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1 #!/bin/bash
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2 ########################################
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3 #### Peak finding Pipeline for NGS Data
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4 #### Bash script
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5 ########################################
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6
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7 export LC_ALL=en_US.UTF-8
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8
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9 ##Finding out the path
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10 sPath="`dirname \"$0\"`"
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11 sPath="`( cd \"$sPath\" && pwd )`"
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12
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13
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14
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15 usage()
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16 {
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17 cat << EOF
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18 Welcome to JAMM v1.0.6rev2 (GNU GPLv3). Copyright (C) 2014 Mahmoud Ibrahim.
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19
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20 This program comes with ABSOLUTELY NO WARRANTY; for details visit http://www.gnu.org/licenses/gpl.html. This is free software, and you are welcome to redistribute it under certain conditions; visit http://www.gnu.org/licenses/gpl.html for details.
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21
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22 OPTIONS:
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23 -s directory containing Sample files (required)
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24 -g Genome size file (required)
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25 -o Output directory (required)
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26 -c directory containing input or Control files
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27 -m Mode, normal or narrow (default: normal)
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28 -r Resolution, peak or region (default: peak)
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29 -f Fragment length(s) (default: estimated)
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30 -w minimum Window size (default: 2 --- Note: this means minimum_window_size = bin_size x the_value_of_-w)
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31 -p Number of processors used by R scripts (default: 1)
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32 -b Bin Size (default: estimated)
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33 -t Type, single or paired (default: single, requires BED files. paired requires BEDPE files)
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34 -e window Enrichment cutoff, auto or any numeric value (default: 1 --- Set this to auto to estimate the appropriate window enrichment cutoff)
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35 EOF
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36 }
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37
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38
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39 # =========================
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40 # Process Input parameters
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41 # =========================
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42
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43
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44 #Defaults -- Change those if you want
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45 mode="normal"
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46 resol="peak"
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47 cores="1"
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48 window="2"
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49 type="single"
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50 windowe="1"
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51
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52
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53 #Defaults -- Do not change
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54 sdir=""
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55 gsize=""
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56 out=""
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57 binsize="ns"
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58 fraglen="ns"
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59 ran=$RANDOM
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60 wdir=$(mktemp -d)
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61
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62 while getopts "s:g:o:c:m:r:f:p:w:b:t:e:" OPTION
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63 do
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64 case $OPTION in
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65 s) sdir=$OPTARG
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66 ;;
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67 g) gsize=$OPTARG
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68 ;;
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69 o) out=$OPTARG
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70 ;;
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71 c) bdir=$OPTARG
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72 ;;
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73 m) mode=$OPTARG
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74 ;;
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75 r) resol=$OPTARG
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76 ;;
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77 f) fraglen=$OPTARG
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78 ;;
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79 p) cores=$OPTARG
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80 ;;
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81 w) window=$OPTARG
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82 ;;
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83 b) binsize=$OPTARG
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84 ;;
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85 t) type=$OPTARG
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86 ;;
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87 e) windowe=$OPTARG
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88 ;;
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89 ?)
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90 usage
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91 exit
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92 ;;
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93 esac
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94 done
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95 if [ "$mode" == "normal" ]; then
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96 clustno="2"
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97 fi
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98 if [ "$mode" == "narrow" ]; then
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99 clustno="3"
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100 fi
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101
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102 if [[ -z $sdir ]] || [[ -z $gsize ]] || [[ -z $out ]]
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103 then
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104 usage
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105 exit 1
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106 fi
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107 #=======================> DONE!
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108
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109
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110
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111 # =============================
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112 # Step One: Initial Processing
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113 # =============================
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114 printf "\n\n========================================\nStarted JAMM Pipeline v1.0.6rev2...Hang on!\n========================================\n\n"
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115
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116 if [ ! -d "$wdir" ]; then
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117 mkdir $wdir #make working directory
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118 fi
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119 if [ ! -d "$out" ]; then
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120 mkdir $out #make output directory
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121 fi
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122 mkdir $wdir/bkgd.$ran/ #directory to store background files
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123 mkdir $wdir/sizes.$ran/ #chromosomes and sizes
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124 mkdir $wdir/samples.$ran/ #store sample files
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125
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126 dupnum=$(ls -1 $sdir | wc -l) #count how many sample files
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127
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128
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129 #separate chromosome sizes
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130 printf "Loading genome size file..."
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131 ext="$wdir/sizes.$ran/"
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132 awk -v ext="$ext" '{ print >> ext"/size." $1 ".bed" }' $gsize
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133 printf "Done!\n"
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134
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135
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136 printf "Processing sample files..."
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137 #load each chromosome from each sample file
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138 for i in $sdir/*.bed; do
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139 samplefile=$(basename $i)
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140 for f in $wdir/sizes.$ran/*; do
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141 sizefile=$(basename $f)
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142 chr=$(echo $sizefile | awk -F"." '{print $2}' | awk -F"." '{print $1}');
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143 awk -v chr="$chr" -v ext="$wdir/samples.$ran/" -v samplefile="$samplefile" -F"\t" '$1 == chr { print $2"\t"$6 >> ext"sample."chr"."samplefile }' "$i"
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144 done
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145 done
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146 printf "Done!\n"
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147
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148
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149 if [ ! -z $bdir ]; then
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150 #concatenate all background files into one file
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151 printf "Processing control files..."
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152 cat $bdir/*.bed > $wdir/bkgd.$ran/ctrl.bed
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153
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154 for f in $wdir/sizes.$ran/*; do
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155 sizefile=$(basename $f)
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156 chr=$(echo $sizefile | awk -F"." '{print $2}' | awk -F"." '{print $1}');
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157 awk -v chr="$chr" -v ext="$wdir/bkgd.$ran/" -F"\t" '$1 == chr { print $2"\t"$6 >> ext"bkgd."chr".ctrl.bed" }' "$wdir/bkgd.$ran/ctrl.bed"
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158 done
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159
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160 printf "Done!\n"
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161 fi
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162
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163 #determine average read lengths
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164 printf "Getting average read lengths..."
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165 if [ ! -z $bdir ]; then
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166 readC=$(awk '{a=$3-$2;print a;}' "$wdir/bkgd.$ran/ctrl.bed" | perl -lane '$a+=$_;END{print $a/$.}' | awk '{a=$1+0.5;print a;}' | cut -d"." -f1)
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167 fi
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168 readL=""
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169 for s in $sdir/*.bed; do #and for each sample file
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170 read=$(awk '{a=$3-$2;print a;}' "$s" | perl -lane '$a+=$_;END{print $a/$.}' | awk '{a=$1+0.5;print a;}' | cut -d"." -f1)
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171 readL="$readL,$read"
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172 done
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173 readL=${readL#","}
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174 printf "Done!\n"
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175 #=======================> DONE!
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176
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177
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178
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179 # =============================
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180 # Step Two: Fragment Length
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181 # =============================
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182 #single-end
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183 if [ $type == "single" ]; then
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184
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185 if [ $fraglen == "ns" ]; then
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186
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187 ##Counting Where Reads Start and Calculating Cross Correlation
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188 mkdir $wdir/stats.$ran/ #store count files
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189 mkdir $out/xcorr #final xcorr results
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190
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191
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192 printf "Calculating Fragment Length(s)...\n"
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193 for f in $wdir/sizes.$ran/*; do #for each chromosome
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194 samplelist=""
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195 readlist=""
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196 sizefile=$(basename $f)
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197 chr=$(echo $sizefile | awk -F"." '{print $2}' | awk -F"." '{print $1}');
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198
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199 #list of sample bed files and read lengths
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200 for s in $wdir/samples.$ran/*.bed; do #and for each sample file
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201 samplefile=$(basename $s)
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202 chr2=$(echo $samplefile | awk -F"." '{print $2}');
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203 if [ $chr == $chr2 ] #belonging to this chromosome
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204 then
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205 samplelist="$samplelist,$wdir/samples.$ran/$samplefile"
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206 fi
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207 done
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208 readlist="$readL"
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209
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210 #list of control bed files and read lengths
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211 if [ ! -z $bdir ]; then
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212 for s in $wdir/bkgd.$ran/*.bed; do #and for each sample file
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213 samplefile=$(basename $s)
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214 chr2=$(echo $samplefile | awk -F"." '{print $2}');
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215 if [ $chr == $chr2 ] #belonging to this chromosome
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216 then
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217 samplelist="$samplelist,$wdir/bkgd.$ran/$samplefile"
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218 readlist="$readL,$readC"
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219 fi
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220 done
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221 fi
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222
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223 #remove leading comma
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224 samplelist=${samplelist#","}
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225
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226 #call R script for xcorr calculation
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227 Rscript "$sPath/xcorr.r" -ibed="$samplelist" -s="$wdir/sizes.$ran/size.$chr.bed" -rl="$readlist" -d="$wdir/stats.$ran" -p="$cores"
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228 done
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229
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230 #report xcorr results (samples)
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231 for f in $sdir/*.bed; do
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232 file=$(basename $f)
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233 samplefile=$(echo $file | awk -F"." '{print $1}');
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234 mkdir "$out/xcorr/$samplefile" #final xcorr results
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235 if [ -f "$wdir/stats.$ran/xc.$samplefile.tab" ]; then
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236 cp $wdir/stats.$ran/xc.$samplefile.tab $out/xcorr/$samplefile/shifts.txt
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237 fi
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238 Rscript "$sPath/xcorrhelper.r" -infile="$out/xcorr/$samplefile/shifts.txt" -out="$out/xcorr/$samplefile"
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239 done
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240 #report xcorr results (control)
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241 if [ ! -z $bdir ]; then
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242 mkdir "$out/xcorr/ctrl" #final xcorr results
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243 if [ -f "$wdir/stats.$ran/xc.ctrl.tab" ]; then
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244 cp $wdir/stats.$ran/xc.ctrl.tab $out/xcorr/ctrl/shifts.txt
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245 fi
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246 Rscript "$sPath/xcorrhelper.r" -infile="$out/xcorr/ctrl/shifts.txt" -out="$out/xcorr/ctrl"
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247 fi
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248
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249 fi
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250 fi
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251
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252 #paired-end
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253 if [ $type == "paired" ]; then
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254 printf "Getting Average Fragment Length(s)...\n"
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255 mkdir $out/xcorr #final xcorr results
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256
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257 for f in $sdir/*.bed; do
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258 file=$(basename $f)
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259 samplefile=$(echo $file | awk -F"." '{print $1}');
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260 mkdir "$out/xcorr/$samplefile"
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261 frag=$(awk '{a=$6-$2;print a;}' $f | perl -lane '$a+=$_;END{print $a/$.}' | awk '{a=$1+0.5;print a;}' | cut -d"." -f1)
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262 echo "Average_from_paired $frag" > $out/xcorr/$samplefile/shifts.txt
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263 Rscript "$sPath/xcorrhelper.r" -infile="$out/xcorr/$samplefile/shifts.txt" -out="$out/xcorr/$samplefile"
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264 done
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265 fi
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266 #=======================> DONE!
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267
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268
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269
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270 # =================================
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271 # Step Three: Calculating Bin Size
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272 # =================================
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273 if [ $binsize == "ns" ]; then
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274 printf "Getting Bin Size: "
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275
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276 chr=$(sort -nr -k2 $gsize | head -n 1 | awk -F"\t" '{print $1}');
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277 samplelist=""
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278 frag=""
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279 if [ $fraglen != "ns" ]; then
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280 frag=$fraglen
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281 k=1
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282 fi
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283
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284 #list of sample bed files and read lengths
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285 for s in $wdir/samples.$ran/*.bed; do
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286 samplefile=$(basename $s)
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287 chr2=$(echo $samplefile | awk -F"." '{print $2}');
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288 if [ $chr == $chr2 ]
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289 then
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290 samplelist="$samplelist,$wdir/samples.$ran/$samplefile"
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291 samplename=$(echo $samplefile | awk -F"." '{ print $3 }')
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292 samplefilename=$(echo $samplefile | cut -d'.' -f 3-)
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293 if [ $fraglen == "ns" ]; then
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294 shift=$(awk -F":" '$1 == "Fragment Length" { print $2 }' "$out/xcorr/$samplename/xcorrsummary.txt")
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295 frag="$frag,$shift"
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296 fi
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297 fi
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298 done
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299 #remove leading comma
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300 samplelist=${samplelist#","}
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301 frag=${frag#","}
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302
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303 Rscript "$sPath/bincalculator.r" -ibed="$samplelist" -s="$gsize" -rl="$readL" -d="$wdir" -p="$cores" -f="$frag" -type="$type"
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304 fi
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305 if [ $binsize != "ns" ]; then
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306 printf "You set a Bin Size: $binsize \n"
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307 fi
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308 #=======================> DONE!
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309
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310
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311
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312 # ===========================
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313 # Step Four: Calling Peaks
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314 # ===========================
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315 mkdir $wdir/peaks.$ran/ #store count files
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316 mkdir $out/peaks #store peak files
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317
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318 printf "Calling Peaks...(mode: $mode, resolution: $resol)\n"
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319
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320
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321 #single-end reads
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322 if [ $type == "single" ]; then
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323
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324 if [ $binsize == "ns" ]; then
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325 binsize=$(cat "$wdir/binsize.txt")
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326 fi
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327
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328 counting=1;
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329 for f in $wdir/sizes.$ran/*; do #for each chromosome
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330 samplelist=""
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331 frag=""
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332 k=1
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333 if [ $fraglen != "ns" ]; then
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334 frag=$fraglen
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335 fi
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336
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337
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338 sizefile=$(basename $f)
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339 chr=$(echo $sizefile | awk -F"." '{print $2}' | awk -F"." '{print $1}');
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340
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341 printf "Chromosome $chr: "
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342
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343 #list of sample bed files and fragment lengths
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344 for s in $wdir/samples.$ran/*.bed; do #and for each sample file
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345 samplefile=$(basename $s)
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346 chr2=$(echo $samplefile | awk -F"." '{print $2}');
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347 if [ $chr == $chr2 ] #belonging to this chromosome
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348 then
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349 samplelist="$samplelist,$wdir/samples.$ran/ext.$samplefile"
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350 samplename=$(echo $samplefile | awk -F"." '{ print $3 }')
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351 samplefilename=$(echo $samplefile | cut -d'.' -f 3-)
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352 if [ $fraglen == "ns" ]; then
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353 shift=$(awk -F":" '$1 == "Fragment Length" { print $2 }' "$out/xcorr/$samplename/xcorrsummary.txt")
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354 frag="$frag,$shift"
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355 read=$(echo $readL | cut -f "$k" -d ",")
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356 k=$(($k+1))
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357 fi
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358 if [ $fraglen != "ns" ]; then
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359 shift=$(echo $frag | cut -f "$k" -d ",")
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360 read=$(echo $readL | cut -f "$k" -d ",")
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361 k=$(($k+1))
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362 fi
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363 perl "$sPath/readshifter.pl" "$wdir/samples.$ran/$samplefile" $shift $read > "$wdir/samples.$ran/ext.$samplefile"
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364 fi
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365 done
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366
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367 #control file
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368 bkgdfile="None"
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369 if [ ! -z $bdir ]; then
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370 if [ $fraglen == "ns" ]; then
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371 bshift=$(awk -F":" '$1 == "Fragment Length" { print $2 }' "$out/xcorr/ctrl/xcorrsummary.txt")
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372 frag="$frag,$bshift"
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373 fi
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374 if [ $fraglen != "ns" ]; then
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375 l=$(($dupnum+1))
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376 bshift=$(echo $frag | cut -f "$l" -d ",")
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377 fi
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378 perl "$sPath/readshifter.pl" "$wdir/bkgd.$ran/bkgd.$chr.ctrl.bed" $bshift $readC > "$wdir/bkgd.$ran/ext.bkgd.$chr.ctrl.bed"
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379 bkgdfile="$wdir/bkgd.$ran/ext.bkgd.$chr.ctrl.bed"
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380 fi
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381
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382 #remove leading comma
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383 samplelist=${samplelist#","}
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384 frag=${frag#","}
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385
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386 #call the peak calling R script
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387 Rscript "$sPath/peakfinder.r" -sfile="$f" -bednames="$samplelist" -frag="$frag" -bkgd=$bkgdfile -out="$wdir/peaks.$ran/" -clustnummer="$clustno" -resolution="$resol" -window="$window" -p="$cores" -bin="$binsize" -type="$type" -chrcount="$counting" -windowe="$windowe"
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388 counting=$(($counting+1));
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389 cp "$wdir/peaks.$ran/$chr.peaks.bed" "$out/peaks/$chr.peaks.bed"
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390 rm "$wdir/peaks.$ran/$chr.peaks.bed"
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391 done
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392 counting=1;
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393 fi
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394
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395
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396 #paired-end reads
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397 if [ $type == "paired" ]; then
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398
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399 if [ $binsize == "ns" ]; then
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400 binsize=$(cat "$wdir/binsize.txt")
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401 fi
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402
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403
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404 counting=1;
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405 for f in $wdir/sizes.$ran/*; do #for each chromosome
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406 samplelist=""
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407
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408 sizefile=$(basename $f)
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409 chr=$(echo $sizefile | awk -F"." '{print $2}' | awk -F"." '{print $1}');
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410
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411 printf "Chromosome $chr: "
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412
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413 #list of sample bed files and fragment lengths
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414 for s in $wdir/samples.$ran/*.bed; do #and for each sample file
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415 samplefile=$(basename $s)
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416 chr2=$(echo $samplefile | awk -F"." '{print $2}');
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417 if [ $chr == $chr2 ] #belonging to this chromosome
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418 then
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419 samplelist="$samplelist,$wdir/samples.$ran/$samplefile"
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420 samplename=$(echo $samplefile | awk -F"." '{ print $3 }')
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421 samplefilename=$(echo $samplefile | cut -d'.' -f 3-)
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422 x="$sdir/$samplefilename"
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423 fi
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424 done
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425
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426 #control file
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427 bkgdfile="None"
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428 if [ ! -z $bdir ]; then
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429 bkgdfile="$wdir/bkgd.$ran/bkgd.$chr.ctrl.bed"
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430 fi
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431
|
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432 #remove leading comma
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433 samplelist=${samplelist#","}
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434 frag=${frag#","}
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435
|
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436 #call the peak calling R script
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437 Rscript "$sPath/peakfinder.r" -sfile=$f -bednames=$samplelist -frag="NA" -bkgd=$bkgdfile -out="$wdir/peaks.$ran/" -clustnummer="$clustno" -resolution="$resol" -window="$window" -p="$cores" -bin="$binsize" -type="$type" -chrcount="$counting" -windowe="$windowe"
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|
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438 counting=$(($counting+1));
|
|
|
439 cp "$wdir/peaks.$ran/$chr.peaks.bed" "$out/peaks/$chr.peaks.bed"
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440 rm "$wdir/peaks.$ran/$chr.peaks.bed"
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|
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441 done
|
|
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442 counting=1;
|
|
|
443 fi
|
|
|
444
|
|
|
445
|
|
|
446 cp $wdir/peaks.$ran/min.peaksize $out/peaks/min.peaksize
|
|
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447
|
|
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448
|
|
|
449 #concatenate, sort and filter
|
|
|
450 cat $out/peaks/*.bed > $out/peaks/all.narrowPeak
|
|
|
451 Rscript "$sPath/peakhelper.r" -filelist="$out/peaks/all.narrowPeak"
|
|
|
452 perl "$sPath/peakfilter.pl" $out/peaks/all.narrowPeak | sort -nr -k7 > $out/peaks/filtered.peaks.narrowPeak
|
|
|
453 cut -f1-10 $out/peaks/all.narrowPeak | awk -F"\t" -v j=0 '$7 > j' | sort -nr -k7 > $out/peaks/all.peaks.narrowPeak
|
|
|
454 rm $out/peaks/all.narrowPeak
|
|
|
455 rm $out/peaks/*.bed
|
|
|
456 rm $out/peaks/min.peaksize
|
|
|
457 #=======================> DONE!
|
|
|
458
|
|
|
459
|
|
|
460
|
|
|
461 rm -rf $wdir
|
|
|
462
|
|
|
463
|
|
|
464
|
|
|
465 printf "\n\n========================================\nWe're done...Congratulations!\n========================================\n\n"
|
