Nov 23, 2018
ADBS Whole Genome Sequencing (WGS) analysis pipeline for Genomic-QC Report
- 1University of Cambridge
Protocol Citation: Ravi Dr More 2018. ADBS Whole Genome Sequencing (WGS) analysis pipeline for Genomic-QC Report. protocols.io https://dx.doi.org/10.17504/protocols.io.vuae6se
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 23, 2018
Last Modified: November 23, 2018
Protocol Integer ID: 18018
Abstract
Whole Genome Sequencing (WGS) analysis pipeline devloped for generating Genomic-QC Report in Accelerator Program for Discovery in Brain Disorders Using Stem Cells (ADBS) program.
Define paths and directories
Define paths and directories
Command
SAMPLE_PATH="/path/to/sample"
SAMPLE_NAME="test_sample"
SOFTWARE_PATH="/path/to/software"
DATABASES_PATH="/path/to/databases"
TEMP_DIR="/path/to/temp"Unzip the raw reads files from .gz to fastq format
Unzip the raw reads files from .gz to fastq format
Command
gunzip $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME*.fastq.gzQC check of R1 and R2 paired-end raw reads using FASTQC, Trimming poor quality reads using Prinseq-lite, and Adapter contimination removal using AfterQC
QC check of R1 and R2 paired-end raw reads using FASTQC, Trimming poor quality reads using Prinseq-lite, and Adapter contimination removal using AfterQC
Software versions used:
FASTQC version 0.10.1
Prinseq-lite version 0.20.4
AfterQC version 0.9.6
Command
$SOFTWARE_PATH/FastQC/fastqc $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_R1.fq
$SOFTWARE_PATH/FastQC/fastqc $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_R2.fq
cd $SAMPLE_PATH/$SAMPLE_NAME/
python $SOFTWARE_PATH/AfterQC-master/after.py -f -1 -t -1 -q 30 -1 $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_R1.fq -2 $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_R2.fq
$SOFTWARE_PATH/prinseq-lite-0.20.4/prinseq-lite.pl -fastq $SAMPLE_PATH/$SAMPLE_NAME/good/$SAMPLE_NAME\_R1.good.fq -fastq2 $SAMPLE_PATH/$SAMPLE_NAME/good/$SAMPLE_NAME\_R2.good.fq -out_good $SAMPLE_PATH/$SAMPLE_NAME/cleaned -out_bad null -min_qual_mean 30
mv $SAMPLE_PATH/$SAMPLE_NAME/cleaned_1.fastq $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_cleaned_R1.fastq
mv $SAMPLE_PATH/$SAMPLE_NAME/cleaned_2.fastq $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_cleaned_R2.fastq
$SOFTWARE_PATH/FastQC/fastqc $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_cleaned_R1.fastq
$SOFTWARE_PATH/FastQC/fastqc $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_cleaned_R2.fastq
mkdir -p $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_4_FASTQC
mv $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_cleaned_R1_fastqc.zip $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_4_FASTQC/$SAMPLE_NAME\_cleaned_R1_fastqc.zip
mv $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_cleaned_R2_fastqc.zip $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_4_FASTQC/$SAMPLE_NAME\_cleaned_R2_fastqc.zipAlignment of clened raw reads against Human Reference Genome hg19 GRCh37.p13 build using BWA and SAMTOOLS.
Alignment of clened raw reads against Human Reference Genome hg19 GRCh37.p13 build using BWA and SAMTOOLS.
BWA version 0.5.9
Samtools version 1.3.1
Command
# Align cleaned R1 reads with hg19
/softwares/bwa-0.5.9/bwa aln -t 30 $DATABASES_PATH/hg19_fa-chrMlast/hg19_chrM-last.fa $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_cleaned_R1.fastq > $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_R1.sai
# Align cleaned R2 reads with hg19
/softwares/bwa-0.5.9/bwa aln -t 30 $DATABASES_PATH/hg19_fa-chrMlast/hg19_chrM-last.fa $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_cleaned_R2.fastq > $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_R2.sai
#convert sai to sam by using cleaned fastq reads
/softwares/bwa-0.5.9/bwa sampe $DATABASES_PATH/hg19_fa-chrMlast/hg19_chrM-last.fa $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_R1.sai $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_R2.sai $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_cleaned_R1.fastq $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_cleaned_R2.fastq > $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME.sam
#convert sam to bam
/softwares/samtools1.3.1/bin/samtools view -bS $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME.sam > $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME.bam
#bam to sort file
/softwares/samtools1.3.1/bin/samtools sort $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME.bam -o $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_sorted.bam
#sort to flagstat
/softwares/samtools1.3.1/bin/samtools flagstat $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_sorted.bam > $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_sorted_flagstat.txt
#index the sorted bam
/softwares/samtools1.3.1/bin/samtools index $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_sorted.bam > $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_sortedbam.baiMark PCR duplicates and sorting BAM using PICARD Tools
Mark PCR duplicates and sorting BAM using PICARD Tools
Picard version 2.0.1
Samtools version 1.3.1
Command
#Remove PCR duplicates
java -Djava.io.tmpdir=$TEMP_DIR/ -Xmx50g -jar $SOFTWARE_PATH/picard/build/libs/picard.jar AddOrReplaceReadGroups I="$SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_sorted.bam" O="$SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_coordsort.bam" ID="1" LB="libraryname" PL="Illumina" PU="platform unit" SM=samplenname SO=coordinate VALIDATION_STRINGENCY=SILENT
java -Djava.io.tmpdir=$TEMP_DIR/ -Xmx50g -jar $SOFTWARE_PATH/picard/build/libs/picard.jar MarkDuplicates I="$SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_coordsort.bam" O="$SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam" M="metrics" REMOVE_DUPLICATES=true ASSUME_SORTED=true VALIDATION_STRINGENCY=LENIENT
#Index the coordinate sorted bam file
/softwares/samtools1.3.1/bin/samtools index $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bamINDEL re-alignment using GATK tools
INDEL re-alignment using GATK tools
GATK version 3.6
Command
java -Xmx8g -jar $SOFTWARE_PATH/GenomeAnalysisTK-3.6/GenomeAnalysisTK.jar -T RealignerTargetCreator -R $DATABASES_PATH/hg19_fa-chrMlast/hg19_chrM-last.fa -I $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam --known $DATABASES_PATH/REF_GENOME_hg19/1000G_phase1.indels.hg19.vcf -o $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_IndelRealigner.intervalsSNP and INDEL variant calling using Isaac Variant Caller tool and filter SNP and INDEL using rtg-tools
SNP and INDEL variant calling using Isaac Variant Caller tool and filter SNP and INDEL using rtg-tools
Isaac Variant Caller -- 1.0.7
rtg-tools version 3.7.1
Command
$SOFTWARE_PATH/isaac_variant_caller-master/bin/configureWorkflow.pl --bam=$SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam --ref=$DATABASES_PATH/hg19_fa-chrMlast/hg19_chrM-last.fa --config=$SAMPLE_PATH/config.ini --output-dir=$SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/
cd $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/
make -j 16
gzip -dc $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/results/$SAMPLE_NAME\_RMDUP.genome.vcf.gz | $SOFTWARE_PATH/gvcftools-0.16/bin/extract_variants | awk '/^#/ || $7 == "PASS"' > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/results/$SAMPLE_NAME\_RMDUP_all_passed_variants.vcf
$SOFTWARE_PATH/rtg-tools-3.7.1/rtg vcffilter --snps-only -i $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/results/$SAMPLE_NAME\_RMDUP_all_passed_variants.vcf -o $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/results/$SAMPLE_NAME\_snp_issac.vcf
$SOFTWARE_PATH/rtg-tools-3.7.1/rtg vcffilter --non-snps-only -i $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/results/$SAMPLE_NAME\_RMDUP_all_passed_variants.vcf -o $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/results/$SAMPLE_NAME\_indel_issac.vcf
cp $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/results/$SAMPLE_NAME\_snp_issac.vcf.gz $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/$SAMPLE_NAME\_snp.vcf.gz
cp $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/results/$SAMPLE_NAME\_indel_issac.vcf.gz $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/$SAMPLE_NAME\_indel.vcf.gz
gunzip $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/$SAMPLE_NAME\_snp.vcf.gz
gunzip $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/$SAMPLE_NAME\_indel.vcf.gzCheck the alignment QC of the bam file using Qualimap
Check the alignment QC of the bam file using Qualimap
Qualimap version 2.2.1
Command
mkdir -p $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_5_ALIGNMENT_QC
$SOFTWARE_PATH/qualimap_v2.2.1/qualimap bamqc -bam $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam -gff $DATABASES_PATH/trueseq1.bed -outdir $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_5_ALIGNMENT_QC/QualiMap_$SAMPLE_NAME\_trueseq1_bed -outfile $SAMPLE_NAME\_trueseq1.pdf --java-mem-size=500GVCF QC of SNP and INDEL files using rtg-tools
VCF QC of SNP and INDEL files using rtg-tools
rtg-tools version 3.7.1
Command
mkdir -p $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/VCF_QC
$SOFTWARE_PATH/rtg-tools-3.7.1/rtg vcfstats $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/$SAMPLE_NAME\_snp.vcf > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/VCF_QC/$SAMPLE_NAME\_snp.vcf.stat
$SOFTWARE_PATH/rtg-tools-3.7.1/rtg vcfstats $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/$SAMPLE_NAME\_indel.vcf > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/VCF_QC/$SAMPLE_NAME\_indel.vcf.statSNP AND INDEL variant annotation using ANNOVAR
SNP AND INDEL variant annotation using ANNOVAR
ANNOVAR reference assembly 65 with reference hg19
Command
mkdir -p $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/annotated_annovar
perl $SOFTWARE_PATH/annovar/convert2annovar.pl -format vcf4 $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/$SAMPLE_NAME\_snp.vcf > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/annotated_annovar/$SAMPLE_NAME\_snp.vcf.avinput
perl $SOFTWARE_PATH/annovar/convert2annovar.pl -format vcf4 $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/$SAMPLE_NAME\_indel.vcf > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/annotated_annovar/$SAMPLE_NAME\_indel.vcf.avinput
#perl $SOFTWARE_PATH/annovar/table_annovar.pl $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/$SAMPLE_NAME\_snp.vcf.avinput $SOFTWARE_PATH/annovar/humandb/ -buildver hg19 -out $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/annotated_annovar/$SAMPLE_NAME\_snp_annovar_annotation -remove -protocol refGene,cytoBand,genomicSuperDups,esp6500siv2_all,1000g2015aug_all,1000g2015aug_eur,exac03,avsnp147,dbnsfp30a -operation g,r,r,f,f,f,f,f,f -nastring . -csvout
perl $SOFTWARE_PATH/annovar/table_annovar.pl $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/$SAMPLE_NAME\_snp.vcf $SOFTWARE_PATH/annovar/humandb/ -buildver hg19 -out $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/annotated_annovar/$SAMPLE_NAME\_snp_annovar_annotation -remove -protocol refGene,cytoBand,genomicSuperDups,esp6500siv2_all,1000g2015aug_all,1000g2015aug_eur,exac03,avsnp147,dbnsfp30a -operation g,r,r,f,f,f,f,f,f -nastring . -vcfinput
perl $SOFTWARE_PATH/annovar/table_annovar.pl $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/$SAMPLE_NAME\_indel.vcf $SOFTWARE_PATH/annovar/humandb/ -buildver hg19 -out $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/annotated_annovar/$SAMPLE_NAME\_indel_annovar_annotation -remove -protocol refGene,cytoBand,genomicSuperDups,esp6500siv2_all,1000g2015aug_all,1000g2015aug_eur,exac03,avsnp147,dbnsfp30a -operation g,r,r,f,f,f,f,f,f -nastring . -vcfinputMitochondria analysis
Mitochondria analysis
Extracting mitochondrial reads from BAM file and creatiing anather BAM file to input mtDNA-Server tool for Mitochondria analysis
Samtools version 1.3
Command
mkdir -p $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_7_MITOCHONDRIA
/softwares/samtools1.3.1/bin/samtools view -b $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam chrM: -o $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_7_MITOCHONDRIA/$SAMPLE_NAME\_MT.bam
/softwares/samtools1.3.1/bin/samtools index $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_7_MITOCHONDRIA/$SAMPLE_NAME\_MT.bamBlood Group Prediction
Blood Group Prediction
BOOGIE - Phenotype prediction from NGS data Version: 1.0
Command
mkdir -p $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_10_blood_group
perl $SAMPLE_PATH/rename_phase2_blood_group_detection.pl $SAMPLE_NAME
perl $SAMPLE_PATH/rename_phase2_blood_group_summary.pl $SAMPLE_NAME
perl $SAMPLE_PATH/rename_phase2_blood_group_genes_extracter.pl $SAMPLE_NAME
chmod 755 $SAMPLE_PATH/$SAMPLE_NAME/*
perl $SAMPLE_PATH/$SAMPLE_NAME/phase2_blood_group_genes_extracter.pl
$SAMPLE_PATH/$SAMPLE_NAME/phase2_blood_group_detection.sh
perl $SAMPLE_PATH/$SAMPLE_NAME/phase2_blood_group_summary.plSNP-Chip rsID comparison with WGS rsID
SNP-Chip rsID comparison with WGS rsID
Command
mkdir -p $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_14_VIRTUAL_SNP
perl $SAMPLE_PATH/rename_phase2_1rsid_get.pl $SAMPLE_NAME
perl $SAMPLE_PATH/rename_phase2_2rsid_filter.pl $SAMPLE_NAME
perl $SAMPLE_PATH/rename_phase2_3rsid_venn.pl $SAMPLE_NAME
perl $SAMPLE_PATH/rename_phase2_4rsid_venn.pl $SAMPLE_NAME
perl $SAMPLE_PATH/rename_exonic_extract_common_indel.pl $SAMPLE_NAME
perl $SAMPLE_PATH/rename_exonic_extract_common_snp.pl $SAMPLE_NAME
perl $SAMPLE_PATH/rename_exonic_extract_rsid_indel.pl $SAMPLE_NAME
perl $SAMPLE_PATH/rename_exonic_extract_rsid_snp.pl $SAMPLE_NAME
perl $SAMPLE_PATH/rename_exonic_extract_unique_Illimina_snp.pl $SAMPLE_NAME
perl $SAMPLE_PATH/rename_exonic_extract_unique_indel_Illimina.pl $SAMPLE_NAME
perl $SAMPLE_PATH/rename_exonic_extract_unique_indel_sample.pl $SAMPLE_NAME
perl $SAMPLE_PATH/rename_exonic_extract_unique_sample_snp.pl $SAMPLE_NAME
perl $SAMPLE_PATH/rename_exonic_venn_snp_indel.pl $SAMPLE_NAME
chmod 755 $SAMPLE_PATH/$SAMPLE_NAME/*
perl $SAMPLE_PATH/$SAMPLE_NAME/phase2_1rsid_get.pl
perl $SAMPLE_PATH/$SAMPLE_NAME/phase2_2rsid_filter.pl
perl $SAMPLE_PATH/$SAMPLE_NAME/phase2_3rsid_venn.pl
perl $SAMPLE_PATH/$SAMPLE_NAME/phase2_4rsid_venn.pl
mkdir -p $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_14_VIRTUAL_SNP/exonic_rsid
perl $SAMPLE_PATH/$SAMPLE_NAME/exonic_extract_rsid_indel.pl
perl $SAMPLE_PATH/$SAMPLE_NAME/exonic_extract_rsid_snp.pl
perl $SAMPLE_PATH/$SAMPLE_NAME/exonic_extract_unique_indel_sample.pl
perl $SAMPLE_PATH/$SAMPLE_NAME/exonic_extract_unique_indel_Illimina.pl
perl $SAMPLE_PATH/$SAMPLE_NAME/exonic_extract_common_indel.pl
perl $SAMPLE_PATH/$SAMPLE_NAME/exonic_extract_unique_sample_snp.pl
perl $SAMPLE_PATH/$SAMPLE_NAME/exonic_extract_unique_Illimina_snp.pl
perl $SAMPLE_PATH/$SAMPLE_NAME/exonic_extract_common_snp.plExtract Damaging Varaints (SIFT, PolyPhen) from SNP file
Extract Damaging Varaints (SIFT, PolyPhen) from SNP file
Command
mkdir -p $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/damaging
mkdir -p $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_13_VARIANT_CALLING/damaging/snp
perl $SAMPLE_PATH/$SAMPLE_NAME/phase2_damaging_1_get_snv_snp.pl
perl $SAMPLE_PATH/$SAMPLE_NAME/phase2_damaging_2_merge.plHLA Analysis using HLAVBSeq
HLA Analysis using HLAVBSeq
Read data aligned to GRCh37/hg19 using HLA-VBSeq Software to predict HLA types
BWA version 0.5.9
Command
erl $SAMPLE_PATH/rename_hla_calculation.pl $SAMPLE_NAME
$SOFTWARE_PATH/bwa.kit/bwa mem -t 8 -P -L 10000 -a $SOFTWARE_PATH/HLA/hla_all.fasta $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_cleaned_R1.fastq $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_cleaned_R2.fastq > $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_part.sam
mkdir -p $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/
java -jar $SOFTWARE_PATH/HLA/HLAVBSeq.jar $SOFTWARE_PATH/HLA/hla_all.fasta $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_part.sam $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result --alpha_zero 0.01 --is_paired
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^A\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_A.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^B\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_B.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^C\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_C.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^DMA\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_DMA.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^DMB\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_DMB.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^DOA\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_DOA.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^DOB\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_DOB.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^DPA1\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_DPA1.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^DPB1\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_DPB1.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^DQA1\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_DQA1.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^DQB1\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_DQB1.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^DRA\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_DRA.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^DRB1\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_DRB1.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^DRB2\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_DRB2.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^DRB3\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_DRB3.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^DRB4\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_DRB4.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^DRB5\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_DRB5.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^DRB6\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_DRB6.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^DRB7\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_DRB7.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^DRB8\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_DRB8.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^DRB9\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_DRB9.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^E\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_E.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^F\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_F.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^G\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_G.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^H\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_H.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^J\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_J.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^K\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_K.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^L\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_L.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^MICA\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_MICA.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^MICB\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_MICB.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^P\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_P.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^TAP1\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_TAP1.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^TAP2\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_TAP2.txt
perl $SOFTWARE_PATH/HLA/parse_result.pl $SOFTWARE_PATH/HLA/Allelelist.txt $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/$SAMPLE_NAME\_part_result | grep "^V\*" | sort -k2 -n -r > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_11_HLA/HLA_V.txt
perl $SAMPLE_PATH/$SAMPLE_NAME/hla_calculation.plStructual Variants (SV) Analysis using GASV
Structual Variants (SV) Analysis using GASV
Geometric Analysis of Structural Variants (GASV) Version: 2.0
Command
mkdir -p $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_15_SV
perl $SAMPLE_PATH/rename_SV_gasv.pl $SAMPLE_NAME
perl $SAMPLE_PATH/$SAMPLE_NAME/SV_gasv.sh
cp /home/odity/ravim/$SAMPLE_NAME\_RMDUP.bam.gasv.in.clusters $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_15_SV/$SAMPLE_NAME\_RMDUP.bam.gasv.in.clusters
mv $SAMPLE_PATH/$SAMPLE_NAME/*_null* $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_15_SV/
mv $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam.gasv.in $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_15_SV/
perl $SAMPLE_PATH/rename_SV_count_type.pl $SAMPLE_NAME
perl $SAMPLE_PATH/$SAMPLE_NAME/SV_count_type.plGene Integration detection using string search
Gene Integration detection using string search
Samtools version 1.3
Command
#cmyc gene end (GE) 65
#TGTTGCGGAAACGACGAGAACAGTTGAAACACAAACTTGAACAGCTACGGAACTCTTGTGCGTAA
#vector start (VS) 15
#GAATTCGCTAGCGAT
#cmyc
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep TGTTGCGGAAACGACGAGAACAGTTGAAACACAAACTTGAACAGCTACGGAACTCTTGTGCGTAAGAATTCGCTAGCGAT > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_cmyc_GE65-VS15.sam
#rc
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep ATCGCTAGCGAATTCTTACGCACAAGAGTTCCGTAGCTGTTCAAGTTTGTGTTTCAACTGTTCTCGTCGTTTCCGCAACA > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_cmyc_GE65-VS15_rc.sam
#######################
#bmi
#gene end CTTCTTTTGCCAATAGACCTCGAAAATCATCAGTAAATGGGTCATCAGCAACTTCTTCTGGTTGA
#vec start GAATTCGCTAGCGAT
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep CTTCTTTTGCCAATAGACCTCGAAAATCATCAGTAAATGGGTCATCAGCAACTTCTTCTGGTTGAGAATTCGCTAGCGAT > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_bmi_GE65-VS15.sam
#rc
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep ATCGCTAGCGAATTCTCAACCAGAAGAAGTTGCTGATGACCCATTTACTGATGATTTTCGAGGTCTATTGGCAAAAGAAG > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_bmi_GE65-VS15_rc.sam
#####################
#bclxl
#gene end
#GGTTCCTGACGGGCATGACTGTGGCCGGCGTGGTTCTGCTGGGCTCACTCTTCAGTCGGAAATGA
# vec start
#GAATTCGCTAGCGAT
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep GGTTCCTGACGGGCATGACTGTGGCCGGCGTGGTTCTGCTGGGCTCACTCTTCAGTCGGAAATGAGAATTCGCTAGCGAT > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_bclxl_GE65-VS15.sam
#rc
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep ATCGCTAGCGAATTCTCATTTCCGACTGAAGAGTGAGCCCAGCAGAACCACGCCGGCCACAGTCATGCCCGTCAGGAACC > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_bclxl_GE65-VS15_rc.sam
#####################
#KLF4
#gene end GTTTGTATTTTGCATACTCAAGGTGAGAATTAAGTTTTAAATAAACCTATAATATTTTATCTGAA
#vec start GAATTCGCTAGCGAT
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep GTTTGTATTTTGCATACTCAAGGTGAGAATTAAGTTTTAAATAAACCTATAATATTTTATCTGAAGAATTCGCTAGCGAT > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_klf4_GE65-VS15.sam
#rc
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep ATCGCTAGCGAATTCTTCAGATAAAATATTATAGGTTTATTTAAAACTTAATTCTCACCTTGAGTATGCAAAATACAAAC > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_klf4_GE65-VS15.sam
#####################
#Lin28
#gene end TCCCTTCTCCTTTCCCTGGGAAAATACAATGAATAAATAAAGACTTATTGGTACGCAAACTGTCA
# vec start GAATTCGCTAGCGAT
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep TCCCTTCTCCTTTCCCTGGGAAAATACAATGAATAAATAAAGACTTATTGGTACGCAAACTGTCAGAATTCGCTAGCGAT > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_lin28_GE65-VS15.sam
#####################
#rc
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep ATCGCTAGCGAATTCTGACAGTTTGCGTACCAATAAGTCTTTATTTATTCATTGTATTTTCCCAGGGAAAGGAGAAGGGA > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_lin28_GE65-VS15_rc.sam
#####################
#oct
#gene end AAAATGTTGTAGCCAACAAGACTGGGATTCCCACATGTGCCATTCCGGAGCCGGAAAAGCCCTCG
#vec start GAATTCGCTAGCGAT
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep AAAATGTTGTAGCCAACAAGACTGGGATTCCCACATGTGCCATTCCGGAGCCGGAAAAGCCCTCGGAATTCGCTAGCGAT > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_oct_GE65-VS15.sam
#rc
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep ATCGCTAGCGAATTCCGAGGGCTTTTCCGGCTCCGGAATGGCACATGTGGGAATCCCAGTCTTGTTGGCTACAACATTTT > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_oct_GE65-VS15.sam
#####################
#sox2
#gene end ACTTAAGTTTTTACTCCATTATGCACAGTTTGAGATAAATAAATTTTTGAAATATGGACACTGAA
#Vec start GAATTCGCTAGCGAT
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep ACTTAAGTTTTTACTCCATTATGCACAGTTTGAGATAAATAAATTTTTGAAATATGGACACTGAAGAATTCGCTAGCGAT > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_sox2_GE65-VS15.sam
#rc
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep ATCGCTAGCGAATTCTTCAGTGTCCATATTTCAAAAATTTATTTATCTCAAACTGTGCATAATGGAGTAAAAACTTAAGT > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_sox2_GE65-VS15_rc.sam
# vector end 15 and gene start 65 in mapped region
##vector end 15 # TTGCGTACGGCCAGC
mkdir -p $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION
#cmyc
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep TTGCGTACGGCCAGCATGCCCCTCAACGTTAGCTTCACCAACAGGAACTATGACCTCGACTACGACTCGGTGCAGCCGTA > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_cmyc_VE15-GS65.sam
#rc
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep CGCTCTGCTGCTGCTGCTGGTAGAAGTTCTCCTCCTCGTCGCAGTAGAAATACGGCTGCACCGAGTCGTAGTCGAGGTCA > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_cmyc_VE15-GS65_rc.sam
#bmi
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep TTGCGTACGGCCAGCATGCATCGAACAACGAGAATCAAGATCACTGAGCTAAATCCCCACCTGATGTGTGTGCTTTGTGG > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_bmi_VE15-GS65.sam
#rc
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep AGAAGGAATGTAGACATTCTATTATGGTTGTGGCATCAATGAAGTACCCTCCACAAAGCACACACATCAGGTGGGGATTT > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_bmi_VE15-GS65_rc.sam
#bclxl
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep TTGCGTACGGCCAGCATGTCTCAGAGCAACCGGGAGCTGGTGGTTGACTTTCTCTCCTACAAGCTTTCCCAGAAAGGATA > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_bclxl_VE15-GS65.sam
#rc
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep CTGGGGCCTCAGTCCTGTTCTCTTCCACATCACTAAACTGACTCCAGCTGTATCCTTTCTGGGAAAGCTTGTAGGAGAGA > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_bclxl_VE15-GS65_rc.sam
#KLF4
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep TTGCGTACGGCCAGCAGTTTCCCGACCAGAGAGAACGAACGTGTCTGCGGGCGCGCGGGGAGCAGAGGCGGTGGCGGGCG > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_klf4_VE15-GS65.sam
#rc
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep GGGGCCAGAGGGGCGGGGGAGGGTCACTCGGCGGCTCCCGGTGCCGCCGCCGCCCGCCACCGCCTCTGCTCCCCGCGCGC > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_klf4_VE15-GS65.sam
#Lin28
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep TTGCGTACGGCCAGCGTGCGGGGGAAGATGTAGCAGCTTCTTCTCCGAACCAACCCTTTGCCTTCGGACTTCTCCGGGGC > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_lin28_VE15-GS65.sam
#rc
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep GCCCCGGAGAAGTCCGAAGGCAAAGGGTTGGTTCGGAGAAGAAGCTGCTACATCTTCCCCCGCACGCTGGCCGTACGCAA > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_lin28_VE15-GS65_rc.sam
#oct
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep TTGCGTACGGCCAGCTTGCTTTTGCAGATGTACCTTCTTAAAGTTTTTTCTTAAAGTTTGGGAAATATTGAAATACGCTT > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_oct_VE15-GS65.sam
#rc
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep AAGCGTATTTCAATATTTCCCAAACTTTAAGAAAAAACTTTAAGAAGGTACATCTGCAAAAGCAAGCTGGCCGTACGCAA > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_oct_VE15-GS65.sam
#sox2
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep TTGCGTACGGCCAGCGGATGGTTGTCTATTAACTTGTTCAAAAAAGTATCAGGAGTTGTCAAGGCAGAGAAGAGAGTGTT > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_sox2_VE15-GS65.sam
#rc
/softwares/samtools1.3/bin/samtools view $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_RMDUP.bam | grep AACACTCTCTTCTCTGCCTTGACAACTCCTGATACTTTTTTGAACAAGTTAATAGACAACCATCCGCTGGCCGTACGCAA > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_16_GENE_INTEGRATION/$SAMPLE_NAME\_sox2_VE15-GS65_rc.samMycoplasma Contimination detection using BWA
Mycoplasma Contimination detection using BWA
BWA version 0.5.9
Samtools version 1.3.1
Command
mkdir -p $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_18_Mycoplasma
#####Alaidlawii
/softwares/bwa-0.5.9/bwa aln -t 30 $SOFTWARE_PATH/Mycoplasma/Alaidlawii.fa $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_cleaned_R1.fastq > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_18_Mycoplasma/$SAMPLE_NAME\_R1_Alaidlawii.sai
/softwares/bwa-0.5.9/bwa samse $SOFTWARE_PATH/Mycoplasma/Alaidlawii.fa $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_18_Mycoplasma/$SAMPLE_NAME\_R1_Alaidlawii.sai $SAMPLE_PATH/$SAMPLE_NAME/$SAMPLE_NAME\_cleaned_R1.fastq > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_18_Mycoplasma/$SAMPLE_NAME\_R1_Alaidlawii.sam
/softwares/samtools1.3.1/bin/samtools view -bS $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_18_Mycoplasma/$SAMPLE_NAME\_R1_Alaidlawii.sam > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_18_Mycoplasma/$SAMPLE_NAME\_R1_Alaidlawii.bam
/softwares/samtools1.3.1/bin/samtools sort $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_18_Mycoplasma/$SAMPLE_NAME\_R1_Alaidlawii.bam -o $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_18_Mycoplasma/$SAMPLE_NAME\_R1_Alaidlawii_sorted.bam
/softwares/samtools1.3.1/bin/samtools flagstat $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_18_Mycoplasma/$SAMPLE_NAME\_R1_Alaidlawii_sorted.bam > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_18_Mycoplasma/$SAMPLE_NAME\_R1_Alaidlawii_sorted_flagstat.txt
/softwares/samtools1.3.1/bin/samtools index $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_18_Mycoplasma/$SAMPLE_NAME\_R1_Alaidlawii_sorted.bam > $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_18_Mycoplasma/$SAMPLE_NAME\_R1_Alaidlawii_sortedbam.bai
/softwares/samtools1.3.1/bin/samtools idxstats $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_18_Mycoplasma/$SAMPLE_NAME\_R1_Alaidlawii_sorted.bam
for BAM in $SAMPLE_PATH/$SAMPLE_NAME/Report_$SAMPLE_NAME\_18_Mycoplasma/*bam ; do
CNT=`/softwares/samtools1.3.1/bin/samtools view -c -q20 $BAM`
echo $BAM $CNT
done