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

摘要

BackgroundPolymerase chain reaction (PCR) remains a simple, flexible, and inexpensive method for enriching genomic regions of interest for next-generation sequencing. In order to utilize PCR in this context, a major challenge facing researchers is how to generate a very large number of functional PCR primers that will successfully generate useable amplicons. For instance, in an exon-only re-sequencing project targeting 100 genes, each with 10 exons, 1,000 pairs of primers are required. In fact, the reality is often more complex as each gene might have several isoforms and large exons need to be divided to maintain the desired amplicon size. With only a list of gene names, our program Optimus Primer (OP) automatically takes into account all these variables, and can generate primers with no need to provide genome coordinates. More importantly however, OP, unlike other primer design programs, uniquely utilizes Primer3 in an iterative manner that allows the user to progressively design up to four iterations of primer designs. Through a single interface, the user can specify up to four different design parameters with different stringencies, thus increasing the probability that a functional PCR primer pair will be designed for all regions of interest in a single pass of the pipeline.