MAGIC BOX – Automated Primer Assay Design for Haploid, Diploid and Polyploid Species

Genome specificity is integral to any PCR / amplicon-probe technology but is often overlooked. The required skills for basic programs such as BLAST, sequence manipulation and alignment are sometimes missing.  Alternatively, the sheer number of alleles to be designed to makes the analysis a laborious process. Correctly targeting genomic regions for KASP genotyping assays  can be the difference between good, useable data, giving the correct frequency, or a poor functioning assay causing loss of data or misleading data. In order to help create target-specific assays we developed 3’-end anchoring of the common reverse primer in KASP primer sets. To do this quickly and efficiently we have developed MAGIC BOX, an automated sequence analysis, alignment and primer design tool for any genome. The central hub of MAGIC BOX pipeline is its BLAST interpreter, which has been developed to automatically analyse and annotate sequence where unique base(s) or clusters of bases occur. The interpretation time takes around 1 second for 300 bp sequences returning a depth of, on average, 40 aligned sequences. BLAST interpreter then hands over to Primer Picker to design the assays. MAGIC BOX can be connected to an offline server containing private databases and BLAST executables, or BLAST results can be downloaded and imported from other repositories such as NCBI along with target sequences for assay design. Here we outline how MAGIC BOX pipeline functions along with some examples of treated assay designs.