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# Running GATK HaplotypeCaller for variant calling usings bulk sperm sequencing data
# Author Ruqian Lyu
# Date 4/15/2021
# snakemake -s run_vcalling.snk --cores 10 --keep-going -j 1 --use-singularity --singularity-args "-B /mnt/beegfs/mccarthy/scratch:/mnt/beegfs/mccarthy/scratch,/mnt/mcfiles/rlyu/Projects/:/mnt/mcfiles/rlyu/Projects/" -j 1 -c sbatch "--mem {resources.mem} --time=120:00:00 --cpus-per-task={resources.cpus} --error=runDevnoVC.log.out"
outdir = "output/variants/"
rule all:
input: outdir+"denovoVar/SRR8454653.mkdup.sort.rg.filter.snps.castVar.bl6.nonVar.vcf.gz","denovoVar/SRR8454653.mkdup.sort.rg.filter.snps.castVar.bl6.nonVar.sorted.vcf.gz"
rule addRG_gatk:
input:
bulkSpermBam = "output/alignment/cleanBam/SRR8454653.mkdup.sort.bam"
output:
bulkSpermRG = "output/alignment/bulkBam/SRR8454653.mkdup.sort.piccard.rg.bam"
threads: 4
singularity:
"docker://broadinstitute/gatk"
resources:
cpus = 4,
mem = 10240
shell:
"""
gatk AddOrReplaceReadGroups \
-I {input.bulkSpermBam} \
-O {output.bulkSpermRG} \
-ID SRR8454653 \
-PL ILLUMINA \
-SM SRR8454653 \
-LB libx \
-PU SRR8454653 \
"""
rule sam_index:
input:
pcbam="output/alignment/bulkBam/SRR8454653.mkdup.sort.piccard.rg.bam"
output:
pcidx="output/alignment/bulkBam/SRR8454653.mkdup.sort.piccard.rg.bam.bai"
resources:
cpus=4,
mem=2000
shell:
"""
samtools index -@ {resources.cpus} {input.pcbam}
"""
rule run_gatk_hc:
input:
bulkSpermRG = "output/alignment/bulkBam/SRR8454653.mkdup.sort.piccard.rg.bam",
bulkSpermRGIdx="output/alignment/bulkBam/SRR8454653.mkdup.sort.piccard.rg.bam.bai",
ref_fa = "./references/genome.fa"
output:
vcf=outdir+"denovoVar/SRR8454653.mkdup.sort.rg.vcf.gz"
singularity:
"docker://broadinstitute/gatk"
log: "log/hc/SRR8454653.hc.log"
threads:
4
resources:
cpus = 4,
mem = 51200
shell:
"""
gatk HaplotypeCaller -R {input.ref_fa} \
--native-pair-hmm-threads {resources.cpus} \
-I '{input.bulkSpermRG}' -O '{output.vcf}' 2> {log}
"""
rule run_filterVCF:
input:
vcf=outdir+"denovoVar/SRR8454653.mkdup.sort.rg.vcf.gz",
ref_fa = "./references/genome.fa"
output:
filter_vcf="denovoVar/SRR8454653.mkdup.sort.rg.filter.vcf.gz"
singularity:
"docker://broadinstitute/gatk"
log: "log/filterVNs/SRR8454653.vf.log"
threads:
4
resources:
cpus = 4,
mem = 51200
shell:
"""
gatk VariantFiltration -V '{input.vcf}' -O '{output.filter_vcf}' \
-R {input.ref_fa} \
--filter-name "MQ50" \
--filter-expression "MQ < 50.0" \
--filter-name "minDP" --filter-expression "DP < 10" \
--filter-name "maxDP" --filter-expression "DP > 80" \
--genotype-filter-name "NOTHETGeno" \
--genotype-filter-expression isHet!=1 \
--set-filtered-genotype-to-no-call true 2> {log}
"""
rule run_selectSNPs:
input:
filter_vcf=outdir+"denovoVar/SRR8454653.mkdup.sort.rg.filter.vcf.gz",
ref_fa = "./references/genome.fa"
output:
snp_filter_vcf=outdir+"denovoVar/SRR8454653.mkdup.sort.rg.filter.snps.vcf.gz"
singularity:
"docker://broadinstitute/gatk"
log: "log/selectVNs/SRR8454653.vf.log"
threads:
4
resources:
cpus = 4,
mem = 51200
shell:
"""
gatk SelectVariants \
-R {input.ref_fa} \
-V {input.filter_vcf} \
--select-type-to-include SNP \
--exclude-filtered true \
-O {output.snp_filter_vcf} 2> {log}
"""
rule filter_nocalGT:
input:
filter_vcf=outdir+"denovoVar/SRR8454653.mkdup.sort.rg.filter.snps.vcf.gz",
cast_ref = "./references/CAST_EiJ.mgp.v5.snps.dbSNP142.homo.var.chr.vcf.gz"
output:
snp_checked=outdir+"denovoVar/SRR8454653.mkdup.sort.rg.filter.snps.castVar.vcf.gz"
log: "log/crossCAST/SRR8454653.vf.log"
threads:
4
resources:
cpus = 4,
mem = 11200
shell:
"""
bcftools filter --threads {resources.cpus} --exclude "GT=='./.'" --regions-file {input.cast_ref} {input.filter_vcf} -Oz -o {output.snp_checked} 2> {log}
"""
rule filter_bl6variants:
input:
bl6_ref = "./references/C57BL_6NJ.mgp.v5.snps.dbSNP142.vcf.gz",
snp_checked=outdir+"denovoVar/SRR8454653.mkdup.sort.rg.filter.snps.castVar.vcf.gz"
output:
snp_checked_bl6=outdir+"denovoVar/SRR8454653.mkdup.sort.rg.filter.snps.castVar.bl6.nonVar.vcf.gz"
log: "log/crossBL6/SRR8454653.vf.log"
threads:
1
resources:
cpus = 1,
mem = 11200
shell:
"""
bedtools intersect -a {input.snp_checked} -b {input.bl6_ref} -header -sorted -v > {output.snp_checked_bl6} 2> {log}
"""
rule sort_index:
input:
snp_checked_bl6=outdir+"denovoVar/SRR8454653.mkdup.sort.rg.filter.snps.castVar.bl6.nonVar.vcf.gz"
output:
snp_checked_bl6_sorted=outdir+"denovoVar/SRR8454653.mkdup.sort.rg.filter.snps.castVar.bl6.nonVar.sorted.vcf.gz"
resources:
cpus = 4,
mem = 21200
shell:
"""
bcftools view {input.snp_checked_bl6} -Oz -o {input.snp_checked_bl6}.tmp.gz
bcftools sort --threads {resources.cpus} {input.snp_checked_bl6}.tmp.gz -Oz -o {output.snp_checked_bl6_sorted}
bcftools index --threads {resources.cpus} {output.snp_checked_bl6_sorted}
rm {input.snp_checked_bl6}.tmp.gz
"""
.