![]() ![]() As a result, several strategies and tools have been developed to aid in degenerate oligo design. Thus, keeping the degeneracy as low as possible is often advised, but manually identifying such oligo-binding regions across a large set of target sequences is time-consuming and error-prone. Moreover, a larger number of oligos increases the probability of nonspecific interactions with other targets. However, as the degeneracy increases, there is a decrease in the fraction of the sequence variant that perfectly matches a specific target sequence and the synthesised PCR products, which reduces the amplification efficiency. For instance, the IUPAC consensus character K can be either G or T, while N can be A, C, G or T. The degenerate bases are specified by nomenclature determined by IUPAC. A degenerate oligo has at least one position with several possible bases, and the degeneracy refers to the number of unique sequence variants encompassed by the oligo. Furthermore, in various RT-PCR applications (such as genotyping), there is often an interest in amplifying a specific, more variable part of the genome.Ī common approach for amplification of sequence variable targets is to use degenerate oligos. An important challenge in oligo design for viral targets is therefore to identify such conserved regions of the genome, but even the most conserved regions may still have some degree of sequence variability. However, substitutions are not evenly distributed across the viral genome, as functional interactions between viral proteins and between viral and host proteins limit sequence variation in certain regions. ![]() Unlike many DNA polymerases, RdRps generally have no proofreading activity and are therefore unable to correct errors during replication (a notable exception, however, is the RdRp of coronaviruses), resulting in an average of 10 –6 to 10 –4 substitutions per site during replication. The high mutation rate is mainly explained by the RNA-dependent RNA polymerase (RdRp), which is responsible for the replication of the genome. This often remarkable sequence diversity results from high mutation rates combined with short generation times and large population sizes. Many viruses, particularly RNA viruses such as norovirus, hepatitis C virus and hepatitis E virus, evolve rapidly and exhibit considerable sequence variability between different strains. Poorly designed primers can reduce the efficiency of RT or PCR and poorly designed probes may not hybridise properly with the target, leading to false-negative results or underestimation of the target concentration. Successful RT-PCR analysis depends on several factors, but oligo (primer and probe) design is arguably the most critical part of assay development. Reverse transcription (RT) PCR-based methods are the gold standard for detecting and quantifying viral genetic material in many fields, including clinical diagnostics and food safety control. The package provides an efficient, flexible and visual approach to degenerate oligo design, and can therefore assist in virus research and method development. ConclusionsĪn 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. Several features were comparable across the different tools, but important advantages of rprimer were its speed, flexibility in oligo design and capacity for visualisation. Merits and limitations of the package were identified through comparison with three similar freely available software packages. 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. The assays generated were evaluated using stool samples testing positive for norovirus GI. 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. The workflow can be run directly from the R console or through a graphical user interface (Shiny application). A multiple DNA sequence alignment is used as input data, while the outputs consist of comprehensive tables (data frames) and dashboard-like plots. This paper presents a new R/Bioconductor package, rprimer, for design of degenerate oligos and PCR assays for sequence variable viruses. ![]()
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