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Filip Pattyn, Steve Lefever, Frank Speleman, Jo Vandesompele Ghent University, Belgium |
Abstract
Primers are the cornerstone of many modern-age high-throughput nucleic acid analysis techniques ranging from real-time quantitative PCR (qPCR) to amplicon generation for next-generation sequencing. Various tools are currently available for automated primer design, but most lack a thorough downstream evaluation. A proper assessment of the multiple aspects affecting a primer assay’s efficiency is a laborious and repetitive work involving manual interactions with different software applications. We automated the primer design process from top to bottom into a processing pipeline combining experimentally optimized design guidelines with a multi-faceted primer evaluation. This evaluation includes checking for the presence of SNPs in the primer annealing sites to avoid sample dependent amplification efficiencies, evaluating secondary structures in the primer annealing sites hampering efficient primer annealing and testing the primer specificity which results in primers having an good overall efficiency. The pipeline is customizable, making it possible to design primers fitting specific needs. In addition to the development of standard qPCR-primers, the pipeline allows to design primers targeting a specific transcript variant or a subset of transcript variants of a single gene or specific gene loci like putative transcription factor or miRNA binding regions.
Another field in which PrimerXL exceeds concurrent tools is the design of primers used in the preprocessing steps of amplicon based next-generation sequencing. Until now, the application of high-throughput amplicon sequencing has been hindered by the lack of tools capable in developing efficient tiling primers. To fill this void, we have expanded our primer design pipeline with a module to design tiling primers targeting all exonic sequences of a specific gene. The generated primers produce more equimolar amplicon sets leading to a cost-efficient and sensitive variation detection without extra sequencing efforts. The low variation in amplicon molarity outperforms the widely used array capturing technology employed to amplify specific target regions.
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