Multiplex primer prediction software for divergent targets

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

Primer design with the MPP software indicates that relatively few primers are required to amplify all sequenced genomes of HIV-1, FMDV and Norwalk virus (Table 2) , and these can be calculated in minutes. Influenza A HA and NA segments demand large numbers of 10-mer or 17-18-mer primers and hours to calculate, so one could break these into subgroups, possibly by serotype, as shown for several HA serotypes in Table 3 . The percentage of genomes amplified versus the number of primers used, for primers of either 10-mers or 17-18-mers, is shown in Figure 6 . This plot shows that a large fraction of targets are amplified with only 2 primers, and the addition of subsequent primers shows diminishing returns in amplifying fewer, more divergent targets not detected by the initial, more conserved, primer pair, although the true diminishing returns depend on the extent to which available sequence data is an unbiased representation of diversity. The more traditional method of attempting to find primers from a MSA would be problematic, probably requiring manually designed primer multiplexes or highly degenerate primers. GAAGAAGCG starting at 9071. These regions are too far apart to be used as primers for most polymerases used in diagnostic PCR protocols, where amplicons must typically be less than 300 bases long for efficient amplification. A recently published study (35) selected primers from the 5 0 LTR U5 end to the Gag-Pol start (5 0 -TAGC AGTGGCGCCCGA-3 0 and 5 0 -TCTCTCTCCTTCTAGC CTCCGC-3 0 ), but a comparison against available genomic data indicates that 487 of the 1175 genomes (41%) do not contain a sequence match for this primer pair, so may fail to be amplified. For influenza A segment HA, the size of the longest conserved region from the 95% consensus is only 5 bases, and for segment NA, only 6 bases, insufficient for even a single primer. For FMDV and Norwalk virus, the longest 100% conserved regions are 9 and 6 bases, respectively. The MPP software makes it straightforward for a nonexpert to predict a multiplex-compatible set of primers to amplify all targets, even for enormous and heterogeneous target sets that cannot be aligned.