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1 #!/usr/bin/Rscript
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2
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3 xmlfile=as.character(commandArgs()[6])
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4 bedfile=as.character(commandArgs()[7])
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5 outdir=as.character(commandArgs()[8])
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6 genome=as.character(commandArgs()[9])
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7 poor=as.numeric(commandArgs()[10])
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8 high=as.numeric(commandArgs()[11])
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9 samplename=as.character(commandArgs()[12])
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10 targetfile=as.character(commandArgs()[13])
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11 details=as.numeric(commandArgs()[14])
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12 cutoff=0
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13 sdetails=""
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14 #samplename0=strsplit(strsplit(xmlfile,"_")[[1]][1],"/")[[1]]
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15 #samplename<-samplename0[length(samplename0)]
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16 outfile=paste(outdir,"/",samplename,"_enrichment.html",sep="")
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17
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18 ##Read XML summary
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19 cat("Reading XML file ... ")
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20 xmltag<-function(line){
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21 return(strsplit(strsplit(line,">")[[1]][2],"</")[[1]][1])
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22 }
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23 xml<-readLines(xmlfile)
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24 numreads<-numeric(0)
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25 nextfold=0
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26 for(line in xml){
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27 if(length(grep("ReadLength",line)>0)){
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28 readlength<-as.numeric(xmltag(line))
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29 }
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30 if(length(grep("NumberReads",line)>0)){
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31 numreads<-c(numreads,as.numeric(xmltag(line)))
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32 }
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33 if(length(grep("AvTargetCoverage",line)>0)){
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34 averagecov=as.numeric(xmltag(line))
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35 }
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36 if(details==1){
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37 sdetails="<a href=\"chromosomes/chromosomes.html\">[Show Details]</a><br/>"
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38 }
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39 if(length(grep("SDTargetCoverage",line)>0)){
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40 stddevcov=as.numeric(xmltag(line))
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41 }
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42 if(length(grep("TargetSize",line)>0)){
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43 targetsize=as.numeric(xmltag(line))
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44 }
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45 if(length(grep("<from1x>",line)>0)){nextfold=1}
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46 else{
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47 if((nextfold==1) && length(grep("PercBases",line))>0){
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48 sample1<-as.numeric(xmltag(line))
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49 nextfold=0
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50 }
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51 }
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52 if(length(grep("<from5x>",line)>0)){nextfold=5}
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53 else{
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54 if((nextfold==5) && length(grep("PercBases",line))>0){
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55 sample5<-as.numeric(xmltag(line))
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56 nextfold=0
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57 }
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58 }
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59 if(length(grep("<from10x>",line)>0)){nextfold=10}
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60 else{
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61 if((nextfold==10) && length(grep("PercBases",line))>0){
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62 sample10<-as.numeric(xmltag(line))
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63 nextfold=0
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64 }
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65 }
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66 if(length(grep("<from20x>",line)>0)){nextfold=20}
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67 else{
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68 if((nextfold==20) && length(grep("PercBases",line))>0){
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69 sample20<-as.numeric(xmltag(line))
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70 nextfold=0
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71 }
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72 }
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73 if(length(grep("<from30x>",line)>0)){nextfold=30}
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74 else{
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75 if((nextfold==30) && length(grep("PercBases",line))>0){
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76
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77 sample30<-as.numeric(xmltag(line))
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78 nextfold=0
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79 }
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80 }
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81 }
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82
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83 numreads_total<-numreads[1]
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84 numreads_target<-numreads[3]
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85 tpkm=round(numreads_target/((targetsize/1000)*(numreads_total/1000000)),2)
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86
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87 cat("ready.\n")
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88
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89
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90 ##Read BED enrichment file and summarize output
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91 cat("Reading BED file ... ")
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92 bed<-read.table(bedfile,stringsAsFactors=FALSE)
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93 area0_2<-sum(bed$V5<2)
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94 area2_10<-sum((bed$V5>=2) & (bed$V5<10))
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95 area10_20<-sum((bed$V5>=10) & (bed$V5<20))
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96 area20_30<-sum((bed$V5>=20) & (bed$V5<30))
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97 area30_50<-sum((bed$V5>=30) & (bed$V5<50))
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98 area50_100<-sum((bed$V5>=50) & (bed$V5<100))
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99 areagr100<-sum(bed$V5>100)
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100 cat("ready.\n")
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101
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102
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103 ##Create pieplot
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104 cat("Creating coverage pieplot ... ")
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105 png(file=paste(outdir,"/plots/",samplename,"_pieplot.png",sep=""),width=580)
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106 par(mar=c(1,7,1,7))
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107 pie(c(area0_2,area2_10,area10_20,area20_30,area30_50,area50_100,areagr100),
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108 labels=c(paste("0x to 2x (",area0_2,")",sep=""),
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109 paste("2x to 10x (",area2_10,")",sep=""),
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110 paste("10x to 20x (",area10_20,")",sep=""),
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111 paste("20x to 30x (",area20_30,")",sep=""),
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112 paste("30x to 50x (",area30_50,")",sep=""),
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113 paste("50x to 100x (",area50_100,")",sep=""),
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114 paste("above 100x (",areagr100,")",sep="")),
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115 col=c("gray30","gray40","gray50","gray60","gray70","gray80","gray90"),main="") ##Mean Coverage of Target Regions")
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116 garbage<-dev.off()
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117 cat("ready.\n")
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118
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119
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120 cat("Preparing coverage barplots ... ")
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121 maxgenemean=0
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122 genes<-levels(as.factor(bed$V4))
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123 genes<-genes[genes!="unknown"]
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124 ngenes<-length(genes)
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125
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126 for(i in 1:ngenes){
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127 genemean<-mean(bed[bed$V4==genes[i],5])
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128 if(maxgenemean<genemean){maxgenemean=genemean}
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129 }
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130
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131 maxgenemean=maxgenemean-(maxgenemean%%100)+100
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132 if((cutoff>0) && (maxgenemean>cutoff)){
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133 maxgenemean=cutoff
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134 }
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135 cat("ready.\n")
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136
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137 fwidth<-function(genes){
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138 fwidth0=10*length(genes)
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139 if(length(genes)<=100){fwidth0=10*length(genes)}
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140 if(length(genes)<=70){fwidth0=10*length(genes)}
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141 if(length(genes)<=40){fwidth0=25*length(genes)}
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142 if(length(genes)<=20){fwidth0=30*length(genes)}
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143 if(length(genes)<=10){fwidth0=35*length(genes)}
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144 if(length(genes)<=5){fwidth0=50*length(genes)}
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145 return(fwidth0)
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146 }
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147
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148
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149 chromosomes<-levels(as.factor(bed$V1))
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150 if(length(genes)>=2000){
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151 cat("Creating coverage barplots ... ")
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152 for(chr in chromosomes){
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153
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154 chrbed<-bed[bed$V1==chr,]
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155 genemean<-numeric(0)
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156 genes<-levels(as.factor(chrbed$V4))
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157 genes<-genes[genes!="unknown"]
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158
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159 for(i in 1:length(genes)){
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160 if(cutoff>0){genemean[i]<-min(mean(chrbed[chrbed$V4==genes[i],5]),cutoff)}
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161 else{genemean[i]<-mean(chrbed[chrbed$V4==genes[i],5])}
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162 }
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163
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164 png(file=paste(outdir,"/plots/",samplename,"_target_coverage_",chr,".png",sep=""),width=fwidth(genes),height=450)
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165 par(mar=c(7,5,1,2))
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166 barplot(as.numeric(genemean),names.arg=genes,las=2,
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167 ylab=paste("Average Coverage at Gene Locus (",chr,")",sep=""),col="tomato3",xlim=c(0.043*length(genemean),1.2*length(genes)-0.043*length(genemean)),ylim=c(0,maxgenemean))
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168 lineh=0
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169 while(lineh<maxgenemean){
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170 lineh=lineh+100
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171 lines(c(-100,1000+length(genes)),c(lineh,lineh),col="gray")
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172 }
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173 garbage<-dev.off()
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174 }
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175 cat("ready.\n")
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176 } else{
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177 cat("Creating coverage barplot ... ")
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178 for(i in 1:length(genes)){
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179 if(cutoff>0){genemean[i]<-min(mean(bed[bed$V4==genes[i],5]),cutoff)}
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180 else{genemean[i]<-mean(bed[bed$V4==genes[i],5])}
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181 }
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182
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183
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184 png(file=paste(outdir,"/plots/",samplename,"_target_coverage.png",sep=""),width=fwidth(genes),height=450)
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185 par(mar=c(7,5,1,2))
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186 barplot(as.numeric(genemean),names.arg=genes,las=2,
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187 ylab="Average Coverage at Gene Locus",col="tomato3",xlim=c(0,1.2*ngenes),ylim=c(0,maxgenemean))
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188 lineh=0
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189 while(lineh<maxgenemean){
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190 lineh=lineh+100
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191 lines(c(-100,1000+length(genes)),c(lineh,lineh),col="gray")
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192 }
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193 garbage<-dev.off()
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194 cat("ready.\n")
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195 }
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196
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197
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198 ##Searching for poorly/highly covered genes
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199 cat("Searching for poorly (<",poor,"x) and highly (>",high,"x) covered genes ... ",sep="")
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200 genes<-levels(as.factor(bed$V4))
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201 chr<-start<-end<-gene1<-ave<-stddev<-gr1x<-gr5x<-gr10x<-gr20x<-gr30x<-numeric(0)
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202 for(gene in genes){
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203
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204 bed0<-bed[bed$V4==gene,]
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205 len<-bed0[,3]-bed0[,2]
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206 fold1<-fold5<-fold10<-fold20<-fold30<-numeric(0)
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207 for(i in 1:nrow(bed0)){fold1<-c(fold1,as.numeric(bed0$V6[i]))}
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208 for(i in 1:nrow(bed0)){fold5<-c(fold5,as.numeric(bed0$V7[i]))}
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209 for(i in 1:nrow(bed0)){fold10<-c(fold10,as.numeric(bed0$V8[i]))}
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210 for(i in 1:nrow(bed0)){fold20<-c(fold20,as.numeric(bed0$V9[i]))}
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211
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212 for(i in 1:nrow(bed0)){fold30<-c(fold30,as.numeric(bed0$V10[i]))}
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213 fold1_gene<-paste(round(sum(fold1*len)/sum(len),2),"%",sep="")
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214 fold5_gene<-paste(round(sum(fold5*len)/sum(len),2),"%",sep="")
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215 fold10_gene<-paste(round(sum(fold10*len)/sum(len),2),"%",sep="")
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216 fold20_gene<-paste(round(sum(fold20*len)/sum(len),2),"%",sep="")
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217 fold30_gene<-paste(round(sum(fold30*len)/sum(len),2),"%",sep="")
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218
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219 chr<-c(chr,bed0[1,1])
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220 start<-c(start,min(bed0[,2]))
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221 end<-c(end,max(bed0[,3]))
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222 gene1<-c(gene1,gene)
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223 ave<-c(ave,round(mean(bed0[,5]),2))
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224 if(is.na(sd(bed0[,5]))){
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225 stddev<-c(stddev,0)
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226 }
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227 else{
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228 stddev<-c(stddev,round(sd(bed0[,5]),2))
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229 }
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230
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231 gr1x<-c(gr1x,fold1_gene)
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232 gr5x<-c(gr5x,fold5_gene)
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233 gr10x<-c(gr10x,fold10_gene)
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234 gr20x<-c(gr20x,fold20_gene)
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235 gr30x<-c(gr30x,fold30_gene)
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236
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237 }
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238
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239 bed_gene<-data.frame(chr,start,end,gene1,ave,stddev,gr1x,gr5x,gr10x,gr20x,gr30x)
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240
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241 poorly_covered<-bed_gene[bed_gene$ave<poor,]
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242 poorly_covered<-poorly_covered[order(poorly_covered[,5],decreasing=FALSE),]
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243 highly_covered<-bed_gene[bed_gene$ave>high,]
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244 highly_covered<-highly_covered[order(highly_covered[,5],decreasing=TRUE),]
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245
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246 linkpoor<-paste("http://www.genome.ucsc.edu/cgi-bin/hgTracks?&db=",genome,"&position=",
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247 poorly_covered$chr,"%3A",poorly_covered$start,"-",
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248 poorly_covered$end,"&hgt.suggest=&pix=800&Submit=submit&hgsid=183341879",sep="")
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249 linkhigh<-paste("http://www.genome.ucsc.edu/cgi-bin/hgTracks?&db=",genome,"&position=",
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250 highly_covered$chr,"%3A",highly_covered$start,"-",
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251 highly_covered$end,"&hgt.suggest=&pix=800&Submit=submit&hgsid=183341879",sep="")
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252 cat("ready.\n",sep="")
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253
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254
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255 ##Output HTML document
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256 cat("Writing HTML output ... ");
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257
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258 cat(file=outfile,paste(
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259 "<html>\n",
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260 "<head>\n",
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261 "<title>Enrichment Performance</title>\n",
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262 "<style type=\"text/css\">\n",
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263 " body{font-family:sans-serif;}\n",
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264 " h2,h3{color: darkblue;}\n",
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265 " a{color:darkblue;}\n",
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266 " table.output td{",
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267 " padding: 4px; background-color: lightskyblue;",
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268 " border: 1px solid #000; border-color: darkblue;",
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269 " }\n",
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270 "</style>\n",
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271 "\n",
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272 "<script language=\"JavaScript\">\n",
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273 " var questionClass=\"chrView\";\n",
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274 " function collapseAll(){\n",
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275 " var allSections = document.getElementsByTagName(\"div\");\n",
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276 " for(i=0; i<allSections.length; i++){\n",
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277 " if(allSections[i].className==questionClass){\n",
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278 " allSections[i].style.display=\"none\";\n",
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279 " }\n",
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280 " }\n",
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281 " }\n",
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282 " function expand(name){\n",
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283 " collapseAll();\n",
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284 " var newStyle=\"\";\n",
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285 " if(document.getElementById(name).style.display!=\"block\"){\n",
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286 " newStyle=\"block\";\n",
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287 " }\n",
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288 " else{\n",
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289 " newStyle=\"none\";\n",
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290 " }\n",
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291 " document.getElementById(name).style.display=newStyle;\n",
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292 " }\n",
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293 "</script>\n",
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294 "</head>\n",
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295 "\n",
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296 "<body onload=\"expand('",chromosomes[1],"')\">",
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297 "<h2>Enrichment Performance of Sample ",samplename,"</h2>\n",
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298 "<table>\n",
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299 "<tr>\n",
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300 "<td>\n",
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301 "<h3>Summary Statistics</h3>\n",
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302 "<table class=\"output\">\n",
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303 " <tr><td><b># Reads</b></td><td>",numreads_total,"</td></tr>\n",
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304 " <tr><td><b># On Target ± 100 bp</b></td><td>",numreads_target,"</td></tr>\n",
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305 " <tr><td><b>Target Size (bp)</b><td>",targetsize,"</td></tr>\n",
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306 " <tr><td><b># Target Regions</b><td>",nrow(bed),"</td></tr>\n",
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307 " <tr><td><b>Coverage Mean</b></td><td>",averagecov,"</td></tr>\n",
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308 " <tr><td><b>Coverage Std Dev</b></td><td>",stddevcov,"</td></tr>\n",
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309 " <tr><td><b>Covered 1x</b></td><td>",sample1,"</td></tr>\n",
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310 " <tr><td><b>Covered 5x</b></td><td>",sample5,"</td></tr>\n",
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311 " <tr><td><b>Covered 10x</b></td><td>",sample10,"</td></tr>\n",
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312 " <tr><td><b>Covered 20x</b></td><td>",sample20,"</td></tr>\n",
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313 " <tr><td><b>Covered 30x</b></td><td>",sample30,"</td></tr>\n",
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314 " <tr><td><b>TPKM</b></td><td>",tpkm,"</td></tr>\n",
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315 "</table>\n",
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316 "</td>\n",
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317 "<td width=\"10%\"></td>",
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318 "<td>\n",
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319 "<img src=\"plots/",samplename,"_pieplot.png\"></img>\n",
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320 "</td>\n",
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321 "</tr>\n",
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322 "</table>\n",
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323 "<br/>\n",
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324 "<h2>Genewise Target Coverage</h2>",sdetails,"<br/>\n",sep=""))
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325
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326
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327 if(ngenes>=2000){
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328 for(chromosome in chromosomes){
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329 cat(file=outfile,paste("<a href=\"javascript:expand('",chromosome,"')\">",chromosome,"</a>\n",sep=""),append=TRUE)
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330 }
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331 for(chromosome in chromosomes){
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332 cat(file=outfile,paste("<div style=\"height:480px; overflow:auto;\" class=\"chrView\" id=\"",chromosome,"\"><img src=\"plots/",samplename,"_target_coverage_",chromosome,".png\"></img></div>\n",sep=""),append=TRUE)
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333 }
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334 } else{
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335 cat(file=outfile,paste("<div style=\"height:480px; overflow:auto;\"><img src=\"plots/",samplename,"_target_coverage.png\"></img></div>\n",sep=""),append=TRUE)
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336 }
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337
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338 cat(file=outfile,paste("<br/><br/>\n",
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339 "<h2>Poorly Covered Genes (Cutoff: ",poor,"x)</h2>\n",sep=""),append=TRUE)
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340 if(nrow(poorly_covered)==0){
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341 cat(file=outfile,"<p>Nothing found for this cutoff.</p>",append=TRUE)
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342 } else{
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343 cat(file=outfile,paste(
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344 "<table class=\"output\">\n",
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345 " <tr>\n",
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346 " <td><b>Region</b></td><td><b>Gene</b></td>\n",
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347 " <td><b>Coverage Mean</b></td>\n",
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348 " <td><b>Covered 1x</b></td><td><b>Covered 5x</b></td>\n",
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349 " <td><b>Covered 10x</b></td><td><b>Covered 20x</b></td>\n",
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350 " <td><b>Covered 30x</b></td><td><b>External Link</b></td>\n",
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351 " </tr>\n",sep=""),append=TRUE)
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352
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353 for(i in 1:nrow(poorly_covered)){
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354 cat(file=outfile,paste(
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355 " <tr>\n",
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356 " <td>",poorly_covered[i,1],":",poorly_covered[i,2],"-",poorly_covered[i,3],"</td>\n",
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357 " <td>",poorly_covered[i,4],"</td>\n",
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358 " <td>",poorly_covered[i,5],"</td>\n",
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359 " <td>",poorly_covered[i,7],"</td>\n",
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360 " <td>",poorly_covered[i,8],"</td>\n",
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361 " <td>",poorly_covered[i,9],"</td>\n",
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362 " <td>",poorly_covered[i,10],"</td>\n",
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363 " <td>",poorly_covered[i,11],"</td>\n",
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364 " <td><a href=\"",linkpoor[i],"\">Show in Genome Browser</a></td>\n",
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365 " </tr>\n",sep=""),append=TRUE)
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366 }
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367
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368 cat(file=outfile,"</table><br/>\n",append=TRUE)
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369 }
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370
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371 cat(file=outfile,paste("<h2>Highly Covered Genes (Cutoff: ",high,"x)</h2>\n",sep=""),append=TRUE)
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372 if(nrow(highly_covered)==0){
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373 cat(file=outfile,"<p>Nothing found for this cutoff.</p>",append=TRUE)
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374 } else{
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375 cat(file=outfile,paste(
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376 "<table class=\"output\">\n",
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377 " <tr>\n",
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378 " <td><b>Target</b></td><td><b>Gene</b></td><td><b>Coverage Mean</b></td>",
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379 " <td><b>Covered 1x</b></td><td><b>Covered 5x</b></td><td><b>Covered 10x</b></td>",
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380 " <td><b>Covered 20x</b></td><td><b>Covered 30x</b></td><td><b>External Link</b></td>\n",
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381 " </tr>\n",sep=""),append=TRUE)
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382 for(i in 1:nrow(highly_covered)){
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383 cat(file=outfile,paste(" <tr>\n",
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384 " <td>",highly_covered[i,1],":",highly_covered[i,2],"-",highly_covered[i,3],"</td>\n",
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385 " <td>",highly_covered[i,4],"</td>\n",
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386 " <td>",highly_covered[i,5],"</td>\n",
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387 " <td>",highly_covered[i,7],"</td>\n",
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388 " <td>",highly_covered[i,8],"</td>\n",
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|
389 " <td>",highly_covered[i,9],"</td>\n",
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390 " <td>",highly_covered[i,10],"</td>\n",
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391 " <td>",highly_covered[i,11],"</td>\n",
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392 " <td><a href=\"",linkhigh[i],"\">Show in Genome Browser</a></td>\n",
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393 " </tr>",sep=""),append=TRUE)
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394 }
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395
|
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396 cat(file=outfile,"</table>\n",append=TRUE)
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397 }
|
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398 cat(file=outfile,"<br/>BED file used for specification of target regions:<br/>",targetfile,append=TRUE)
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399 cat(file=outfile,"</body>\n</html>\n",append=TRUE)
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400
|
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401 cat("ready.\n")
|
