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purple

performs purity and ploidy estimation

Overview

This workflow integrated some tools from hmftools (https://github.com/hartwigmedical/hmftools), including AMBER, COBALT, GRIPSS, PURPLE and LINX. It combines B-allele frequency (BAF) from AMBER, read depth ratios from COBALT, somatic variants and structural variants to estimate the purity and copy number profile of a tumor sample.

The optional tasks are filterSMALL, which uses bcftools to filter a vcf, provides an optional argument for PURPLE; and task FilterSV, which uses GRIPSS (GRIDSS Post Somatic Software) to apply a set of filtering and post processing steps on GRIDSS (https://github.com/PapenfussLab/gridss) paired tumor-normal output to produce a high confidence set of somatic SV for a tumor sample, provides a somatic vcf to serve as another optional argument for PURPLE.

If performed the optional task filterSV, then the results of PURPLE will go through a step to run on LINX, which is an annotation, interpretation and visualisation tool for structural variants. The primary function of LINX is grouping together individual SV calls into distinct events and properly classify and annotating the event to understand both its mechanism and genomic impact.

The mandatory arguments for this workflow are paired tumor-normal bam files for AMBER and COBALT. The optional arguments are a vcf input from mutect2 (for optional task filterSMALL), and a vcf input from GRIDSS (for optional task filterSV). All vcf file tumor and normal sample names in the header should match the sample names in the tumor bam and normal bam header respectively.

flowchart

Dependencies

Usage

Cromwell

java -jar cromwell.jar run purple.wdl --inputs inputs.json

Inputs

Required workflow parameters:

Parameter Value Description
tumour_bam File Input tumor file (bam)
tumour_bai File Input tumor file index (bai)
normal_bam File Input normal file (bam)
normal_bai File Input normal file index (bai)

Optional workflow parameters:

Parameter Value Default Description
genomeVersion String "38" Genome Version, only 38 supported
doSV Boolean true include somatic structural variant calls, true/false
doSMALL Boolean true include somatic small (SNV+indel) calls, true/false

Optional task parameters:

Parameter Value Default Description
extractTumorName.memory Int 4 Memory allocated for this job (GB)
extractTumorName.timeout Int 4 Hours before task timeout
extractNormalName.memory Int 4 Memory allocated for this job (GB)
extractNormalName.timeout Int 4 Hours before task timeout
amber.amberScript String "java -Xmx32G -cp $HMFTOOLS_ROOT/amber.jar com.hartwig.hmftools.amber.AmberApplication" location of AMBER script
amber.min_mapping_quality Int 30 Minimum mapping quality for an alignment to be used
amber.min_base_quality Int 25 Minimum quality for a base to be considered
amber.threads Int 8 Requested CPU threads
amber.memory Int 32 Memory allocated for this job (GB)
amber.timeout Int 100 Hours before task timeout
cobalt.colbaltScript String "java -Xmx8G -cp $HMFTOOLS_ROOT/cobalt.jar com.hartwig.hmftools.cobalt.CobaltApplication" location of COBALT script
cobalt.gamma String 300 gamma (penalty) value for segmenting
cobalt.min_mapping_quality Int 30 Minimum mapping quality for an alignment to be used
cobalt.threads Int 8 Requested CPU threads
cobalt.memory Int 32 Memory allocated for this job (GB)
cobalt.timeout Int 100 Hours before task timeout
filterSV.vcf File? None VCF file for filtering
filterSV.gripssScript String "java -Xmx80G -jar $HMFTOOLS_ROOT/gripss.jar" location and java call for gripss jar
filterSV.hard_min_tumor_qual Int 500 Any variant with QUAL less than x is filtered
filterSV.filter_sgls String "-filter_sgls" include filtering of single breakends
filterSV.memory Int 80 Memory allocated for this job (GB)
filterSV.threads Int 1 Requested CPU threads
filterSV.timeout Int 100 Hours before task timeout
filterSMALL.vcf File? None VCF file for filtering
filterSMALL.vcf_index File? None index of VCF file for filtering
filterSMALL.bcftoolsScript String "$BCFTOOLS_ROOT/bin/bcftools" location for bcftools
filterSMALL.genome String "$HG38_ROOT/hg38_random.fa" reference fasta
filterSMALL.regions String "chr1,chr10,chr11,chr12,chr13,chr14,chr15,chr16,chr17,chr18,chr19,chr2,chr20,chr21,chr22,chr3,chr4,chr5,chr6,chr7,chr8,chr9,chrX" regions/chromosomes to include
filterSMALL.difficultRegions String "--targets-file $HG38_DAC_EXCLUSION_ROOT/hg38-dac-exclusion.v2.bed" regions to exclude because they are difficult
filterSMALL.tumorVAF String "0.01" minimum variant allele frequency for tumour calls to pass filter
filterSMALL.modules String "bcftools/1.9 hg38/p12 hg38-dac-exclusion/1.0" Required environment modules
filterSMALL.threads Int 8 Requested CPU threads
filterSMALL.memory Int 32 Memory allocated for this job (GB)
filterSMALL.timeout Int 100 Hours before task timeout
runPURPLE.solution_name String "Primary" Name of solution
runPURPLE.outfilePrefix String tumour_name + ".sol" + solution_name Prefix of output file
runPURPLE.min_diploid_tumor_ratio_count Int 60 smooth over contiguous segments which are fewer than this number of depth windows long and which have no SV support on either side and which are bounded on both sides by copy number regions which could be smoothed together using our normal smoothing rules.
runPURPLE.purpleScript String "java -Xmx8G -jar $HMFTOOLS_ROOT/purple.jar" location of PURPLE script
runPURPLE.min_ploidy String? None minimum ploidy
runPURPLE.max_ploidy String? None max ploidy
runPURPLE.min_purity String? None mininimum purity
runPURPLE.max_purity String? None max purity
runPURPLE.ploidy_penalty_factor String? None multiplies aggregate event penalty by this factor
runPURPLE.ploidy_penalty_standard_deviation String? None not entirely sure what this does
runPURPLE.threads Int 8 Requested CPU threads
runPURPLE.memory Int 32 Memory allocated for this job (GB)
runPURPLE.timeout Int 100 Hours before task timeout
expandAlternates.min_alternate_ploidy Int 1 Minimum of alternative ploidy
expandAlternates.max_alternate_ploidy Int 8 Maximum of alternative ploidy
expandAlternates.alternate_ploidy_step Int 1 NUmber of steps
expandAlternates.jobMemory Int 1 Memory allocated for this job (GB)
expandAlternates.timeout Int 1 Hours before task timeout
expandAlternates.modules String "python/3.10.6" Required environment modules
runPURPLEAlternates.outfilePrefix String tumour_name + ".sol" + solution_name Prefix of output file
runPURPLEAlternates.min_diploid_tumor_ratio_count Int 60 smooth over contiguous segments which are fewer than this number of depth windows long and which have no SV support on either side and which are bounded on both sides by copy number regions which could be smoothed together using our normal smoothing rules.
runPURPLEAlternates.purpleScript String "java -Xmx8G -jar $HMFTOOLS_ROOT/purple.jar" location of PURPLE script
runPURPLEAlternates.min_purity String? None mininimum purity
runPURPLEAlternates.max_purity String? None max purity
runPURPLEAlternates.ploidy_penalty_factor String? None multiplies aggregate event penalty by this factor
runPURPLEAlternates.ploidy_penalty_standard_deviation String? None not entirely sure what this does
runPURPLEAlternates.threads Int 8 Requested CPU threads
runPURPLEAlternates.memory Int 32 Memory allocated for this job (GB)
runPURPLEAlternates.timeout Int 100 Hours before task timeout
group_alternates.jobMemory Int 1 Memory allocated for this job (GB)
group_alternates.threads Int 1 Requested CPU threads
group_alternates.timeout Int 2 Hours before task timeout
LINX.linxScript String "java -Xmx32G -cp $HMFTOOLS_ROOT/linx.jar com.hartwig.hmftools.linx.LinxApplication" location of LINX script
LINX.threads Int 8 Requested CPU threads
LINX.memory Int 32 Memory allocated for this job (GB)
LINX.timeout Int 100 Hours before task timeout

Outputs

Output Type Description Labels
purple_directory File? Zipped results from PURPLE vidarr_label: purple_directory
purple_alternate_directory File Directory for alternate solution files vidarr_label: purple_alternate_directory
purple_qc File QC results from PURPLE vidarr_label: purple_qc
purple_purity File tab seperated Purity estimate from PURPLE vidarr_label: purple_purity
purple_purity_range File tab seperated range of Purity estimate from PURPLE vidarr_label: purple_purity_range
purple_segments File tab seperated segments estimated by PURPLE vidarr_label: purple_segments
purple_cnv File tab seperated somatic copy number variants from PURPLE vidarr_label: purple_cnv
purple_cnv_gene File tab seperated somatic gene-level copy number variants from PURPLE vidarr_label: purple_cnv_gene
purple_SV_index File? Structural Variant .vcf index edited by PURPLE vidarr_label: purple_SV_index
purple_SV File? Structural Variant .vcf edited by PURPLE vidarr_label: purple_SV
purple_SMALL_index File? SNV+IN/DEL .vcf index edited by PURPLE vidarr_label: purple_SMALL_index
purple_SMALL File? SNV+IN/DEL .vcf edited by PURPLE vidarr_label: purple_SMALL

Commands

This section lists commands run by the PURPLE workflow

Run AMBER

       ~{amberScript} \
       -reference ~{normal_name} -reference_bam ~{normal_bam} \
       -tumor ~{tumour_name} -tumor_bam ~{tumour_bam} \
       -output_dir ~{tumour_name}.amber/ \
       -loci ~{PON} \
       -ref_genome_version ~{genomeVersion}

Run COBALT

       ~{colbaltScript} \
         -reference ~{normal_name} -reference_bam ~{normal_bam} \
         -tumor ~{tumour_name} -tumor_bam ~{tumour_bam} \
         -output_dir ~{tumour_name}.cobalt/ \
         -gc_profile ~{gcProfile} \
         -pcf_gamma ~{gamma}

Filter structural variant VCFs using GRIPSS, this is optional but recommended

       ~{gripssScript} \
           -vcf ~{vcf}  \
           -sample ~{tumour_name} -reference ~{normal_name} \
           -ref_genome_version ~{genomeVersion} \
           -ref_genome ~{refFasta} \
           -pon_sgl_file ~{pon_sgl_file} \
           -pon_sv_file ~{pon_sv_file} \
           -known_hotspot_file ~{known_hotspot_file} \
           -repeat_mask_file ~{repeat_mask_file} \
           -output_dir gripss/ \
           -hard_min_tumor_qual ~{hard_min_tumor_qual} \
           ~{filter_sgls}

Filter small variant (SNV + in/del) VCFs using bcftools, this is optional but recommended

        ~{bcftoolsScript} view -f "PASS" -S samples.txt -r ~{regions} ~{difficultRegions} ~{vcf} |\
        ~{bcftoolsScript} norm --multiallelics - --fasta-ref ~{genome} |\
        ~{bcftoolsScript} filter -i "(FORMAT/AD[0:1])/(FORMAT/AD[0:0]+FORMAT/AD[0:1]) >= ~{tumorVAF}"  > ~{tumour_name}.PASS.vcf

Run PURPLE

       ~{purpleScript} \
         -ref_genome_version ~{genomeVersion} \
         -ref_genome ~{refFasta}  \
         -gc_profile ~{gcProfile} \
         -ensembl_data_dir ~{ensemblDir}  \
         -reference ~{normal_name} -tumor ~{tumour_name}  \
         -amber ~{tumour_name}.amber -cobalt ~{tumour_name}.cobalt \
         ~{"-somatic_sv_vcf " + SV_vcf} \
         ~{"-somatic_vcf " + smalls_vcf} \
         -output_dir ~{tumour_name}.purple \
         -no_charts

Run LINX, if structural variant calls from GRIDSS are included

       ~{linxScript} \
         -sample ~{tumour_name} \
         -ref_genome_version ~{genomeVersion} \
         -ensembl_data_dir ~{ensemblDir}  \
         -check_fusions \
         -known_fusion_file ~{fusions_file} \
         -purple_dir ~{tumour_name}.purple \
         -output_dir ~{tumour_name}.linx  ## Support

For support, please file an issue on the Github project or send an email to gsi@oicr.on.ca .

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