Multiplex primer prediction software for divergent targets

Gardner, Shea N.; Hiddessen, Amy L.; Williams, Peter L.; Hara, Christine; Wagner, Mark C.; Colston, Bill W.

We set out to determine a set of highly conserved 'universal' primers for viruses, akin to the highly conserved 16S rRNA universal primers for bacteria. Throughout, we use the term 'detected' to mean that there should be at least one PCR product. This is a loose definition of 'detected' adopted for convenience in the following discussions, and we recognize that a PCR product may prove insufficient for viral characterization. We predicted a set of 'universal viral primers' for all available complete genomes of all viruses, and found that the number of universal viral primers would be impractical to implement, even if short, highly conserved priming sequences were used. Then we predicted family-level primer sets for every viral family, as well as for several highly diverse species of RNA viruses, for primers of a traditional length as well as nontraditional, shorter, more highly conserved primers, which are more likely to amplify novel, unsequenced viruses and which could be an alternative to degenerate primers. While the software uses a greedy algorithm that may settle at a local minimum which is above the true minimal set, it generated primer sets for each viral family with fewer than half the number of primers that would be expected without optimization. Although family-level primer sets for some families are too large to be practical, 64% of the families had primer sets of no more than 20 primer pairs, quite feasible for multiplex PCR.