Multiplex primer prediction software for divergent targets

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

Most currently available multiplex primer prediction tools require an MSA (20) (21) (22) (23) (24) . None of those tools build multiplex sets in which no primers in the set are predicted to form primer-dimers, although some avoid homodimers. We attempted to run a number of alternative software tools for multiplex or degenerate primer prediction for several species level target sets ranging in size from 41 to $6000 sequences: Primaclade (24) , FastPCR (www .biocenter.helsinki.fi/bi/programs/fastpcr.htm), GeneUp (25) , PDA-MS/UniQ software (26) , Greene SCPrimer (20) and HYDEN (22) . Only Greene SCPrimer and HYDEN could handle the two smallest, species-level target sets (Norwalk virus and FMDV), and none of the tools completed for the larger target sets that we attempted to run. Therefore, we designed the MPP software to avoid the requirement of an MSA and to scale better for large and diverse target sets. MPP builds a multiplex-compatible set of primers capable of amplifying all target sequences, attempting to minimize the number primers in the set.