W56 Nucleic Acids Research, 2009, Vol. 37, Web Server issue Aravind test set (14) in our original study, it appeared that the incurrence of errors increases dramatically at a FOM of 10 8. We have mentioned this empirical FOM thresh- old of 10 8 in the Manual page as a ‘point of reference’ for users to ‘gauge’ the statistical signiﬁcance of each hit. Users are cautioned to the use of this threshold as this value is expected to depend on the size of the database. Each hit on the results page is shown with its GI number, which has a link to the protein annotation page at the NCBI’s website that allows the user to obtain details of the hit to ascertain whether the hit is indeed a true homolog or not. Along with the GI number and FOM, the E-values from the second and the ﬁnal iterations are provided for each hit. Users can view the PSI-BLAST pairwise alignment between the query and the hit sequence by clicking on the E-value link. Diﬀerent output options sorted by E-value at iteration 2 or E-value at the last iter- ation and in HTML or text format are also available to the users. The results page for these diﬀerent options are organized identically to the default results page. Documentation and runtime The SIB-BLAST manual page connected by a clickable link on the SIB-BLAST front page provides users with a brief overview of the SIB algorithm in addition to a detailed description for each input parameter and an explanation of the result page. A sample sequence is made available for users to test a trial run of the SIB- BLAST server. As reﬂected in the algorithm’s name, the beauty of our approach is in its simplicity—it requires minimal changes to the existing PSI-BLAST algorithm since it uses information already output by PSI-BLAST and the pro- cessing time to calculate the FOM for individual hits and re-sorting them are negligible. Thus, SIB-BLAST improves the search accuracy of PSI-BLAST without compromising its computational eﬃciency. Future development The current FOM provides a relative measure of the statistical signiﬁcance of the resultant hits against one another. It is not an E-value, however, due to SIB’s impli- cit assumption in the calculation of the combined P-value—that the hits identiﬁed in the second and last rounds of PSI-BLAST are independent. It would be more meaningful if it were possible to calculate the com- bined P-value under the condition of the hits being depen- dent. Then the FOM calculated would be the true E-value, and the value obtained would provide an accurate mea- sure of the error probability. Bailey and Grundy’s POP (product of P-values) algorithm, which calculates the product of the P-values of correlated variables may pro- vide some suggestions as to how to achieve this as a direc- tion for future study (16). To improve on the functionality of SIB-BLAST, it would also be useful to provide a mul- tiple sequence alignment of the hits (as is being done now in PSI-BLAST) in future versions of SIB-BLAST. ACKNOWLEDGEMENTS NSF (DBI-0317335 to R.B., partial); OSU internal funds (to M.K.C.); OSU Presidential Fellowship (to M.M.L.). FUNDING Funding for open access charge: Ohio State University. Conﬂict of interest statement. None declared. REFERENCES 1. Altschul,S.F., Gish,W., Miller,W., Myers,E.W. and Lipman,D.J. (1990) Basic local alignment search tool. J. Mol. Biol., 215, 403–410. 2. Altschul,S.F., Madden,T.L., Schaﬀer,A.A., Zhang,J., Zhang,Z., Miller,W. and Lipman,D.J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res., 25, 3389–3402. 3. Schaﬀer,A.A., Aravind,L., Madden,T.L., Shavirin,S., Spouge,J.L., Wolf,Y.I., Koonin,E.V. and Altschul,S.F. 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