diff --git a/bamUtil_Coverage_Tool/MVL_Coverage_Tool.sh b/bamUtil_Coverage_Tool/MVL_Coverage_Tool.sh new file mode 100644 index 0000000..f894c26 --- /dev/null +++ b/bamUtil_Coverage_Tool/MVL_Coverage_Tool.sh @@ -0,0 +1,205 @@ +#!/bin/bash +set -u +set -e +underline=`tput smul` +normal=`tput sgr0` +bold=`tput bold` + +function usage () { +echo " +${bold}This script is calculating coverage per position of pathogenic and likely pathogenic varaints from the MVL. +AvgCoverage, SD, avarage cov <1x,<10x,<30x, +${bold}Arguments${normal} + example sh MVL_Coverage_Tool.sh -s /groups/umcg-gd/prm02/projects/QXTR_90-Exoom_v1/run01/results/alignment/ OPTIONS + Required: + -s|--sampledir Directory of of bam files of the used samples + -t|--tmpdir Give tmpfolder location for inbetween and final files + Optional: + -b|--bamstat path to the ban stats (your/directory/to/bamUtil-1.0.14/bin/bam stats) + -m|--mvl location of MVL_per_bp_unique.txt (default: $EBROOTNGSMINUTILS/bamUtil_Coverage_Tool/MVL_per_bp_unique.txt) + -x|--mvltabix location of MVL_per_bp_unique_tabix.txt (default: $EBROOTNGSMINUTILS/bamUtil_Coverage_Tool/MVL_per_bp_unique_tabix.txt) + -q|--qcmvldir location of MerdgeQCofMVL_file.py necessary for final txt generation (default:$EBROOTNGSMINUTILS/bamUtil_Coverage_Tool/) +" +} + +module load HTSlib +module load Python + +PARSED_OPTIONS=$(getopt -n "$0" -o s:b:o:m:x:t:f:z: --long "sampledir:bamstat:outputdir:mvl:mvltabix:tmpdir:outputfilter:finaloutput:" -- "$@") + +# +# Bad arguments, something has gone wrong with the getopt command. +# +if [ $? -ne 0 ]; then + usage + echo "FATAL: Wrong arguments." + exit 1 +fi + +eval set -- "$PARSED_OPTIONS" + +# +# Now goes through all the options with a case and using shift to analyse 1 argument at a time. +# $1 identifies the first argument, and when we use shift we discard the first argument, so $2 becomes $1 and goes again through the case. +# +while true; do + case "$1" in + -s|--sampledir) + case "$2" in + "") shift 2 ;; + *) SAMPLEDIR=$2 ; shift 2 ;; + esac ;; + -b|--bamstat) + case "$2" in + *) BAMSTAT=$2 ; shift 2 ;; + esac ;; + -m|--mvl) + case "$2" in + *) MVL=$2 ; shift 2 ;; + esac ;; + -x|--mvltabix) + case "$2" in + *) MVLTABIX=$2 ; shift 2 ;; + esac ;; + -t|--tmpdir) + case "$2" in + *) TmpDirBam=$2 ; shift 2 ;; + esac ;; + -q|--qcmvl) + case "$2" in + *) QCMVLDIR=$2 ; shift 2 ;; + esac ;; + --) shift ; break ;; + *) echo "Internal error!" ; exit 1 ;; + esac +done + + +# +#Check required options were provided. +# +if [[ -z "${SAMPLEDIR-}" ]]; then + usage + exit 1 +fi +if [[ -z "${BAMSTAT-}" ]]; then + #BAMSTAT="your/directry/to/bamUtil_Coverage_Tool/bamUtil-1.0.14/bin/bam" + usage +fi +if [[ -z "${MVL-}" ]]; then + MVL="$EBROOTNGSMINUTILS/bamUtil_Coverage_Tool/MVL_per_bp_unique.txt" +fi +if [[ -z "${MVLTABIX-}" ]]; then + MVLTABIX="$EBROOTNGSMINUTILS/bamUtil_Coverage_Tool/MVL_per_bp_unique_tabix.txt" +fi +if [[ -z "${TmpDirBam=-}" ]]; then + usage + exit 1 +fi +if [[ -z "${QCMVLDIR-}" ]]; then + QCMVLDIR="$EBROOTNGSMINUTILS/bamUtil_Coverage_Tool/" +fi +echo "SAMPLEDIR: ${SAMPLEDIR}" +echo "BAMSTAT: ${BAMSTAT}" +echo "MVL: ${MVL}" +echo "MVLTABIX: ${MVLTABIX}" +echo "TMPDIR: ${TmpDirBam}" +echo "QCMVLDIR: ${QCMVLDIR}" +echo "starting" + + +rm -rf "${TmpDirBam}" +mkdir -p "${TmpDirBam}"/input/ +mkdir -p "${TmpDirBam}"/outputfilter/ +mkdir -p "${TmpDirBam}"/output_final/ +mkdir -p "${TmpDirBam}"/output_final_txt_files/ + + +#runBamStatWithRegion3.sh run the BAM stat tool to generate QC report per position +echo "start Bam stats" + +count=0 + +for i in $(ls "${SAMPLEDIR}"/*.bam) +do + echo "processingBamstat ${i}" + "${BAMSTAT}" stats \ + --in "${i}" \ + --cBaseQC "${TmpDirBam}"/input/outputBamUtil_${count}.txt \ + --regionlist "${MVL}" \ + --withinRegion \ + --nophonehome + + count=$((count+1)) + +done +echo "finisched Bam stats" + + +# test.sh, make QC report complete by adding also position with 0x coverage + +echo "start tabix 0x coverage" +for i in $(ls "${TmpDirBam}"/input/output*.txt) +do + b=$(basename "${i}") + bedfile=${b%.*}.bed.txt + awk 'BEGIN{OFS="\t"}{print $1,$2,($3-1),$4}' ${i} > "${TmpDirBam}"/"${bedfile}" + +sed -i '1d' "${TmpDirBam}"/"${bedfile}" +bgzip -c "${TmpDirBam}"/"${bedfile}" > "${TmpDirBam}"/"${bedfile}".gz + tabix -0 -p bed "${TmpDirBam}"/"${bedfile}".gz +rm -f "${TmpDirBam}"/outputfilter/${b%.*}_filter.txt + while read line + do + if tabix -0 "${TmpDirBam}"/"${bedfile}".gz ${line} | grep -q "^" + then + tabix -0 "${TmpDirBam}"/"${bedfile}".gz ${line} + else + echo $line | awk -F":" '{print $1"\t"$2}' | awk -F"-" '{print $1"\t"$2"\t0"}' + fi + done < "${MVLTABIX}" >> "${TmpDirBam}"/outputfilter/${b%.*}_filter.txt +echo "${i} done tabix 0x coverage" +done + +# test_chr_pos_cov.sh, make files usable for python script including 0x +echo "starting making final txt files per patient" +for i in $(ls ${TmpDirBam}/outputfilter/output*.txt) +do + b=$(basename ${i}) + filename=${b%.*} + + awk '{ + if ($4==0) { + print $1"\t"($2)"\t"$4 + } + else{ + print $1"\t"$2"\t"$4 + } + }' $i > "${TmpDirBam}/output_final/${filename}_final.txt" +echo "${i} done" +done +echo "finisched making final txt per patient" + +#add gene name to coverage file. +echo "add gene name" +sort -V "${MVL}" > "${MVL}".sorted +awk '{print $4}' "${MVL}" > "${MVL}".genes + +for i in $(ls "${TmpDirBam}"/output_final/outputBamUtil_*_filter_final.txt) +do + + sortedFile=${i%.*}.sorted.txt + sort -V ${i} > ${sortedFile} + a=$(diff <(awk '{print $2}' "${MVL}.sorted") <(awk '{print $2}' "${sortedFile}")) + if [ "${a}" == "" ] + then + paste -d"\t" $i "${MVL}".genes > ${i%.*}_gene.txt + else + echo 'positions in MVL and BAMutil output do not match' + fi +echo "${i} done adding gene name" +done +echo "start python script" + +python "${QCMVLDIR}"/MerdgeQCofMVL_file.py --in "${TmpDirBam}"/output_final/ --ff "${TmpDirBam}"/output_final_txt_files/ + diff --git a/bamUtil_Coverage_Tool/MVL_per_bp_unique.txt 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compare GT to ASE effect') +parser.add_argument('--inn', help='BAM util output file, input for python script, ${TMPDIR}/output_final/') +parser.add_argument('--ff', help='directory to store final output txt files ${TMPDIR}/output_final_txt_files/') + +args = parser.parse_args() +print('inputfiles:', args.inn) +print ('directory output files:', args.ff) + +outputlist = glob.glob((args.inn)+'/*_gene.txt') +print ('number of files:', len(outputlist)) +## Makes a dictionary with key variant position and values the coverage of all patient in inputlist. +QCdict = {} + +for inputQCfile in outputlist: + with open(inputQCfile) as h: + for line in h: + splitlineA = line.rstrip('\n') + splitlineB = splitlineA.split('\t') + VariantPosition = splitlineB[3]+'_'+splitlineB[0]+':'+splitlineB[1] + if VariantPosition not in QCdict: + QCdict[VariantPosition] = [] + QCdict[VariantPosition].append(int(splitlineB[2])) + else: + QCdict[VariantPosition].append(int(splitlineB[2])) +#print (QCdict) + +## makes a dictionary with key variant position and the sum of the coverage of all patients in the input list divided by 50. +## and print the variants with coverage < 1, <10, <30 +gemdict = {} +Covdict30 = {} + + +for pos in QCdict: + count = 0 + SDposition = statistics.pstdev(QCdict[pos]) + SEposition = SDposition/math.sqrt(len(outputlist)) + medianpos = statistics.median(QCdict[pos]) + tellerpos = 0 + teller30 = 0 + teller20 = 0 + teller10 = 0 + for cov in QCdict[pos]: + count+=int(cov) + if cov > 30: + teller30+=1 + if cov > 20: + teller20+=1 + if cov > 10: + teller10+=1 + tellerpos+=1 + per30x = (float(teller30)/float(tellerpos))*100 + per20x = (float(teller20)/float(tellerpos))*100 + per10x = (float(teller10)/float(tellerpos))*100 + minSDcov = (count/len(outputlist))-(SDposition*3) + maxSDcov = (count/len(outputlist))+(SDposition*3) + gemcount = count/len(outputlist) + gemdict[pos] = ["%.2f" % gemcount, "%.2f" % SDposition, round(minSDcov, 0), round(maxSDcov, 0), "%.2f" % SEposition, "%.2f" % int(per30x), "%.2f" % int(per20x), "%.2f" % int(per10x), medianpos] + +#print (gemdict) + + +# it will write a txt file with the gene, position, average coverage, SD , and percentage of samples with coverage above 30x, 20x and 10x. +with open((args.ff)+'/Avrg_SD_moreThan30x20x10x.txt', 'w') as averagefile: + averagefile.write('Gene\tPosition\tAverageCov\tSD\tMoreThan30x\tMoreThan20x\tMoreThan10x') + for pos in gemdict: + name = pos.split('_') + gene = name[0] + position = name[1] + averagefile.write('\n'+gene+'\t'+position+'\t'+str(gemdict[pos][0])+'\t'+str(gemdict[pos][1])+'\t'+str(gemdict[pos][5])+'\t'+str(gemdict[pos][6])+'\t'+str(gemdict[pos][7])) + +#file with all the position with average coverage of below 1x, 10x and 30x. +with open((args.ff)+'/PosBelow_30x_10x_1x.txt', 'w') as outfile: + outfile.write('Gene\tPosition\tAverageCov\tCov<') + for pos in gemdict: + name = pos.split('_') + gene = name[0] + position = name[1] + if float(gemdict[pos][0]) < 1.00: + outfile.write('\n'+gene+'\t'+position+'\t'+str(gemdict[pos][0])+'\t'+'<1x') + elif float(gemdict[pos][0]) < 10.00: + outfile.write('\n'+gene+'\t'+position+'\t'+str(gemdict[pos][0])+'\t'+'<10x') + elif float(gemdict[pos][0]) < 30.00: + outfile.write('\n'+gene+'\t'+position+'\t'+str(gemdict[pos][0])+'\t'+'<30x') + +# file for Roan in Molgenis +with open((args.ff)+'/Avrg_SD_morethan20x10x_Molgenis.txt', 'w') as averagefile: + averagefile.write('Chr\tStart\tStop\tGene\tAvgCoverage\tSD\tMoreThan10x\tMoreThan20x\tMedian') + for pos in gemdict: + name = pos.split('_') + gene = name[0] + positionchr = name[1].split(':') + chr = positionchr[0] + positionstart = int(positionchr[1])+1 + positionstop = int(positionchr[1])+1 + averagefile.write('\n'+chr+'\t'+str(positionstart)+'\t'+str(positionstop)+'\t'+gene+'\t'+str(gemdict[pos][0])+'\t'+str(gemdict[pos][1])+'\t'+str(gemdict[pos][7])+'\t'+str(gemdict[pos][6])+'\t'+str(gemdict[pos][8])) + +# makes a txt file with position of patient with a minimal or maximal coverage 3xSD outside the average coverage. +with open((args.ff)+'/MVL_outliers.txt', 'w') as SDfile: + SDfile.write('Gene\tPosition\tminCov3xSD\tmaxCov3xSD\tCoverage\tavgCov\tSD\tSE') + for pos in gemdict: + name = pos.split('_') + gene = name[0] + position = name[1] + for cov in QCdict[pos]: + if cov not in range(int(gemdict[pos][2]), int(gemdict[pos][3])): + #print (cov, pos, range(int(gemdict[pos][2]), int(gemdict[pos][3]))) + SDfile.write('\n'+gene+'\t'+position+'\t'+str(gemdict[pos][2])+'\t'+str(gemdict[pos][3])+'\t'+str(cov)+'\t'+str(gemdict[pos][0])+'\t'+str(gemdict[pos][1])+'\t'+str(gemdict[pos][4])) + + + + + diff --git a/bamUtil_Coverage_Tool/README.md b/bamUtil_Coverage_Tool/README.md new file mode 100644 index 0000000..25dc705 --- /dev/null +++ b/bamUtil_Coverage_Tool/README.md @@ -0,0 +1,27 @@ +This tool is calculating coverage per position of pathogenic and likely pathogenic varaints from the Managed Variant List (MVL) (UMCG-MVL_VKGLconsensusMVL-2017-11-27). + +to test this tool version:bamUtil-1.0.14 was used. + +run MVL_Coverage_Tool.sh to execute + +required input is a folder containing BAM files. + -s| locaion of folder with bam files + -t| Give loaction of tmp folder for inbetween and final files +optional input + -b|--bamstat path to the bam stats (not availeble on cluster) + -m|--mvl location of MVL_per_bp_unique.txt (default:$EBROOTNGSMINUTILS/bamUtil_Coverage_Tool//MVL_per_bp_unique.txt) + -x|--mvltabix location of MVL_per_bp_unique_tabix.txt (default: $EBROOTNGSMINUTILS/bamUtil_Coverage_Tool/MVL_per_bp_unique_tabix.txt) + -q|--qcmvldir location of MerdgeQCofMVL_file.py necessary for final txt generation (default:$EBROOTNGSMINUTILS/bamUtil_Coverage_Tool/) + + -b The bam stats tool is located here /bamUtil_Coverage_Tool//bamUtil-1.0.14/bin/bam stats. + +-mx you can make your own position list or MVL, make sure you also make a tabix.txt of this file. even if you give regions, the output will be per bp. + +-t location for in-between and final files. + +-q a python scrip generating the 4 txt files. + Avrg_SD_moreThan30x20x10x.txt: all postions with average coverage, SD, percenateg more than 30x, 20x and 10x. + Avrg_SD_moretha20x10x_Molgenis.txt: like Avrg_SD_moreThan30x20x10x.txt but fit for Molgenis96 + MVL_outliers.txt: all the outliers of the input sample set, if coverage is above or below 3x the SD from the average, it is in this txt file. + PosBelow_30x_10x_1x.txt: all position with an average coverage below 30x, 10x and 1x are in this txt file. +