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#' Bacth assay analysis for last five Nr of primers
#'
#' @description Last five nucleotides of primer mutation count/type for any reverse transcription
#' polymerase chain reaction (RT-PCR) primer.
#'
#' @param nucmerr Mutation information containing group list(derived from "nucmer" object using "nucmerRMD" function).
#' @param assays Assays dataframe including the detection ranges of mutations.
#' @param totalsample Total sample number, total cleared GISAID fasta data.
#' @param outdir The output directory. if the figurelist = TRUE, output the figure in the R session.
#' @param figurelist Whether to output the integrated plot list for each assay.
#'
#' @return Plot the mutation counts(last five nucleotides for each primer) for each assay as output.
#' @export
#' @importFrom ggplot2 aes ggplot geom_point theme_bw scale_x_continuous labs ggsave theme geom_vline element_blank
#' @importFrom cowplot plot_grid
#'
#' @examples
#' data("nucmerr")
#' data("assays")
#' totalsample <- 434
#' #outdir <- tempdir()
#' LastfiveNrMutation(nucmerr = nucmerr,
#' assays = assays,
#' totalsample = totalsample,
#' figurelist = FALSE,
#' outdir = NULL)
LastfiveNrMutation <- function(nucmerr = nucmerr, assays = assays, totalsample = totalsample, figurelist = FALSE, outdir = NULL){
assays$MR_Five=0
plist<-list()
if(figurelist == TRUE){
for (i in seq_along(assays$Assay)){
#i=2
F1=assays[i,"F1"]
F2five=assays[i,"F2"]-5
F2=assays[i,"F2"]
R1=assays[i,"R1"]
R1five=assays[i,"R1"]+5
R2=assays[i,"R2"]
sub_nucmer= nucmerr[nucmerr$rpos %in% c(F2five:F2,R1:R1five),]
TMN<- length(unique(sub_nucmer$sample))
#Total=29183 # Total Cleared GISAID fasta data
Mutation_Ratio<- round(TMN/totalsample*100,5)
assays[i,"MR_Five"]<- Mutation_Ratio
rpos <- sub_nucmer$rpos
sample<- sub_nucmer$sample
M_type<- sub_nucmer$M_type
plist[[i]]<- ggplot(data =sub_nucmer,aes(x=rpos,y=sample, color=M_type))+
geom_point()+
theme_bw()+
scale_x_continuous(breaks=seq(F1,R2,2),limits =c(F1,R2))+
labs(x="SARS-CoV-2 Genomic position",
title=paste0(assays$Assay[i],"(Samples:", TMN,"/Ratio:",Mutation_Ratio,"%)"))+
theme(axis.text.x = element_text(angle=90, hjust=1, vjust=.5),
axis.text.y=element_blank(),
axis.title.y=element_blank(),
plot.title = element_text(size=8),
legend.position="none")+
geom_vline(aes(xintercept=F1),color="blue", linetype="dashed", size=0.5)+
geom_vline(aes(xintercept=F2),color="blue", linetype="dashed", size=0.5)+
geom_vline(aes(xintercept=R1),color="red", linetype="dashed", size=0.5)+
geom_vline(aes(xintercept=R2),color="red", linetype="dashed", size=0.5)+
geom_vline(aes(xintercept=F2five),color="gray", linetype="dashed", size=0.5)+
geom_vline(aes(xintercept=R1five),color="gray", linetype="dashed", size=0.5)
}
p <- plot_grid(plotlist=plist,ncol=4)
print(p)
}
if(is.null(outdir) == FALSE){
if(figurelist == FALSE){
for (i in seq_along(assays$Assay)){
#i=2
F1=assays[i,"F1"]
F2five=assays[i,"F2"]-5
F2=assays[i,"F2"]
R1=assays[i,"R1"]
R1five=assays[i,"R1"]+5
R2=assays[i,"R2"]
sub_nucmer= nucmerr[nucmerr$rpos %in% c(F2five:F2,R1:R1five),]
TMN<- length(unique(sub_nucmer$sample))
#Total=29183 # Total Cleared GISAID fasta data
Mutation_Ratio<- round(TMN/totalsample*100,5)
assays[i,"MR_Five"]<- Mutation_Ratio
rpos <- sub_nucmer$rpos
sample<- sub_nucmer$sample
M_type<- sub_nucmer$M_type
p<- ggplot(data =sub_nucmer,aes(x=rpos,y=sample, color=M_type))+
geom_point()+
theme_bw()+
scale_x_continuous(breaks=seq(F1,R2,2),limits =c(F1,R2))+
labs(x="SARS-CoV-2 Genomic position",
title=paste0(assays$Assay[i],"(Samples:", TMN,"/Ratio:",Mutation_Ratio,"%)"))+
theme(axis.text.x = element_text(angle=90, hjust=1, vjust=.5),
axis.text.y=element_blank(),
axis.title.y=element_blank(),
plot.title = element_text(size=8),
legend.position="none")+
geom_vline(aes(xintercept=F1),color="blue", linetype="dashed", size=0.5)+
geom_vline(aes(xintercept=F2),color="blue", linetype="dashed", size=0.5)+
geom_vline(aes(xintercept=R1),color="red", linetype="dashed", size=0.5)+
geom_vline(aes(xintercept=R2),color="red", linetype="dashed", size=0.5)+
geom_vline(aes(xintercept=F2five),color="gray", linetype="dashed", size=0.5)+
geom_vline(aes(xintercept=R1five),color="gray", linetype="dashed", size=0.5)
ggsave(p,filename=paste0(assays$Assay[i],'_Mutation_Count.png'),width = 12, height = 8, dpi=300,path = outdir)
}
}
}
}
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