Multiplex primer prediction software for divergent targets

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

Predicting a set of universal viral primers that are all mixed in a single large reaction, we predict that 1008 primers of length 10 nt would be required to ensure amplification of at least one fragment of length 80-620 bp from every viral genome. These are predicted to generate a mean and maximum number of amplicons per viral genome of 13.2 and 948, respectively. Binning the primers into smaller sets of 20 primers/bin doubled the number of primers required, as Figure 1 shows the fraction of viral genomes amplified versus the number of primers, for primers ranging from 6 to 18 nt in length. About 2000 binned 10-mers are required to amplify 100% of sequenced viruses, generating a mean of 1 and maximum of 2.9 amplicons per genome, on average. Using traditional-length 18-mer primers nearly doubles the number to $3700 primers. The incomplete curve for 7-mers was from inappropriate settings for this oligo length, since some genomes do not contain pairs of 7-mers with the required T m , as given in the Supplementary Tables S1 and S2, within the desired amplicon length range. The concave curves showing diminishing returns reflect biased sequence availability rather than any particularly highly conserved primers: the primers that amplify the largest number of sequences are all from influenza, as far more sequences are available for this species than for any other.