Multiplex Degenerate Primer Design for Targeted Whole Genome Amplification of Many Viral Genomes

Gardner, Shea N.; Jaing, Crystal J.; Elsheikh, Maher M.; Peña, José; Hysom, David A.; Borucki, Monica K.

Input to run tiled primers was an alignment of 22 MHV genomes (genome identities provided as supplementary information) created using MUSCLE [5] . Regions tiled were the Nsp1, Nsp3, Nsp14, and several genes at the 3 end of the genome (regions file provided in supplementary information), using the primer parameters in Table 2 . Primer sets were predicted to produce overlapping amplicons for these regions from all MHV genomes, and a subset of primers predicted to amplify the MHV-1 or MHV strain JHM genome was selected. Some primers that were predicted to amplify the JHM strain but not the MHV-1 strain were included in the multiplex, to check for possible evolutionary change of the original sequence toward the annotated reference JHM sequence or cross reactions with primer-genome mismatches. Table 3 : Number of nontarget amplicons predicted in a multiplex reaction of tiled primers for 3 kb amplicons. In a multiplex of the 3 kbamplicon tiled primers for a given organism, of the possible reactions producing products, only a small number of primer combinations are predicted to amplify regions in nontarget organisms. Counts show the number of unique primer combinations in a multiplex that yield products for any sequence in the NCBI nt nucleotide database. The numerator is for any nontarget organism in nt and the denominator is for any target or nontarget organism in nt, that is, nonspecific/total of the possible primer combinations in the multiplex predicted to yield product when compared against nt. Vastly more amplicons are produced from target organisms, indicating any contaminating nontarget species should be a small minority of amplified product.