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.

The few cases of off-target amplification come from closely related organisms in the same family such as Old World (OW) and New World (NW) Arenaviruses or other Flaviviruses amplified by the Japanese encephalitis virus (JEV) multiplex. The three exceptions were a single amplicon of 2830 bp from a BAC clone of Zea mays (maize) from the Ebola 3 kb multiplex, a single amplicon of 3610 bp from Methylococcus capsulatus str. Bath from the OW Arena S segment 3 kb multiplex and a single amplicon of 851 bp from a human BAC from a library at CalTech. All three of these predicted nontarget amplicons result from a single primer in each of those reactions performing as both forward primer (FP) and reverse primer (RP). Nonetheless, the primer multiplexes described here should strongly favor the preferential enrichment of desired targets. Deriving each primer set required multiple sequence alignment and a call to run tile primers in the current PriMux software distribution (http://sourceforge.net/projects/primux/). In comparison, primer design with the JCVI pipeline for any of these target sets would require the following steps: (1) inspection of a phylogeny for the full target set to build multiple smaller clade-level sets with no more than 10% sequence variation, (2) realignment of the clade-level sets, (3) running of the JCVI pipeline on each clade set, (4) assessing which target sequences are not amplified after one design round and rerun the pipeline on those sequences for each clade, (5) and repeating step 4 until all target sequences are predicted to be amplified.