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.

in multiplex should facilitate whole genome amplification in a single reaction. It may yield longer amplicons from the reaction of forward and reverse primers from different parts (FP from 0part reacting with RP from 1part gives product ∼2 times the split size), depending on the polymerase processivity and the duration of the extension step, and should facilitate assembly across amplified regions. This helps alleviate cases where a primer cannot be found for one part in an outlier genome due to , homopolymers, primer dimer Δ , and so forth, since primers from different parts may amplify across the region. However, since primers of overlapping regions can also produce amplicons shorter (less than bp) than the desired amplicon of length between − and bp (e.g., RP of 0part with the FP from 1part), a step to remove short amplicons before sequencing may be desired. In our experimental test with MHV, the primers from parts 0, 2, and 4 were combined in one reaction and the primers from parts 1 and 3 were combined in another, so that short products would not be produced.