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1 ## This is a sample PBS script for profiling STR from short read using STR-FM version 2.0.0 (April 20, 2015)
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2 ##
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3 ##requirement
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4 ##1 fastq input in sangerfq Phred scale --> ${INPUT}.fastq
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5 ##2 index of mapping program (bwa, bowtie, etc)
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6 ##3 location of all STR in reference genome (use PBS script name "sampleSTR_reference_profiling.txt) --> /path/to/STR/in/reference/genome.TR (you can make 4 separated TR files for 4 types of STRs)
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7 ##4 reference genome in FASTA and in 2bit file --> /path/to/2bit/ref.2bit (use utility from UCSC genome browser to create 2bit file version of reference genome)
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8 ##5 local Galaxy (available from Galaxy website for Mac and Unix computer)
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9 ##6 STR error rates (can be downloaded from https://usegalaxy.org/u/guru%40psu.edu/h/error-rates-files) --> errorrate.bymajorallele
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10 ##
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11 echo " "
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12 echo " "
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13 echo "Job started on `hostname` at `date`"
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14 ref=/path/to/reference/sequence/and/bwa/index/ref.fa
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15 export PYTHONPATH=/path/to/galaxy-dist/lib/
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16 galaxydir=/path/to/galaxy-dist/tools
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17 cd /working/directory/
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18 echo " "
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19 echo " detect STR in short read" ## See detail in microsatellite.xml on https://github.com/Arkarachai/STR-FM
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20 python microsatellite.py ${INPUT}.fastq --fastq --period=1 --partialmotifs --minlength=5 --prefix=20 --suffix=20 --hamming=0 --multipleruns >${INPUT}.mono.out
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21 python microsatellite.py ${INPUT}.fastq --fastq --period=2 --partialmotifs --minlength=6 --prefix=20 --suffix=20 --hamming=0 --multipleruns >${INPUT}.di.out
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22 python microsatellite.py ${INPUT}.fastq --fastq --period=3 --partialmotifs --minlength=9 --prefix=20 --suffix=20 --hamming=0 --multipleruns >${INPUT}.tri.out
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23 python microsatellite.py ${INPUT}.fastq --fastq --period=4 --partialmotifs --minlength=12 --prefix=20 --suffix=20 --hamming=0 --multipleruns >${INPUT}.tetra.out
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24
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25 echo "change read name at " ## See detail in space2underscore_readname.xml on https://github.com/Arkarachai/STR-FM
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26 python changespacetounderscore_readname.py ${INPUT}.mono.out ${INPUT}.mono.new 6
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27 python changespacetounderscore_readname.py ${INPUT}.di.out ${INPUT}.di.new 6
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28 python changespacetounderscore_readname.py ${INPUT}.tri.out ${INPUT}.tri.new 6
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29 python changespacetounderscore_readname.py ${INPUT}.tetra.out ${INPUT}.tetra.new 6
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30
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31 echo "start fetch flanking at `date`" ## See detail in fetchflank.xml on https://github.com/Arkarachai/STR-FM
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32 python pair_fetch_DNA_ff.py ${INPUT}.mono.new ${INPUT}.mono_ff_L.txt ${INPUT}.mono_ff_R.txt 20 20
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33 python pair_fetch_DNA_ff.py ${INPUT}.di.new ${INPUT}.di_ff_L.txt ${INPUT}.di_ff_R.txt 20 20
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34 python pair_fetch_DNA_ff.py ${INPUT}.tri.new ${INPUT}.tri_ff_L.txt ${INPUT}.tri_ff_R.txt 20 20
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35 python pair_fetch_DNA_ff.py ${INPUT}.tetra.new ${INPUT}.tetra_ff_L.txt ${INPUT}.tetra_ff_R.txt 20 20
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36
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37 echo "BWA uniquely mapped no indel no deletion "
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38 bwa aln -n 0 -o 0 ${ref} ${INPUT}.mono_ff_L.txt > ${INPUT}.mono_ff_L.sai
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39 bwa aln -n 0 -o 0 ${ref} ${INPUT}.mono_ff_R.txt > ${INPUT}.mono_ff_R.sai
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40 bwa sampe ${ref} ${INPUT}.mono_ff_L.sai ${INPUT}.mono_ff_R.sai ${INPUT}.mono_ff_L.txt ${INPUT}.mono_ff_R.txt > ${INPUT}.mono.sam
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41 samtools view -Sb -F 12 -q 1 ${INPUT}.mono.sam > ${INPUT}.mono.n.all.bam
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42 bwa aln -n 0 -o 0 ${ref} ${INPUT}.di_ff_L.txt > ${INPUT}.di_ff_L.sai
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43 bwa aln -n 0 -o 0 ${ref} ${INPUT}.di_ff_R.txt > ${INPUT}.di_ff_R.sai
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44 bwa sampe ${ref} ${INPUT}.di_ff_L.sai ${INPUT}.di_ff_R.sai ${INPUT}.di_ff_L.txt ${INPUT}.di_ff_R.txt > ${INPUT}.di.sam
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45 samtools view -Sb -F 12 -q 1 ${INPUT}.di.sam > ${INPUT}.di.n.all.bam
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46 bwa aln -n 0 -o 0 ${ref} ${INPUT}.tri_ff_L.txt > ${INPUT}.tri_ff_L.sai
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47 bwa aln -n 0 -o 0 ${ref} ${INPUT}.tri_ff_R.txt > ${INPUT}.tri_ff_R.sai
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48 bwa sampe ${ref} ${INPUT}.tri_ff_L.sai ${INPUT}.tri_ff_R.sai ${INPUT}.tri_ff_L.txt ${INPUT}.tri_ff_R.txt > ${INPUT}.tri.sam
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49 samtools view -Sb -F 12 -q 1 ${INPUT}.tri.sam > ${INPUT}.tri.n.all.bam
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50 bwa aln -n 0 -o 0 ${ref} ${INPUT}.tetra_ff_L.txt > ${INPUT}.tetra_ff_L.sai
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51 bwa aln -n 0 -o 0 ${ref} ${INPUT}.tetra_ff_R.txt > ${INPUT}.tetra_ff_R.sai
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52 bwa sampe ${ref} ${INPUT}.tetra_ff_L.sai ${INPUT}.tetra_ff_R.sai ${INPUT}.tetra_ff_L.txt ${INPUT}.tetra_ff_R.txt > ${INPUT}.tetra.sam
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53 samtools view -Sb -F 12 -q 1 ${INPUT}.tetra.sam > ${INPUT}.tetra.n.all.bam
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54
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55 echo "sort result by read name"
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56 samtools sort -n ${INPUT}.mono.n.all.bam ${INPUT}.mono.n.sorted.all
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57 samtools sort -n ${INPUT}.di.n.all.bam ${INPUT}.di.n.sorted.all
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58 samtools sort -n ${INPUT}.tri.n.all.bam ${INPUT}.tri.n.sorted.all
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59 samtools sort -n ${INPUT}.tetra.n.all.bam ${INPUT}.tetra.n.sorted.all
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60 samtools view -h -o ${INPUT}.mono.n.sorted.all.sam ${INPUT}.mono.n.sorted.all.bam
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61 samtools view -h -o ${INPUT}.di.n.sorted.all.sam ${INPUT}.di.n.sorted.all.bam
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62 samtools view -h -o ${INPUT}.tri.n.sorted.all.sam ${INPUT}.tri.n.sorted.all.bam
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63 samtools view -h -o ${INPUT}.tetra.n.sorted.all.sam ${INPUT}.tetra.n.sorted.all.bam
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64
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65 echo "merge faux paired end reads" ## See detail in PEsortedSAM2readprofile.xml on https://github.com/Arkarachai/STR-FM
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66 python PEsortedSAM2readprofile.py ${INPUT}.mono.n.sorted.all.sam /path/to/2bit/ref.2bit 100 250 ${INPUT}.mono.RF
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67 python PEsortedSAM2readprofile.py ${INPUT}.di.n.sorted.all.sam /path/to/2bit/ref.2bit 100 250 ${INPUT}.mono.RF
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68 python PEsortedSAM2readprofile.py ${INPUT}.tri.n.sorted.all.sam /path/to/2bit/ref.2bit 100 250 ${INPUT}.mono.RF
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69 python PEsortedSAM2readprofile.py ${INPUT}.tetra.n.sorted.all.sam /path/to/2bit/ref.2bit 100 250 ${INPUT}.mono.RF
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70
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71 echo "join mapped coordinate with STR length using read name"
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72 python ${galaxydir}/filters/join.py ${INPUT}.mono.new ${INPUT}.mono.RF 6 1 ${INPUT}.mono.RF.j "" "" --index_depth=3 --buffer=50000000 --fill_options_file='None'
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73 python ${galaxydir}/filters/join.py ${INPUT}.di.new ${INPUT}.di.RF 6 1 ${INPUT}.mono.RF.j "" "" --index_depth=3 --buffer=50000000 --fill_options_file='None'
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74 python ${galaxydir}/filters/join.py ${INPUT}.tri.new ${INPUT}.tri.RF 6 1 ${INPUT}.mono.RF.j "" "" --index_depth=3 --buffer=50000000 --fill_options_file='None'
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75 python ${galaxydir}/filters/join.py ${INPUT}.tetra.new ${INPUT}.tetra.RF 6 1 ${INPUT}.mono.RF.j "" "" --index_depth=3 --buffer=50000000 --fill_options_file='None'
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76
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77 echo "join mapped coordinate and STR length with STR location in genome"
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78 python ${galaxydir}/new_operations/gops_join.py /path/to/STR/in/reference/genome.TR ${INPUT}.mono.RF.j ${INPUT}.mono.gop -1 1,2,3,0 -2 10,13,14,0 -m 1 -f
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79 python ${galaxydir}/new_operations/gops_join.py /path/to/STR/in/reference/genome.TR ${INPUT}.di.RF.j ${INPUT}.di.gop -1 1,2,3,0 -2 10,13,14,0 -m 1 -f
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80 python ${galaxydir}/new_operations/gops_join.py /path/to/STR/in/reference/genome.TR ${INPUT}.tri.RF.j ${INPUT}.tri.gop -1 1,2,3,0 -2 10,13,14,0 -m 1 -f
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81 python ${galaxydir}/new_operations/gops_join.py /path/to/STR/in/reference/genome.TR ${INPUT}.tetra.RF.j ${INPUT}.tetra.gop -1 1,2,3,0 -2 10,13,14,0 -m 1 -f
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82
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83 echo "remove incompatible motif (remove incorrect mapped reads given that there is no STR motif difference from reference genome)" ## See detail in microsatcompat.xml on https://github.com/Arkarachai/STR-FM
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84 python microsatcompat.py ${INPUT}.mono.gop 4 10 > ${INPUT}.mono.fulltable1
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85 python microsatcompat.py ${INPUT}.di.gop 4 10 > ${INPUT}.di.fulltable1
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86 python microsatcompat.py ${INPUT}.tri.gop 4 10 > ${INPUT}.tri.fulltable1
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87 python microsatcompat.py ${INPUT}.tetra.gop 4 10 > ${INPUT}.tetra.fulltable1
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88
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89 echo "remove shifting flanking location (remove cases that come from STR interruption or flanking bases are misread as STRs)"
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90 cat ${INPUT}.mono.fulltable1 | awk '($19==$2) && ($20==$3) {print $0}' > ${INPUT}.mono.fulltable2
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91 cat ${INPUT}.di.fulltable1 | awk '($19==$2) && ($20==$3) {print $0}' > ${INPUT}.di.fulltable2
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92 cat ${INPUT}.tri.fulltable1 | awk '($19==$2) && ($20==$3) {print $0}' > ${INPUT}.tri.fulltable2
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93 cat ${INPUT}.tetra.fulltable1 | awk '($19==$2) && ($20==$3) {print $0}' > ${INPUT}.tetra.fulltable2
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94
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95 echo "keep only column that are necessary for profiling"
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96 cat ${INPUT}.mono.fulltable2| cut -f 1,2,3,4,5,7 | sort -k 1n,1 -k 2n,2 -k 3n,3 > ${INPUT}.mono.cuttmp0
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97 cat ${INPUT}.di.fulltable2| cut -f 1,2,3,4,5,7 | sort -k 1n,1 -k 2n,2 -k 3n,3 > ${INPUT}.di.cuttmp0
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98 cat ${INPUT}.tri.fulltable2| cut -f 1,2,3,4,5,7 | sort -k 1n,1 -k 2n,2 -k 3n,3 > ${INPUT}.tri.cuttmp0
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99 cat ${INPUT}.tetra.fulltable2| cut -f 1,2,3,4,5,7 | sort -k 1n,1 -k 2n,2 -k 3n,3 > ${INPUT}.tetra.cuttmp0
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100
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101 echo "If you multiple analysis by splitting initial fastq, you should merge (cat) all results from the same sample after this step"
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102
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103 echo "create genomic coordinate column and group by that column"
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104 perl ${galaxydir}/filters/fixedValueColumn.pl ${INPUT}.mono.cuttmp0 ${INPUT}.mono.cuttmp1 "_" "no"
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105 python ${galaxydir}/filters/mergeCols.py ${INPUT}.mono.cuttmp1 ${INPUT}.mono.cuttmp2 1 7 2 7 3
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106 python ${galaxydir}/stats/grouping.py ${INPUT}.mono.cuttmp3 ${INPUT}.mono.cuttmp2 8 0 'cat 6 0' 'cat_uniq 4 0'
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107 perl ${galaxydir}/filters/fixedValueColumn.pl ${INPUT}.di.cuttmp0 ${INPUT}.di.cuttmp1 "_" "no"
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108 python ${galaxydir}/filters/mergeCols.py ${INPUT}.di.cuttmp1 ${INPUT}.di.cuttmp2 1 7 2 7 3
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109 python ${galaxydir}/stats/grouping.py ${INPUT}.di.cuttmp3 ${INPUT}.di.cuttmp2 8 0 'cat 6 0' 'cat_uniq 4 0'
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110 perl ${galaxydir}/filters/fixedValueColumn.pl ${INPUT}.tri.cuttmp0 ${INPUT}.tri.cuttmp1 "_" "no"
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111 python ${galaxydir}/filters/mergeCols.py ${INPUT}.tri.cuttmp1 ${INPUT}.tri.cuttmp2 1 7 2 7 3
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112 python ${galaxydir}/stats/grouping.py ${INPUT}.tri.cuttmp3 ${INPUT}.tri.cuttmp2 8 0 'cat 6 0' 'cat_uniq 4 0'
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113 perl ${galaxydir}/filters/fixedValueColumn.pl ${INPUT}.tetra.cuttmp0 ${INPUT}.tetra.cuttmp1 "_" "no"
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114 python ${galaxydir}/filters/mergeCols.py ${INPUT}.tetra.cuttmp1 ${INPUT}.tetra.cuttmp2 1 7 2 7 3
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115 python ${galaxydir}/stats/grouping.py ${INPUT}.tetra.cuttmp3 ${INPUT}.tetra.cuttmp2 8 0 'cat 6 0' 'cat_uniq 4 0'
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116
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117 echo "you may filter for minimum sequencing depth here"
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118
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119 echo "genotyping using error correction model" ## See detail in GenotypingSTR.xml on https://github.com/Arkarachai/STR-FM
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120 cat ${INPUT}.mono.cuttmp2 ${INPUT}.di.cuttmp2 ${INPUT}.tri.cuttmp2 ${INPUT}.tetra.cuttmp2 > ${INPUT}.step5
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121 python GenotypeTRcorrection.py ${INPUT}.step5 errorrate.bymajorallele ${INPUT}.step5.result 0.5
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122 ## final output is ${INPUT}.step5.result
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123
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124 echo "Job end on `hostname` at `date`"
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