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.

Multiplexed primers were tested in the lab as primer pairs in individual reactions then as multiplexed reactions. Twentytwo of the primer pairs worked and four failed to give a product and were paired with other primers in subsequent testing or if necessary, replaced with an alternative primer. Amplicons were detected in the expected size ranges, confirming amplification of the expected regions from the multiplexed sets ( Figure S1 ). In some cases extra bands were present, but they were generally smaller than the targeted size; this was common when the template cDNA was obtained from a clinical sample rather than high titer cell culture derived viral stock from this study. The PCR products generated with these highly multiplexed assays were then sequenced using Illumina ultradeep sequencing with a high fidelity polymerase. These primers yielded high coverage averaging 150,000x of the genomic regions amplified by the multiplex primers.