Detection of homologous proteins by an intermediate sequence search

We developed a variant of the intermediate sequence search method (ISS_new) for detection and alignment of weakly similar pairs of protein sequences. ISS_new relates two query sequences by an intermediate sequence that is potentially homologous to both queries. The improvement was achieved by a more robust overlap score for a match between the queries through an intermediate. The approach was benchmarked on a data set of 2369 sequences of known structure with insignificant sequence similarity to each other (BLAST E‐value larger than 0.001); 2050 of these sequences had a related structure in the set. ISS_new performed significantly better than both PSI‐BLAST and a previously described intermediate sequence search method. PSI‐BLAST could not detect correct homologs for 1619 of the 2369 sequences. In contrast, ISS_new assigned a correct homolog as the top hit for 121 of these 1619 sequences, while incorrectly assigning homologs for only nine targets; it did not assign homologs for the remainder of the sequences. By estimate, ISS_new may be able to assign the folds of domains in ∼29,000 of the ∼500,000 sequences unassigned by PSI‐BLAST, with 90% specificity (1 − false positives fraction). In addition, we show that the 15 alignments with the most significant BLAST E‐values include the nearly best alignments constructed by ISS_new.