Why is it that my main rhAmpSeq™ CRISPR Panel does not contain assays against all my targets?

The rhAmpSeq Design Tool aims to maximize PCR target specificity at intended loci and minimize any amplification of unintended loci that might consume valuable sequencing reads. Therefore, the design output can sometimes include not only the main panel, but also a secondary pool and possibly individual (singleton) primer sets requiring independent amplification. 

Several factors can lead to more than one panel being designed: 

• The PCR targets may be too close together. The rhAmpSeq Design Tool requires a minimum distance of 600 base pairs (bp) between targets. Targets that do not meet this minimum distance requirement impede having two unique primer pairs in a single pool. Typically, this results in an adjacent target needing to be covered by either a secondary pool, or as a singleton assay, if the assays cannot be merged. 
• The algorithm may not be able to select a primer pair that is both specific enough to cover the PCR target of interest, and distinct enough to allow pooling (multiplexing) without creating primer dimers. 
• The ideal primer pair for a given target may amplify a repetitive region. In this case, the optimal primer pair must remain a singleton primer pair to avoid impacting the operation of the primary pool.
• An assay with potential issues is less likely to be pooled due to the risk of impacting the overall panel results. Examples of these types of potential issues include the following:
• A primer overlaps common single nucleotide polymorphisms (SNPs) leading to potential inefficient amplification.
• The GC content of a primer or amplicon significantly differs from the panel’s average GC content.
• The T_m or secondary structure of the primers is not ideal.
• The amplicons contain homopolymers.
• The locus and design constraints do not permit the design of a primer pair that is expected to uniformly amplify with the assays in the primary pool.