diff report_clonality/RScript.r.old @ 20:9185c3dfc679 draft

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author davidvanzessen
date Fri, 27 Jan 2017 03:44:18 -0500
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--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/report_clonality/RScript.r.old	Fri Jan 27 03:44:18 2017 -0500
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+# ---------------------- load/install packages ----------------------
+
+if (!("gridExtra" %in% rownames(installed.packages()))) {
+  install.packages("gridExtra", repos="http://cran.xl-mirror.nl/") 
+}
+library(gridExtra)
+if (!("ggplot2" %in% rownames(installed.packages()))) {
+  install.packages("ggplot2", repos="http://cran.xl-mirror.nl/") 
+}
+library(ggplot2)
+if (!("plyr" %in% rownames(installed.packages()))) {
+  install.packages("plyr", repos="http://cran.xl-mirror.nl/") 
+}
+library(plyr)
+
+if (!("data.table" %in% rownames(installed.packages()))) {
+  install.packages("data.table", repos="http://cran.xl-mirror.nl/") 
+}
+library(data.table)
+
+if (!("reshape2" %in% rownames(installed.packages()))) {
+  install.packages("reshape2", repos="http://cran.xl-mirror.nl/")
+}
+library(reshape2)
+
+if (!("lymphclon" %in% rownames(installed.packages()))) {
+  install.packages("lymphclon", repos="http://cran.xl-mirror.nl/")
+}
+library(lymphclon)
+
+# ---------------------- parameters ----------------------
+
+args <- commandArgs(trailingOnly = TRUE)
+
+infile = args[1] #path to input file
+outfile = args[2] #path to output file
+outdir = args[3] #path to output folder (html/images/data)
+clonaltype = args[4] #clonaltype definition, or 'none' for no unique filtering
+ct = unlist(strsplit(clonaltype, ","))
+species = args[5] #human or mouse
+locus = args[6] # IGH, IGK, IGL, TRB, TRA, TRG or TRD
+filterproductive = ifelse(args[7] == "yes", T, F) #should unproductive sequences be filtered out? (yes/no)
+clonality_method = args[8]
+
+
+# ---------------------- Data preperation ----------------------
+
+print("Report Clonality - Data preperation")
+
+inputdata = read.table(infile, sep="\t", header=TRUE, fill=T, comment.char="", stringsAsFactors=F)
+
+print(paste("nrows: ", nrow(inputdata)))
+
+setwd(outdir)
+
+# remove weird rows
+inputdata = inputdata[inputdata$Sample != "",]
+
+print(paste("nrows: ", nrow(inputdata)))
+
+#remove the allele from the V,D and J genes
+inputdata$Top.V.Gene = gsub("[*]([0-9]+)", "", inputdata$Top.V.Gene)
+inputdata$Top.D.Gene = gsub("[*]([0-9]+)", "", inputdata$Top.D.Gene)
+inputdata$Top.J.Gene = gsub("[*]([0-9]+)", "", inputdata$Top.J.Gene)
+
+print(paste("nrows: ", nrow(inputdata)))
+
+#filter uniques
+inputdata.removed = inputdata[NULL,]
+
+print(paste("nrows: ", nrow(inputdata)))
+
+inputdata$clonaltype = 1:nrow(inputdata)
+
+#keep track of the count of sequences in samples or samples/replicates for the front page overview
+input.sample.count = data.frame(data.table(inputdata)[, list(All=.N), by=c("Sample")])
+input.rep.count = data.frame(data.table(inputdata)[, list(All=.N), by=c("Sample", "Replicate")])
+
+PRODF = inputdata
+UNPROD = inputdata
+if(filterproductive){
+  if("Functionality" %in% colnames(inputdata)) { # "Functionality" is an IMGT column
+    #PRODF = inputdata[inputdata$Functionality == "productive" | inputdata$Functionality == "productive (see comment)", ]
+    PRODF = inputdata[inputdata$Functionality %in% c("productive (see comment)","productive"),]
+    
+    PRODF.count = data.frame(data.table(PRODF)[, list(count=.N), by=c("Sample")])
+    
+    UNPROD = inputdata[inputdata$Functionality %in% c("unproductive (see comment)","unproductive"), ]
+  } else {
+    PRODF = inputdata[inputdata$VDJ.Frame != "In-frame with stop codon" & inputdata$VDJ.Frame != "Out-of-frame" & inputdata$CDR3.Found.How != "NOT_FOUND" , ]
+    UNPROD = inputdata[!(inputdata$VDJ.Frame != "In-frame with stop codon" & inputdata$VDJ.Frame != "Out-of-frame" & inputdata$CDR3.Found.How != "NOT_FOUND" ), ]
+  }
+}
+
+for(i in 1:nrow(UNPROD)){
+    if(!is.numeric(UNPROD[i,"CDR3.Length"])){
+        UNPROD[i,"CDR3.Length"] = 0
+    }
+}
+
+prod.sample.count = data.frame(data.table(PRODF)[, list(Productive=.N), by=c("Sample")])
+prod.rep.count = data.frame(data.table(PRODF)[, list(Productive=.N), by=c("Sample", "Replicate")])
+
+unprod.sample.count = data.frame(data.table(UNPROD)[, list(Unproductive=.N), by=c("Sample")])
+unprod.rep.count = data.frame(data.table(UNPROD)[, list(Unproductive=.N), by=c("Sample", "Replicate")])
+
+clonalityFrame = PRODF
+
+#remove duplicates based on the clonaltype
+if(clonaltype != "none"){
+  clonaltype = paste(clonaltype, ",Sample", sep="") #add sample column to clonaltype, unique within samples
+  PRODF$clonaltype = do.call(paste, c(PRODF[unlist(strsplit(clonaltype, ","))], sep = ":"))
+  PRODF = PRODF[!duplicated(PRODF$clonaltype), ]
+    
+  UNPROD$clonaltype = do.call(paste, c(UNPROD[unlist(strsplit(clonaltype, ","))], sep = ":"))
+  UNPROD = UNPROD[!duplicated(UNPROD$clonaltype), ]
+  
+  #again for clonalityFrame but with sample+replicate
+  clonalityFrame$clonaltype = do.call(paste, c(clonalityFrame[unlist(strsplit(clonaltype, ","))], sep = ":"))
+  clonalityFrame$clonality_clonaltype = do.call(paste, c(clonalityFrame[unlist(strsplit(paste(clonaltype, ",Replicate", sep=""), ","))], sep = ":"))
+  clonalityFrame = clonalityFrame[!duplicated(clonalityFrame$clonality_clonaltype), ]
+}
+
+print("SAMPLE TABLE:")
+print(table(PRODF$Sample))
+
+prod.unique.sample.count = data.frame(data.table(PRODF)[, list(Productive_unique=.N), by=c("Sample")])
+prod.unique.rep.count = data.frame(data.table(PRODF)[, list(Productive_unique=.N), by=c("Sample", "Replicate")])
+
+unprod.unique.sample.count = data.frame(data.table(UNPROD)[, list(Unproductive_unique=.N), by=c("Sample")])
+unprod.unique.rep.count = data.frame(data.table(UNPROD)[, list(Unproductive_unique=.N), by=c("Sample", "Replicate")])
+
+PRODF$freq = 1
+
+if(any(grepl(pattern="_", x=PRODF$ID))){ #the frequency can be stored in the ID with the pattern ".*_freq_.*"
+  PRODF$freq = gsub("^[0-9]+_", "", PRODF$ID)
+  PRODF$freq = gsub("_.*", "", PRODF$freq)
+  PRODF$freq = as.numeric(PRODF$freq)
+  if(any(is.na(PRODF$freq))){ #if there was an "_" in the ID, but not the frequency, go back to frequency of 1 for every sequence
+    PRODF$freq = 1
+  }
+}
+
+#make a names list with sample -> color
+naive.colors = c('blue4', 'darkred', 'olivedrab3', 'red', 'gray74', 'darkviolet', 'lightblue1', 'gold', 'chartreuse2', 'pink', 'Paleturquoise3', 'Chocolate1', 'Yellow', 'Deeppink3', 'Mediumorchid1', 'Darkgreen', 'Blue', 'Gray36', 'Hotpink', 'Yellow4')
+unique.samples = unique(PRODF$Sample)
+
+if(length(unique.samples) <= length(naive.colors)){
+	sample.colors = naive.colors[1:length(unique.samples)]
+} else {
+	sample.colors = rainbow(length(unique.samples))
+}
+
+names(sample.colors) = unique.samples
+
+print("Sample.colors")
+print(sample.colors)
+
+
+#write the complete dataset that is left over, will be the input if 'none' for clonaltype and 'no' for filterproductive
+write.table(PRODF, "allUnique.txt", sep="\t",quote=F,row.names=F,col.names=T)
+write.table(PRODF, "allUnique.csv", sep=",",quote=F,row.names=F,col.names=T)
+write.table(UNPROD, "allUnproductive.csv", sep=",",quote=F,row.names=F,col.names=T)
+
+#write the samples to a file
+sampleFile <- file("samples.txt")
+un = unique(inputdata$Sample)
+un = paste(un, sep="\n")
+writeLines(un, sampleFile)
+close(sampleFile)
+
+# ---------------------- Counting the productive/unproductive and unique sequences ----------------------
+
+print("Report Clonality - counting productive/unproductive/unique")
+
+#create the table on the overview page with the productive/unique counts per sample/replicate
+#first for sample
+sample.count = merge(input.sample.count, prod.sample.count, by="Sample", all.x=T)
+sample.count$perc_prod = round(sample.count$Productive / sample.count$All * 100)
+sample.count = merge(sample.count, prod.unique.sample.count, by="Sample", all.x=T)
+sample.count$perc_prod_un = round(sample.count$Productive_unique / sample.count$All * 100)
+
+sample.count = merge(sample.count , unprod.sample.count, by="Sample", all.x=T)
+sample.count$perc_unprod = round(sample.count$Unproductive / sample.count$All * 100)
+sample.count = merge(sample.count, unprod.unique.sample.count, by="Sample", all.x=T)
+sample.count$perc_unprod_un = round(sample.count$Unproductive_unique / sample.count$All * 100)
+
+#then sample/replicate
+rep.count = merge(input.rep.count, prod.rep.count, by=c("Sample", "Replicate"), all.x=T)
+rep.count$perc_prod = round(rep.count$Productive / rep.count$All * 100)
+rep.count = merge(rep.count, prod.unique.rep.count, by=c("Sample", "Replicate"), all.x=T)
+rep.count$perc_prod_un = round(rep.count$Productive_unique / rep.count$All * 100)
+
+rep.count = merge(rep.count, unprod.rep.count, by=c("Sample", "Replicate"), all.x=T)
+rep.count$perc_unprod = round(rep.count$Unproductive / rep.count$All * 100)
+rep.count = merge(rep.count, unprod.unique.rep.count, by=c("Sample", "Replicate"), all.x=T)
+rep.count$perc_unprod_un = round(rep.count$Unproductive_unique / rep.count$All * 100)
+
+rep.count$Sample = paste(rep.count$Sample, rep.count$Replicate, sep="_")
+rep.count = rep.count[,names(rep.count) != "Replicate"]
+
+count = rbind(sample.count, rep.count)
+
+
+
+write.table(x=count, file="productive_counting.txt", sep=",",quote=F,row.names=F,col.names=F)
+
+# ---------------------- V+J+CDR3 sequence count ----------------------
+
+VJCDR3.count = data.frame(table(clonalityFrame$Top.V.Gene, clonalityFrame$Top.J.Gene, clonalityFrame$CDR3.Seq.DNA))
+names(VJCDR3.count) = c("Top.V.Gene", "Top.J.Gene", "CDR3.Seq.DNA", "Count")
+
+VJCDR3.count = VJCDR3.count[VJCDR3.count$Count > 0,]
+VJCDR3.count = VJCDR3.count[order(-VJCDR3.count$Count),]
+
+write.table(x=VJCDR3.count, file="VJCDR3_count.txt", sep="\t",quote=F,row.names=F,col.names=T)
+
+# ---------------------- Frequency calculation for V, D and J ----------------------
+
+print("Report Clonality - frequency calculation V, D and J")
+
+PRODFV = data.frame(data.table(PRODF)[, list(Length=sum(freq)), by=c("Sample", "Top.V.Gene")])
+Total = ddply(PRODFV, .(Sample), function(x) data.frame(Total = sum(x$Length)))
+PRODFV = merge(PRODFV, Total, by.x='Sample', by.y='Sample', all.x=TRUE)
+PRODFV = ddply(PRODFV, c("Sample", "Top.V.Gene"), summarise, relFreq= (Length*100 / Total))
+
+PRODFD = data.frame(data.table(PRODF)[, list(Length=sum(freq)), by=c("Sample", "Top.D.Gene")])
+Total = ddply(PRODFD, .(Sample), function(x) data.frame(Total = sum(x$Length)))
+PRODFD = merge(PRODFD, Total, by.x='Sample', by.y='Sample', all.x=TRUE)
+PRODFD = ddply(PRODFD, c("Sample", "Top.D.Gene"), summarise, relFreq= (Length*100 / Total))
+
+PRODFJ = data.frame(data.table(PRODF)[, list(Length=sum(freq)), by=c("Sample", "Top.J.Gene")])
+Total = ddply(PRODFJ, .(Sample), function(x) data.frame(Total = sum(x$Length)))
+PRODFJ = merge(PRODFJ, Total, by.x='Sample', by.y='Sample', all.x=TRUE)
+PRODFJ = ddply(PRODFJ, c("Sample", "Top.J.Gene"), summarise, relFreq= (Length*100 / Total))
+
+# ---------------------- Setting up the gene names for the different species/loci ----------------------
+
+print("Report Clonality - getting genes for species/loci")
+
+Vchain = ""
+Dchain = ""
+Jchain = ""
+
+if(species == "custom"){
+	print("Custom genes: ")
+	splt = unlist(strsplit(locus, ";"))
+	print(paste("V:", splt[1]))
+	print(paste("D:", splt[2]))
+	print(paste("J:", splt[3]))
+	
+	Vchain = unlist(strsplit(splt[1], ","))
+	Vchain = data.frame(v.name = Vchain, chr.orderV = 1:length(Vchain))
+	
+	Dchain = unlist(strsplit(splt[2], ","))
+	if(length(Dchain) > 0){
+		Dchain = data.frame(v.name = Dchain, chr.orderD = 1:length(Dchain))
+	} else {
+		Dchain = data.frame(v.name = character(0), chr.orderD = numeric(0))
+	}
+	
+	Jchain = unlist(strsplit(splt[3], ","))
+	Jchain = data.frame(v.name = Jchain, chr.orderJ = 1:length(Jchain))
+
+} else {
+	genes = read.table("genes.txt", sep="\t", header=TRUE, fill=T, comment.char="")
+
+	Vchain = genes[grepl(species, genes$Species) & genes$locus == locus & genes$region == "V",c("IMGT.GENE.DB", "chr.order")]
+	colnames(Vchain) = c("v.name", "chr.orderV")
+	Dchain = genes[grepl(species, genes$Species) & genes$locus == locus & genes$region == "D",c("IMGT.GENE.DB", "chr.order")]
+	colnames(Dchain) = c("v.name", "chr.orderD")
+	Jchain = genes[grepl(species, genes$Species) & genes$locus == locus & genes$region == "J",c("IMGT.GENE.DB", "chr.order")]
+	colnames(Jchain) = c("v.name", "chr.orderJ")
+}
+useD = TRUE
+if(nrow(Dchain) == 0){
+  useD = FALSE
+  cat("No D Genes in this species/locus")
+}
+print(paste(nrow(Vchain), "genes in V"))
+print(paste(nrow(Dchain), "genes in D"))
+print(paste(nrow(Jchain), "genes in J"))
+
+# ---------------------- merge with the frequency count ----------------------
+
+PRODFV = merge(PRODFV, Vchain, by.x='Top.V.Gene', by.y='v.name', all.x=TRUE)
+
+PRODFD = merge(PRODFD, Dchain, by.x='Top.D.Gene', by.y='v.name', all.x=TRUE)
+
+PRODFJ = merge(PRODFJ, Jchain, by.x='Top.J.Gene', by.y='v.name', all.x=TRUE)
+
+# ---------------------- Create the V, D and J frequency plots and write the data.frame for every plot to a file ----------------------
+
+print("Report Clonality - V, D and J frequency plots")
+
+pV = ggplot(PRODFV)
+pV = pV + geom_bar( aes( x=factor(reorder(Top.V.Gene, chr.orderV)), y=relFreq, fill=Sample), stat='identity', position="dodge") + theme(axis.text.x = element_text(angle = 90, hjust = 1))
+pV = pV + xlab("Summary of V gene") + ylab("Frequency") + ggtitle("Relative frequency of V gene usage") + scale_fill_manual(values=sample.colors)
+pV = pV + theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), axis.text.x = element_text(angle = 45, hjust = 1), panel.grid.major.y = element_line(colour = "black"), panel.grid.major.x = element_blank())
+write.table(x=PRODFV, file="VFrequency.csv", sep=",",quote=F,row.names=F,col.names=T)
+
+png("VPlot.png",width = 1280, height = 720)
+pV
+dev.off();
+
+if(useD){
+  pD = ggplot(PRODFD)
+  pD = pD + geom_bar( aes( x=factor(reorder(Top.D.Gene, chr.orderD)), y=relFreq, fill=Sample), stat='identity', position="dodge") + theme(axis.text.x = element_text(angle = 90, hjust = 1))
+  pD = pD + xlab("Summary of D gene") + ylab("Frequency") + ggtitle("Relative frequency of D gene usage") + scale_fill_manual(values=sample.colors)
+  pD = pD + theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), axis.text.x = element_text(angle = 45, hjust = 1), panel.grid.major.y = element_line(colour = "black"), panel.grid.major.x = element_blank())
+  write.table(x=PRODFD, file="DFrequency.csv", sep=",",quote=F,row.names=F,col.names=T)
+  
+  png("DPlot.png",width = 800, height = 600)
+  print(pD)
+  dev.off();
+}
+
+pJ = ggplot(PRODFJ)
+pJ = pJ + geom_bar( aes( x=factor(reorder(Top.J.Gene, chr.orderJ)), y=relFreq, fill=Sample), stat='identity', position="dodge") + theme(axis.text.x = element_text(angle = 90, hjust = 1))
+pJ = pJ + xlab("Summary of J gene") + ylab("Frequency") + ggtitle("Relative frequency of J gene usage") + scale_fill_manual(values=sample.colors)
+pJ = pJ + theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), axis.text.x = element_text(angle = 45, hjust = 1), panel.grid.major.y = element_line(colour = "black"), panel.grid.major.x = element_blank())
+write.table(x=PRODFJ, file="JFrequency.csv", sep=",",quote=F,row.names=F,col.names=T)
+
+png("JPlot.png",width = 800, height = 600)
+pJ
+dev.off();
+
+# ---------------------- Now the frequency plots of the V, D and J families ----------------------
+
+print("Report Clonality - V, D and J family plots")
+
+VGenes = PRODF[,c("Sample", "Top.V.Gene")]
+VGenes$Top.V.Gene = gsub("-.*", "", VGenes$Top.V.Gene)
+VGenes = data.frame(data.table(VGenes)[, list(Count=.N), by=c("Sample", "Top.V.Gene")])
+TotalPerSample = data.frame(data.table(VGenes)[, list(total=sum(.SD$Count)), by=Sample])
+VGenes = merge(VGenes, TotalPerSample, by="Sample")
+VGenes$Frequency = VGenes$Count * 100 / VGenes$total
+VPlot = ggplot(VGenes)
+VPlot = VPlot + geom_bar(aes( x = Top.V.Gene, y = Frequency, fill = Sample), stat='identity', position='dodge' ) + theme(axis.text.x = element_text(angle = 90, hjust = 1)) + 
+  ggtitle("Distribution of V gene families") + 
+  ylab("Percentage of sequences") +
+  scale_fill_manual(values=sample.colors) +
+  theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), axis.text.x = element_text(angle = 45, hjust = 1), panel.grid.major.y = element_line(colour = "black"), panel.grid.major.x = element_blank())
+png("VFPlot.png")
+VPlot
+dev.off();
+write.table(x=VGenes, file="VFFrequency.csv", sep=",",quote=F,row.names=F,col.names=T)
+
+if(useD){
+  DGenes = PRODF[,c("Sample", "Top.D.Gene")]
+  DGenes$Top.D.Gene = gsub("-.*", "", DGenes$Top.D.Gene)
+  DGenes = data.frame(data.table(DGenes)[, list(Count=.N), by=c("Sample", "Top.D.Gene")])
+  TotalPerSample = data.frame(data.table(DGenes)[, list(total=sum(.SD$Count)), by=Sample])
+  DGenes = merge(DGenes, TotalPerSample, by="Sample")
+  DGenes$Frequency = DGenes$Count * 100 / DGenes$total
+  DPlot = ggplot(DGenes)
+  DPlot = DPlot + geom_bar(aes( x = Top.D.Gene, y = Frequency, fill = Sample), stat='identity', position='dodge' ) + theme(axis.text.x = element_text(angle = 90, hjust = 1)) + 
+    ggtitle("Distribution of D gene families") + 
+    ylab("Percentage of sequences") + 
+    scale_fill_manual(values=sample.colors) +
+    theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), axis.text.x = element_text(angle = 45, hjust = 1), panel.grid.major.y = element_line(colour = "black"), panel.grid.major.x = element_blank())
+  png("DFPlot.png")
+  print(DPlot)
+  dev.off();
+  write.table(x=DGenes, file="DFFrequency.csv", sep=",",quote=F,row.names=F,col.names=T)
+}
+
+# ---------------------- Plotting the cdr3 length ----------------------
+
+print("Report Clonality - CDR3 length plot")
+
+CDR3Length = data.frame(data.table(PRODF)[, list(Count=.N), by=c("Sample", "CDR3.Length")])
+TotalPerSample = data.frame(data.table(CDR3Length)[, list(total=sum(.SD$Count)), by=Sample])
+CDR3Length = merge(CDR3Length, TotalPerSample, by="Sample")
+CDR3Length$Frequency = CDR3Length$Count * 100 / CDR3Length$total
+CDR3LengthPlot = ggplot(CDR3Length)
+CDR3LengthPlot = CDR3LengthPlot + geom_bar(aes( x = factor(reorder(CDR3.Length, as.numeric(CDR3.Length))), y = Frequency, fill = Sample), stat='identity', position='dodge' ) + theme(axis.text.x = element_text(angle = 90, hjust = 1)) + 
+  ggtitle("Length distribution of CDR3") + 
+  xlab("CDR3 Length") + 
+  ylab("Percentage of sequences") +
+  scale_fill_manual(values=sample.colors) +
+  theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), axis.text.x = element_text(angle = 45, hjust = 1), panel.grid.major.y = element_line(colour = "black"), panel.grid.major.x = element_blank())
+png("CDR3LengthPlot.png",width = 1280, height = 720)
+CDR3LengthPlot
+dev.off()
+write.table(x=CDR3Length, file="CDR3LengthPlot.csv", sep=",",quote=F,row.names=F,col.names=T)
+
+# ---------------------- Plot the heatmaps ----------------------
+
+#get the reverse order for the V and D genes
+revVchain = Vchain
+revDchain = Dchain
+revVchain$chr.orderV = rev(revVchain$chr.orderV)
+revDchain$chr.orderD = rev(revDchain$chr.orderD)
+
+if(useD){
+  print("Report Clonality - Heatmaps VD")
+  plotVD <- function(dat){
+    if(length(dat[,1]) == 0){
+      return()
+    }
+    
+    img = ggplot() + 
+      geom_tile(data=dat, aes(x=factor(reorder(Top.D.Gene, chr.orderD)), y=factor(reorder(Top.V.Gene, chr.orderV)), fill=relLength)) + 
+      theme(axis.text.x = element_text(angle = 90, hjust = 1)) + 
+      scale_fill_gradient(low="gold", high="blue", na.value="white") + 
+      ggtitle(paste(unique(dat$Sample), " (N=" , sum(dat$Length, na.rm=T) ,")", sep="")) + 
+      xlab("D genes") + 
+      ylab("V Genes") +
+      theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), panel.grid.major = element_line(colour = "gainsboro"))
+    
+    png(paste("HeatmapVD_", unique(dat[3])[1,1] , ".png", sep=""), width=150+(15*length(Dchain$v.name)), height=100+(15*length(Vchain$v.name)))
+    print(img)
+    dev.off()
+    write.table(x=acast(dat, Top.V.Gene~Top.D.Gene, value.var="Length"), file=paste("HeatmapVD_", unique(dat[3])[1,1], ".csv", sep=""), sep=",",quote=F,row.names=T,col.names=NA)
+  }
+  
+  VandDCount = data.frame(data.table(PRODF)[, list(Length=.N), by=c("Top.V.Gene", "Top.D.Gene", "Sample")])
+  
+  VandDCount$l = log(VandDCount$Length)
+  maxVD = data.frame(data.table(VandDCount)[, list(max=max(l)), by=c("Sample")])
+  VandDCount = merge(VandDCount, maxVD, by.x="Sample", by.y="Sample", all.x=T)
+  VandDCount$relLength = VandDCount$l / VandDCount$max
+  check = is.nan(VandDCount$relLength)
+  if(any(check)){
+	VandDCount[check,"relLength"] = 0
+  }
+  
+  cartegianProductVD = expand.grid(Top.V.Gene = Vchain$v.name, Top.D.Gene = Dchain$v.name)
+  
+  completeVD = merge(VandDCount, cartegianProductVD, by.x=c("Top.V.Gene", "Top.D.Gene"), by.y=c("Top.V.Gene", "Top.D.Gene"), all=TRUE)
+ 
+  completeVD = merge(completeVD, revVchain, by.x="Top.V.Gene", by.y="v.name", all.x=TRUE)
+ 
+  completeVD = merge(completeVD, Dchain, by.x="Top.D.Gene", by.y="v.name", all.x=TRUE)
+  
+  fltr = is.nan(completeVD$relLength)
+  if(all(fltr)){
+	  completeVD[fltr,"relLength"] = 0
+  }
+  
+  VDList = split(completeVD, f=completeVD[,"Sample"])
+  lapply(VDList, FUN=plotVD)
+}
+
+print("Report Clonality - Heatmaps VJ")
+
+plotVJ <- function(dat){
+  if(length(dat[,1]) == 0){
+    return()
+  }
+  cat(paste(unique(dat[3])[1,1]))
+  img = ggplot() + 
+    geom_tile(data=dat, aes(x=factor(reorder(Top.J.Gene, chr.orderJ)), y=factor(reorder(Top.V.Gene, chr.orderV)), fill=relLength)) + 
+    theme(axis.text.x = element_text(angle = 90, hjust = 1)) + 
+    scale_fill_gradient(low="gold", high="blue", na.value="white") + 
+    ggtitle(paste(unique(dat$Sample), " (N=" , sum(dat$Length, na.rm=T) ,")", sep="")) + 
+    xlab("J genes") + 
+    ylab("V Genes") +
+    theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), panel.grid.major = element_line(colour = "gainsboro"))
+  
+  png(paste("HeatmapVJ_", unique(dat[3])[1,1] , ".png", sep=""), width=150+(15*length(Jchain$v.name)), height=100+(15*length(Vchain$v.name)))
+  print(img)
+  dev.off()
+  write.table(x=acast(dat, Top.V.Gene~Top.J.Gene, value.var="Length"), file=paste("HeatmapVJ_", unique(dat[3])[1,1], ".csv", sep=""), sep=",",quote=F,row.names=T,col.names=NA)
+}
+
+VandJCount = data.frame(data.table(PRODF)[, list(Length=.N), by=c("Top.V.Gene", "Top.J.Gene", "Sample")])
+
+VandJCount$l = log(VandJCount$Length)
+maxVJ = data.frame(data.table(VandJCount)[, list(max=max(l)), by=c("Sample")])
+VandJCount = merge(VandJCount, maxVJ, by.x="Sample", by.y="Sample", all.x=T)
+VandJCount$relLength = VandJCount$l / VandJCount$max
+
+check = is.nan(VandJCount$relLength)
+if(any(check)){
+	VandJCount[check,"relLength"] = 0
+}
+
+cartegianProductVJ = expand.grid(Top.V.Gene = Vchain$v.name, Top.J.Gene = Jchain$v.name)
+
+completeVJ = merge(VandJCount, cartegianProductVJ, all.y=TRUE)
+completeVJ = merge(completeVJ, revVchain, by.x="Top.V.Gene", by.y="v.name", all.x=TRUE)
+completeVJ = merge(completeVJ, Jchain, by.x="Top.J.Gene", by.y="v.name", all.x=TRUE)
+
+fltr = is.nan(completeVJ$relLength)
+if(any(fltr)){
+	completeVJ[fltr,"relLength"] = 1
+}
+
+VJList = split(completeVJ, f=completeVJ[,"Sample"])
+lapply(VJList, FUN=plotVJ)
+
+
+
+if(useD){
+  print("Report Clonality - Heatmaps DJ")	
+  plotDJ <- function(dat){
+    if(length(dat[,1]) == 0){
+      return()
+    }
+    img = ggplot() + 
+      geom_tile(data=dat, aes(x=factor(reorder(Top.J.Gene, chr.orderJ)), y=factor(reorder(Top.D.Gene, chr.orderD)), fill=relLength)) + 
+      theme(axis.text.x = element_text(angle = 90, hjust = 1)) + 
+      scale_fill_gradient(low="gold", high="blue", na.value="white") + 
+      ggtitle(paste(unique(dat$Sample), " (N=" , sum(dat$Length, na.rm=T) ,")", sep="")) + 
+      xlab("J genes") + 
+      ylab("D Genes") +
+      theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), panel.grid.major = element_line(colour = "gainsboro"))
+    
+    png(paste("HeatmapDJ_", unique(dat[3])[1,1] , ".png", sep=""), width=150+(15*length(Jchain$v.name)), height=100+(15*length(Dchain$v.name)))
+    print(img)
+    dev.off()
+    write.table(x=acast(dat, Top.D.Gene~Top.J.Gene, value.var="Length"), file=paste("HeatmapDJ_", unique(dat[3])[1,1], ".csv", sep=""), sep=",",quote=F,row.names=T,col.names=NA)
+  }
+  
+  
+  DandJCount = data.frame(data.table(PRODF)[, list(Length=.N), by=c("Top.D.Gene", "Top.J.Gene", "Sample")])
+  
+  DandJCount$l = log(DandJCount$Length)
+  maxDJ = data.frame(data.table(DandJCount)[, list(max=max(l)), by=c("Sample")])
+  DandJCount = merge(DandJCount, maxDJ, by.x="Sample", by.y="Sample", all.x=T)
+  DandJCount$relLength = DandJCount$l / DandJCount$max
+  
+  check = is.nan(DandJCount$relLength)
+  if(any(check)){
+    DandJCount[check,"relLength"] = 0
+  }
+  
+  cartegianProductDJ = expand.grid(Top.D.Gene = Dchain$v.name, Top.J.Gene = Jchain$v.name)
+  
+  completeDJ = merge(DandJCount, cartegianProductDJ, all.y=TRUE)
+  completeDJ = merge(completeDJ, revDchain, by.x="Top.D.Gene", by.y="v.name", all.x=TRUE)
+  completeDJ = merge(completeDJ, Jchain, by.x="Top.J.Gene", by.y="v.name", all.x=TRUE)
+  
+  fltr = is.nan(completeDJ$relLength)
+  if(any(fltr)){
+	  completeDJ[fltr, "relLength"] = 1
+  }
+  
+  DJList = split(completeDJ, f=completeDJ[,"Sample"])
+  lapply(DJList, FUN=plotDJ)
+}
+
+
+# ---------------------- output tables for the circos plots ----------------------
+
+print("Report Clonality - Circos data")
+
+for(smpl in unique(PRODF$Sample)){
+	PRODF.sample = PRODF[PRODF$Sample == smpl,]
+	
+	fltr = PRODF.sample$Top.V.Gene == ""
+	if(any(fltr, na.rm=T)){
+	  PRODF.sample[fltr, "Top.V.Gene"] = "NA"
+	}
+	
+	fltr = PRODF.sample$Top.D.Gene == ""
+	if(any(fltr, na.rm=T)){
+	  PRODF.sample[fltr, "Top.D.Gene"] = "NA"
+	}
+
+	fltr = PRODF.sample$Top.J.Gene == ""
+	if(any(fltr, na.rm=T)){
+	  PRODF.sample[fltr, "Top.J.Gene"] = "NA"
+	}
+	
+	v.d = table(PRODF.sample$Top.V.Gene, PRODF.sample$Top.D.Gene)
+	v.j = table(PRODF.sample$Top.V.Gene, PRODF.sample$Top.J.Gene)
+	d.j = table(PRODF.sample$Top.D.Gene, PRODF.sample$Top.J.Gene)
+
+	write.table(v.d, file=paste(smpl, "_VD_circos.txt", sep=""), sep="\t", quote=F, row.names=T, col.names=NA)
+	write.table(v.j, file=paste(smpl, "_VJ_circos.txt", sep=""), sep="\t", quote=F, row.names=T, col.names=NA)
+	write.table(d.j, file=paste(smpl, "_DJ_circos.txt", sep=""), sep="\t", quote=F, row.names=T, col.names=NA)
+}
+
+# ---------------------- calculating the clonality score ----------------------
+
+if("Replicate" %in% colnames(inputdata)) #can only calculate clonality score when replicate information is available
+{
+  print("Report Clonality - Clonality")
+  write.table(clonalityFrame, "clonalityComplete.csv", sep=",",quote=F,row.names=F,col.names=T)
+  if(clonality_method == "boyd"){
+    samples = split(clonalityFrame, clonalityFrame$Sample, drop=T)
+   
+    for (sample in samples){
+      res = data.frame(paste=character(0))
+      sample_id = unique(sample$Sample)[[1]]
+      for(replicate in unique(sample$Replicate)){
+        tmp = sample[sample$Replicate == replicate,]
+        clone_table = data.frame(table(tmp$clonaltype))
+        clone_col_name = paste("V", replicate, sep="")
+        colnames(clone_table) = c("paste", clone_col_name)
+        res = merge(res, clone_table, by="paste", all=T)
+      }
+      
+      res[is.na(res)] = 0      
+      infer.result = infer.clonality(as.matrix(res[,2:ncol(res)]))
+      
+      #print(infer.result)
+      
+      write.table(data.table(infer.result[[12]]), file=paste("lymphclon_clonality_", sample_id, ".csv", sep=""), sep=",",quote=F,row.names=F,col.names=F)
+      
+      res$type = rowSums(res[,2:ncol(res)])
+      
+      coincidence.table = data.frame(table(res$type))
+      colnames(coincidence.table) = c("Coincidence Type",  "Raw Coincidence Freq")
+      write.table(coincidence.table, file=paste("lymphclon_coincidences_", sample_id, ".csv", sep=""), sep=",",quote=F,row.names=F,col.names=T)
+    }
+  } else {
+    clonalFreq = data.frame(data.table(clonalityFrame)[, list(Type=.N), by=c("Sample", "clonaltype")])
+    
+    #write files for every coincidence group of >1
+    samples = unique(clonalFreq$Sample)
+    for(sample in samples){
+		clonalFreqSample = clonalFreq[clonalFreq$Sample == sample,]
+		if(max(clonalFreqSample$Type) > 1){
+			for(i in 2:max(clonalFreqSample$Type)){
+				clonalFreqSampleType = clonalFreqSample[clonalFreqSample$Type == i,]
+				clonalityFrame.sub = clonalityFrame[clonalityFrame$clonaltype %in% clonalFreqSampleType$clonaltype,]
+				clonalityFrame.sub = clonalityFrame.sub[order(clonalityFrame.sub$clonaltype),]
+				write.table(clonalityFrame.sub, file=paste("coincidences_", sample, "_", i, ".txt", sep=""), sep="\t",quote=F,row.names=F,col.names=T)
+			}
+		}
+	}
+	
+    clonalFreqCount = data.frame(data.table(clonalFreq)[, list(Count=.N), by=c("Sample", "Type")])
+    clonalFreqCount$realCount = clonalFreqCount$Type * clonalFreqCount$Count
+    clonalSum = data.frame(data.table(clonalFreqCount)[, list(Reads=sum(realCount)), by=c("Sample")])
+    clonalFreqCount = merge(clonalFreqCount, clonalSum, by.x="Sample", by.y="Sample")
+    
+    ct = c('Type\tWeight\n2\t1\n3\t3\n4\t6\n5\t10\n6\t15')
+    tcct = textConnection(ct)
+    CT  = read.table(tcct, sep="\t", header=TRUE)
+    close(tcct)
+    clonalFreqCount = merge(clonalFreqCount, CT, by.x="Type", by.y="Type", all.x=T)
+    clonalFreqCount$WeightedCount = clonalFreqCount$Count * clonalFreqCount$Weight
+    
+    ReplicateReads = data.frame(data.table(clonalityFrame)[, list(Type=.N), by=c("Sample", "Replicate", "clonaltype")])
+    ReplicateReads = data.frame(data.table(ReplicateReads)[, list(Reads=.N), by=c("Sample", "Replicate")])
+    clonalFreqCount$Reads = as.numeric(clonalFreqCount$Reads)
+    ReplicateReads$Reads = as.numeric(ReplicateReads$Reads)
+    ReplicateReads$squared = as.numeric(ReplicateReads$Reads * ReplicateReads$Reads)
+    
+    ReplicatePrint <- function(dat){
+      write.table(dat[-1], paste("ReplicateReads_", unique(dat[1])[1,1] , ".csv", sep=""), sep=",",quote=F,na="-",row.names=F,col.names=F)
+    }
+    
+    ReplicateSplit = split(ReplicateReads, f=ReplicateReads[,"Sample"])
+    lapply(ReplicateSplit, FUN=ReplicatePrint)
+    
+    ReplicateReads = data.frame(data.table(ReplicateReads)[, list(ReadsSum=sum(as.numeric(Reads)), ReadsSquaredSum=sum(as.numeric(squared))), by=c("Sample")])
+    clonalFreqCount = merge(clonalFreqCount, ReplicateReads, by.x="Sample", by.y="Sample", all.x=T)
+    
+    ReplicateSumPrint <- function(dat){
+      write.table(dat[-1], paste("ReplicateSumReads_", unique(dat[1])[1,1] , ".csv", sep=""), sep=",",quote=F,na="-",row.names=F,col.names=F)
+    }
+    
+    ReplicateSumSplit = split(ReplicateReads, f=ReplicateReads[,"Sample"])
+    lapply(ReplicateSumSplit, FUN=ReplicateSumPrint)
+    
+    clonalFreqCountSum = data.frame(data.table(clonalFreqCount)[, list(Numerator=sum(WeightedCount, na.rm=T)), by=c("Sample")])
+    clonalFreqCount = merge(clonalFreqCount, clonalFreqCountSum, by.x="Sample", by.y="Sample", all.x=T)
+    clonalFreqCount$ReadsSum = as.numeric(clonalFreqCount$ReadsSum) #prevent integer overflow
+    clonalFreqCount$Denominator = (((clonalFreqCount$ReadsSum * clonalFreqCount$ReadsSum) - clonalFreqCount$ReadsSquaredSum) / 2)
+    clonalFreqCount$Result = (clonalFreqCount$Numerator + 1) / (clonalFreqCount$Denominator + 1)
+    
+    ClonalityScorePrint <- function(dat){
+      write.table(dat$Result, paste("ClonalityScore_", unique(dat[1])[1,1] , ".csv", sep=""), sep=",",quote=F,na="-",row.names=F,col.names=F)
+    }
+    
+    clonalityScore = clonalFreqCount[c("Sample", "Result")]
+    clonalityScore = unique(clonalityScore)
+    
+    clonalityScoreSplit = split(clonalityScore, f=clonalityScore[,"Sample"])
+    lapply(clonalityScoreSplit, FUN=ClonalityScorePrint)
+    
+    clonalityOverview = clonalFreqCount[c("Sample", "Type", "Count", "Weight", "WeightedCount")]
+    
+    
+    
+    ClonalityOverviewPrint <- function(dat){
+	  dat = dat[order(dat[,2]),]
+      write.table(dat[-1], paste("ClonalityOverView_", unique(dat[1])[1,1] , ".csv", sep=""), sep=",",quote=F,na="-",row.names=F,col.names=F)
+    }
+    
+    clonalityOverviewSplit = split(clonalityOverview, f=clonalityOverview$Sample)
+    lapply(clonalityOverviewSplit, FUN=ClonalityOverviewPrint)
+  }
+}
+
+bak = PRODF
+
+imgtcolumns = c("X3V.REGION.trimmed.nt.nb","P3V.nt.nb", "N1.REGION.nt.nb", "P5D.nt.nb", "X5D.REGION.trimmed.nt.nb", "X3D.REGION.trimmed.nt.nb", "P3D.nt.nb", "N2.REGION.nt.nb", "P5J.nt.nb", "X5J.REGION.trimmed.nt.nb", "X3V.REGION.trimmed.nt.nb", "X5D.REGION.trimmed.nt.nb", "X3D.REGION.trimmed.nt.nb", "X5J.REGION.trimmed.nt.nb", "N1.REGION.nt.nb", "N2.REGION.nt.nb", "P3V.nt.nb", "P5D.nt.nb", "P3D.nt.nb", "P5J.nt.nb")
+if(all(imgtcolumns %in% colnames(inputdata)))
+{
+  print("found IMGT columns, running junction analysis")
+  
+  if(locus %in% c("IGK","IGL", "TRA", "TRG")){
+	  print("VJ recombination, no filtering on absent D")
+  } else {
+	  print("VDJ recombination, using N column for junction analysis")
+	  fltr = nchar(PRODF$Top.D.Gene) < 4
+	  print(paste("Removing", sum(fltr), "sequences without a identified D"))
+	  PRODF = PRODF[!fltr,]
+  }
+  
+  
+  #ensure certain columns are in the data (files generated with older versions of IMGT Loader)
+  col.checks = c("N.REGION.nt.nb", "N1.REGION.nt.nb", "N2.REGION.nt.nb", "N3.REGION.nt.nb", "N4.REGION.nt.nb")
+  for(col.check in col.checks){
+	  if(!(col.check %in% names(PRODF))){
+		  print(paste(col.check, "not found adding new column"))
+		  if(nrow(PRODF) > 0){ #because R is anoying...
+			PRODF[,col.check] = 0
+		  } else {
+			PRODF = cbind(PRODF, data.frame(N3.REGION.nt.nb=numeric(0), N4.REGION.nt.nb=numeric(0)))
+		  }
+		  if(nrow(UNPROD) > 0){
+			UNPROD[,col.check] = 0
+		  } else {
+			UNPROD = cbind(UNPROD, data.frame(N3.REGION.nt.nb=numeric(0), N4.REGION.nt.nb=numeric(0)))
+		  }
+	  }
+  }
+  
+  num_median = function(x, na.rm=T) { as.numeric(median(x, na.rm=na.rm)) }
+  
+  newData = data.frame(data.table(PRODF)[,list(unique=.N, 
+                                               VH.DEL=mean(.SD$X3V.REGION.trimmed.nt.nb, na.rm=T),
+                                               P1=mean(.SD$P3V.nt.nb, na.rm=T),
+                                               N1=mean(rowSums(.SD[,c("N.REGION.nt.nb", "N1.REGION.nt.nb"), with=F], na.rm=T)),
+                                               P2=mean(.SD$P5D.nt.nb, na.rm=T),
+                                               DEL.DH=mean(.SD$X5D.REGION.trimmed.nt.nb, na.rm=T),
+                                               DH.DEL=mean(.SD$X3D.REGION.trimmed.nt.nb, na.rm=T),
+                                               P3=mean(.SD$P3D.nt.nb, na.rm=T),
+                                               N2=mean(rowSums(.SD[,c("N2.REGION.nt.nb", "N3.REGION.nt.nb", "N4.REGION.nt.nb"), with=F], na.rm=T)),
+                                               P4=mean(.SD$P5J.nt.nb, na.rm=T),
+                                               DEL.JH=mean(.SD$X5J.REGION.trimmed.nt.nb, na.rm=T),
+                                               Total.Del=mean(rowSums(.SD[,c("X3V.REGION.trimmed.nt.nb", "X5D.REGION.trimmed.nt.nb", "X3D.REGION.trimmed.nt.nb", "X5J.REGION.trimmed.nt.nb"), with=F], na.rm=T)),
+                                               Total.N=mean(rowSums(.SD[,c("N.REGION.nt.nb", "N1.REGION.nt.nb", "N2.REGION.nt.nb", "N3.REGION.nt.nb", "N4.REGION.nt.nb"), with=F], na.rm=T)),
+                                               Total.P=mean(rowSums(.SD[,c("P3V.nt.nb", "P5D.nt.nb", "P3D.nt.nb", "P5J.nt.nb"), with=F], na.rm=T)),
+                                               Median.CDR3.l=as.double(median(.SD$CDR3.Length))),
+                                         by=c("Sample")])
+  newData[,sapply(newData, is.numeric)] = round(newData[,sapply(newData, is.numeric)],1)
+  write.table(newData, "junctionAnalysisProd_mean.csv" , sep=",",quote=F,na="-",row.names=F,col.names=F)
+  
+  newData = data.frame(data.table(PRODF)[,list(unique=.N, 
+                                               VH.DEL=num_median(.SD$X3V.REGION.trimmed.nt.nb, na.rm=T),
+                                               P1=num_median(.SD$P3V.nt.nb, na.rm=T),
+                                               N1=num_median(rowSums(.SD[,c("N.REGION.nt.nb", "N1.REGION.nt.nb"), with=F], na.rm=T)),
+                                               P2=num_median(.SD$P5D.nt.nb, na.rm=T),
+                                               DEL.DH=num_median(.SD$X5D.REGION.trimmed.nt.nb, na.rm=T),
+                                               DH.DEL=num_median(.SD$X3D.REGION.trimmed.nt.nb, na.rm=T),
+                                               P3=num_median(.SD$P3D.nt.nb, na.rm=T),
+                                               N2=num_median(rowSums(.SD[,c("N2.REGION.nt.nb", "N3.REGION.nt.nb", "N4.REGION.nt.nb"), with=F], na.rm=T)),
+                                               P4=num_median(.SD$P5J.nt.nb, na.rm=T),
+                                               DEL.JH=num_median(.SD$X5J.REGION.trimmed.nt.nb, na.rm=T),
+											   Total.Del=num_median(rowSums(.SD[,c("X3V.REGION.trimmed.nt.nb", "X5D.REGION.trimmed.nt.nb", "X3D.REGION.trimmed.nt.nb", "X5J.REGION.trimmed.nt.nb"), with=F], na.rm=T)),
+											   Total.N=num_median(rowSums(.SD[,c("N.REGION.nt.nb", "N1.REGION.nt.nb", "N2.REGION.nt.nb", "N3.REGION.nt.nb", "N4.REGION.nt.nb"), with=F], na.rm=T)),
+											   Total.P=num_median(rowSums(.SD[,c("P3V.nt.nb", "P5D.nt.nb", "P3D.nt.nb", "P5J.nt.nb"), with=F], na.rm=T)),
+											   Median.CDR3.l=as.double(median(.SD$CDR3.Length))),
+                                         by=c("Sample")])
+  newData[,sapply(newData, is.numeric)] = round(newData[,sapply(newData, is.numeric)],1)
+  write.table(newData, "junctionAnalysisProd_median.csv" , sep=",",quote=F,na="-",row.names=F,col.names=F)
+  
+  newData = data.frame(data.table(UNPROD)[,list(unique=.N, 
+                                                VH.DEL=mean(.SD$X3V.REGION.trimmed.nt.nb, na.rm=T),
+                                                P1=mean(.SD$P3V.nt.nb, na.rm=T),
+                                                N1=mean(rowSums(.SD[,c("N.REGION.nt.nb", "N1.REGION.nt.nb"), with=F], na.rm=T)),
+                                                P2=mean(.SD$P5D.nt.nb, na.rm=T),
+                                                DEL.DH=mean(.SD$X5D.REGION.trimmed.nt.nb, na.rm=T),
+                                                DH.DEL=mean(.SD$X3D.REGION.trimmed.nt.nb, na.rm=T),
+                                                P3=mean(.SD$P3D.nt.nb, na.rm=T),
+                                                N2=mean(rowSums(.SD[,c("N2.REGION.nt.nb", "N3.REGION.nt.nb", "N4.REGION.nt.nb"), with=F], na.rm=T)),
+                                                P4=mean(.SD$P5J.nt.nb, na.rm=T),
+                                                DEL.JH=mean(.SD$X5J.REGION.trimmed.nt.nb, na.rm=T),
+                                                Total.Del=mean(rowSums(.SD[,c("X3V.REGION.trimmed.nt.nb", "X5D.REGION.trimmed.nt.nb", "X3D.REGION.trimmed.nt.nb", "X5J.REGION.trimmed.nt.nb"), with=F], na.rm=T)),
+                                                Total.N=mean(rowSums(.SD[,c("N.REGION.nt.nb", "N1.REGION.nt.nb", "N2.REGION.nt.nb", "N3.REGION.nt.nb", "N4.REGION.nt.nb"), with=F], na.rm=T)),
+                                                Total.P=mean(rowSums(.SD[,c("P3V.nt.nb", "P5D.nt.nb", "P3D.nt.nb", "P5J.nt.nb"), with=F], na.rm=T)),
+                                                Median.CDR3.l=as.double(median(.SD$CDR3.Length))),
+                                          by=c("Sample")])
+  newData[,sapply(newData, is.numeric)] = round(newData[,sapply(newData, is.numeric)],1)
+  write.table(newData, "junctionAnalysisUnProd_mean.csv" , sep=",",quote=F,na="-",row.names=F,col.names=F)
+  
+    newData = data.frame(data.table(UNPROD)[,list(unique=.N, 
+                                                VH.DEL=num_median(.SD$X3V.REGION.trimmed.nt.nb, na.rm=T),
+                                                P1=num_median(.SD$P3V.nt.nb, na.rm=T),
+                                                N1=num_median(rowSums(.SD[,c("N.REGION.nt.nb", "N1.REGION.nt.nb"), with=F], na.rm=T)),
+                                                P2=num_median(.SD$P5D.nt.nb, na.rm=T),
+                                                DEL.DH=num_median(.SD$X5D.REGION.trimmed.nt.nb, na.rm=T),
+                                                DH.DEL=num_median(.SD$X3D.REGION.trimmed.nt.nb, na.rm=T),
+                                                P3=num_median(.SD$P3D.nt.nb, na.rm=T),
+                                                N2=num_median(rowSums(.SD[,c("N2.REGION.nt.nb", "N3.REGION.nt.nb", "N4.REGION.nt.nb"), with=F], na.rm=T)),
+                                                P4=num_median(.SD$P5J.nt.nb, na.rm=T),
+                                                DEL.JH=num_median(.SD$X5J.REGION.trimmed.nt.nb, na.rm=T),
+                                                Total.Del=num_median(rowSums(.SD[,c("X3V.REGION.trimmed.nt.nb", "X5D.REGION.trimmed.nt.nb", "X3D.REGION.trimmed.nt.nb", "X5J.REGION.trimmed.nt.nb"), with=F], na.rm=T)),
+                                                Total.N=num_median(rowSums(.SD[,c("N.REGION.nt.nb", "N1.REGION.nt.nb", "N2.REGION.nt.nb", "N3.REGION.nt.nb", "N4.REGION.nt.nb"), with=F], na.rm=T)),
+                                                Total.P=num_median(rowSums(.SD[,c("P3V.nt.nb", "P5D.nt.nb", "P3D.nt.nb", "P5J.nt.nb"), with=F], na.rm=T)),
+                                                Median.CDR3.l=as.double(median(.SD$CDR3.Length))),
+															by=c("Sample")])
+															
+  newData[,sapply(newData, is.numeric)] = round(newData[,sapply(newData, is.numeric)],1)
+  write.table(newData, "junctionAnalysisUnProd_median.csv" , sep=",",quote=F,na="-",row.names=F,col.names=F)
+}
+
+PRODF = bak
+
+
+# ---------------------- D reading frame ----------------------
+
+D.REGION.reading.frame = PRODF[,c("Sample", "D.REGION.reading.frame")]
+
+chck = is.na(D.REGION.reading.frame$D.REGION.reading.frame)
+if(any(chck)){
+	D.REGION.reading.frame[chck,"D.REGION.reading.frame"] = "No D"
+}
+
+D.REGION.reading.frame = data.frame(data.table(D.REGION.reading.frame)[, list(Freq=.N), by=c("Sample", "D.REGION.reading.frame")])
+
+write.table(D.REGION.reading.frame, "DReadingFrame.csv" , sep="\t",quote=F,row.names=F,col.names=T)
+
+D.REGION.reading.frame = ggplot(D.REGION.reading.frame)
+D.REGION.reading.frame = D.REGION.reading.frame + geom_bar(aes( x = D.REGION.reading.frame, y = Freq, fill=Sample), stat='identity', position='dodge' ) + ggtitle("D reading frame") + xlab("Frequency") + ylab("Frame")
+D.REGION.reading.frame = D.REGION.reading.frame + scale_fill_manual(values=sample.colors)
+D.REGION.reading.frame = D.REGION.reading.frame + theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), axis.text.x = element_text(angle = 45, hjust = 1), panel.grid.major.y = element_line(colour = "black"), panel.grid.major.x = element_blank())
+
+png("DReadingFrame.png")
+D.REGION.reading.frame
+dev.off()
+
+
+
+
+# ---------------------- AA composition in CDR3 ----------------------
+
+AACDR3 = PRODF[,c("Sample", "CDR3.Seq")]
+
+TotalPerSample = data.frame(data.table(AACDR3)[, list(total=sum(nchar(as.character(.SD$CDR3.Seq)))), by=Sample])
+
+AAfreq = list()
+
+for(i in 1:nrow(TotalPerSample)){
+	sample = TotalPerSample$Sample[i]
+  AAfreq[[i]] = data.frame(table(unlist(strsplit(as.character(AACDR3[AACDR3$Sample == sample,c("CDR3.Seq")]), ""))))
+  AAfreq[[i]]$Sample = sample
+}
+
+AAfreq = ldply(AAfreq, data.frame)
+AAfreq = merge(AAfreq, TotalPerSample, by="Sample", all.x = T)
+AAfreq$freq_perc = as.numeric(AAfreq$Freq / AAfreq$total * 100)
+
+
+AAorder = read.table(sep="\t", header=TRUE, text="order.aa\tAA\n1\tR\n2\tK\n3\tN\n4\tD\n5\tQ\n6\tE\n7\tH\n8\tP\n9\tY\n10\tW\n11\tS\n12\tT\n13\tG\n14\tA\n15\tM\n16\tC\n17\tF\n18\tL\n19\tV\n20\tI")
+AAfreq = merge(AAfreq, AAorder, by.x='Var1', by.y='AA', all.x=TRUE)
+
+AAfreq = AAfreq[!is.na(AAfreq$order.aa),]
+
+AAfreqplot = ggplot(AAfreq)
+AAfreqplot = AAfreqplot + geom_bar(aes( x=factor(reorder(Var1, order.aa)), y = freq_perc, fill = Sample), stat='identity', position='dodge' )
+AAfreqplot = AAfreqplot + annotate("rect", xmin = 0.5, xmax = 2.5, ymin = 0, ymax = Inf, fill = "red", alpha = 0.2)
+AAfreqplot = AAfreqplot + annotate("rect", xmin = 3.5, xmax = 4.5, ymin = 0, ymax = Inf, fill = "blue", alpha = 0.2)
+AAfreqplot = AAfreqplot + annotate("rect", xmin = 5.5, xmax = 6.5, ymin = 0, ymax = Inf, fill = "blue", alpha = 0.2)
+AAfreqplot = AAfreqplot + annotate("rect", xmin = 6.5, xmax = 7.5, ymin = 0, ymax = Inf, fill = "red", alpha = 0.2)
+AAfreqplot = AAfreqplot + ggtitle("Amino Acid Composition in the CDR3") + xlab("Amino Acid, from Hydrophilic (left) to Hydrophobic (right)") + ylab("Percentage") + scale_fill_manual(values=sample.colors)
+AAfreqplot = AAfreqplot + theme(panel.background = element_rect(fill = "white", colour="black"),text = element_text(size=15, colour="black"), axis.text.x = element_text(angle = 45, hjust = 1), panel.grid.major.y = element_line(colour = "black"), panel.grid.major.x = element_blank())
+
+png("AAComposition.png",width = 1280, height = 720)
+AAfreqplot
+dev.off()
+write.table(AAfreq, "AAComposition.csv" , sep=",",quote=F,na="-",row.names=F,col.names=T)
+
+# ---------------------- AA median CDR3 length ----------------------
+
+median.aa.l = data.frame(data.table(PRODF)[, list(median=as.double(median(.SD$CDR3.Length))), by=c("Sample")])
+write.table(median.aa.l, "AAMedianBySample.csv" , sep=",",quote=F,na="-",row.names=F,col.names=F)
+