Performing Evolutionary Trace Analysis

Evolutionary Trace method involves identifying evolutionarily important residue positions in an alignment. A trace residue is one that, when the tree is subdivided at a given level of overall sequence divergence, is identically conserved within each group into which the tree is divided, but may vary from one group to another. To obtain a measure of the evolutionary importance of each trace residue, we used the number of groups into which the tree must be subdivided to have conservation at that residue position within each group. This measure was referred to in previous publications (refs.) as the "rank." By this measure a residue that is absolutely conserved over all 330 sequences in the global alignment has a "rank" of 1, indicating the highest evolutionary importance, and a residue that is not conserved within all subfamilies unless each individual sequence is considered as a "group" has a "rank" of 330, indicating the lowest evolutionary importance. Residues important to each family (e.g opsin family) were determined by tracing the alignment of that family separately. These trace residues include many of those which are obtained using all class A sequences (globally important) and additional residues which are conserved within groups into which the specific family subdivides but have no such conservation pattern within other families of class A GPCRs.