DeGenPrime provides robust primer design and optimization unlocking the biosphere

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.Availability and implementation DeGenPrime is written in C++ and distributed under a BSD-3-Clause license. The source code for DeGenPrime is freely available on www.github.com/raw-lab/degenprime.