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R script.txt
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qpcrplot = function(x,df,y,z,hk, f) {
library(ggplot2)
dat = read.csv(choose.files(), header = T)
colnames(dat)[1] = "Well"
as.factor = dat$Well
UsedWells = dat[dat$Well %in% df$well,]
QPCRDATA = replicate(length(df$well),data.frame(well= replicate(x,1),Cycle = factor(1:x),GREEN=(replicate(x,1)),normalised=(replicate(x,1)),gene =(replicate(x,1)), CT=(replicate(x,1))),simplify = FALSE)
QPCRDATA = setNames(QPCRDATA, df$well)
well_list = df$well
gene_list = df$gene
ct = data.frame(CT=1:length(df$well))
rownames(ct) = df$well
ct$gene = df$gene
ct$sample = factor(df$sample)
ctlist = list()
dat2 = dat[(dat$Well %in% well_list),]
dat2$gene = gene_list
dat2$sample = factor(rep(df$sample, x))
dat2$threshold = y
cycle_list = unique(dat2$Cycle)
for (i in unique(well_list)) {
for (cy in cycle_list) {
dat2$normalised[dat2$Well == i & dat2$Cycle == cycle_list[cy]]= dat2$GREEN[dat2$Well == i & dat2$Cycle == cycle_list[cy+5]] / (dat2$GREEN[dat2$Cycle == cycle_list[cy] & dat2$Well == i ])
} }
for (t in 1:length(df$well)) {
QPCRDATA[[t]]$well = replicate(x, well_list[t])
QPCRDATA[[t]]$gene = replicate(x, gene_list[t])
QPCRDATA[[t]]$GREEN = UsedWells$GREEN[UsedWells$Well == well_list[t]]
QPCRDATA[[t]]$normalised = QPCRDATA[[t]]$GREEN - QPCRDATA[[t]]$GREEN[QPCRDATA[[t]]$Cycle==25]
QPCRDATA[[t]]$CT = approx(QPCRDATA[[t]]$normalised, QPCRDATA[[t]]$Cycle, xout=y)[2]
ct$CT[[t]] = unique(QPCRDATA[[t]]$CT)
}
dat_list = replicate(length(unique(ct$gene)), data.frame(CT=(replicate(9,1)), sample = factor(c(rep(1,3),rep(2,3),rep(3,3)))), simplify = FALSE)
dat_list2 = replicate(length(unique(ct$gene)), data.frame(meanCT=(replicate(3,1)), sample = factor(c(rep(1,1),rep(2,1),rep(3,1))), deltactActin = rep(3,1),deltactElfa1 = rep(3,1)), simplify = FALSE)
dat_list = setNames(dat_list, unique(ct$gene))
dat_list2 = setNames(dat_list2, unique(ct$gene))
for (i in unique(ct$gene)){
dat_list[[i]]$CT = unlist(ct$CT[ct$gene == i])
dat_list2[[i]]$meanCT[dat_list2[[hk]]$sample == 1] = mean(dat_list[[i]]$CT[dat_list[[hk]]$sample == 1])
dat_list2[[i]]$meanCT[dat_list2[[i]]$sample == 2] = mean(dat_list[[i]]$CT[dat_list[[i]]$sample == 2])
dat_list2[[i]]$meanCT[dat_list2[[i]]$sample == 3] = mean(dat_list[[i]]$CT[dat_list[[i]]$sample == 3])
dat_list2[[i]]$deltact = dat_list2[[i]]$meanCT - dat_list2[[hk]]$meanCT
ctlist[[i]] = (dat_list2[[i]]$deltact)}
for (i in df$well) {
df$max[df$well == i] = max(na.omit(dat2$normalised[dat2$Well == i & dat2$Cycle > 20]))
df$ct[df$well == i] = dat2$Cycle[dat2$normalised == df$max & dat2$Well == i]
}
dat3 = data.frame(Well = df$well)
dat3$gene = df$gene
dat3$sample = df$sample
dat3$ct = df$ct
dat3$normalised = df$max
dat3$condition = f
#dat3 = dat3[dat3$ct > 20,]
for (gene in df$gene) {
dat3$meanct[dat3$gene == gene & dat3$sample == 1] = mean(dat3$ct[dat3$gene == gene & dat3$sample == 1])
dat3$meanct[dat3$gene == gene & dat3$sample == 2] = mean(dat3$ct[dat3$gene == gene & dat3$sample == 2])
dat3$meanct[dat3$gene == gene & dat3$sample == 3] = mean(dat3$ct[dat3$gene == gene & dat3$sample == 3])
dat3$deltact[dat3$gene == gene & dat3$sample == 1] = dat3$meanct[dat3$gene == gene & dat3$sample == 1] - dat3$meanct[dat3$gene == hk & dat3$sample == 1]
dat3$deltact[dat3$gene == gene & dat3$sample == 2] = dat3$meanct[dat3$gene == gene & dat3$sample == 2] - dat3$meanct[dat3$gene == hk & dat3$sample == 2]
dat3$deltact[dat3$gene == gene & dat3$sample == 3] = dat3$meanct[dat3$gene == gene & dat3$sample == 3] - dat3$meanct[dat3$gene == hk & dat3$sample == 3]
}
dat4 = data.frame(gene = rep(unique(df$gene),3))
dat4$sample = c(rep(1,10),rep(2,10),rep(3,10))
dat4$condition = f
for (i in df$gene) {
dat4$deltact[dat4$gene == i & dat4$sample == 1] = mean(dat3$deltact[dat3$gene == i & dat3$sample == 1])
dat4$deltact[dat4$gene == i & dat4$sample == 2] = mean(dat3$deltact[dat3$gene == i & dat3$sample == 2])
dat4$deltact[dat4$gene == i & dat4$sample == 3] = mean(dat3$deltact[dat3$gene == i & dat3$sample == 3])
}
ctlist = data.frame(ctlist)
if(missing(z)) {
return(dat2)
}
else {
if (z == "deltact"){
return(ctlist)
}}
if (z == "graph"){
return(ggplot(data = dat2, aes(x=Cycle, y= normalised)) + geom_line(aes(col = Well), size =1.25) +
theme_minimal() + facet_wrap(~gene + sample, scales = "free") + theme(legend.position = "NONE") + labs(title = f)) }
if ( z == "new"){
return(dat3)
}
if ( z == "newdelta"){
return(dat4)
}
else {
print("please select one of the following: 'graph' or 'deltact' or 'new'")
}
}