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 run tiled primers process can be summarized as follows: split a multiple sequence alignment into overlapping regions, and for each region design a degenerate multiplex set of primers that in combination amplify that region in all strains with as few primers as possible. Run tiled primers takes as input a multiple sequence alignment (MSA). Run tiled primers splits the alignment into regions of size " " bases that overlap by " " bases ( Figure 1 ). When splitting the alignment into regions of size , if the last "remainder" piece of an alignment is less than half of , then is increased by the amount that evenly divides the alignment without any remainder to , and the split regions are recalculated with . If a user desires to tile across only selected regions instead of tiling across the entire sequence, then an optional regions file may be specified which contains the regions (e.g., genes) and their start and end positions in the alignment. For each region, the PriMux software [3] is used to search for conserved, degenerate, and multiplex compatible primer sets to amplify that region in all target sequences with as few primers as possible. The PriMux "max" algorithm is used. Primers should be multiplex compatible since the primers for a given region are predicted not to form primer dimers and all to have 's in a range specified by the user. As run tiled primers is a wrapper script around the PriMux workhorse, all the primer design characteristics are specified in a PriMux options file. The minimum and maximum amplicon lengths are determined by the ( , ) parameters to run tiled primers (Table 2) , so these parameters may be omitted in the input options file or if they are present, their values will be replaced with values appropriate for the specified values of ( , ). Run tiled primers requires that primers must anneal within 0.5 of either end of the region. If the value of is 36 bp or less, it is too short for two nonoverlapping primers, typically at least 18 bp long. In this case, the code does not require that adjacent regions overlap and amplicons are allowed from anywhere in each region. Small overlaps (e.g., 40-80) do not leave much room to find good priming regions that pass the filters on , entropy, free energy, and homopolymers as specified in the options file, and consequently it may not be possible to find primers for all targets. When this happens, increasing the overlap and relaxing the primer specifications may be necessary.