rprimer: an R/bioconductor package for design of degenerate oligos for sequence variable viruses

Abstract

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.

A tool to automatically design multiplex PCR primer pairs for specific targets using diverse templates

“…To demonstrate the performance of PMPrimer, we compared this software tool with DECIPHER 11 , PrimerDesign-M 12 , PhyloPrimer 14 , rprimer 15 , and openPrimeR 13 in terms of the analysis process, degree of automation, run time, and results. For the analysis process, modular design allows users to conveniently choose an appropriate module for subsequent analysis.…”

“…DECIPHER is used to design primer pairs targeting a specific group of sequences from the whole sequence set, but its R-based package is time-consuming and its web-based tool cannot currently be accessed 11 . Many tools design primers using consensus sequences while ignoring minor alleles in consensus regions, such as PrimerDesign-M 12 , openPrimeR 13 , PhyloPrimer 14 , and rprimer 15 . PrimerDesign-M and rprimer require previous multiple sequence alignment coupled with information about the intended target regions 12 , 15 .…”

“…Many tools design primers using consensus sequences while ignoring minor alleles in consensus regions, such as PrimerDesign-M 12 , openPrimeR 13 , PhyloPrimer 14 , and rprimer 15 . PrimerDesign-M and rprimer require previous multiple sequence alignment coupled with information about the intended target regions 12 , 15 . The rprimer tool cannot identify conserved regions and produces many candidate multiplex PCR primer pairs, which makes it almost impossible for the user to directly obtain the optimal primer pairs 15 .…”

“…PrimerDesign-M and rprimer require previous multiple sequence alignment coupled with information about the intended target regions 12 , 15 . The rprimer tool cannot identify conserved regions and produces many candidate multiplex PCR primer pairs, which makes it almost impossible for the user to directly obtain the optimal primer pairs 15 . PhyloPrimer is used to design primers for microbial sequences and preferentially produces non-degenerate primer pairs 14 .…”

“…Furthermore, most primer design tools strip gaps for convenience, especially software based on consensus sequences. Finally, tools based on the R language, such as openPrimeR 13 and rprimer 15 , are inefficient at processing massive amounts of data.…”