Bioinformatic Pipelines to the Rescue of Primer Design: Development of DrPrimer, EOSAL-Primer and ExonSurfer for Specific PCR Applications

Despite the rise in new PCR-based technologies and applications, primer design is still a challenge and acts as a bottleneck for many set- ups, requiring several rounds of optimization. Primers largely dictate the specificity and efficiency of the amplification and, to a lesser degree, reproducibility. All primers need to be specific to their target and not amplify spurious sequences, such as primer-dimers or regions outside of the region of interest (ROI). In this work, we present three different primer design pipelines, aimed at three PCR applications with specific requirements. Firstly, a primer design pipeline aimed for the creation of amplicon-based libraries for targeted gene panels: DrPrimer. A key step of targeted sequencing is the enrichment of ROIs, usually carried out via a multiplex PCR, creating an amplicon-based library. In the cases where long ROIs are targeted, tiled amplicons need to be designed; this, coupled with the extra requirements of a multiplex PCR, make manual design a troublesome process. Primers that produce overlapping amplicons need to be placed in separate reaction pools, and since the least amount of pools is preferable to reduce costs and pipetting, all primers from a same pool need to be verified for the amplification of dimer or off-target products. DrPrimers designs multiple candidate primers, which are filtered based on their dimer-creating tendency and specificity. A selection algorithm selects one candidate pair per amplicon, minimizing the pool#s dimer-creating tendency and specificity. DrPrimer incorporates a novel coverage-predicting model that takes into account all primers and their designated pools, and reports which of these will produce amplicons with sub-par coverage. As a final evaluation of the complete pipeline, we chose 10 genes from a diabetes-related panel and compared the naive primers against the panel fully-designed with DrPrimer, demonstrating that primers returned by DrPrimer yield a higher percent of on target reads. Secondly, we developed EOSAL-Primers, an application focused at the primer design required for the EOSAL-CNV technique. EOSAL-CNV panels need to be designed so as to each amplicon has a unique length. To solve this issue, we create multiple differently sized candidate primers for each ROI and use a branch-and-bound algorithm that greatly reduces the list of candidates to only compatible combinations. Finally, we present ExonSurfer, an en-to-end web tool aimed at primer design for RT-qPCR with intercalating dyes. This primer design pipeline focuses on gene expression experiments where genomic DNA amplification is a contaminant. Primers are designed to span exon-exon junctions, and filtered according to their specificity and dimer-tendency. The pipeline is embedded in a user-friendly web application, with amplicon view tools. ExonSurfer has been tested in a real-case scenario, demonstrating high accuracy and specificity, making it a key tool in gene expression analysis.