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.

All the primers for both ( , ) settings are provided as Supplementary data as are the predicted amplicon start and end positions in each target genome from a multiplex of the primers for a given viral target set. Tiled amplification of these viruses required from 2 to 116 primers (Table 1) . Primers are predicted to be specific to the target organisms for the most part, although not exclusively (Tables 3 and 4) . In separate seminested PCR reactions, forward primers were paired with a reverse primer from an overlapping reaction to verify that product was generated for each overlapping region. 5 3 Figure 2 : Diagram of the murine hepatitis virus (MHV) genome regions for which primer sets were tested. The approximate position of each region amplified by primer sets is shown (MHV genome is not drawn to scale). Each multiplex reaction consisted of primer sets that do not overlap in regions amplified. Each region is amplified using 3 forward primers and 3 reverse primers (Table S1 ; see Supplementary Material available online at http://dx.doi.org/10.1155/2014/101894). For example, the A primer set consists of 3 forward primers (A1F, A2F, and A3F) and 3 reverse primers (A1R, A2R, and A3R). To verify that each region is amplified in the multiplex reaction, a second set of seminested PCRs were performed using the amplicons from the multiplex reaction as a template. For example, to ensure region A was amplified, the PCR product from the A mix multiplex was diluted 1 : 10,000 and used as template in a PCR reaction with AR1 primer paired with BF2 (Table S2) . Primers are labeled according to genome region (A-I) and primer direction (F = forward, R = reverse).