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make_alignment_VC_CNV.sh
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make_alignment_VC_CNV.sh
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#!/bin/bash
###########################################
## WES Pipeline for somatic and germline ##
###########################################
# script to run the actual analysis
# Version 20.12.2018
case=$1; # somatic or somaticGermline
task=$2; # GD or TD (alignment)
num=$3; # 73
sex=$4;
##################################################################################################################
#### Parameters which have to be adjusted accoridng the the environment or the users needs
## General
homedata="/path/to/data" # folder contatining the raw data (.fastq files)
annot="/path/to/annotation" # folder containing annotation files like captureRegions.bed
motherpath="/path/to/output"
mtb="${motherpath}/${case}_${num}" # folder containing output
wes="${mtb}/WES"
ana="${mtb}/Analysis"
RscriptPath="${motherpath}/RScripts"
DatabasePath="${motherpath}/Databases"
tempdir="/scratch" # temporary folder
## Genome
GENOME="/path/to/ref/genome/including/index/hg19.fa"
Chromosomes="/path/to/ref/genome/chromosomes"
ChromoLength="/path/to/ref/chromosomes/length/hg19_chr.len"
## SureSelect (Capture Kit)
CaptureRegions="${annot}/Agilent/SureSelectV5UTR/V5UTR.bed"
## dbSNP vcf File
dbSNPvcf="/path/to/dbSNP/dbSNP/snp150hg19.vcf.gz "
### Software
## Parameters
## General
# Cores to use
nCore=12
minBaseQual="28"
minVAF="0.10"
# VarScan somatic
minCoverage="8"
TumorPurity="0.5"
minFreqForHom="0.75" # VAF to call homozygote
# VarScan fpfilter
minVarCount="4"
# ANNOVAR Databases
protocol='refGene,gnomad_exome,exac03,esp6500siv2_ea,1000g2015aug_eur,avsnp150,clinvar_20180603,intervar_20180118,dbnsfp35a,cosmic86_coding,cosmic86_noncoding'
argop='g,f,f,f,f,f,f,f,f,f,f'
## Tools and paths
# Paths
soft="/path/to/tools/software" # folder containing all used tools
java="${soft}/bin/java -Djava.io.tmpdir=${tempdir} " # path to java
# Pre-Processing
FASTQC="${soft}/FastQC/fastqc -t ${nCore} --extract "
TRIM="${java} -Xmx150g -jar ${soft}/Trimmomatic-0.36/trimmomatic-0.36.jar PE -threads ${nCore} -phred33 "
CUT="cut -f1,2,3"
# Alignment
BWAMEM="${soft}/bin/bwa mem -M "
# BAM-Readcount
BamReadcount="${soft}/bin/bam-readcount -q 1 -b 20 -w 1 -f ${GENOME} "
# SAMTOOLS
SAMTOOLS="${soft}/bin/samtools" # path to samtools
SAMVIEW="${SAMTOOLS} view -@ ${nCore} "
SAMSORT="${SAMTOOLS} sort -@ ${nCore} "
SAMRMDUP="${SAMTOOLS} rmdup "
SAMINDEX="${SAMTOOLS} index "
MPILEUP="${SAMTOOLS} mpileup -B -C 50 -f ${GENOME} -q 1 --min-BQ ${minBaseQual}"
STATS="${SAMTOOLS} stats "
# GATK
GATK="${soft}/bin/gatk"
RealignerTargetCreator="${GATK} -T RealignerTargetCreator -R ${GENOME} -nt ${nCore} "
IndelRealigner="${GATK} -R ${GENOME} -T IndelRealigner "
BaseRecalibrator="${GATK} -T BaseRecalibrator -l INFO -R ${GENOME} -knownSites ${dbSNPvcf} -nct ${nCore} "
PrintReads="${GATK} -T PrintReads -R ${GENOME} -nct ${nCore} "
# PICARD
FixMate="${soft}/bin/picard FixMateInformation "
# VARSCAN
VarScan="${soft}/anaconda2/bin/varscan"
SOMATIC="${VarScan} somatic"
PROCESSSOMATIC="${VarScan} processSomatic"
# ANNOVAR
CONVERT2ANNOVAR2="${soft}/annovar/convert2annovar.pl --format vcf4old --outfile "
CONVERT2ANNOVAR3="${soft}/annovar/convert2annovar.pl --format vcf4old --includeinfo --comment --outfile "
CONVERT2ANNOVAR="${soft}/annovar/convert2annovar.pl --format vcf4 --includeinfo --comment --withzyg --outfile "
TABLEANNOVAR="${soft}/annovar/table_annovar.pl"
# COVERAGE
COVERAGE="${soft}/bedtools2/bin/bedtools coverage -hist -g ${GENOME}.fai -sorted "
# SNPEFF
SNPEFF="${java} -Xmx150g -jar ${soft}/snpEff/snpEff.jar GRCh37.75 -c ${soft}/snpEff/snpEff.config -canon -v"
# ControlFREEC
freec="${soft}/bin/freec "
gemMappabilityFile="${soft}/FREEC-11.0/mappability/out100m2_hg19.gem"
# R
Rscript="${soft}/bin/Rscript"
##################################################################################################################
##########
## MAIN ##
##########
# SAMPLE
NameD=${case}_${num}_${task}
xx=${4};
InputPath=${homedata}/ngs/$xx/fastq ## change later !!!
Input1File=${5}1; # filename without extension
Input2File=${5}2; # filename without extension
# temp files
fastq1=${InputPath}/${Input1File}.fastq.gz
fastq2=${InputPath}/${Input2File}.fastq.gz
fastq_o1_p_t=${tempdir}/${NameD}_output1_paired_trimmed.fastq.gz
fastq_o1_u_t=${tempdir}/${NameD}_output1_unpaired_trimmed.fastq.gz
fastq_o2_p_t=${tempdir}/${NameD}_output2_paired_trimmed.fastq.gz
fastq_o2_u_t=${tempdir}/${NameD}_output2_unpaired_trimmed.fastq.gz
bam=${tempdir}/${NameD}_output.bam
prefixsort=${tempdir}/${NameD}_output.sort
sortbam=${tempdir}/${NameD}_output.sort.bam
rmdupbam=${tempdir}/${NameD}_output.sort.filtered.rmdup.bam
bai=${tempdir}/${NameD}_output.sort.filtered.rmdup.bai
bamlist=${tempdir}/${NameD}_output.sort.filtered.rmdup.bam.list
realignedbam=${tempdir}/${NameD}_output.sort.filtered.rmdup.realigned.bam
realignedbai=${tempdir}/${NameD}_output.sort.filtered.rmdup.realigned.bai
fixedbam=${tempdir}/${NameD}_output.sort.filtered.rmdup.realigned.fixed.bam
fixedbai=${tempdir}/${NameD}_output.sort.filtered.rmdup.realigned.fixed.bai
csv=${tempdir}/${NameD}_output.sort.filtered.rmdup.realigned.fixed.recal_data.csv
recalbam=${wes}/${NameD}_output.sort.filtered.rmdup.realigned.fixed.recal.bam
statstxt=${wes}/${NameD}_stats.txt
coveragetxt=${wes}/${NameD}_coverage.all.txt
### program calls
## alignment -----------------------------------------------------------------------------------------------------
if [ ${task} = GD ] || [ ${task} = TD ]
then
# fastqc zip to WES
${FASTQC} ${fastq1} -o ${wes}
${FASTQC} ${fastq2} -o ${wes}
# trim fastq
${TRIM} ${fastq1} ${fastq2} ${fastq_o1_p_t} ${fastq_o1_u_t} ${fastq_o2_p_t} ${fastq_o2_u_t} ILLUMINACLIP:${soft}/Trimmomatic-0.36/adapters/TruSeq3-PE-2.fa:2:30:10 HEADCROP:3 TRAILING:10 MINLEN:25
${FASTQC} ${fastq_o1_p_t} -o ${wes}
${FASTQC} ${fastq_o2_p_t} -o ${wes}
# make bam
${BWAMEM} -R "@RG\tID:${NameD}\tSM:${NameD}\tPL:illumina\tLB:lib1\tPU:unit1" -t 12 ${GENOME} ${fastq_o1_p_t} ${fastq_o2_p_t} | ${SAMVIEW} -bS - > ${bam}
rm ${fastq_o1_p_t} ${fastq_o1_u_t}
rm ${fastq_o2_p_t} ${fastq_o2_u_t}
# stats
${STATS} ${bam} > ${statstxt}
# sort bam
${SAMSORT} ${bam} -T ${prefixsort} -o ${sortbam}
rm ${bam}
# rmdup bam
${SAMVIEW} -b -f 0x2 -q1 ${sortbam} | ${SAMRMDUP} - ${rmdupbam}
rm ${sortbam}
# make bai
${SAMINDEX} ${rmdupbam} ${bai}
# make bam list
${RealignerTargetCreator} -o ${bamlist} -I ${rmdupbam}
# realign bam
${IndelRealigner} -I ${rmdupbam} -targetIntervals ${bamlist} -o ${realignedbam}
rm ${bamlist} ${bai} ${rmdupbam}
rm -r ${tempdir}
# fix bam
${FixMate} INPUT=${realignedbam} OUTPUT=${fixedbam} SO=coordinate VALIDATION_STRINGENCY=LENIENT CREATE_INDEX=true
rm ${realignedbam} ${realignedbai}
rm -r ${tempdir}
# make csv
${BaseRecalibrator} -I ${fixedbam} -cov ReadGroupCovariate -cov QualityScoreCovariate -cov CycleCovariate -cov ContextCovariate -o ${csv}
# recal bam
${PrintReads} -I ${fixedbam} -BQSR ${csv} -o ${recalbam}
rm ${csv} ${fixedbam} ${fixedbai}
rm -r ${tempdir}
# coverage
${COVERAGE} -b ${recalbam} -a ${CaptureRegions} | grep '^all' > ${coveragetxt}
# zip
${FASTQC} ${recalbam} -o ${wes}
fi
# eo alignment
## variantCalling ------------------------------------------------------------------------------------------------
if [ $task = VC ]
then
NameD=${case}_${num}_${task}
NameGD=${case}_${num}_GD
NameTD=${case}_${num}_TD
recalbamGD=${wes}/${NameGD}_output.sort.filtered.rmdup.realigned.fixed.recal.bam
recalbamTD=${wes}/${NameTD}_output.sort.filtered.rmdup.realigned.fixed.recal.bam
snpvcf=${wes}/${NameD}.output.snp.vcf
indelvcf=${wes}/${NameD}.output.indel.vcf
${MPILEUP} ${recalbamGD} ${recalbamTD} | ${SOMATIC} --output-snp ${snpvcf} --output-indel ${indelvcf} --min-coverage ${minCoverage} --tumor-purity ${TumorPurity} --min-var-freq ${minVAF} --min-freq-for-hom ${minFreqForHom} --min-avg-qual ${minBaseQual} --output-vcf 1 --mpileup 1
# Processing of somatic mutations
${PROCESSSOMATIC} ${snpvcf} --min-tumor-freq ${minVAF}
${PROCESSSOMATIC} ${indelvcf} --min-tumor-freq ${minVAF}
# FP Filter: snp.Somatic.hc snp.LOH.hc snp.Germline.hc
# FP Filter: indel.Somatic.hc indel.LOH.hc indel.Germline.hc
names1="snp indel"
for name1 in ${names1}
do
if [ ${case} = somatic ]; then
names2="Somatic LOH"
else
names2="Somatic LOH Germline"
fi
for name2 in ${names2}
do
hc_vcf=${wes}/${NameD}.output.${name1}.${name2}.hc.vcf
hc_avi=${wes}/${NameD}.output.${name1}.${name2}.hc.avinput
hc_rci=${wes}/${NameD}.output.${name1}.${name2}.hc.readcount.input
hc_rcs=${wes}/${NameD}.output.${name1}.${name2}.hc.readcounts
hc_fpf=${wes}/${NameD}.output.${name1}.${name2}.hc.fpfilter.vcf
if [ ${name2} = Somatic ]; then
recalbam=${recalbamTD}
else
recalbam=${recalbamGD}
fi
${CONVERT2ANNOVAR2} ${hc_avi} ${hc_vcf}
${CUT} ${hc_avi} > ${hc_rci}
${BamReadcount} -l ${hc_rci} ${recalbam} > ${hc_rcs}
${VarScan} fpfilter ${hc_vcf} ${hc_rcs} --output-file ${hc_fpf} --keep-failures 1 --min-ref-basequal ${minBaseQual} --min-var-basequal ${minBaseQual} --min-var-count ${minVarCount} --min-var-freq ${minVAF}
done
done
data=${wes}
for name1 in ${names1}
do
# Annotation snp.Somatic.hc $data/NameD.output.snp.Somatic.hc.fpfilter.vcf
# Annotation indel.Somatic.hc $data/NameD.output.indel.Somatic.hc.fpfilter.vcf
hc_=${data}/${NameD}.output.${name1}.Somatic.hc
hc_fpf=${data}/${NameD}.output.${name1}.Somatic.hc.fpfilter.vcf
hc_T_avi=${data}/${NameD}.output.${name1}.Somatic.hc.TUMOR.avinput
hc_T_avi_multi=${data}/${NameD}.output.${name1}.Somatic.hc.TUMOR.avinput.hg19_multianno.csv
${CONVERT2ANNOVAR} ${hc_} ${hc_fpf} -allsample
${TABLEANNOVAR} ${hc_T_avi} ${soft}/annovar/humandb/ -protocol ${protocol} -buildver hg19 -operation ${argop} -csvout -otherinfo -remove -nastring NA
hc_snpeff=$data/${NameD}.output.$name1.Somatic.SnpEff.vcf
${SNPEFF} ${hc_fpf} > ${hc_snpeff}
if [ $case = somaticGermline ]; then
# Annotation snp.Germline.hc $data/NameD.output.snp.Germline.hc.fpfilter.vcf
# Annotation indel.Germline.hc $data/NameD.output.indel.Germline.hc.fpfilter.vcf
hc_=${data}/${NameD}.output.${name1}.Germline.hc
hc_fpf=${data}/${NameD}.output.${name1}.Germline.hc.fpfilter.vcf
hc_N_avi=${data}/${NameD}.output.${name1}.Germline.hc.NORMAL.avinput
hc_N_avi_multi=${data}/${NameD}.output.${name1}.Germline.hc.NORMAL.avinput.hg19_multianno.csv
${CONVERT2ANNOVAR} ${hc_} ${hc_fpf} -allsample
${TABLEANNOVAR} ${hc_N_avi} ${soft}/annovar/humandb/ -protocol ${protocol} -buildver hg19 -operation ${argop} -csvout -otherinfo -remove -nastring NA
hc_N_snpeff=${data}/${NameD}.output.${name1}.NORMAL.SnpEff.vcf
${SNPEFF} ${hc_fpf} > ${hc_N_snpeff}
fi
# Annotation snp.LOH.hc
# Annotation indel.LOH.hc
hc_vcf=${data}/${NameD}.output.${name1}.LOH.hc.vcf
hc_fpf=${data}/${NameD}.output.${name1}.LOH.hc.fpfilter.vcf
hc_avi=${data}/${NameD}.output.${name1}.LOH.hc.avinput
hc_avi_multi=${data}/${NameD}.output.${name1}.LOH.hc.avinput.hg19_multianno.csv
${CONVERT2ANNOVAR3} ${hc_avi} ${hc_fpf}
${TABLEANNOVAR} ${hc_avi} ${soft}/annovar/humandb/ -protocol ${protocol} -buildver hg19 -operation ${argop} -csvout -otherinfo -remove -nastring NA
hc_L_snpeff=${data}/${NameD}.output.${name1}.LOH.SnpEff.vcf
${SNPEFF} ${hc_fpf} > ${hc_L_snpeff}
done
fi
# eo VC
## CNV ----------------------------------------------------------------------------------------------------------
if [ ${task} = CNV ]; then
output="${wes}/CNV"
if [ ! -d ${output} ]; then
mkdir ${output}
fi
cat > ${wes}/CNV_config.txt <<EOI
[general]
chrFiles = ${Chromosomes}
chrLenFile = ${ChromoLength}
breakPointType = 4
breakPointThreshold = 1.2
forceGCcontentNormalization = 1
gemMappabilityFile = ${gemMappabilityFile}
intercept = 0
minCNAlength = 3
maxThreads = 12
noisyData = TRUE
outputDir = ${output}
ploidy = 2
printNA = FALSE
readCountThreshold = 50
samtools = ${SAMTOOLS}
sex = ${sex}
step = 0
window = 0
uniqueMatch = TRUE
contaminationAdjustment = TRUE
[sample]
mateFile = ${wes}/${case}_${num}_TD_output.sort.filtered.rmdup.realigned.fixed.recal.bam
inputFormat = BAM
mateOrientation = FR
[control]
mateFile = ${wes}/${case}_${num}_GD_output.sort.filtered.rmdup.realigned.fixed.recal.bam
inputFormat = BAM
mateOrientation = FR
[target]
captureRegions = ${CaptureRegions}
EOI
export PATH=${PATH}:${SAMTOOLS}
${freec}-conf ${wes}/CNV_config.txt
fi
# eo CNV
## Report -------------------------------------------------------------------------------------------------------
if [ ${task} = Report ]; then
cd ${ana}
${Rscript} ${ana}/Main.R ${case} ${num} ${4} ${5} ${mtb} ${RscriptPath} ${DatabasePath}
${Rscript} -e "library(knitr); knit('Report.Rnw')"
${soft}/pdflatex -interaction=nonstopmode Report.tex
${soft}/pdflatex -interaction=nonstopmode Report.tex
fi
# eo Report
echo "task ${task} for ${num} finished"
rm .STARTING_MARKER_${task}
exit