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calling_handson [2012/12/05 15:41]
jjimeneza
calling_handson [2017/05/24 11:26] (current)
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 +====== Variant calling and annotation: Practical session ======
  
 +\\
 +
 +<code bash>
 +#!/bin/bash
 +
 +# Working directory
 +
 +cd ~/mda12/calling
 +
 +
 +# Run program and see options
 +~/mda12/calling/software/GenomeAnalysisTK-1.4-15-gcd43f01/GenomeAnalysisTK.jar
 +
 +# UnifiedGenotyper
 +
 +~/mda12/calling/software/GenomeAnalysisTK-1.4-15-gcd43f01/GenomeAnalysisTK.jar\
 + -T UnifiedGenotyper
 +
 +# Checking the reference
 +
 +head ~/mda12/resources/ref/human_g1k_v37.chr20.fasta
 +
 +head -3000 ~/mda12/resources/ref/human_g1k_v37.chr20.fasta | tail
 +
 +#Checking the bed file
 +
 +head ~/mda12/resources/ref/Exon_50mb_hg19_chr20.bed
 +
 +#SNV Calling of all sites
 +~/mda12/calling/software/GenomeAnalysisTK-1.4-15-gcd43f01/GenomeAnalysisTK.jar \
 +-T UnifiedGenotyper \
 +-R ~/mda12/resources/ref/human_g1k_v37.chr20.fasta \
 +-L ~/mda12/resources/ref/Exon_50mb_hg19_chr20.bed \
 +-I ~/mda12/resources/mapping/test_final.bam \
 +-glm SNP \
 +-out_mode EMIT_ALL_SITES \
 +-o all_sites.vcf
 +
 +
 +# Checking file
 +less all_sites.vcf
 +
 +#Counting lines
 +du -hs all_sites.vcf
 +wc -l all_sites.vcf
 +
 +# Executing SNVs calling of variants
 +~/mda12/calling/software/GenomeAnalysisTK-1.4-15-gcd43f01/GenomeAnalysisTK.jar \
 +-T UnifiedGenotyper \
 +-R ~/mda12/resources/ref/human_g1k_v37.chr20.fasta \
 +-L ~/mda12/resources/ref/Exon_50mb_hg19_chr20.bed \
 +-I ~/mda12/resources/mapping/test_final.bam \
 +-glm SNP \
 +-o snvs.vcf
 +
 +# Checking file
 +less snvs.vcf
 +
 +# Counting lines
 +du -hs snvs.vcf
 +wc -l snvs.vcf
 +
 +# Executing indels calling of variants
 +~/mda12/calling/software/GenomeAnalysisTK-1.4-15-gcd43f01/GenomeAnalysisTK.jar \
 +-T UnifiedGenotyper \
 +-R ~/mda12/resources/ref/human_g1k_v37.chr20.fasta \
 +-L ~/mda12/resources/ref/Exon_50mb_hg19_chr20.bed \
 +-I ~/mda12/resources/mapping/test_final.bam \
 +-glm INDEL \
 +-o indels.vcf
 +
 +# Checking file
 +less indels.vcf
 +
 +# Counting lines
 +du -hs indels.vcf
 +wc -l indels.vcf
 +
 +# Labeling VCF files
 +# VariantFiltration
 +~/mda12/calling/software/GenomeAnalysisTK-1.4-15-gcd43f01/GenomeAnalysisTK.jar \
 +-T VariantFiltration
 +
 +
 +# Labeling SNVs VCF file
 +~/mda12/calling/software/GenomeAnalysisTK-1.4-15-gcd43f01/GenomeAnalysisTK.jar \
 +-T VariantFiltration \
 +-filter "QD < 2.0 || MQ < 40.0 || FS > 60.0 || HaplotypeScore > 13.0 || MQRankSum < -12.5 || ReadPosRankSum < -8.0" \
 +-filterName "STD_FILTER" \
 +-R ~/mda12/resources/ref/human_g1k_v37.chr20.fasta \
 +-V snvs.vcf \
 +-o snvs_labeled.vcf
 +
 +# Checking files
 +wc -l snvs_labeled.vcf
 +wc -l snvs.vcf
 +grep PASS snvs_labeled.vcf | wc -l
 +
 +# Labeling indels VCF file
 +~/mda12/calling/software/GenomeAnalysisTK-1.4-15-gcd43f01/GenomeAnalysisTK.jar \
 +-T VariantFiltration \
 +-filter "QD < 2.0 || ReadPosRankSum < -20.0 || FS > 200.0" \
 +-filterName "STD_FILTER" \
 +-R ~/mda12/resources/ref/human_g1k_v37.chr20.fasta \
 +-V indels.vcf \
 +-o indels_labeled.vcf
 +
 +# Checking files
 +wc -l indels.vcf
 +wc -l indels_labeled.vcf
 +grep PASS indels_labeled.vcf | wc -l
 +
 +# Converting SNV VCF file to annovar format file
 +~/mda12/calling/software/annovar/convert2annovar.pl \
 +-format vcf4 \
 +-filter PASS snvs_labeled.vcf > \
 +snvs_labeled.vcf.annovar
 +
 +# Checking files
 +wc -l snvs_labeled.vcf.annovar
 +grep PASS snvs_labeled.vcf | wc -l
 +head snvs_labeled.vcf.annovar
 +
 +# Annotating
 +~/mda12/calling/software/annovar/annotate_variation.pl \
 +--geneanno \
 +--buildver hg19 \
 +--dbtype gene \
 +snvs_labeled.vcf.annovar \
 +~/mda12/calling/software/annovar/humandb/
 +
 +# Output
 +ls -latr
 +head snvs_labeled.vcf.annovar.exonic_variant_function
 +head snvs_labeled.vcf.annovar.variant_function
 +head snvs_labeled.vcf.annovar.log
 +
 +# Converting indels VCF file to annovar format file
 +~/mda12/calling/software/annovar/convert2annovar.pl \
 +-format vcf4 \
 +-filter PASS indels_labeled.vcf > \
 +indels_labeled.vcf.annovar
 +
 +# Checking files
 +wc -l indels_labeled.vcf.annovar
 +grep PASS indels_labeled.vcf | wc -l
 +head indels_labeled.vcf.annovar
 +
 +# Annotating
 +~/mda12/calling/software/annovar/annotate_variation.pl \
 +--geneanno \
 +--buildver hg19 \
 +--dbtype gene \
 +indels_labeled.vcf.annovar \
 +~/mda12/calling/software/annovar/humandb/
 +
 +# Output
 +ls -latr
 +head indels_labeled.vcf.annovar.exonic_variant_function
 +head indels_labeled.vcf.annovar.variant_function
 +head indels_labeled.vcf.annovar.log
 +
 +
 +# IGV
 +igv
 +</code>