DeGenPrime provides robust primer design and optimization unlocking the biosphere

Abstract

Motivation: Polymerase chain reaction (PCR) is the world's most important molecular diagnostic with applications ranging from medicine to ecology. PCR can fail because of poor primer design. The nearest-neighbor thermodynamic properties, picking conserved regions, and filtration via penalty of oligonucleotides form the basis for good primer design. Results: DeGenPrime is a console-based high-quality PCR primer design tool that can utilize MSA formats and degenerate bases expanding the target range for a single primer set. Our software utilizes thermodynamic properties, filtration metrics, penalty scoring, and conserved region finding of any proposed primer. It has degeneracy, repeated k-mers, relative GC content, and temperature range filters. Minimal penalty scoring is included according to secondary structure self-dimerization metrics, GC clamping, tri- and tetra-loop hairpins, and internal repetition. We compared PrimerDesign-M, DegePrime, ConsensusPrimer, and DeGenPrime on acceptable primer yield. PrimerDesign-M, DegePrime, and ConsensusPrimer provided 0%, 11%, and 17% yield, respectively, for the alternative iron nitrogenase (anfD) gene target. DeGenPrime successfully identified quality primers within the conserved regions of the T4-like phage major capsid protein (g23), conserved regions of molybdenum-based nitrogenase (nif), and its alternatives vanadium (vnf) and iron (anf) nitrogenase. DeGenPrime provides a universal and scalable primer design tool for the entire tree of life.