#!/bin/bash/
fcid=$1 ##################################################### ########### preparo genoma di riferimento ########### cat /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/*.fa > /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.fa 
/home/vm-gen-ngs/Scrivania/PROGRAMMI/bwa-0.7.10/bwa index -p /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.fa -a bwtsw /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.fa

 ###################################################### ########### creo files bam indicizzati ########### creo SAM FILES con BWA mem ###################################################### /home/vm-gen-ngs/Scrivania/PROGRAMMI/bwa-0.7.10/bwa mem -R "@RG ID:IDa SM:SM PL:Illumina" -t 1 /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.fa /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1_R1_001.fastq.gz /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1_R2_001.fastq.gz > /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1_001.sam ###################################################### ## ALTERNATIVA creo SAM FILES con BWA aln # #################################################### /home/vm-gen-ngs/Scrivania/PROGRAMMI/bwa-0.7.10/bwa aln -q 20 -t 4 /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.fa /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1_R1_001.fastq.gz > /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1_R1_001.sai /home/vm-gen-ngs/Scrivania/PROGRAMMI/bwa-0.7.10/bwa aln -q 20 -t 4 /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.fa /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1_R2_001.fastq.gz > /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1_R2_001.sai /home/vm-gen-ngs/Scrivania/PROGRAMMI/bwa-0.7.10/bwa sampe -r "@RG ID:IDa SM:SM PL:Illumina" /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.fa /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1_R1_001.sai /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1_R2_001.sai /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1_R1_001.fastq.gz /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1_R2_001.fastq.gz > /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1_001.sam ##################################################### # CONVERTO SAM in BAM ##################################################### samtools view -bt /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.fa -o /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.bam /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1_001.sam samtools sort /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.bam /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1 samtools index /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.bam
 ######################################################################### ### ALTERNATIVA: CONVERTO SAM in BAM sorted con picard-tools ######################################################################### java -jar -Xms4g -Xmx4g /home/vm-gen-ngs/Scrivania/PROGRAMMI/picard-tools-1.119/SortSam.jar INPUT= /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1_001.sam OUTPUT= /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.sorted.bam SORT_ORDER=coordinate 
java -jar -Xms4g -Xmx4g /home/vm-gen-ngs/Scrivania/PROGRAMMI/picard-tools-1.119/BuildBamIndex.jar INPUT= /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.sorted.bam ##################################################### ### riallineamento locale ##################################################### samtools faidx /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.fa picard-tools CreateSequenceDictionary R= /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.fa O= /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.dict java -d64 -Xms4g -Xmx4g -jar /home/vm-gen-ngs/Scrivania/PROGRAMMI/GenomeAnalysisTK.jar -T RealignerTargetCreator -R /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.fa -I /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.sorted.bam -L:capture,BED /home/vm-gen-ngs/Scrivania/CORSO_NGS/TARGETS_AND_DATABASES/targets.interval_list -o /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.sorted.targets.list java -d64 -Xms4g -Xmx4g -jar /home/vm-gen-ngs/Scrivania/PROGRAMMI/GenomeAnalysisTK.jar -T IndelRealigner -I /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.sorted.bam -targetIntervals /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.sorted.targets.list -R /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.fa -known /home/vm-gen-ngs/Scrivania/CORSO_NGS/TARGETS_AND_DATABASES/1000G_phase1.indels.hg19.vcf -known /home/vm-gen-ngs/Scrivania/CORSO_NGS/TARGETS_AND_DATABASES/dbsnp_135.hg19.vcf --consensusDeterminationModel KNOWNS_ONLY -LOD 0.4 -o /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.realigned.bam ##################################################### ### RICALIBRAZIONE ##################################################### 
java -d64 -Xms4g -Xmx4g -jar /home/vm-gen-ngs/Scrivania/PROGRAMMI/GenomeAnalysisTK.jar -T BaseRecalibrator --maximum_cycle_value 600 -I /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.realigned.bam -R /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.fa -knownSites /home/vm-gen-ngs/Scrivania/CORSO_NGS/TARGETS_AND_DATABASES/dbsnp_135.hg19.vcf -cov QualityScoreCovariate -cov CycleCovariate -o /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.realigned.grp java -d64 -Xms4g -Xmx4g -jar /home/vm-gen-ngs/Scrivania/PROGRAMMI/GenomeAnalysisTK.jar -T PrintReads -I /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.realigned.bam -R /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.fa -BQSR /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.realigned.grp -o /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.bam rm -f, yes | rm /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/*.realigned*
rm -f, yes | rm /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/*.sorted*
 ##################################################### ########### chiamata varianti GATK ########### ##################################################### 
java -jar /home/vm-gen-ngs/Scrivania/PROGRAMMI/GenomeAnalysisTK.jar -T UnifiedGenotyper -I /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.bam -R /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.fa -o /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1_UNI.vcf -nct 200 -L:capture,BED /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/targets.interval_list -glm BOTH -dt NONE -stand_emit_conf 20
 java -jar /home/vm-gen-ngs/Scrivania/PROGRAMMI/GenomeAnalysisTK.jar -T HaplotypeCaller -I /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.bam -R /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.fa -o /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1_HAPLO.vcf -nct 200 -L:capture,BED /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/targets.interval_list -stand_emit_conf 20 ##################################################### ########### chiamata varianti VARSCAN ########## ##################################################### samtools mpileup -B -q 1 -f /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.fa /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.bam > /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.pileup java -jar /home/vm-gen-ngs/Scrivania/CORSO_NGS/PROGRAMMI/VarScan.v2.3.9.jar mpileup2snp /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.pileup --min-coverage 4 --min-reads2 4 --min-avg-qual 20 --min-var-freq 0.05 --p-value 0.05 --output-vcf 1 > /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1_varscan_snp.vcf java -jar /home/vm-gen-ngs/Scrivania/CORSO_NGS/PROGRAMMI/VarScan.v2.3.9.jar mpileup2indel /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.pileup --min-coverage 4 --min-reads2 4 --min-avg-qual 20 --min-var-freq 0.05 --p-value 0.05 --output-vcf 1 > /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1_varscan_indel.vcf java -jar /home/vm-gen-ngs/Scrivania/CORSO_NGS/PROGRAMMI/VarScan.v2.3.9.jar mpileup2cns /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.pileup --min-coverage 4 --min-reads2 4 --min-avg-qual 20 --min-var-freq 0.05 --p-value 0.05 --output-vcf 1 > /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1_varscan_cns.vcf ##################################################################### ########### chiamata varianti SOMATICHE VARSCAN ########### ##################################################################### ### Passaggio Fastq BAM saltando per ragioni di tempo la parte della ricalibrazione e allineamento locale /home/vm-gen-ngs/Scrivania/PROGRAMMI/bwa-0.7.10/bwa mem -R "@RG ID:IDa SM:SM PL:Illumina" -t 1 /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.fa /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/SOMATIC/PT_05_CK19p_S12_L001_R1_001.fastq.gz /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/SOMATIC/PT_05_CK19p_S12_L001_R2_001.fastq.gz > /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/SOMATIC/SOMATIC_SANE.sam ###### /home/vm-gen-ngs/Scrivania/PROGRAMMI/bwa-0.7.10/bwa mem -R "@RG ID:IDa SM:SM PL:Illumina" -t 1 /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.fa /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/SOMATIC/T_05_CK19p_S1_L001_R1_001.fastq.gz /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/SOMATIC/T_05_CK19p_S1_L001_R2_001.fastq.gz > /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/SOMATIC/SOMATIC_TUMORAL.sam ###### java -jar -Xms4g -Xmx4g /home/vm-gen-ngs/Scrivania/PROGRAMMI/picard-tools-1.119/SortSam.jar INPUT= /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/SOMATIC/SOMATIC_TUMORAL.sam OUTPUT= /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/SOMATIC/SOMATIC_TUMORAL.bam SORT_ORDER=coordinate ###### java -jar -Xms4g -Xmx4g /home/vm-gen-ngs/Scrivania/PROGRAMMI/picard-tools-1.119/BuildBamIndex.jar INPUT= /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/SOMATIC/SOMATIC_TUMORAL.bam ###### java -jar -Xms4g -Xmx4g /home/vm-gen-ngs/Scrivania/PROGRAMMI/picard-tools-1.119/SortSam.jar INPUT= /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/SOMATIC/SOMATIC_SANE.sam OUTPUT= /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/SOMATIC/SOMATIC_SANE.bam SORT_ORDER=coordinate
 ###### java -jar -Xms4g -Xmx4g /home/vm-gen-ngs/Scrivania/PROGRAMMI/picard-tools-1.119/BuildBamIndex.jar INPUT= /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/SOMATIC/SOMATIC_SANE.bam ### Generazione files Pileup samtools mpileup -B -q 1 -f /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.fa /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/SOMATIC/SOMATIC_TUMORAL.bam > /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/SOMATIC/SOMATIC_TUMORAL.pileup ###### samtools mpileup -B -q 1 -f /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/reference.fa /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/SOMATIC/SOMATIC_SANE.bam > /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/SOMATIC/SOMATIC_SANE.pileup ### analisi con varscan java -jar /home/vm-gen-ngs/Scrivania/CORSO_NGS/PROGRAMMI/VarScan.v2.3.9.jar somatic /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/SOMATIC/SOMATIC_SANE.pileup /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/SOMATIC/SOMATIC_TUMORAL.pileup /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/SOMATIC/SOMATIC.output -min-coverage 10 -min-var-freq 0.08 -somatic-p-value 0.05 ###### java -jar /home/vm-gen-ngs/Scrivania/CORSO_NGS/PROGRAMMI/VarScan.v2.3.9.jar processSomatic /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/SOMATIC/SOMATIC.output.snp
java -jar /home/vm-gen-ngs/Scrivania/CORSO_NGS/PROGRAMMI/VarScan.v2.3.9.jar processSomatic /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/SOMATIC/SOMATIC.output.indel ### calcolo delle COPERTURE # mkdir /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/DEPTH/ samtools depth /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.bam > /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/DEPTH/EXAMPLE_1.deep.txt bamToBed -i /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.bam > /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/DEPTH/EXAMPLE_1.TOT.bed awk -F" " '$3 < '20' { print $1" "$2" "$2 }' /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.deep.txt > /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/DEPTH/EXAMPLE_1.underT.bed subtractBed -a /home/vm-gen-ngs/Scrivania/CORSO_NGS/TARGETS_AND_DATABASES/targets.interval_list.bed -b /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/DEPTH/EXAMPLE_1.TOT.bed > /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/DEPTH/EXAMPLE_1.zero.bed intersectBed -a /home/vm-gen-ngs/Scrivania/CORSO_NGS/TARGETS_AND_DATABASES/targets.interval_list.bed -b /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/DEPTH/EXAMPLE_1.underT.bed > /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/DEPTH/EXAMPLE_1.underTHRESHOLD.bed cat /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/DEPTH/EXAMPLE_1.underTHRESHOLD.bed /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/DEPTH/EXAMPLE_1.zero.bed > /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/DEPTH/EXAMPLE_1.RECOVER.bed sortBed -i /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/DEPTH/EXAMPLE_1.RECOVER.bed > /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/DEPTH/EXAMPLE_1.SORT.RECOVER.bed mergeBed -i /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/DEPTH/EXAMPLE_1.SORT.RECOVER.bed > /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/DEPTH/EXAMPLE_1.MERGED.RECOVER.bed ##################################################### ########### ANALISI COPERTURE con NGSRICH ########### ##################################################### java -Xms2g -Xmx4g -cp /home/vm-gen-ngs/Scrivania/PROGRAMMI/NGSRICH/NGSrich_0.7.8/bin NGSrich evaluate -r /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/EXAMPLE_1.bam -u hg19 -a /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/refGene.txt -t /home/vm-gen-ngs/Scrivania/CORSO_NGS/GENOME_REF/targets.interval_list.bed -o /home/vm-gen-ngs/Scrivania/CORSO_NGS/FASTQ_FILES/EXAMPLE_1/output.coverage -p 20 -h 200 --no-details
