’ matches, where degenerate IUPAC codes must be identical between the two sequences, thus requiring degenerate consensus sequences to be aligned in regions of high sequence identity. The last three dataﬁles also require an exact match and extend the overall length of sequence identity. To achieve this, the IUPAC code ‘N’ (any nucleotide) is added to both ends of the consensus, resulting in three ﬁles with 2, 4 and 6 conserved nucleotides ﬂanking the core sequence. Functional binding sites are likely to be located in highly conserved sequence regions. Therefore, our method of increasing the degree of conservation in our TFBS dataﬁles aims to enrich functional binding sites whilst decreasing the number of false positive sites. The identiﬁcation of completely or near completely conserved binding sites is a method that has been successfully used in a variety of other appro- aches (28). The second set of IUPAC codes was taken from a recently published study (29), which identiﬁed common regulatory motifs conserved in human, dog, mouse and rat genomes. We have now taken the top 50 IUPAC consensus sequences from this study and determined their positions in whole genome alignments (human–mouse, human–dog, human– opossum, mouse–human, mouse–dog and mouse–opossum). The third set of IUPAC codes was taken from a similar study (30) that identiﬁed a ‘dictionary’ of conserved consensus sequences in the promoter regions of orthologous human and mouse genes. Again, we have determined the genome- wide positions of the non-degenerate IUPAC consensus sequences detailed in this second study, which differentiated between those located in CpG rich regions (35 consensus sequences) and those in non-CpG rich regions (19 consensus sequences). Finally for the human version of TFBScluster, we have incorporated the genome-wide positions of TFBSs matching 410 positional weight matrices conserved in human, mouse and rat whole genome alignments. We obtained these from the UCSC genome browser (http://genome.ucsc. edu/). The positional weight matrices originate from the TRANSFAC database v8.3. Whole genome alignments Human and mouse versions of TFBScluster have been imple- mented to serve these two large research communities. Both versions of TFBScluster incorporate the genome-wide positions of TFBS consensus sequences conserved in a series of pair-wise genome alignments, where either the human or mouse genome is the reference sequence. The pair-wise genome comparisons were downloaded from Genome Bioinformatics at the UCSC (http://genome.ucsc.edu/ downloads.html). For the human centric version (NCBI v35/hg17) TFBSs conserved in the mouse genome (NCBI 33/mm5) represent the default level of conservation. Positions of TFBSs are also catalogued in human–dog (canFam1) and human–opossum (monDom1) alignments. Human–mouse conserved sites have then been ﬁltered to retain only those that are also present in the latter two pair-wise alignments. For the mouse version (NCBI 34/mm6) the default level of con- servation uses sites conserved in the human (NCBI 35/hg17)