Optimus Primer: A PCR enrichment primer design program for next-generation sequencing of human exonic regions

To demonstrate the effectiveness of the program, we designed PCR primers against 77 genes located in loci associated with ulcerative colitis as part of a candidate gene re-sequencing experiment. We achieved an experimental success rate of 93% or 472 out of 508 amplicons spanning the exonic regions of the 77 genes. Moreover, by automatically passing amplicons that failed primer design through three additional iterations of design parameters, we achieved an additional 170 successful primer pairs or 34% more in a single pass of OP than by conventional methods.With only a gene list and PCR parameters, a user can produce hundreds of PCR primer designs for regions of interest with a high probability of success in a very short amount of time. Optimus Primer is an essential tool for researchers who want to pursue PCR-based enrichment strategies for next-generation re-sequencing applications. The program can be accessed via website at http://op.pgx.ca webcite.The development of next-generation sequencing (NGS) technologies has dramatically increased the size and scale of sequencing experiments. It is now possible to produce several gigabases of DNA sequence in a short period of time [1]. To date, the cost of whole human genome sequencing remains prohibitive. Focusing NGS experiments to specific genomic regions is an alternative, cost-effective approach to whole genome sequencing but it requires the enrichment of the targeted regions before library construction. Several hybridization-based methods - each with its own strengths and weaknesses - have been developed [2]. While these DNA enrichment methods continue to be developed and improved, a simple and inexpensive alternative is PCR. PCR is a robust, well-understood, very accessible and flexible strategy for DNA enrichment. It also allows for the very specific amplification of targeted regions without the high background found in hybridization-based methods. Hundreds or even thousands of PCR amplicons that span selected geno