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0.ParseBamReadcount.R

+# To parse files generated by Bam-readcount
+
+# Details in field of bam read count
+
+#base:count:avg_mapping_quality:avg_basequality:avg_se_mapping_quality:num_plus_strand:num_minus_strand:avg_pos_as_fraction:avg_num_mismatches_as_fraction:avg_sum_mismatch_qualities:num_q2_containing_reads:avg_distance_to_q2_start_in_q2_reads:avg_clipped_length:avg_distance_to_effective_3p_end
+rm(list=ls())
+bam_rc.parse <- function(x){
+  columns = "base:count:avg_mapping_quality:avg_basequality:avg_se_mapping_quality:num_plus_strand:num_minus_strand:avg_pos_as_fraction:avg_num_mismatches_as_fraction:avg_sum_mismatch_qualities:num_q2_containing_reads:avg_distance_to_q2_start_in_q2_reads:avg_clipped_length:avg_distance_to_effective_3p_end"
+  column.names = strsplit(columns, ":")[[1]];rm(columns)
+  # get line
+  Row = strsplit(x, split = "\t")[[1]]
+  # get base counts
+  temp <- lapply(Row[-c(1:4)], function(basecnt){
+    basecnt = strsplit(basecnt, split = ":")[[1]]
+  }) %>% do.call(rbind, .)
+  
+  colnames(temp) = column.names
+  
+  dat = data.frame(chr =Row[1],pos = Row[2],ref = Row[3], dp = Row[4],
+                   temp)
+  
+}
+strandBias=function(arg1,arg2){
+  if(length(arg1)!=length(arg2)){
+    error=paste("Parameters with unequal lengths passed as argument!\narg1 =",length(arg1),"; arg2 =",length(arg2))
+    message(error)
+    return(NA)
+  }
+  SB=c()
+  for (i in seq(1,length(arg1))){
+    if(is.na(arg1[i]) | is.na(arg2[i])){
+      calc=NA
+    }
+    else if(arg1[i]<arg2[i]){
+      calc=(arg2[i]/arg1[i])*-1
+    }
+    else{
+      calc=(arg1[i]/arg2[i])
+    }
+    SB=c(SB,calc)
+  }
+  return(SB)
+}
+
+library(dplyr)
+setwd("~/BaseSpace/20180112 EGFR Trimmed HiDP/Bam-readcount")
+
+files=list.files(".",pattern = "bamcount")
+for (file in files){
+  #file="EGFR.Lib.S1.bam.tsv"
+  #file="EGFREGFP5.S1.bam.tsv"
+  #file="EGFREGFP1.S1.bam.tsv"
+  temp=scan(file, what = "character", sep = "\n")
+  dat = lapply(temp, bam_rc.parse) %>% bind_rows()
+  dat$chr=as.character(dat$chr)
+  dat$pos=as.numeric(dat$pos)
+  dat$dp=as.numeric(dat$dp)
+  dat$count=as.numeric(dat$count)
+  dat[,7:18]=sapply(dat[,7:18],as.numeric)
+  dat=dat[dat$base!="=",]
+  dat$strandbias=strandBias(dat$num_plus_strand,dat$num_minus_strand)
+  dat$AlleleFreq=dat$count/dat$dp
+  saveRDS(dat,file=paste("Parsed/parsed.",gsub(".tsv","",file),".RDS",sep=""))
+  write.table(dat,file=paste("Parsed/parsed.",file,sep=""),sep="\t",row.names = F,col.names = T,quote = F,na = "NA")
+  
+}
+

1.ParseVCF.R

+# To parse VCF files generated from samtools mpileup & bcftools
+rm(list=ls())
+setwd("~/BaseSpace/20180112 EGFR Trimmed HiDP/vcf")
+# file="20180112 EGFR_lib.EGFR.locus.HiDP.vcf"
+file="20180112 EGFR.No.Lig.EGFR.locus.HiDP.vcf"
+
+temptext=readLines(paste(file,sep=""))
+temptext=temptext[-grep("^##",x = temptext)]
+require(progress)
+pb <- progress_bar$new( format = "  Processing [:bar] :percent in :elapsed", total = length(temptext), clear = FALSE, width= 60)
+temptext=gsub(";","\t",temptext)
+temptext=temptext[-seq(1:49)]
+parsing=matrix(NA,length(temptext),19)
+colnames(parsing)=c("chr","pos","dot1","Ref","Alt","zero","dot2","DP","I16","QS","VDB","SGB","RPB","MQB","MQSB","BQB","MQ0F","Format","DATA")
+for( i in seq(1,length(temptext))){
+#for( i in seq(69801,69810)){
+  line=temptext[i]
+  words=unlist(strsplit(line,"\t"))
+  parsing[i,1:10]=words[1:10]
+  for (j in seq(11,19)){
+    if(is.na(words[j])|words[j]==""){
+      break
+    }
+    #print(regexpr("VDB",words[j]))
+    if(regexpr("VDB",words[j])==1){
+      parsing[i,"VDB"]=words[j]
+    }
+    else if(regexpr("SGB",words[j])==1){
+      parsing[i,"SGB"]=words[j]
+    }
+    else if(regexpr("RPB",words[j])==1){
+      parsing[i,"RPB"]=words[j]
+    }
+    else if(regexpr("MQB",words[j])==1){
+      parsing[i,"MQB"]=words[j]
+    }
+    else if(regexpr("MQSB",words[j])==1){
+      parsing[i,"MQSB"]=words[j]
+    }
+    else if(regexpr("BQB",words[j])==1){
+      parsing[i,"BQB"]=words[j]
+    }
+    else if(regexpr("MQ0F",words[j])==1){
+      parsing[i,"MQ0F"]=words[j]
+    }
+    else if(regexpr("PL:AD",words[j])==1){
+      parsing[i,"Format"]=words[j]
+      parsing[i,"DATA"]=words[j+1]
+    }
+  }
+  if(i%%1000==0){
+    pb$tick(1000)
+  }
+}
+temp=data.frame(parsing,stringsAsFactors = F)
+
+pb <- progress_bar$new( format = "  Processing [:bar] :percent in :elapsed", total = length(temp$DATA), clear = FALSE, width= 60)
+PL=c();AD=c()
+for(i in seq(1,length(temp$DATA))){
+#for(i in seq(1,10)){
+  #print(temp$DATA[i])
+  var=unlist(strsplit(temp$DATA[i],":"))
+  PL=c(PL,var[1])
+  AD=c(AD,var[2])
+  if(i%%1000==0){
+    pb$tick(1000)
+  }
+}
+temp=cbind(temp,PL,AD)
+temp=temp[,c(-18,-19)]
+colnames(temp)[c(19)]="Allelic.Depth"
+parsing=temp;rm(temp)
+
+assign(file,parsing);rm(parsing)
+file.s=paste("Parsed/P-",file,sep="")
+saveRDS(object =get(file),file = paste(file.s,".RDS",sep=""))
+write.table(get(file),file=paste(file.s,".tsv",sep=""),sep="\t",row.names = F,col.names = T,quote = F,na = "NA")
+
+

2.FilterVCF.R

+bdir="~/BaseSpace/20180112 EGFR Trimmed HiDP/vcf/Parsed/";setwd(bdir)
+
+# rm(list=ls());file="P-20180112 EGFR_lib.EGFR.locus.HiDP.vcf.tsv"
+rm(list=ls());file="P-20180112 EGFR.No.Lig.EGFR.locus.HiDP.vcf.tsv"
+
+#setwd(paste(bdir,dir,sep=""))
+source("~/Documents/OneDrive.UTU/OneDrive - O365 Turun yliopisto//Git/GitLab.UTU/Personal/str.extra-for-R.R/str.extra.R")
+tab=read.table(file,sep="\t",header = T,as.is = T,stringsAsFactors = F)
+tab=tab[c(-3,-6,-7)] # Removing zero and dot (.) columns
+#Change Depth to numeric
+tab$DP=as.numeric(gsub("DP=","",tab$DP))
+
+# Subsetting to EGFR locus #chr7:55,084,725-55,277,031
+subtab=subset(tab,tab$chr=="chr7" & tab$pos>55084725 & tab$pos < 55277031)
+
+#Subsetting to get Depth > 1000
+# subtab=subset(subtab,subtab$DP>1000)
+
+# Make New Only Mutation Matrix
+
+require(progress);pb <- progress_bar$new( format = "  Processing [:bar] :current SNVs processed in - :elapsed", total = length(unlist(strsplit(subtab$Alt,","))), clear = FALSE, width= 65)
+chr=c();pos=c();ref=c();alt=c();DP=c();AD=c();count=0
+for(i in seq(1,length(subtab$DP))){
+#for(i in seq(1,100)){
+  datarow=subtab[i,]
+  Alts=unlist(strsplit(datarow[,"Alt"],",",fixed = T),use.names = F)
+  ADs=unlist(strsplit(datarow[,"Allelic.Depth"],",",fixed = T),use.names = T)
+  for(j in seq(1,length(Alts))){
+    count=count+1
+    #chr=unlist(c(chr,datarow[1]))
+    chr=append(chr,unlist(datarow[1]))
+    pos=append(pos,unlist(datarow[2]))
+    ref=append(ref,unlist(datarow[3]))
+    alt=append(alt,Alts[j])
+    DP=append(DP,unlist(datarow[5]))
+    AD=append(AD,ADs[j+1])
+    if(count%%100==0){
+      pb$tick(100)
+    }
+  }
+}
+AD=as.numeric(AD)
+SNV.matrix=data.frame(chr,pos,ref,alt,DP,AD,stringsAsFactors = F)
+rm(chr,pos,ref,alt,DP,AD,Alts,ADs,datarow,i,j)
+submat=subset(SNV.matrix,SNV.matrix$alt!="<*>")
+
+fnm=gsub("P-","../Variants/V-",gsub(".vcf.tsv","",file))
+saveRDS(object = submat,file = paste(fnm,".RDS",sep="")) # use var=readRDS("FileName")
+write.table(submat,file = paste(fnm,".tsv",sep=""),sep="\t",row.names = F,col.names = T,quote = F,na = "NA")
+
+

3.FoldChange & Plots.R

+
+#Subsetting to get Depth > 1000
+#subtab=subset(subtab,subtab$DP>1000)
+
+setwd("~/BaseSpace/20180112 EGFR Trimmed HiDP/vcf/Variants/")
+
+rm(list=ls())
+source("~/Documents/OneDrive.UTU/OneDrive - O365 Turun yliopisto//Git/GitLab.UTU/Personal/str.extra-for-R.R/str.extra.R")
+
+
+
+file1="V-20180112 EGFR_lib.EGFR.locus.HiDP.RDS";tab=readRDS(file1)
+VF=(tab$AD*100)/tab$DP
+Rel.Ab=(tab$AD*100)/sum(tab$AD)
+MutID=paste(tab$chr,":",tab$pos,tab$ref,">",tab$alt,sep="")
+tab=data.frame(tab,VF,Rel.Ab,MutID,stringsAsFactors = F)
+rm(VF,Rel.Ab,MutID)
+Library=tab;rm(tab,file1)
+colnames(Library)=paste("Lib.",colnames(Library),sep="")
+
+file1="V-20180112 EGFR.No.Lig.EGFR.locus.HiDP.RDS";tab=readRDS(file1)
+VF=(tab$AD*100)/tab$DP
+Rel.Ab=(tab$AD*100)/sum(tab$AD)
+MutID=paste(tab$chr,":",tab$pos,tab$ref,">",tab$alt,sep="")
+tab=data.frame(tab,VF,Rel.Ab,MutID,stringsAsFactors = F)
+rm(VF,Rel.Ab,MutID)
+NoLigand=tab;rm(tab,file1)
+colnames(NoLigand)=paste("NoLig.",colnames(NoLigand),sep="")
+
+allMuts=union(NoLigand$NoLig.MutID,Library$Lib.MutID)
+idx=match(allMuts,Library$Lib.MutID)
+Mutation.Table=Library[idx,]
+idx=match(allMuts,NoLigand$NoLig.MutID)
+Mutation.Table=data.frame(allMuts,Mutation.Table,NoLigand[idx,],stringsAsFactors = F)
+rm(Library,NoLigand)
+
+FoldChange=function(arg1,arg2){
+  if(length(arg1)!=length(arg2)){
+    error=paste("Parameters with unequal lengths passed as argument!\narg1 =",length(arg1),"; arg2 =",length(arg2))
+    message(error)
+    return(NA)
+  }
+  FC=c()
+  for (i in seq(1,length(arg1))){
+    if(is.na(arg1[i]) | is.na(arg2[i])){
+      calc=NA
+    }
+    else if(arg1[i]<arg2[i]){
+      calc=(arg2[i]/arg1[i])
+    }
+    else{
+      calc=(arg1[i]/arg2[i])*-1
+    }
+    FC=c(FC,calc)
+  }
+  return(FC)
+}
+
+Mutation.Table$FC=FoldChange(Mutation.Table$Lib.VF,Mutation.Table$NoLig.VF)
+
+AnnoDF=Mutation.Table[,c(2,3,3,4,5,1)]
+# write.table(AnnoDF,file = "../20180131.InputAnnovar.txt",col.names = F,quote = F,row.names = F)
+
+#- run following line in annovar
+# annotate_variation.pl -out EGFR.muts --build hg19 20180131.InputAnnovar.txt humandb
+#- more annotations
+# table_annovar.pl "20180131.InputAnnovar.txt" ~/NGS_Seq_Tools/annovar2/humandb -buildver hg19 -out EGFR.iSCREAM -remove -protocol refgene,gnomad_genome,gnomad_exome,avsnp150,clinvar_20170905 -operation g,f,f,f,f -nastring . -csvout -polish
+rm(AnnoDF)
+var=read.table("../EGFR.iSCREAM.hg19_multianno.csv",sep=",",stringsAsFactors = F,header = T)
+var$MutID=paste(var$Chr,":",var$Start,var$Ref,">",var$Alt,sep="")
+
+idx=match(Mutation.Table$allMuts,var$MutID)
+Mutation.Table=data.frame(Mutation.Table,var[idx,c(6:26)])
+source("~/Documents/OneDrive.UTU/OneDrive - O365 Turun yliopisto//Git/GitLab.UTU/EleniusGroup/DvsP.EGFR.HiDP/AnnovarMutCodeFind.R")
+
+Mutation.Table$AAchange=annovarMutCodeFind(Mutation.Table$AAChange.refgene,isoform = "NM_005228")
+temp=gsub("^.","",x = Mutation.Table$AAchange);temp=gsub(".$","",x=temp)
+Mutation.Table$AAPos=as.numeric(temp);rm(temp)
+
+Mutation.Table=Mutation.Table[Mutation.Table$ExonicFunc.refgene!="synonymous SNV",]
+
+# Add BamReadCount
+rm(var,idx)
+Lib.Count=readRDS("../../Bam-readcount/Parsed/parsed.EGFR.Lib.bamcount.RDS")
+Lib.Count$MutID=paste(Lib.Count$chr,":",Lib.Count$pos,Lib.Count$ref,">",Lib.Count$base,sep="")
+colnames(Lib.Count)=paste("bamRC.Lib.",colnames(Lib.Count),sep="")
+idx=match(Mutation.Table$allMuts,Lib.Count$bamRC.Lib.MutID)
+test=cbind.data.frame(Mutation.Table,Lib.Count[idx,])
+
+NoLig.Count=readRDS("../../Bam-readcount/Parsed/parsed.EGFR.NoLig.bamcount.RDS")
+NoLig.Count$MutID=paste(NoLig.Count$chr,":",NoLig.Count$pos,NoLig.Count$ref,">",NoLig.Count$base,sep="")
+colnames(NoLig.Count)=paste("bamRC.NoLig.",colnames(NoLig.Count),sep="")
+idx=match(Mutation.Table$allMuts,NoLig.Count$bamRC.NoLig.MutID)
+test=cbind.data.frame(test,NoLig.Count[idx,])
+
+Mutation.Table=test;rm(test,Lib.Count,NoLig.Count,idx)
+
+# write.table(Mutation.Table,file = "../20180206.Result.tsv",sep="\t",col.names = T,quote = F,row.names = F)
+# saveRDS(Mutation.Table,file = "../20180206.Result.RDS")
+
+
+#------------------------- 
+library(ggplot2)
+source("~/Seafile/My Library/Git/GitLab.UTU/Misc Code/Master ggplot2 Theme/theme.DC.plot.R")
+# All data. Massive plot!!!
+FC.data.cut=droplevels.data.frame(na.exclude(Mutation.Table))
+# View(FC.data.cut[FC.data.cut$AAchange%in%c("L858R","T790M","A702V","A1118E","L184P"),])
+
+# str.extra(FC.data.cut)
+toRemove=c(10,11,12,13,14,19,21,23,26,28,29,30,31,32,33,34,36,38,39,40,seq(44,46),48,seq(50,52),seq(55,61),64,seq(65,67),69,seq(71,73),seq(76,82),85)
+colnames(FC.data.cut)[toRemove]
+
+FC.data.cut=FC.data.cut[,-toRemove]
+# Removed columns
+# [1] "Lib.MutID"          "NoLig.chr"         
+# [3] "NoLig.pos"          "NoLig.ref"         
+# [5] "NoLig.alt"          "NoLig.MutID"       
+# [7] "Func.refgene"       "GeneDetail.refgene"
+# [9] "Xref.refgene"       "gnomAD_genome_AFR" 
+# [11] "gnomAD_genome_AMR"  "gnomAD_genome_ASJ" 
+# [13] "gnomAD_genome_EAS"  "gnomAD_genome_FIN" 
+# [15] "gnomAD_genome_NFE"  "gnomAD_genome_OTH" 
+# [17] "CLINSIG"            "CLNACC"            
+# [19] "CLNDSDB"            "CLNDSDBID"         
+# [21] "MutID"       
+
+FC.data.cut$Direction=FC.data.cut$FC
+FC.data.cut$Direction[FC.data.cut$Direction<0]="Decrease"
+FC.data.cut$Direction[FC.data.cut$Direction!="Decrease"]="Increase"
+FC.data.cut$Direction=as.factor(FC.data.cut$Direction)
+
+ggplot(data = FC.data.cut,aes(x=bamRC.NoLig.strandbias,y=FC.data.cut$bamRC.Lib.strandbias))+geom_point(alpha=0.4,size=2)+theme_dc.plot.xaxis.regular+geom_vline(xintercept = 0)+geom_hline(yintercept = 0)+xlab("StrandBias NoLigand")+ylab("Strand Bias Plasmid Library")+ggtitle("Strand Bias distribution")+ggtitle(paste("Strand Bias distribution N=",length(FC.data.cut$allMuts)))
+
+# removing infinite strand bias AA changes
+FC.data.cut=FC.data.cut[!((FC.data.cut$bamRC.NoLig.strandbias==Inf | FC.data.cut$bamRC.NoLig.strandbias==-Inf)|(FC.data.cut$bamRC.Lib.strandbias==Inf | FC.data.cut$bamRC.Lib.strandbias==-Inf)),]
+                        
+ggplot(data = FC.data.cut,aes(x=bamRC.NoLig.strandbias,y=FC.data.cut$bamRC.Lib.strandbias))+geom_point(alpha=0.4,size=2)+theme_dc.plot.xaxis.regular+geom_vline(xintercept = 0)+geom_hline(yintercept = 0)+xlab("StrandBias NoLigand")+ylab("Strand Bias Plasmid Library")+ggtitle("Strand Bias distribution (Inf removed)")+ggtitle(paste("Strand Bias distribution N=",length(FC.data.cut$allMuts)))
+
+
+FC.data.cut=FC.data.cut[FC.data.cut$bamRC.NoLig.strandbias<=10&FC.data.cut$bamRC.NoLig.strandbias>=-10|FC.data.cut$bamRC.Lib.strandbias<=10&FC.data.cut$bamRC.Lib.strandbias>=-10,]
+
+ggplot(data = FC.data.cut,aes(x=bamRC.NoLig.strandbias,y=FC.data.cut$bamRC.Lib.strandbias))+geom_point(alpha=0.4,size=2)+theme_dc.plot.xaxis.regular+geom_vline(xintercept = 0)+geom_hline(yintercept = 0)+xlab("StrandBias NoLigand")+ylab("Strand Bias Plasmid Library")+ggtitle(paste("Strand Bias distribution N=",length(FC.data.cut$allMuts)))
+
+
+# write.table(FC.data.cut,file = "../20180206.Final.Table.tsv",sep="\t",col.names = T,quote = F,row.names = F)
+# saveRDS(FC.data.cut,file = "../20180206.Final.table.RDS")
+
+library(RColorBrewer)
+cols=colorRampPalette(c("#003366","firebrick1"))(20)
+#cols=colorRampPalette(c("#ffeaea","#ff3030"))(20)
+
+# #temp=temp[order(temp$VF.NoLigand,decreasing = F),]
+# p=ggplot(FC.data.cut,aes(y=FC,x=AAPos,size=NoLig.VF,color=NoLig.VF))+geom_point(alpha=0.9)+scale_colour_gradient(low="#003366",high="firebrick1")+theme(panel.border = element_rect(linetype = 1, colour = "black",fill=NA,size=0.15),panel.background=element_rect(fill = NA, colour = NA),axis.text.y= element_text(size = rel(1),color="black"),legend.key= element_rect(fill=NA,colour = NA), axis.ticks =element_line(colour = "black"),axis.text.x = element_text(angle = 90, hjust = 1,size = rel(0.75)))+ggtitle("Fold Change Map of Mutations (From Library to Ligand Independent)")+xlab("Amino Acid Position")+ylab("Fold Change")+scale_x_continuous(breaks = c(1,seq(50,1210,by = 50),1210))+geom_hline(yintercept = 0,color="black")+geom_text(data=FC.data.cut[FC.data.cut$FC>25,],aes(label=FC.data.cut[FC.data.cut$FC>25,"AAchange"]),hjust=1.3,size=3)+scale_y_continuous(breaks=seq(-240,100,by = 20))+geom_hline(yintercept = 0,color="black")+geom_text(data=FC.data.cut[FC.data.cut$FC<(-50),],aes(label=FC.data.cut[FC.data.cut$FC<(-50),"AAchange"]),hjust=1.3,size=3)
+# pdf(file = "../FC.plot.pdf",width = 10,height = 7);print(p);dev.off()
+# 
+# p=ggplot(FC.data.cut,aes(y=FC,x=AAPos,size=NoLig.VF,color=NoLig.VF))+geom_point(alpha=0.1)+scale_colour_gradient(low="#003366",high="firebrick1")+theme(panel.border = element_rect(linetype = 1, colour = "black",fill=NA,size=0.15),panel.background=element_rect(fill = NA, colour = NA),axis.text.y= element_text(size = rel(1),color="black"),legend.key= element_rect(fill=NA,colour = NA), axis.ticks =element_line(colour = "black"),axis.text.x = element_text(angle = 90, hjust = 1,size = rel(0.75)))+ggtitle("Fold Change Map of Mutations (From Library to Ligand Independent)")+xlab("Amino Acid Position")+ylab("Fold Change")+scale_x_continuous(breaks = c(1,seq(50,1210,by = 50),1210))+geom_hline(yintercept = 0,color="black")+geom_text(data=FC.data.cut[FC.data.cut$FC>25,],aes(label=FC.data.cut[FC.data.cut$FC>25,"AAchange"]),hjust=1.3,size=3)+scale_y_continuous(breaks=seq(-240,100,by = 20))+geom_hline(yintercept = 0,color="black")+geom_text(data=FC.data.cut[FC.data.cut$FC<(-50),],aes(label=FC.data.cut[FC.data.cut$FC<(-50),"AAchange"]),hjust=1.3,size=3)
+# pdf(file = "../FC.plot.alpha.pdf",width = 10,height = 7);print(p);dev.off()
+# ggplotly(p)
+
+# p=ggplot(FC.data.cut,aes(y=FC,x=AAPos,size=NoLig.VF,color=NoLig.VF))+geom_point(alpha=0.9)+scale_colour_gradient(low="#003366",high="firebrick1")+theme(panel.border = element_rect(linetype = 1, colour = "black",fill=NA,size=0.15),panel.background=element_rect(fill = NA, colour = NA),axis.text.y= element_text(size = rel(1),color="black"),legend.key= element_rect(fill=NA,colour = NA), axis.ticks =element_line(colour = "black"),axis.text.x = element_text(angle = 90, hjust = 1,size = rel(0.75)))+ggtitle("Fold Change Map of Mutations (From Library to Ligand Independent)")+xlab("Amino Acid Position")+ylab("Fold Change")+scale_x_continuous(breaks = c(1,seq(50,1210,by = 50),1210))+geom_hline(yintercept = 0,color="black")+geom_text(data=FC.data.cut[FC.data.cut$FC>25,],aes(label=FC.data.cut[FC.data.cut$FC>25,"AAchange"]),hjust=1.3,size=3)+scale_y_continuous(breaks=seq(-240,100,by = 20))+geom_hline(yintercept = 0,color="black")+geom_text(data=FC.data.cut[FC.data.cut$FC<(-50),],aes(label=FC.data.cut[FC.data.cut$FC<(-50),"AAchange"]),hjust=1.3,size=3)+geom_rug(sides="r",alpha=0.5,size=0.2,color="black");p
+# ggsave(plot = p,filename = "../FC.plot.w.rug.pdf",width = 10,height = 7);print(p);dev.off()
+
+
+# Only FC not my signed FC.
+
+FC.data.cut$FC.reg=(FC.data.cut$NoLig.VF/FC.data.cut$Lib.VF)
+p=ggplot(FC.data.cut,aes(y=FC.reg,x=AAPos,size=NoLig.VF,color=NoLig.VF))+geom_point(alpha=0.9)+scale_colour_gradient(low="#003366",high="firebrick1")+theme(panel.border = element_rect(linetype = 1, colour = "black",fill=NA,size=0.15),panel.background=element_rect(fill = NA, colour = NA),axis.text.y= element_text(size = rel(1),color="black"),legend.key= element_rect(fill=NA,colour = NA), axis.ticks =element_line(colour = "black"),axis.text.x = element_text(angle = 90, hjust = 1,size = rel(0.75)))+ggtitle("Fold Change Map of Mutations (From Library to Ligand Independent)")+xlab("Amino Acid Position")+ylab("Fold Change")+scale_x_continuous(breaks = c(1,seq(50,1210,by = 50),1210))+geom_text(data=FC.data.cut[FC.data.cut$FC>25,],aes(label=FC.data.cut[FC.data.cut$FC.reg>25,"AAchange"]),hjust=1.3,size=3)+geom_hline(yintercept = 0,color="black")+geom_hline(yintercept = 1,linetype="dashed",color="red",alpha=0.5);p
+pdf(file = "../FCreg.plot.pdf",width = 10,height = 5);print(p);dev.off()
+# 
+# p=ggplot(FC.data.cut,aes(y=FC.reg,x=AAPos,size=NoLig.VF,color=NoLig.VF))+geom_point(alpha=0.1)+scale_colour_gradient(low="#003366",high="firebrick1")+theme(panel.border = element_rect(linetype = 1, colour = "black",fill=NA,size=0.15),panel.background=element_rect(fill = NA, colour = NA),axis.text.y= element_text(size = rel(1),color="black"),legend.key= element_rect(fill=NA,colour = NA), axis.ticks =element_line(colour = "black"),axis.text.x = element_text(angle = 90, hjust = 1,size = rel(0.75)))+ggtitle("Fold Change Map of Mutations (From Library to Ligand Independent)")+xlab("Amino Acid Position")+ylab("Fold Change")+scale_x_continuous(breaks = c(1,seq(50,1210,by = 50),1210))+geom_text(data=FC.data.cut[FC.data.cut$FC>25,],aes(label=FC.data.cut[FC.data.cut$FC.reg>25,"AAchange"]),hjust=1.3,size=3)+geom_hline(yintercept = 0,color="black")+geom_hline(yintercept = 1,linetype="dashed",color="red",alpha=0.5);p
+# pdf(file = "../FCreg.alpha.plot.pdf",width = 10,height = 5);print(p);dev.off()
+
+
+# base=c(1,2,4,6,8)
+# y.breaks=sort(c(0.01,0.1,base*1,base*10,100))
+# 
+# p=ggplot(FC.data.cut,aes(y=log2(FC.reg),x=AAPos,size=NoLig.VF,color=NoLig.VF))+geom_point(alpha=0.9)+scale_colour_gradient(low="#003366",high="firebrick1")+theme(panel.border = element_rect(linetype = 1, colour = "black",fill=NA,size=0.15),panel.background=element_rect(fill = NA, colour = NA),axis.text.y= element_text(size = rel(1),color="black"),legend.key= element_rect(fill=NA,colour = NA), axis.ticks =element_line(colour = "black"),axis.text.x = element_text(angle = 90, hjust = 1,size = rel(0.75)))+ggtitle("Fold Change Map of Mutations (From Library to Ligand Independent)")+xlab("Amino Acid Position")+ylab("log2(Fold Change)")+scale_x_continuous(breaks = c(1,seq(50,1210,by = 50),1210))+geom_text(data=FC.data.cut[FC.data.cut$FC>25,],aes(label=FC.data.cut[FC.data.cut$FC.reg>25,"AAchange"]),hjust=1.3,size=3)+scale_y_continuous(breaks = log2(y.breaks),labels=c(0.01,0.1,1,rep("",4),10,rep("",4),100))+geom_hline(yintercept = 0,color="black");p
+# pdf(file = "../FCreg.log.plot.pdf",width = 10,height = 7);print(p);dev.off()
+
+
+# ggplotly(p)
+
+# Browsable plot
+mini=FC.data.cut[,c("AAchange","Lib.VF","NoLig.VF","Lib.AD","NoLig.AD","FC.reg","AAPos")]
+colnames(mini)[c(3)]=c("VariantFrequency")
+
+library(plotly)
+
+X.A <- list(
+  title = "EGFR amino acid",
+  showticklabels = TRUE,
+  dtick=100,
+  ticklen = 5,
+  gridcolor = toRGB("white"),
+  gridwidth = 0.5,
+  tickwidth = 1,
+  range = c(-10, 1220),
+  tickangle = 0,
+  zerolinecolor = toRGB("black"),
+  zerolinewidth = 1.5)
+
+Y.A <- list(
+  title = "Fold Change",
+  showticklabels = TRUE,
+  dtick=25,
+  ticklen = 5,
+  gridcolor = toRGB("white"),
+  gridwidth = 0.5,
+  tickwidth = 1,
+  zerolinecolor = toRGB("black"),
+  zerolinewidth = 1.5)
+
+p=plot_ly(mini,alpha=0.7,x=~AAPos,y=~FC.reg,color=~VariantFrequency,size=~VariantFrequency,mode="text",text=~paste('EGFR ',AAchange,'</br> FC: ',round(FC.reg,digits = 3),'</br>- - - -</br>Variant frequency</br>Library: ',round(Lib.VF,digits = 3),'</br>Surving cells: ',round(VariantFrequency,digits=3),'</br>- - - -</br>Number of reads</br>Library: ',Lib.AD,'</br>Surving cells: ',NoLig.AD),colors = cols,sizes=c(50,300))%>%
+  add_markers()%>%
+  layout(title = "EGFR iSREAM",xaxis = X.A,yaxis = Y.A)
+print(p)
+# 
+# 
+# library(htmlwidgets)
+# library(htmltools)
+# 
+# p <- htmlwidgets::appendContent(p, htmltools::tags$input(id='inputText', value='L858R', ''), htmltools::tags$button(id='buttonSearch', 'Search'))
+# p <- htmlwidgets::appendContent(p, htmltools::tags$script(HTML(
+#   'document.getElementById("buttonSearch").addEventListener("click", function()
+#   {        
+#   var i = 0;
+#   var j = 0;
+#   var found = [];
+#   var myDiv = document.getElementsByClassName("js-plotly-plot")[0]
+#   var data = JSON.parse(document.querySelectorAll("script[type=\'application/json\']")[0].innerHTML);
+#   for (i = 0 ;i < data.x.data.length; i += 1) {
+#   for (j = 0; j < data.x.data[i].text.length; j += 1) {
+#   if (data.x.data[i].text[j].indexOf(document.getElementById("inputText").value) !== -1) {
+#   found.push({curveNumber: i, pointNumber: j});
+#   }
+#   }
+#   }
+#   Plotly.Fx.hover(myDiv, found);
+#   }  
+# );')))                                                    
+# 
+# htmlwidgets::saveWidget(p, paste('../FoldChangeRegulariSCREAM', ".html", sep=""))
+# p
+# 
+# # ,text=~paste('EGFR Mut: ',AAchange,'</br> Reads_Library: ',Lib.AD,'</br> VF_Lib: ',round(Lib.VF,digits = 3),'</br> Reads_NoLig: ',NoLig.AD,'</br> VF_NoLig: ',round(NoLig.VF,digits=3),'</br> FC: ',round(FC.reg,digits = 3))

3b.Animate Foldchange plot.R

+setwd("~/BaseSpace/20180112 EGFR Trimmed HiDP/vcf/Variants/")
+
+rm(list=ls())
+source("~/Documents/OneDrive.UTU/OneDrive - O365 Turun yliopisto/Git/GitLab.UTU/Personal/str.extra-for-R.R/str.extra.R")
+
+FC.data.cut=readRDS("../20180401.MutationTable.RDS")
+FC.data.cut$FC.reg=(FC.data.cut$NoLig.VF/FC.data.cut$Lib.VF)
+
+selected=FC.data.cut[,c("AAchange","AAPos","Lib.VF","NoLig.VF","FC.reg")]
+
+expandFrames=function(FC,VF.Begin,VF.End,frames=30){
+  # FC is foldchange column
+  # frames is the number of frames you want for a smooth animation
+  DF=matrix(data = 0,nrow =length(FC),ncol = frames)
+  VF=matrix(data = 0,nrow =length(VF.Begin),ncol = frames)
+  for(i in seq(1,length(FC))){
+    DF[i,]=seq(from = 0,to = FC[i],length.out = frames)
+    VF[i,]=seq(from = VF.Begin[i],to = VF.End[i],length.out = frames)
+  }
+  DF=as.data.frame(DF)
+  colnames(DF)=paste("FC.frame.",seq(1,frames,by = 1),sep="")
+  VF=as.data.frame(VF)
+  colnames(VF)=paste("VF.frame.",seq(1,frames,by = 1),sep="")
+  out.DF=cbind(DF,VF)
+  return(out.DF)
+}
+
+DF=expandFrames(FC = selected$FC,VF.Begin = selected$Lib.VF,VF.End = selected$NoLig.VF,frames = 60)
+300
+# setwd("~/BaseSpace/20180112 EGFR Trimmed HiDP/vcf/Variants/")
+# 
+# rm(list=ls())
+# source("~/Documents/OneDrive.UTU/OneDrive - O365 Turun yliopisto/Git/GitLab.UTU/Personal/str.extra-for-R.R/str.extra.R")
+# # saveRDS(Mutation.Table,file = "../20180206.Result.RDS")
+# 
+# FC.data.cut=readRDS("../20180206.Final.table.RDS")
+# selected=FC.data.cut[,c("AAchange","AAPos","Lib.VF","NoLig.VF","FC")]
+# 
+# expandFrames=function(FC,VF.Begin,VF.End,frames=30){
+#   # FC is foldchange column
+#   # frames is the number of frames you want for a smooth animation
+#   DF=matrix(data = 0,nrow =length(FC),ncol = frames)
+#   VF=matrix(data = 0,nrow =length(VF.Begin),ncol = frames)
+#   for(i in seq(1,length(FC))){
+#     DF[i,]=seq(from = 0,to = FC[i],length.out = frames)
+#     VF[i,]=seq(from = VF.Begin[i],to = VF.End[i],length.out = frames)
+#   }
+#   DF=as.data.frame(DF)
+#   colnames(DF)=paste("FC.frame.",seq(1,frames,by = 1),sep="")
+#   VF=as.data.frame(VF)
+#   colnames(VF)=paste("VF.frame.",seq(1,frames,by = 1),sep="")
+#   out.DF=cbind(DF,VF)
+#   return(out.DF)
+# }
+# 
+# DF=expandFrames(FC = selected$FC,VF.Begin = selected$Lib.VF,VF.End = selected$NoLig.VF,frames = 60)
+# selected=cbind(selected,DF)
+# # FC = selected$FC;VF.Begin = selected$Lib.VF;VF.End = selected$NoLig.VF;frames = 20
+# 
+# FC.data=selected[,1:5];colnames(FC.data)
+# # [1] "AAchange" "AAPos"    "Lib.VF"   "NoLig.VF" "FC"    
+# FC.data$FC=rep(0,length(FC.data$FC))
+# FC.data$VF=rep(0,length(FC.data$Lib.VF))
+# 
+# frames=grep("FC.frame",colnames(selected))
+# VF.frames=grep("VF.frame",colnames(selected))
+# j=1
+# VF.range=seq(0,5,by = 0.01)
+# color.range=colorRampPalette(c("#003366","firebrick1","firebrick1","firebrick1","firebrick1","firebrick1","firebrick1"))(length(VF.range))
+# for(i in frames){
+#   print(paste("Frame",i))
+#   FC.data$FC=selected[,i]
+#   FC.data$VF=selected[,VF.frames[j]];j=j+1
+#   p=ggplot(FC.data,aes(y=FC,x=AAPos,size=VF,color=VF))+geom_point(alpha=0.9)+scale_colour_gradientn(colours = color.range,values=VF.range)+theme(panel.border = element_rect(linetype = 1, colour = "black",fill=NA,size=0.15),panel.background=element_rect(fill = NA, colour = NA),axis.text.y= element_text(size = rel(1),color="black"),legend.key= element_rect(fill=NA,colour = NA), axis.ticks =element_line(colour = "black"),axis.text.x = element_text(angle = 90, hjust = 1,size = rel(0.75)))+ggtitle("Fold Change Map of Mutations (From Library to Ligand Independent)")+xlab("Amino Acid Position")+ylab("Fold Change")+scale_x_continuous(breaks = c(1,seq(50,1210,by = 50),1210))+geom_hline(yintercept = 0,color="black")+scale_y_continuous(breaks=seq(-240,100,by = 20),limits = c(-240,110))+geom_hline(yintercept = 0,color="black")
+#  ggsave(filename = paste("../Animated.FC/FC.plot.frame",stringr::str_pad(i,2,pad = "0"),".png",sep = ""),plot = p,dpi = 200,width = 10,height = 7)
+# };j=1
+# # Final frame
+# FC.data.cut$VF=FC.data.cut$NoLig.VF
+# p=ggplot(FC.data.cut,aes(y=FC,x=AAPos,size=VF,color=VF))+geom_point(alpha=0.9)+scale_colour_gradientn(colours = color.range,values=VF.range)+theme(panel.border = element_rect(linetype = 1, colour = "black",fill=NA,size=0.15),panel.background=element_rect(fill = NA, colour = NA),axis.text.y= element_text(size = rel(1),color="black"),legend.key= element_rect(fill=NA,colour = NA), axis.ticks =element_line(colour = "black"),axis.text.x = element_text(angle = 90, hjust = 1,size = rel(0.75)))+ggtitle("Fold Change Map of Mutations (From Library to Ligand Independent)")+xlab("Amino Acid Position")+ylab("Fold Change")+scale_x_continuous(breaks = c(1,seq(50,1210,by = 50),1210))+geom_hline(yintercept = 0,color="black")+geom_text(data=FC.data.cut[FC.data.cut$FC>25,],aes(label=FC.data.cut[FC.data.cut$FC>25,"AAchange"]),hjust=1.3,size=3)+scale_y_continuous(breaks=seq(-240,100,by = 20),limits = c(-240,110))+geom_hline(yintercept = 0,color="black")+geom_text(data=FC.data.cut[FC.data.cut$FC<(-50),],aes(label=FC.data.cut[FC.data.cut$FC<(-50),"AAchange"]),hjust=1.3,size=3)
+# ggsave(filename = paste("../Animated.FC/FC.plot.frame",paste("../Animated.FC/FC.plot.frame",stringr::str_pad(i+1,2,pad = "0"),".png",sep = ""),".png",sep = ""),plot = p,dpi = 200,width = 10,height = 7)
+# 
+# 
+# # run this: convert -delay 0.1 -loop 1 *.png animation.gif
+# 

4.SankeyStats.R

+
+load("~/Documents/OneDrive.UTU/OneDrive - O365 Turun yliopisto//Git/GitLab.UTU/EleniusGroup/ERBB.Miscellaneous/Posssible mutants/EGFR/egfr_Variants.bin")
+print(paste("Unique EGFR cDNA=",length(unique(Variants$mutants)),"Unique EGFR proteins=",length(unique(Variants$mutantprotein))))
+
+EGFR.WT=Variants$mutantprotein[1]
+non.syn=Variants[Variants$mutantprotein!=EGFR.WT,]
+length(unique(non.syn$mutants))+1 # +1 for WT
+length(unique(non.syn$mutantprotein))+1 # +1 for WT
+
+print(paste("Non-synonymous EGFR muts =",length(non.syn$mutants)))
+
+#Subsetting to get Depth > 1000
+#subtab=subset(subtab,subtab$DP>1000)
+
+setwd("~/BaseSpace/20180112 EGFR Trimmed HiDP/vcf/Variants/")
+
+rm(list=ls())
+source("~/Documents/OneDrive.UTU/OneDrive - O365 Turun yliopisto//Git/GitLab.UTU/Personal/str.extra-for-R.R/str.extra.R")
+
+
+
+file1="V-20180112 EGFR_lib.EGFR.locus.HiDP.RDS";tab=readRDS(file1)
+VF=(tab$AD*100)/tab$DP
+Rel.Ab=(tab$AD*100)/sum(tab$AD)
+MutID=paste(tab$chr,":",tab$pos,tab$ref,">",tab$alt,sep="")
+tab=data.frame(tab,VF,Rel.Ab,MutID,stringsAsFactors = F)
+rm(VF,Rel.Ab,MutID)
+Library=tab;rm(tab,file1)
+colnames(Library)=paste("Lib.",colnames(Library),sep="")
+
+file1="V-20180112 EGFR.No.Lig.EGFR.locus.HiDP.RDS";tab=readRDS(file1)
+VF=(tab$AD*100)/tab$DP
+Rel.Ab=(tab$AD*100)/sum(tab$AD)
+MutID=paste(tab$chr,":",tab$pos,tab$ref,">",tab$alt,sep="")
+tab=data.frame(tab,VF,Rel.Ab,MutID,stringsAsFactors = F)
+rm(VF,Rel.Ab,MutID)
+NoLigand=tab;rm(tab,file1)
+colnames(NoLigand)=paste("NoLig.",colnames(NoLigand),sep="")
+
+allMuts=union(NoLigand$NoLig.MutID,Library$Lib.MutID)
+idx=match(allMuts,Library$Lib.MutID)
+Mutation.Table=Library[idx,]