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rprimer is an R package that designs degenerate oligos and PCR assaysfrom a multiple DNA sequence alignment of target sequences of interest.The package is specifically designed for sequence variable viruses.

To install rprimer fromBioconductor,start R (version 4.2) and enter:

if (!requireNamespace("BiocManager",quietly=TRUE))  install.packages("BiocManager")BiocManager::install("rprimer")

Attach the package by calling:

library(rprimer)

The package contains five main functions:

• consensusProfile()
• designOligos()
• designAssays()
• checkMatch()
• plotData()

The package can be run through a Shiny application (a graphical userinterface). To start the application, typerunRprimerApp() from withinR upon installing and attaching the package.

The application can also be found online,here.

The first step is to import an alignment with target sequences ofinterest. This is done by usingreadDNAMultipleAlignment().

The file “example_alignment.txt” contains an alignment of 50 hepatitisE virus sequences.

infile<- system.file("extdata","example_alignment.txt",package="rprimer")myAlignment<- readDNAMultipleAlignment(infile,format="fasta")

consensusProfile() takes aDNAMultipleAlignment as input and returnsall the information needed for the subsequent design process.

myConsensusProfile<- consensusProfile(myAlignment,ambiguityThreshold=0.05)

Results (row 100-106):

The results can be visualized withplotData():

plotData(myConsensusProfile)

The next step is to design oligos. You can either use the defaultsettings as below, or adjust them as preferred (see the package vignetteor?designOligos for more information). The default settings allow amaximum degeneracy of four, which means that only the most conservedregions of the genome will be considered as oligo binding sites.

myOligos<- designOligos(myConsensusProfile)

Results (first six rows):

The results can be visualized as a dashboard, usingplotData():

plotData(myOligos)

designAssays() finds pairs of forward and reverse primers and combinethem with probes, if probes are present in the input dataset. You caneither use the default settings as below, or adjust the designconstraints (see the package vignette or?designAssays for moreinformation).

myAssays<- designAssays(myOligos)

Results (first six rows):

The assays can be visualized usingplotData():

plotData(myAssays)

checkMatch() shows the proportion and names of the target sequences inthe input alignment that match with the generated oligos or assays. Seethe package vignette or?checkMatch for more information.

## Randomly select six oligos to illustrate an exampleselection<- sample(seq_len(nrow(myOligos)),size=6)matchTableOligos<- checkMatch(myOligos[selection, ],target=myAlignment)

Results:

The match table can be visualized usingplotData():

plotData(matchTableOligos)

Please see thepackagevignettefor more information on how to use the package.

Persson S., Larsson C., Simonsson M., Ellström P. (2022) rprimer: anR/bioconductor package for design of degenerate oligos for sequencevariable viruses.BMC Bioinformatics23:239