Background
This paper presents a new R/Bioconductor package, rprimer, for design of degenerate oligos and PCR assays for sequence variable viruses. A multiple DNA sequence alignment is used as input data, while the outputs consist of comprehensive tables (data frames) and dashboard-like plots. The workflow can be run directly from the R console or through a graphical user interface (Shiny application). Here, rprimer is demonstrated and evaluated by using it to design two norovirus genogroup I (GI) assays: one RT-qPCR assay for quantitative detection and one RT‑PCR assay for Sanger sequencing and polymerase-capsid based genotyping.
Results
The assays generated were evaluated using stool samples testing positive for norovirus GI. The RT-qPCR assay accurately amplified and quantified all samples and showed comparable performance to a widely-used standardised assay, while the RT-PCR assay resulted in successful sequencing and genotyping of all samples. Merits and limitations of the package were identified through comparison with three similar freely available software packages. Several features were comparable across the different tools, but important advantages of rprimer were its speed, flexibility in oligo design and capacity for visualisation.
Conclusions
An R/Bioconductor package, rprimer, was developed and shown to be successful in designing primers and probes for quantitative detection and genotyping of a sequence-variable virus. The package provides an efficient, flexible and visual approach to degenerate oligo design, and can therefore assist in virus research and method development.