How does Primaclade work exactly?
Put simply, Primaclade takes a brute force approach to the problem of finding multiple primers. Each individual sequence in the alignment is fed into the Primer3 program multiple times to generate a comprehensive “meta-list” of possible primers for the alignment. Specifically, Primaclade runs Primer3 ten times starting with a primer length of 18 nucleotides and then increasing the length of the primer by one base pair at a time until a maximum length of 27 nucleotides. During each of these runs a maximum of 20 primers are returned creating a meta-list of 10 x 20 x (# of sequences) primers. This meta-list is then sorted, duplicates are removed, and the remaining results are checked to see if they meet the users input criteria: degenerate nucleotides and gapped-lines. If a primer falls in a region of the alignment that has the correct number of degenerate nucleotides (or less) it gets passed along. Next the primer is checked to see if the region it lies in has the correct number (or less
