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1 #!/bin/bash
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2
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3 #Preprocessing workflow - trimming adapters, barcodes etc
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4
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5 # Barcode in the oligo used: NWTRYSNNNN
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6 # Which means that each proper read must begin with: NNNN(C|G)(A|G)(C|T)A(A|T)N
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7 # As regex:
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8 # ^[ACGT][ACGT][ACGT][ACGT][CG][AG][CT][A][AT][ACGT]
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9
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10 function print_help {
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11 cat <<End-of-message
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12 RNA probing data preprocessing.
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13 Read trimming and debarcoding.
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14 -------------------------------------
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15 Input arguments:
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16 -h: Help
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17 -1: Read1 (FASTQ) - Required
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18 -2: Read2 (FASTQ) - Optional
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19 -b: Barcode signature
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20 -t: Trimming length
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21 -o: Output folder (default: "output_dir")
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22 -------------------------------------
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23 Usage : preprocessing.sh -f <READ1> -r <READ2> -b <BARCODE_SEQ> -t <TRIM_LENGTH> -o <output_dir>
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24 End-of-message
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25 exit
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26 }
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27
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28 #defaults
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29 output_dir="./output_dir"
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30 read2=""
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31 trim_length=0
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32
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33 #parse input
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34 while getopts h1:2:b:t:o: myarg
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35 do case "$myarg" in
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36 h) print_help
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37 exit ;;
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38 1) read1="$OPTARG" ;; #required
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39 2) read2="$OPTARG" ;; #optional
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40 b) barcode="$OPTARG" ;; #optional
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41 t) trim_length="$OPTARG" ;; #optional
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42 o) output_dir="$OPTARG" ;; #optional
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43 [?]) echo "ERROR: Unknown parameter"
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44 print_help
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45 exit 1 ;;
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46 esac
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47 done
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48
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49 if [[ -z $read1 ]]; then
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50 echo "ERROR: Read1 file required!"
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51 print_help
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52 exit 1
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53 fi
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54
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55 # Create the output folder
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56 mkdir -p $output_dir
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57
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58 if [ -z "$barcode" ]; then
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59 barcode=''
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60 fi
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61 BAR_LENGTH=`eval echo ${#barcode}`
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62
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63 #########################################################################################
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64
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65 # Reverse complement the barcode sequence and create a regular expression.
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66
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67 REV_COMPLEMENT=$(perl -0777ne's/\n //g; tr/ATGCatgcNnYyRrKkMmBbVvDdHh/TACGtacgNnRrYyMmKkVvBbHhDd/; print scalar reverse $_;' <(echo $barcode))
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68
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69 REG_EXP=$(sed 's/A/[A]/g;s/C/[C]/g;s/G/[G]/g;s/T/[T]/g;s/R/[AG]/g;s/Y/[CT]/g;s/S/[GC]/g;s/W/[AT]/g;s/K/[GT]/g;s/M/[AC]/g;s/B/[CGT]/g;s/D/[AGT]/g;s/H/[ACT]/g;s/V/[ACG]/g;s/N/[AGCTN]/g;' <<< $REV_COMPLEMENT)
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70
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71 ########################################################################
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72 #1. Remove all the reads that do not start with the signature
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73
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74 # (first awk removes them, second awk removes corresponding quality strings) followed by cutadapt.
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75
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76 # After cutadapt, remove last 15 nt - may be derived from the random primer [should be optional, as the user may: 1) use different random primer length, 2) have short reasd and would lose too much info] and remove all the reads that are shorter than 30 nt
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77
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78 # (10 nt barcode + 20 nt for mapping)
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79
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80 awk '{if(NR%4==2){if(/^'"$REG_EXP"'/){print}else{print ""}}else{print}}' $read1 |
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81
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82 awk 'BEGIN{trim_flag=0; trimming_stats=0; all_processed=0}
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83 {
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84 if(NR%4==1){print; all_processed++}
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85 if(NR%4==2){if(length($1)==0){trim_flag=1;trimming_stats++}else{trim_flag=0};print}
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86 if(NR%4==3){print}
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87 if(NR%4==0){if(trim_flag==1){print ""}else{print $0}}
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88 }END{print(trimming_stats, all_processed) > "trimming_stats.error"}' |
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89
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90 awk -v len="${trim_length}" '{if(NR%2==0){print(substr($1,0,length($1)-len))}else{print}}' |
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91
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92 awk -v len="${BAR_LENGTH}" '{if(NR%2==0 && length($1)<20+len){printf("\n")}else{print}}' | gzip > R1.fastq.gz &
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93
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94 wait
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95
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96 mv trimming_stats.error $output_dir/trimming_stats.txt
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97
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98 ########################################################################
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99 #2. Trim the adapter, primer and possible random barcode from the second read
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100
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101 if [ -z "$read2" ]; then
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102 #single-end
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103
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104 #3. Remove empty reads - Unecessary
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105 #4. Extract the barcode sequence from the first read:
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106 zcat R1.fastq.gz | awk -v len="${BAR_LENGTH}" '{if(NR%2==0 && length($1)<20+len){printf("\n")}else{if(NR%2==0){print(substr($0,len+1,length($0)))}else{print($0)}}}' | awk '{print($1)}' > $output_dir/read1.fastq &
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107
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108 zcat R1.fastq.gz | awk -v len="${BAR_LENGTH}" '{if(NR%4==1){print($1)}else{if(NR%4==2){print(substr($0,0,len))}}}' | paste - - > $output_dir/barcodes.txt &
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109
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110 wait
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111
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112 ########################################################################
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113 #5. Remove temp files
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114
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115 rm R1.fastq.gz
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116
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117 else
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118 #paired-end
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119
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120 awk -v len1="${trim_length}" -v len2="${BAR_LENGTH}" '{if(NR%2==0){print(substr($0,len1+1,(length($0)-len1-len2)))}else{print($0)}}' $read2 |
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121
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122 awk '{if(NR%2==0 && length($1)<20){printf("\n")}else{print}}' | gzip > R2.fastq.gz &
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123
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124 wait
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125
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126 ########################################################################
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127 #3. Remove empty reads - remove each pair from for which at least one read of the pair got removed (they are problematic for tophat mapping)
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128 #first define which lines to keep from both fastq files (k for keep, d for discard in the lines_to_keep file)
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129
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130 paste <(zcat R1.fastq.gz) <(zcat R2.fastq.gz) | awk 'BEGIN{OFS="\n"}{if(NR%4==2 && NF==2){print("k","k","k","k")}else{if(NR%4==2 && NF<2){print("d","d","d","d")}}}' > lines_to_keep
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131
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132 paste lines_to_keep <(zcat R1.fastq.gz) | awk '{if($1=="k")print($2,$3)}' | gzip > R1_readsANDbarcodes.fastq.gz &
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133
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134 paste lines_to_keep <(zcat R2.fastq.gz) | awk '{if($1=="k")print($2,$3)}' | awk '{print($1)}' > $output_dir/read2.fastq &
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135
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136 wait
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137
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138 rm lines_to_keep
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139
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140 ########################################################################
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141 #4. Extract the barcode sequence from the first read:
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142 zcat R1_readsANDbarcodes.fastq.gz | awk -v len="${BAR_LENGTH}" '{if(NR%2==0 && length($1)<20+len){printf("\n")}else{if(NR%2==0){print(substr($0,len+1,length($0)))}else{print($0)}}}' | awk '{print($1)}' > $output_dir/read1.fastq &
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143
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144 zcat R1_readsANDbarcodes.fastq.gz | awk -v len="${BAR_LENGTH}" '{if(NR%4==1){print($1)}else{if(NR%4==2){print(substr($0,0,len))}}}' | paste - - > $output_dir/barcodes.txt &
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145
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146 wait
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147
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148 ########################################################################
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149 #5. Remove temp files
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150
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151 rm R1_readsANDbarcodes.fastq.gz R1.fastq.gz R2.fastq.gz
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152
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153 fi
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