Iterated profile search methods have led to biologically important observations but, for many years, were quite slow and generally did not provide precise means for evaluating the significance of their results. This limited their utility for systematic mining of the protein databases. The principal design goals in developing the Position-Specific Iterated BLAST (PSI-BLAST) program [10] were speed, simplicity and automatic operation. The procedure PSI-BLAST uses can be summarized in five steps:
(1) PSI-BLAST takes as an input a single protein sequence and compares it to a protein database, using the gapped BLAST program [10].
(2) The program constructs a multiple alignment, and then a profile, from any significant local alignments found. The original query sequence serves as a template for the multiple alignment and profile, whose lengths are identical to that of the query. Different numbers of sequences can be aligned in different template positions.
(3) The profile is compared to the protein database, again seeking local alignments. After a few minor modifications, the BLAST algorithm [10,12] can be used for this directly.
(4) PSI-BLAST estimates the statistical significance of the local alignments found. Because profile substitution scores are constructed to a fixed scale [13], and gap scores remain independent of position, the statistical theory and parameters for gapped BLAST alignments [14] remain applicable to profile alignments [10].
(5) Finally, PSI-BLAST iterates, by returning to step (2), an arbitrary number of times or until convergence.
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