Hotspots pipeline

Single pool designs represent cost and handling advantages over panels that require two or more pools of primers. Designs targeting small genomic regions such as SNPs and small INDELs are well suited to fit in a single pool because those regions can be covered by non-overlapping amplicons.

In standard designs, the "tiling" stage of the pipeline is when amplicons are selected to cover the target regions. Because the regions are often larger than a single amplicon, several amplicons are required to cover one particular target. This process results in a set of overlapping amplicons that, if put together, are prone to interact with each other during the sequencing process. The "pooling" stage is when as many pools as needed are created to avoid these interactions.

The hotspots pipeline includes special algorithms that allow in the pooling stage the creation of a single pool of primers with maximum coverage for targets as small as one base, and as large as 50 bases.

The idea behind the hotspots designer can be better understood by looking at the following diagram in which the long line represents a genomic region; a, b, and c are the amplicons available for covering that region. The red portions of the amplicons represent the primers.

In a standard (non-hotspots) Ion AmpliSeq™ panel, the targets are long regions (delimited by arrows in the diagram). The algorithm attempts to find a set of primers at minimum cost to cover the target (amplicons a and b). Amplicons a and b overlap, so this would require two pools to cover the region in question. Forcing the panel to be in one pool would eliminate amplicon a or b resulting in reduced coverage for the one-pool solution. Using amplicons c and b is not an option because they fail to cover a portion of the region between the arrows. In contrast, for a hotspots panel, the targets are small genomic regions (marked with dots over the line representing the genome), the hotspots pipeline would actually select non-overlapping amplicons c and b to cover the submitted targets. Non-overlapping amplicons can be put together in a single pool of primers.