Nucleic Acids Research, 2007, Vol. 35, Web Server issue W445 Table 1. Databases used in the diﬀerent pipelines, and the programs and parameters used to search them Server ProtSweep 2DSweep Databases Uniprot/Swissprot Uniprot/Trembl Refseq Ensembl DSSP Nrpep (non-redundant NCBI protein database) Uniprot/Swissprot þUniprot/Trembl þupdates DomainSweep Prosite Pfamhmm Prints Smart Tigrfams SCOP Blocks Interpro Prodom Links Program Parameters http://www.ebi.ac.uk/swissprot BlastP http://www.ncbi.nlm.nih.gov/RefSeq/ BlastP ftp://ftp.ebi.ac.uk/pub/databases/trembl/sptrembl/ BlastP http://www.ensembl.org/ BlastP http://www.sander.ebi.ac.uk/dssp/ BlastP ftp://ftp.ncbi.nih.gov/blast/db/FASTA PsiBlast -NOFILTER -EXP ¼ 10.0 -NOFILTER -EXP ¼ 10.0 -NOFILTER -EXP ¼ 10.0 -NOFILTER -EXP ¼ 10.0 -EXP ¼ 0.001 -EXTension ¼ 10 - NOGAPPEDalign -b20000 -a5 -j2 -e0.001 http://www.expasy.ch/sprot/sprot-top.html http://www.ebi.ac.uk/swissprot ftp://ftp.expasy.ch/databases/prosite http://www.sanger.ac.uk/Software/Pfam/ ftp.bioinf.man.ac.uk http://smart.embl-heidelberg.de/ http://www.tigr.org/TIGRFAMs/index.shtml/ http://scop.mrc-lmb.cam.ac.uk/scop ftp://ftp.ncbi.nih.gov/repository/blocks/unix http://www.ebi.ac.uk/interpro/ http://www.toulouse.inra.fr/prodom.html MSFGenerator -EXP ¼ 0.001 -OVERLAP ¼ 75 -CUSTOMRANGE ¼ 5 CUSTOMPERCENTAGE ¼ 80,60,50,45,40,35 Motifs & Pfscan Default HMMscan -lib ¼ pfam.hmm -d HMMscan -lib ¼ prints.hmm -d HMMscan -lib ¼ smart.hmm -d HMMscan -lib ¼ tigrfams.hmm -d SCOPscan Default Blockssearcher -cutoﬀ ¼ 0.01 –d SRS queries Prodomblast Default available. Concerning EnsEMBL, the situation is more complex. Due to possible inconsistencies between the diﬀerent EnsEMBL API versions, which are used in the tasks, it is not possible to automatically update the EnsEMBL data and this needs to be done by hand. The diﬀerent databases and the way they were used in these pipelines are described in Table 1. APPLICATION PIPELINES ProtSweep is an approach to the functional characteriza- tion of unknown proteins based on a cascade of similarity searches. It is well known that protein databases do not completely overlap and diﬀer in their annotation quality (5). This task takes into account the signiﬁcant diﬀerences among databases (Supplementary Table 1) to improve the quality of the protein characterization. It selects the order in which the databases have to be searched and combines the annotation found depending on the results. Protsweep classiﬁes proteins into the following cate- gories: identical, homolog, similar, weakly similar and putative proteins. The query protein starts the BLAST (6) cascade against Swissprot (7) ﬁrst (Figure 2). We do take into account three parameters to classify the BLAST hits: (i) percentage of identity, (ii) ‘qpercent’ and (iii) ‘spercent’. The two last parameters are related to the length of the total alignment, being ‘qpercent’ the percentage of the query sequence length covered in the alignment with the database hit and ‘spercent’ the percentage of the hit (subject) sequence length covered by the alignment (Figure 2). Depending on the classiﬁcation of the BLAST hits according to these parameters and the hit protein annotation, three diﬀerent approaches will be followed. If the hit has 100% ‘qpercent’ and ‘spercent’ and more than 98% identity, it is considered an identical protein and the Swissprot ID will be searched in Ensembl (8). If it is successful, all information from both databases will be combined (Supplementary Table 2) and stored in the XML output. If the ID cannot be found in Ensembl then a BLAST search is performed with the query protein against Ensembl. The best Ensembl hit is selected and compared against the Swissprot hit using the Smith– Waterman algorithm implemented in Water (EMBOSS) (9). If the identity between sequences is greater than 98%, then the information from both sources and the BLAST alignment will be added to the ﬁnal output, if the identity is less, only Swissprot annotation and the alignments will be added to the XML. If the ‘qpercent’ and ‘spercent’ is between 80% and 98% and the identity is between 85% and 98%, the hit is classiﬁed as homologous and follows the same strategy with Ensembl as already described (Figure 2). In case, the identity is between 20% and 85% and ‘qpercent’ and ‘spercent’ are greater than 85%, then the BLAST cascade continues with SpTrembl and RefseqProt. In the case that no identical or homologous hits can be found in any of the databases, the best similar hit among the three databases is selected and classiﬁed as similar, weakly similar or putative (Figure 2). Depending on the classiﬁcation, the task displays diﬀerent kinds of information. If the protein is character- ized, information concerning the coding gene, about the splicing variants and orthologous genes is also provided. Depending on the degree of homology, protein function, transcript of origin, genomic localization, and GO annotation or partial similarities will also be shown. Proteins annotated as ‘hypothetical’ are further analysed. Hypothetical proteins will only be presented in the result when no other information about identical or homologous proteins can be found in any of the databases (Supplementary Figure 1). The web output of ProtSweep (Supplementary Figure 1) is divided in ﬁve sections: (i) General Information,