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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
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7 # Which means that each proper read must begin with: NNNN(C|G)(A|G)(C|T)A(A|T)N
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8
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9 # As regex:
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10
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11 # ^[ACGT][ACGT][ACGT][ACGT][CG][AG][CT][A][AT][ACGT]
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12
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13
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14 READ1=${1}
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15 READ2=${2}
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16 BARCODE=$3
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17 ADAPTER1=$4
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18 ADAPTER2=$5
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19 CUTOFF=$6
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20 CUTADAPT_LOG=${7}
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21 TRIM_LENGTH=$8
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22 OVERLAP_LENGTH=$9
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23 OUT_R1=${10}
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24 OUT_R2=${11}
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25 OUT_BARCODES=${12}
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26
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27 BAR_LENGTH=`eval echo ${#BARCODE}`
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28
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29 #########################################################################################
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30
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31 # Reverse complement the barcode sequence and create a regular expression.
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32
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33 REV_COMPLEMENT=$(perl -0777ne's/\n //g; tr/ATGCatgcNnYyRrKkMmBbVvDdHh/TACGtacgNnRrYyMmKkVvBbHhDd/; print scalar reverse $_;' <(echo $BARCODE))
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34
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35 REG_EXP=$(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/[AGCT]/g;' <<< $REV_COMPLEMENT)
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36
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37 ########################################################################
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38 #1. Remove all the reads that do not start with the signature
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39
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40 # (first awk removes them, second awk removes corresponding quality strings) followed by cutadapt.
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41
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42 # 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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43
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44 # (10 nt barcode + 20 nt for mapping)
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45
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46 echo -e '------------------------Read1------------------------\n' > $CUTADAPT_LOG
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47
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48 awk '{if(NR%4==2){if(/^'"$REG_EXP"'/){print}else{print ""}}else{print}}' $READ1 |
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49
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50 awk 'BEGIN{trim_flag=0; trimming_stats=0; all_processed=0}
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51 {
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52 if(NR%4==1){print; all_processed++}
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53 if(NR%4==2){if(length($1)==0){trim_flag=1;trimming_stats++}else{trim_flag=0};print}
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54 if(NR%4==3){print}
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55 if(NR%4==0){if(trim_flag==1){print ""}else{print $0}}
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56 }END{print(trimming_stats, all_processed) > "trimming_stats.error"}' |
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57
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58 cutadapt -a $ADAPTER1 -q $CUTOFF --format=fastq -O $OVERLAP_LENGTH - 2>>$CUTADAPT_LOG |
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59
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60 awk -v len="${TRIM_LENGTH}" '{if(NR%2==0){print(substr($1,0,length($1)-len))}else{print}}' |
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61
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62 awk -v len="${BAR_LENGTH}" '{if(NR%2==0 && length($1)<20+len){printf("\n")}else{print}}' | gzip > R1.fastq.gz &
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63
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64 wait
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65
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66 ########################################################################
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67 #2. Trim the adapter, primer and possible random barcode from the second read
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68
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69 echo -e '------------------------Read2------------------------\n' >> $CUTADAPT_LOG
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70
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71 cutadapt -a $ADAPTER2 -q $CUTOFF --format=fastq -O $OVERLAP_LENGTH $READ2 2>>$CUTADAPT_LOG |
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72
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73 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)}}' - |
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74
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75 awk '{if(NR%2==0 && length($1)<20){printf("\n")}else{print}}' | gzip > R2.fastq.gz &
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76
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77 wait
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78
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79 ########################################################################
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80 #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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81
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82 #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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83
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84 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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85
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86 paste lines_to_keep <(zcat R1.fastq.gz) | awk '{if($1=="k")print($2,$3)}' | gzip > R1_readsANDbarcodes.fastq.gz &
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87
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88 paste lines_to_keep <(zcat R2.fastq.gz) | awk '{if($1=="k")print($2,$3)}' > $OUT_R2 &
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89
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90 wait
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91
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92 ########################################################################
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93 #4. Extract the barcode sequence from the first read:
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94 zcat R1_readsANDbarcodes.fastq.gz | awk -v len="${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)}}}' > $OUT_R1 &
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95
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96 zcat R1_readsANDbarcodes.fastq.gz | awk -v len="${BAR_LENGTH}" '{if(NR%4==1){print($1)}else{if(NR%4==2){print(substr($0,0,len))}}}' | paste - - > ${OUT_BARCODES} &
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97
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98 wait
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99
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100 ########################################################################
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101 #6. Remove temporary fastq files"
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102
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103 rm R1.fastq.gz R2.fastq.gz R1_readsANDbarcodes.fastq.gz
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104 rm lines_to_keep
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105
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106 ########################################################################
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107 #7. problem! Spaces added at the end of the strings in fastq files (AWK induced). I will remove them:
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108
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109 mv $OUT_R1 R1.temp.fastq
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110 mv $OUT_R2 R2.temp.fastq
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111
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112 awk '{print($1)}' R1.temp.fastq > $OUT_R1 &
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113 awk '{print($1)}' R2.temp.fastq > $OUT_R2 &
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114
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115 wait
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116 rm R?.temp.fastq
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