varscan

Varscan 2.3, workflow for calling SNVs and CVs Creation of mpileups and calling variants are done with parallel processing

Dependencies

Usage

Cromwell

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

Inputs

Required workflow parameters:

Parameter	Value	Description
`inputTumor`	File	input .bam file for tumor sample
`inputNormal`	File	input .bam file for normal sample
`inputTumorIndex`	File	input .bai file for tumor sample
`inputNormalIndex`	File	input .bai file for normal sample
`reference`	String	Reference assembly id, hg19 hg38 or mm10
`outputFileNamePrefix`	String	Output file(s) prefix
`runVarscanCNV.scaleCoefficient`	Float	Scaling coefficient for RAM allocation, depends on chromosome size
`getSnvNative.scaleCoefficient`	Float	Scaling coefficient for RAM allocation, depends on chromosome size
`getSnvVcf.scaleCoefficient`	Float	Scaling coefficient for RAM allocation, depends on chromosome size

Optional workflow parameters:

Parameter	Value	Default	Description
`bedIntervalsPath`	String	""	Path to a .bed file used for splitting pileup job/limiting analysis to selected regions

Optional task parameters:

Parameter	Value	Default	Description
`expandRegions.modules`	String	"hg38-dac-exclusion/1.0"	required modules (This is to allow modularized data for bed path)
`expandRegions.jobMemory`	Int	4	Memory for this task in GB
`expandRegions.timeout`	Int	12	Timeout in hours, needed to override imposed limits
`getChrCoefficient.memory`	Int	1	Memory allocated for this job
`getChrCoefficient.timeout`	Int	1	Hours before task timeout
`makePileups.samtools`	String	"$SAMTOOLS_ROOT/bin/samtools"	path to samtools
`makePileups.jobMemory`	Int	18	memory for this job, in Gb
`makePileups.minMemory`	Int	6	Minimal amount of memory to assign to the task
`makePileups.timeout`	Int	40	Timeout in hours, needed to override imposed limits
`runVarscanCNV.pValue`	Float	0.05	p-value for cnv calling, default is 0.05
`runVarscanCNV.jobMemory`	Int	20	Memory in Gb for this job
`runVarscanCNV.minMemory`	Int	4	A minimum amount of memory allocated to the task, overrides the scaled RAM setting
`runVarscanCNV.javaMemory`	Int	6	Memory in Gb for Java
`runVarscanCNV.logFile`	String	"VARSCAN_CNV.log"	File for logging Varscan messages
`runVarscanCNV.varScan`	String	"$VARSCAN_ROOT/VarScan.jar"	path to varscan .jar file
`runVarscanCNV.modules`	String	"varscan/2.4.2 java/8"	Names and versions of modules
`runVarscanCNV.timeout`	Int	40	Timeout in hours, needed to override imposed limits
`getSnvNative.pValue`	Float	0.05	somatic p-value for SNV calling, default is 0.05
`getSnvNative.jobMemory`	Int	20	Memory in Gb for this job
`getSnvNative.minMemory`	Int	4	A minimum amount of memory allocated to the task, overrides the scaled RAM setting
`getSnvNative.javaMemory`	Int	6	Memory in Gb for Java
`getSnvNative.minCoverage`	Int	8	Minimum coverage in normal and tumor to call variant [8]
`getSnvNative.minCoverageNormal`	Int	8	Minimum coverage in normal to call somatic [8]
`getSnvNative.minCoverageTumor`	Int	6	Minimum coverage in tumor to call somatic [6]
`getSnvNative.minVarFreq`	Float	0.1	Minimum variant frequency to call a heterozygote [0.10]
`getSnvNative.minFreqForHom`	Float	0.75	Minimum frequency to call homozygote [0.75]
`getSnvNative.normalPurity`	Float	1.0	Estimated purity (non-tumor content) of normal sample [1.00]
`getSnvNative.tumorPurity`	Float	1.0	Estimated purity (tumor content) of normal sample [1.00]
`getSnvNative.pValueHet`	Float	0.99	p-value threshold to call a heterozygote [0.99]
`getSnvNative.strandFilter`	Int	0	If set to 1, removes variants with >90% strand bias
`getSnvNative.validation`	Int	0	If set to 1, outputs all compared positions even if non-variant
`getSnvNative.outputVcf`	Int	0	Flag that when set to 1 indicates that we need results in vcf format
`getSnvNative.logFile`	String	"VARSCAN_SNV.log"	File for logging Varscan messages
`getSnvNative.varScan`	String	"$VARSCAN_ROOT/VarScan.jar"	path to varscan .jar file
`getSnvNative.modules`	String	"varscan/2.4.2 java/8"	Names and versions of modules
`getSnvNative.timeout`	Int	40	Timeout in hours, needed to override imposed limits
`getSnvVcf.pValue`	Float	0.05	somatic p-value for SNV calling, default is 0.05
`getSnvVcf.jobMemory`	Int	20	Memory in Gb for this job
`getSnvVcf.minMemory`	Int	4	A minimum amount of memory allocated to the task, overrides the scaled RAM setting
`getSnvVcf.javaMemory`	Int	6	Memory in Gb for Java
`getSnvVcf.minCoverage`	Int	8	Minimum coverage in normal and tumor to call variant [8]
`getSnvVcf.minCoverageNormal`	Int	8	Minimum coverage in normal to call somatic [8]
`getSnvVcf.minCoverageTumor`	Int	6	Minimum coverage in tumor to call somatic [6]
`getSnvVcf.minVarFreq`	Float	0.1	Minimum variant frequency to call a heterozygote [0.10]
`getSnvVcf.minFreqForHom`	Float	0.75	Minimum frequency to call homozygote [0.75]
`getSnvVcf.normalPurity`	Float	1.0	Estimated purity (non-tumor content) of normal sample [1.00]
`getSnvVcf.tumorPurity`	Float	1.0	Estimated purity (tumor content) of normal sample [1.00]
`getSnvVcf.pValueHet`	Float	0.99	p-value threshold to call a heterozygote [0.99]
`getSnvVcf.strandFilter`	Int	0	If set to 1, removes variants with >90% strand bias
`getSnvVcf.validation`	Int	0	If set to 1, outputs all compared positions even if non-variant
`getSnvVcf.logFile`	String	"VARSCAN_SNV.log"	File for logging Varscan messages
`getSnvVcf.varScan`	String	"$VARSCAN_ROOT/VarScan.jar"	path to varscan .jar file
`getSnvVcf.modules`	String	"varscan/2.4.2 java/8"	Names and versions of modules
`getSnvVcf.timeout`	Int	40	Timeout in hours, needed to override imposed limits
`mergeCNV.jobMemory`	Int	6	memory in GB for this job
`mergeCNV.timeout`	Int	10	Timeout in hours, needed to override imposed limits
`mergeSNP.jobMemory`	Int	6	memory in GB for this job
`mergeSNP.timeout`	Int	10	Timeout in hours, needed to override imposed limits
`mergeIND.jobMemory`	Int	6	memory in GB for this job
`mergeIND.timeout`	Int	10	Timeout in hours, needed to override imposed limits
`mergeSNPvcf.jobMemory`	Int	12	memory in GB for this job
`mergeSNPvcf.javaMemory`	Int	8	memory in GB for java VM
`mergeSNPvcf.timeout`	Int	10	Timeout in hours, needed to override imposed limits
`mergeINDvcf.jobMemory`	Int	12	memory in GB for this job
`mergeINDvcf.javaMemory`	Int	8	memory in GB for java VM
`mergeINDvcf.timeout`	Int	10	Timeout in hours, needed to override imposed limits
`smoothData.varScan`	String	"$VARSCAN_ROOT/VarScan.jar"	Path to VarScan jar file
`smoothData.modules`	String	"varscan/2.4.2 java/8 rstats/3.6"	Modules for this job
`smoothData.min_coverage`	Int	20	Fine-tuning parameter for VarScan
`smoothData.max_homdel_coverage`	Int	5	Max coverage form homozygous deletion, default is 5
`smoothData.min_tumor_coverage`	Int	10	Min coverage in tumor sample, default is 10
`smoothData.del_threshold`	Float	0.25	Fine-tuning parameter for VarScan
`smoothData.amp_threshold`	Float	0.25	Amplification threshold to report, default is 0.25
`smoothData.min_region_size`	Int	10	Fine-tuning parameter for VarScan
`smoothData.recenter_up`	Int	0	Fine-tuning parameter for VarScan
`smoothData.recenter_down`	Int	0	Fine-tuning parameter for VarScan
`smoothData.jobMemory`	Int	16	Memory in Gb for this job
`smoothData.javaMemory`	Int	6	Memory in Gb for Java

Outputs

Output	Type	Description
`resultCnvFile`	File?	file with CNV calls, smoothed
`resultSnpFile`	File	file with SNPs, native varscan format
`resultIndelFile`	File	file with Indel calls, native varscan format
`resultSnpVcfFile`	File	file with SNPs, vcf format
`resultIndelVcfFile`	File	file with Indels, vcf format

Commands

This section lists command(s) run by varscan workflow

Produce a scaling coefficient for allocating RAM

   CHROM=$(echo ~{region} | sed 's/:.*//')
   grep -w SN:$CHROM ~{refDict} | cut -f 3 | sed 's/.*://' | awk '{print int(($1/~{largestChrom} + 0.1) * 10)/10}'

Preprocessing

bed file re-format to be used with scattered pileup creation. Note that it should be a resonable ( <100 perhaps? ) intervals so that we do not end up with a million jobs running. Use wisely, as it may result in grabbing a lot of compute nodes.

  In this embedded script we reformat bed lines into varscan-friendly intervals
 
  import os
  if os.path.exists("~{bedPath}"):
     with open("~{bedPath}") as f:
         for line in f:
            line = line.rstrip()
            tmp = line.split("\t")
            r = " " + tmp[0] + ":" + tmp[1] + "-" + tmp[2]
            print(r)
     f.close()

Run samtools mpileup

  samtools mpileup -q 1 -r REGION -f REF_FASTA INPUT_NORMAL INPUT_TUMOR | awk -F "\t" '$4 > 0 && $7 > 0' | gzip -c > normtumor_sorted.pileup.gz

Remove mitochondrial chromosome:

  head -n 1 ~{filePaths[0]} > "~{outputFile}.~{outputExtension}"
  cat ~{sep=' ' filePaths} | sort -V -k 1,2 | grep -v ^chrom | grep -v ^chrM >> "~{outputFile}.~{outputExtension}"
  cat ~{sep=' ' filePaths} | awk '{if($1 == "chrM"){print $0}}' | sort -V -k 1,2 >> "~{outputFile}.~{outputExtension}"
  if [ ! -s ~{outputFile}.~{outputExtension} ] ; then
   rm ~{outputFile}.~{outputExtension}
  fi

Sort vcf using sequence dictionary

  java -Xmx[MEMORY]G -jar picard.jar SortVcf I=INPUT_VCFS SD=SEQ_DICTIONARY O=OUTPUT_FILE.SUFFIX.vcf

SNP/Indel Calling:

   See the full source code in .wdl, here we run this command:
 
   zcat INPUT_PILEUP | java -Xmx[MEMORY]G -jar varscan somatic -mpileup 1 
      --somatic-p-value P_VALUE
 
      Optional parameters:
 
     --min-coverage-normal MIN_COVERAGE_NORMAL
     --min-coverage-tumor  MIN_COVERAGE_TUMOR
     --min-var-freq MIN_VAR_FREQUENCY
     --min-freq-for-hom MIN_FREQUENCY_FOR_HOM
     --normal-purity NORMAL_PURITY
     --tumor-purity TUMOR_PURIY
     --p-value P_VALUE_HET
     --strand-filter 1
     --validation 1
     --output-vcf 1
     
     Settings for output format:
 
     --output-snp SAMPLE_ID.snp.vcf --output-indel SAMPLE_ID.indel.vcf
   
     or:
 
     --output-snp SAMPLE_ID.snp --output-indel SAMPLE_ID.indel

Find minimum coverage threshold for CV analysis:

 
 A python code configures and runs this command: 
 
 zcat ~{inputPileup} | java -Xmx~{javaMemory}G -jar " + varscan + " copynumber --output-file ~{sampleID} -mpileup 1 --p-value ~{pValue}"
 
 Varscan reports if the coverage threshold was sufficient for the analysis. We use this coverage setting in the next step

Run copy number change analysis:

 
 A python code configures and runs this command:
  
 java -Xmx[MEMORY]G -jar varscan copyCaller CV_FILE 
                    --output-file SAMPLE_ID.copynumber.filtered
                    --min-coverage MIN_COVERAGE # Value found in the previous step
 
                     Optional parameters:
 
                    --min-tumor-coverage  MIN_TUMOR_COVERAGE
                    --max-homdel-coverage MAX_HOMDEL_COVERAGE
                    --del-threshold   DEL_THRESHOLD
                    --amp-threshold   AMP_THRESHOLD
                    --min-region-size MIN_REGION_SIZE
                    --recenter-up     RECENTER_UP
                    --recenter-down   RECENTER_DOWN

Support

For support, please file an issue on the Github project or send an email to [email protected] .

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