VarScan - Variant Detection in Massively Parallel Sequencing Data (original) (raw)
VarScan User's Manual
VarScan is coded in Java, and should be executed from the command line (Terminal, in Linux/UNIX/OSX, or Command Prompt in MS Windows). For variant calling, you will need a pileup file. See the How to Build A Pileup File section for details. Running VarScan with no arguments prints the usage information. Because some fields changed as of VarScan v2.2.3, we are providing updated documentations for the current release. For documentation of v2.2.2 and prior, see below.
VarScan Documentation (v2.2.3 and later)
USAGE: java -jar VarScan.jar [COMMAND] [OPTIONS]
COMMANDS:
**Single-sample Calling:**
[**pileup2snp**](#v2.3%5Fpileup2snp) [pileup file]
[**pileup2indel**](#v2.3%5Fpileup2indel) [pileup file]
[**pileup2cns**](#v2.3%5Fpileup2cns) [pileup file]
**Multi-sample Calling:**
[**mpileup2snp**](#v2.3%5Fmpileup2snp) [mpileup file]
[**mpileup2indel**](#v2.3%5Fmpileup2indel) [mpileup file]
[**mpileup2cns**](#v2.3%5Fmpileup2cns) [mpileup file]
**Tumor-normal Comparison:**
[**somatic**](#v2.3%5Fsomatic) [normal pileup] [tumor pileup] or [normal-tumor mpileup]
[**copynumber**](#v2.3%5Fcopynumber) [normal pileup] [tumor pileup] or [normal-tumor mpileup]
**Variant Filtering:**
[**filter**](#v2.3%5Ffilter) [variants file]
[**somaticFilter**](#v2.3%5FsomaticFilter) [mutations file]
**Utility Functions:**
[**limit**](#v2.3%5Flimit) [variants file]
[**readcounts**](#v2.3%5Freadcounts) [pileup file]
[**compare**](#v2.3%5Fcompare) [file1] [file2]pileup2snp
This command calls SNPs from a pileup file based on user-defined parameters:
USAGE: java -jar VarScan.jar pileup2snp [pileup file] OPTIONS
pileup file - The SAMtools pileup file
OPTIONS:
--min-coverage Minimum read depth at a position to make a call [8]
--min-reads2 Minimum supporting reads at a position to call variants [2]
--min-avg-qual Minimum base quality at a position to count a read [15]
--min-var-freq Minimum variant allele frequency threshold [0.01]
--p-value Default p-value threshold for calling variants [99e-02]
OUTPUT
Tab-delimited SNP calls with the following columns:
Chrom chromosome name
Position position (1-based)
Ref reference allele at this position
Cons Consensus genotype of sample in IUPAC format.
Reads1 reads supporting reference allele
Reads2 reads supporting variant allele
VarFreq frequency of variant allele by read count
Strands1 strands on which reference allele was observed
Strands2 strands on which variant allele was observed
Qual1 average base quality of reference-supporting read bases
Qual2 average base quality of variant-supporting read bases
Pvalue Significance of variant read count vs. expected baseline error
MapQual1 Average map quality of ref reads (only useful if in pileup)
MapQual2 Average map quality of var reads (only useful if in pileup)
Reads1Plus Number of reference-supporting reads on + strand
Reads1Minus Number of reference-supporting reads on - strand
Reads2Plus Number of variant-supporting reads on + strand
Reads2Minus Number of variant-supporting reads on - strand
VarAllele Most frequent non-reference allele observed pileup2indel
This command calls indels from a pileup file based on user-defined parameters:
USAGE: java -jar VarScan.jar pileup2indel [pileup file] OPTIONS
pileup file - The SAMtools pileup file
OPTIONS:
--min-coverage Minimum read depth at a position to make a call [8]
--min-reads2 Minimum supporting reads at a position to call variants [2]
--min-avg-qual Minimum base quality at a position to count a read [15]
--min-var-freq Minimum variant allele frequency threshold [0.01]
--p-value Default p-value threshold for calling variants [99e-02]
OUTPUT
Tab-delimited indel calls with the following columns:
Chrom chromosome name
Position position (1-based)
Ref reference allele at this position
Cons Consensus genotype of sample; */(var) indicates heterozygous
Reads1 reads supporting reference allele
Reads2 reads supporting variant allele
VarFreq frequency of variant allele by read count
Strands1 strands on which reference allele was observed
Strands2 strands on which variant allele was observed
Qual1 average base quality of reference-supporting read bases
Qual2 average base quality of variant-supporting read bases
Pvalue Significance of variant read count vs. expected baseline error
MapQual1 Average map quality of ref reads (only useful if in pileup)
MapQual2 Average map quality of var reads (only useful if in pileup)
Reads1Plus Number of reference-supporting reads on + strand
Reads1Minus Number of reference-supporting reads on - strand
Reads2Plus Number of variant-supporting reads on + strand
Reads2Minus Number of variant-supporting reads on - strand
VarAllele Most frequent non-reference allele observed pileup2cns
This command makes consensus calls (SNP/Indel/Reference) from a pileup file based on user-defined parameters:
USAGE: java -jar VarScan.jar pileup2cns [pileup file] OPTIONS
pileup file - The SAMtools pileup file
OPTIONS:
--min-coverage Minimum read depth at a position to make a call [8]
--min-reads2 Minimum supporting reads at a position to call variants [2]
--min-avg-qual Minimum base quality at a position to count a read [15]
--min-var-freq Minimum variant allele frequency threshold [0.01]
--p-value Default p-value threshold for calling variants [99e-02]
OUTPUT
Tab-delimited consensus calls with the following columns:
Chrom chromosome name
Position position (1-based)
Ref reference allele at this position
Cons Consensus genotype of sample; */(var) indicates heterozygous
Reads1 reads supporting reference allele
Reads2 reads supporting variant allele
VarFreq frequency of variant allele by read count
Strands1 strands on which reference allele was observed
Strands2 strands on which variant allele was observed
Qual1 average base quality of reference-supporting read bases
Qual2 average base quality of variant-supporting read bases
Pvalue Significance of variant read count vs. expected baseline error
MapQual1 Average map quality of ref reads (only useful if in pileup)
MapQual2 Average map quality of var reads (only useful if in pileup)
Reads1Plus Number of reference-supporting reads on + strand
Reads1Minus Number of reference-supporting reads on - strand
Reads2Plus Number of variant-supporting reads on + strand
Reads2Minus Number of variant-supporting reads on - strand
VarAllele Most frequent non-reference allele observed mpileup2snp
This command calls SNPs from an mpileup file based on user-defined parameters:
USAGE: java -jar VarScan.jar mpileup2snp [mpileup file] OPTIONS
mpileup file - The SAMtools mpileup file
OPTIONS:
--min-coverage Minimum read depth at a position to make a call [8]
--min-reads2 Minimum supporting reads at a position to call variants [2]
--min-avg-qual Minimum base quality at a position to count a read [15]
--min-var-freq Minimum variant allele frequency threshold [0.01]
--min-freq-for-hom Minimum frequency to call homozygote [0.75]
--p-value Default p-value threshold for calling variants [99e-02]
--strand-filter Ignore variants with >90% support on one strand [1]
--output-vcf If set to 1, outputs in VCF format
--variants Report only variant (SNP/indel) positions (mpileup2cns only) [0]
OUTPUT
Tab-delimited SNP calls with the following columns:
Chrom chromosome name
Position position (1-based)
Ref reference allele at this position
Var variant allele observed
PoolCall Cross-sample call using all data (Cons:Cov:Reads1:Reads2:Freq:P-value)
Cons - consensus genotype in IUPAC format
Cov - total depth of coverage
Reads1 - number of reads supporting reference
Reads2 - number of reads supporting variant
Freq - the variant allele frequency by read count
P-value - FET p-value of observed reads vs expected non-variant
StrandFilt Information to look for strand bias using all reads (R1+:R1-:R2+:R2-:pval)
R1+ = reference supporting reads on forward strand
R1- = reference supporting reads on reverse strand
R2+ = variant supporting reads on forward strand
R2- = variant supporting reads on reverse strand
pval = FET p-value for strand distribution, R1 versus R2
SamplesRef Number of samples called reference (wildtype)
SamplesHet Number of samples called heterozygous-variant
SamplesHom Number of samples called homozygous-variant
SamplesNC Number of samples not covered / not called
SampleCalls The calls for each sample in the mpileup, space-delimited
Each sample has six values separated by colons:
Cons - consensus genotype in IUPAC format
Cov - total depth of coverage
Reads1 - number of reads supporting reference
Reads2 - number of reads supporting variant
Freq - the variant allele frequency by read count
P-value - FET p-value of observed reads vs expected non-variant mpileup2indel
This command calls indels from a mpileup file based on user-defined parameters:
USAGE: java -jar VarScan.jar mpileup2indel [mpileup file] OPTIONS
mpileup file - The SAMtools mpileup file
OPTIONS:
--min-coverage Minimum read depth at a position to make a call [8]
--min-reads2 Minimum supporting reads at a position to call variants [2]
--min-avg-qual Minimum base quality at a position to count a read [15]
--min-var-freq Minimum variant allele frequency threshold [0.01]
--min-freq-for-hom Minimum frequency to call homozygote [0.75]
--p-value Default p-value threshold for calling variants [99e-02]
--strand-filter Ignore variants with >90% support on one strand [1]
--output-vcf If set to 1, outputs in VCF format
--variants Report only variant (SNP/indel) positions (mpileup2cns only) [0]
OUTPUT
Tab-delimited SNP calls with the following columns:
Chrom chromosome name
Position position (1-based)
Ref reference allele at this position
Var variant allele observed
PoolCall Cross-sample call using all data (Cons:Cov:Reads1:Reads2:Freq:P-value)
Cons - consensus genotype in IUPAC format
Cov - total depth of coverage
Reads1 - number of reads supporting reference
Reads2 - number of reads supporting variant
Freq - the variant allele frequency by read count
P-value - FET p-value of observed reads vs expected non-variant
StrandFilt Information to look for strand bias using all reads, format R1+:R1-:R2+:R2-:pval
R1+ = reference supporting reads on forward strand
R1- = reference supporting reads on reverse strand
R2+ = variant supporting reads on forward strand
R2- = variant supporting reads on reverse strand
pval = FET p-value for strand distribution, R1 versus R2
SamplesRef Number of samples called reference (wildtype)
SamplesHet Number of samples called heterozygous-variant
SamplesHom Number of samples called homozygous-variant
SamplesNC Number of samples not covered / not called
SampleCalls The calls for each sample in the mpileup, space-delimited
Each sample has six values separated by colons:
Cons - consensus genotype in IUPAC format
Cov - total depth of coverage
Reads1 - number of reads supporting reference
Reads2 - number of reads supporting variant
Freq - the variant allele frequency by read count
P-value - FET p-value of observed reads vs expected non-variant mpileup2cns
This command makes consensus calls (SNP/Indel/Reference) from a mpileup file based on user-defined parameters:
USAGE: java -jar VarScan.jar mpileup2cns [mpileup file] OPTIONS
mpileup file - The SAMtools mpileup file
OPTIONS:
--min-coverage Minimum read depth at a position to make a call [8]
--min-reads2 Minimum supporting reads at a position to call variants [2]
--min-avg-qual Minimum base quality at a position to count a read [15]
--min-var-freq Minimum variant allele frequency threshold [0.01]
--min-freq-for-hom Minimum frequency to call homozygote [0.75]
--p-value Default p-value threshold for calling variants [99e-02]
--strand-filter Ignore variants with >90% support on one strand [1]
--output-vcf If set to 1, outputs in VCF format
--variants Report only variant (SNP/indel) positions (mpileup2cns only) [0]
OUTPUT
Tab-delimited SNP calls with the following columns:
Chrom chromosome name
Position position (1-based)
Ref reference allele at this position
Var variant allele observed
PoolCall Cross-sample call using all data (Cons:Cov:Reads1:Reads2:Freq:P-value)
Cons - consensus genotype in IUPAC format
Cov - total depth of coverage
Reads1 - number of reads supporting reference
Reads2 - number of reads supporting variant
Freq - the variant allele frequency by read count
P-value - FET p-value of observed reads vs expected non-variant
StrandFilt Information to look for strand bias using all reads, format R1+:R1-:R2+:R2-:pval
R1+ = reference supporting reads on forward strand
R1- = reference supporting reads on reverse strand
R2+ = variant supporting reads on forward strand
R2- = variant supporting reads on reverse strand
pval = FET p-value for strand distribution, R1 versus R2
SamplesRef Number of samples called reference (wildtype)
SamplesHet Number of samples called heterozygous-variant
SamplesHom Number of samples called homozygous-variant
SamplesNC Number of samples not covered / not called
SampleCalls The calls for each sample in the mpileup, space-delimited
Each sample has six values separated by colons:
Cons - consensus genotype in IUPAC format
Cov - total depth of coverage
Reads1 - number of reads supporting reference
Reads2 - number of reads supporting variant
Freq - the variant allele frequency by read count
P-value - FET p-value of observed reads vs expected non-variant somatic
This command calls variants and identifies their somatic status (Germline/LOH/Somatic) usingpileup files from a matched tumor-normal pair.
USAGE: java -jar VarScan.jar somatic [normal_pileup] [tumor_pileup] [output] OPTIONS
normal_pileup - The SAMtools pileup file for Normal
tumor_pileup - The SAMtools pileup file for Tumor
output - Output base name for SNP and indel outputYou can also give it a single mpileup file with normal and tumor data.
USAGE: java -jar VarScan.jar somatic [normal-tumor.mpileup] [output] --mpileup 1 OPTIONS
normal-tumor.mpileup - The SAMtools mpileup file with normal and then tumor
output - Output base name for SNP and indel outputBoth formats of the command share these common options:
OPTIONS:
--output-snp - Output file for SNP calls [default: output.snp]
--output-indel - Output file for indel calls [default: output.indel]
--min-coverage - Minimum coverage in normal and tumor to call variant [8]
--min-coverage-normal - Minimum coverage in normal to call somatic [8]
--min-coverage-tumor - Minimum coverage in tumor to call somatic [6]
--min-var-freq - Minimum variant frequency to call a heterozygote [0.10]
--min-freq-for-hom Minimum frequency to call homozygote [0.75]
--normal-purity - Estimated purity (non-tumor content) of normal sample [1.00]
--tumor-purity - Estimated purity (tumor content) of tumor sample [1.00]
--p-value - P-value threshold to call a heterozygote [0.99]
--somatic-p-value - P-value threshold to call a somatic site [0.05]
--strand-filter - If set to 1, removes variants with >90% strand bias
--validation - If set to 1, outputs all compared positions even if non-variantNote that more specific options (e.g. min-coverage-normal) will override the default or specificied value of less specific options (e.g. min-coverage).
The normal and tumor purity values should be a value between 0 and 1. The default (1) implies that the normal is 100% pure with no contaminating tumor cells, and the tumor is 100% pure with no contaminating stromal or other non-malignant cells. You would change tumor-purity to something less than 1 if you have a low-purity tumor sample and thus expect lower variant allele frequencies for mutations. You would change normal-purity to something less than 1 only if it's possible that there will be some tumor content in your "normal" sample, e.g. adjacent normal tissue for a solid tumor, malignant blood cells in the skin punch normal for some liquid tumors, etc.
There are two p-value options. One (p-value) is the significance threshold for the first-pass algorithm that determines, for each position, if either normal or tumor is variant at that position. The second (somatic-p-value) is more important; this is the threshold below which read count differences between tumor and normal are deemed significant enough to classify the sample as a somatic mutation or an LOH event. In the case of a shared (germline) variant, this p-value is used to determine if the combined normal and tumor evidence differ significantly enough from the null hypothesis (no variant with same coverage) to report the variant. See the somatic mutation calling section for details.
OUTPUT
Two tab-delimited files (SNPs and Indels) with the following columns:
chrom chromosome name
position position (1-based from the pileup)
ref reference allele at this position
var variant allele at this position
normal_reads1 reads supporting reference allele
normal_reads2 reads supporting variant allele
normal_var_freq frequency of variant allele by read count
normal_gt genotype call for Normal sample
tumor_reads1 reads supporting reference allele
tumor_reads2 reads supporting variant allele
tumor_var_freq frequency of variant allele by read count
tumor_gt genotype call for Tumor sample
somatic_status status of variant (Germline, Somatic, or LOH)
variant_p_value Significance of variant read count vs. baseline error rate
somatic_p_value Significance of tumor read count vs. normal read count
tumor_reads1_plus Ref-supporting reads from + strand in tumor
tumor_reads1_minus Ref-supporting reads from - strand in tumor
tumor_reads2_plus Var-supporting reads from + strand in tumor
tumor_reads2_minus Var-supporting reads from - strand in tumorcopynumber
This command calls variants and identifies their somatic status (Germline/LOH/Somatic) usingpileup files from a matched tumor-normal pair.
USAGE: java -jar VarScan.jar copynumber [normal_pileup] [tumor_pileup] [output] OPTIONS
normal_pileup - The SAMtools pileup file for Normal
tumor_pileup - The SAMtools pileup file for Tumor
output - Output base name for SNP and indel outputYou can also give it a single mpileup file with normal and tumor data.
USAGE: java -jar VarScan.jar copynumber [normal-tumor.mpileup] [output] --mpileup 1 OPTIONS
normal-tumor.mpileup - The SAMtools mpileup file with normal and then tumor
output - Output base name for SNP and indel outputBoth formats of the command share these common options:
OPTIONS:
--min-base-qual - Minimum base quality to count for coverage [20]
--min-map-qual - Minimum read mapping quality to count for coverage [20]
--min-coverage - Minimum coverage threshold for copynumber segments [20]
--min-segment-size - Minimum number of consecutive bases to report a segment [10]
--max-segment-size - Max size before a new segment is made [100]
--p-value - P-value threshold for significant copynumber change-point [0.01]
--data-ratio - The normal/tumor input data ratio for copynumber adjustment [1.0]Note: The data ratio is intended to help you account for overall differences in the amount of sequencing coverage between normal and tumor, which might otherwise give the appearance of global copy number differences. If normal has more data than tumor, set this to something greater than 1. If tumor has more data than normal, adjust it to something below 1. A basic formula for data ratio might be something like _ratio = normal_unique_bp / tumor_unique_bp_where unique base pairs are computed as mapped_non_dup_reads * read_length.
OUTPUT
chrom Chromosome name
chr_start Region start position (1-based from the pileup)
chr_stop Region stop position (1-based from the pileup)
num_positions Size of the region in base pairs
normal_depth Average normal sequence depth for the region
tumor_depth Average tumor sequence depth for the region
log2_ratio Log-base-2 ratio of: adjusted tumor depth over normal depth
gc_content Estimated GC content of the region (0-100)The raw regions reported by VarScan are delineated by drops in coverage or changes in the tumor/normal ratio, so there are many small, nearby regions with similar copy number. It is therefore recommended that raw VarScan copynumber output be processed with circular binary segmentation (CBS) or a similar algorithm, which will generate larger segments delineated by statistically significant change points. See the copy number calling section for details.
filter
This command filters variants in a file by coverage, supporting reads, variant frequency, or average base quality. It is for use with output from pileup2snp or pileup2indel.
USAGE: java -jar VarScan.jar filter [variants file] OPTIONS
variants file - A file of SNP or indel calls from VarScan pileup2snp or pileup2indel
OPTIONS:
--min-coverage Minimum read depth at a position to make a call [10]
--min-reads2 Minimum supporting reads at a position to call variants [2]
--min-strands2 Minimum # of strands on which variant observed (1 or 2) [1]
--min-avg-qual Minimum average base quality for variant-supporting reads [20]
--min-var-freq Minimum variant allele frequency threshold [0.20]
--p-value Default p-value threshold for calling variants [1e-01]
--indel-file File of indels for filtering nearby SNPs, from pileup2indel command
--output-file File to contain variants passing filterssomaticFilter
This command filters somatic mutation calls to remove clusters of false positives and SNV calls near indels. Note: this is a basic filter. More advanced filtering strategies consider mapping quality, read mismatches, soft-trimming, and other factors when deciding whether or not to filter a variant. See the VarScan 2 publication (Koboldt et al, Genome Research, Feb 2012) for details.
USAGE: java -jar VarScan.jar somaticFilter [mutations file] OPTIONS
mutations file - A file of SNVs from VarScan somatic
OPTIONS:
--min-coverage Minimum read depth [10]
--min-reads2 Minimum supporting reads for a variant [2]
--min-strands2 Minimum # of strands on which variant observed (1 or 2) [1]
--min-avg-qual Minimum average base quality for variant-supporting reads [20]
--min-var-freq Minimum variant allele frequency threshold [0.20]
--p-value Default p-value threshold for calling variants [1e-01]
--indel-file File of indels for filtering nearby SNPs
--output-file Optional output file for filtered variantslimit
This command limits variants in a file to a set of positions or regions
USAGE: java -jar VarScan.jar limit [infile] OPTIONS infile - A file of chromosome-positions, tab-delimited
OPTIONS
--positions-file - a file of chromosome-positions, tab delimited
--regions-file - a file of chromosome-start-stops, tab delimited
--output-file - Output file for the matching variantsreadcounts
This command reports the read counts for each base at positions in a pileup file
USAGE: java -jar VarScan.jar readcounts [pileup file] OPTIONS pileup file - The SAMtools pileup file
OPTIONS:
--variants-file A list of variants at which to report readcounts
--output-file Output file to contain the readcounts
--min-coverage Minimum read depth at a position to make a call [8]
--min-base-qual Minimum base quality at a position to count a read [30]compare
This command performs set-comparison operations on two files of variants.
USAGE: java -jar VarScan.jar compare [file1] [file2] [type] [output] OPTIONS file1 - A file of chromosome-positions, tab-delimited file2 - A file of chromosome-positions, tab-delimited type - Type of comparison [intersect|merge|unique1|unique2] output - Output file for the comparison result
For detailed usage information, see the VarScan JavaDoc.
VarScan Documentation (v2.2.2 and before)
USAGE: java -jar VarScan.jar [COMMAND] [OPTIONS]
COMMANDS
[**pileup2snp**](#v2.2%5Fpileup2snp) [pileup file]
[**pileup2indel**](#v2.2%5Fpileup2indel) [pileup file]
[**pileup2cns**](#v2.2%5Fpileup2cns) [pileup file]
[**somatic**](#v2.2%5Fsomatic) [normal pileup] [tumor pileup]
[**filter**](#v2.2%5Ffilter) [variants file]
[**somaticFilter**](#v2.2%5FsomaticFilter) [mutations file]
[**limit**](#v2.2%5Flimit) [variants file]
[**readcounts**](#v2.2%5Freadcounts) [pileup file]
[**compare**](#v2.2%5Fcompare) [file1] [file2]pileup2snp
This command calls SNPs from a pileup file based on user-defined parameters:
USAGE: java -jar VarScan.jar pileup2snp [pileup file] OPTIONS
pileup file - The SAMtools pileup file
OPTIONS:
--min-coverage Minimum read depth at a position to make a call [10]
--min-reads2 Minimum supporting reads at a position to call variants [2]
--min-avg-qual Minimum base quality at a position to count a read [15]
--min-var-freq Minimum variant allele frequency threshold [0.01]
--p-value Default p-value threshold for calling variants [99e-02]
OUTPUT
Tab-delimited SNP calls with the following columns:
Chrom chromosome name
Position position (1-based)
Ref reference allele at this position
Var variant allele at this position
Reads1 reads supporting reference allele
Reads2 reads supporting variant allele
VarFreq frequency of variant allele by read count
Strands1 strands on which reference allele was observed
Strands2 strands on which variant allele was observed
Qual1 average base quality of reference-supporting read bases
Qual2 average base quality of variant-supporting read bases
Pvalue Significance of variant read count vs. expected baseline errorpileup2indel
This command calls indels from a pileup file based on user-defined parameters:
USAGE: java -jar VarScan.jar pileup2indel [pileup file] OPTIONS
pileup file - The SAMtools pileup file
OPTIONS:
--min-coverage Minimum read depth at a position to make a call [8]
--min-reads2 Minimum supporting reads at a position to call variants [2]
--min-avg-qual Minimum base quality at a position to count a read [15]
--min-var-freq Minimum variant allele frequency threshold [0.01]
--p-value Default p-value threshold for calling variants [99e-02]
OUTPUT
Tab-delimited indel calls with the following columns:
Chrom chromosome name
Position position (1-based)
Ref reference allele at this position
Var variant allele at this position
Reads1 reads supporting reference allele
Reads2 reads supporting variant allele
VarFreq frequency of variant allele by read count
Strands1 strands on which reference allele was observed
Strands2 strands on which variant allele was observed
Qual1 average base quality of reference-supporting read bases
Qual2 average base quality of variant-supporting read bases
Pvalue Significance of variant read count vs. expected baseline errorpileup2cns
This command makes consensus calls (SNP/Indel/Reference) from a pileup file based on user-defined parameters:
USAGE: java -jar VarScan.jar pileup2cns [pileup file] OPTIONS
pileup file - The SAMtools pileup file
OPTIONS:
--min-coverage Minimum read depth at a position to make a call [8]
--min-reads2 Minimum supporting reads at a position to call variants [2]
--min-avg-qual Minimum base quality at a position to count a read [15]
--min-var-freq Minimum variant allele frequency threshold [0.01]
--p-value Default p-value threshold for calling variants [99e-02]
OUTPUT
Tab-delimited consensus calls with the following columns:
Chrom chromosome name
Position position (1-based)
Ref reference allele at this position
Var consensus call (reference, IUPAC SNP code, or indel)
Reads1 reads supporting reference allele
Reads2 reads supporting variant allele
VarFreq frequency of variant allele by read count
Strands1 strands on which reference allele was observed
Strands2 strands on which variant allele was observed
Qual1 average base quality of reference-supporting read bases
Qual2 average base quality of variant-supporting read bases
Pvalue Significance of variant read count vs. expected baseline errorsomatic
This command calls variants and identifies their somatic status (Germline/LOH/Somatic) usingpileup files from a matched tumor-normal pair.
USAGE: java -jar VarScan.jar somatic [normal_pileup] [tumor_pileup] [output] OPTIONS
normal_pileup - The SAMtools pileup file for Normal
tumor_pileup - The SAMtools pileup file for Tumor
output - Output base name for SNP and indel output
OPTIONS:
--output-snp Output file for SNP calls [output.snp]
--output-indel Output file for indel calls [output.indel]
--min-coverage Minimum coverage in normal and tumor to call variant [10]
--min-coverage-normal Minimum coverage in normal to call somatic [10]
--min-coverage-tumor Minimum coverage in tumor to call somatic [5]
--min_var_freq Minimum variant frequency to call a heterozygote [0.20]
--p-value P-value threshold to call a heterozygote [1.0e-01]
--somatic-p-value P-value threshold to call a somatic site [1.0e-04]
OUTPUT
Two tab-delimited files (SNPs and Indels) with the following columns:
Chrom chromosome name
Position position (1-based)
Ref reference allele at this position
Var variant allele at this position
Normal_Reads1 reads supporting reference allele
Normal_Reads2 reads supporting variant allele
Normal_VarFreq frequency of variant allele by read count
Normal_Gt genotype call for Normal sample
Tumor_Reads1 reads supporting reference allele
Tumor_Reads2 reads supporting variant allele
Tumor_VarFreq frequency of variant allele by read count
Tumor_Gt genotype call for Tumor sample
Somatic_Status status of variant (Germline, Somatic, or LOH)
Pvalue Significance of variant read count vs. expected baseline error
Somatic_Pvalue Significance of tumor read count vs. normal read countfilter
This command filters variants in a file by coverage, supporting reads, variant frequency, or average base quality
USAGE: java -jar VarScan.jar filter [variants file] OPTIONS
variants file - A file of SNP or indel calls from VarScan
OPTIONS:
--min-coverage Minimum read depth at a position to make a call [8]
--min-reads2 Minimum supporting reads at a position to call variants [2]
--min-avg-qual Minimum base quality at a position to count a read [15]
--min-var-freq Minimum variant allele frequency threshold [0.01]
--p-value Default p-value threshold for calling variants [99e-02]somaticFilter
This command filters somatic mutation calls to remove clusters of false positives and SNV calls near indels.
USAGE: java -jar VarScan.jar somaticFilter [mutations file] OPTIONS
mutations file - A file of SNVs from VarScan somatic
OPTIONS:
--min-coverage Minimum read depth [10]
--min-reads2 Minimum supporting reads for a variant [2]
--min-strands2 Minimum # of strands on which variant observed (1 or 2) [1]
--min-avg-qual Minimum average base quality for variant-supporting reads [20]
--min-var-freq Minimum variant allele frequency threshold [0.20]
--p-value Default p-value threshold for calling variants [1e-01]
--indel-file File of indels for filtering nearby SNPs
--output-file Optional output file for filtered variantslimit
This command limits variants in a file to a set of positions or regions
USAGE: java -jar VarScan.jar limit [infile] OPTIONS infile - A file of chromosome-positions, tab-delimited
OPTIONS
--positions-file - a file of chromosome-positions, tab delimited
--regions-file - a file of chromosome-start-stops, tab delimited
--output-file - Output file for the matching variantsreadcounts
This command reports the read counts for each base at positions in a pileup file
USAGE: java -jar VarScan.jar readcounts [pileup file] OPTIONS pileup file - The SAMtools pileup file
OPTIONS:
--variants-file A list of variants at which to report readcounts
--output-file Output file to contain the readcounts
--min-coverage Minimum read depth at a position to make a call [8]
--min-base-qual Minimum base quality at a position to count a read [30]compare
This command performs set-comparison operations on two files of variants.
USAGE: java -jar VarScan.jar compare [file1] [file2] [type] [output] OPTIONS file1 - A file of chromosome-positions, tab-delimited file2 - A file of chromosome-positions, tab-delimited type - Type of comparison [intersect|merge|unique1|unique2] output - Output file for the comparison result
For detailed usage information, see the VarScan JavaDoc.
How to Build a SAMtools (m)pileup File
The variant calling features of VarScan for single samples (pileup2snp, pileup2indel, pileup2cns) and multiple samples (mpileup2snp, mpileup2indel, mpileup2cns, and somatic) expect input in SAMtools pileup or mpileup format. In current versions of SAMtools, the "pileup" command has now been replaced with the "mpileup" command. For a single sample, these operate in a very similar fashion, except that mpileup applies BAQ adjustments by default, and the output is identical. When you give it multiple BAM files, however, SAMtools mpileup generates a multi-sample pileup format that must be processed with the mpileup2* commands in VarScan. To build a mpileup file, you will need:
- One or more BAM files ("myData.bam") that have been sorted using the sort command of SAMtools.
- The reference sequence ("reference.fasta") to which reads were aligned, in FASTA format.
- The SAMtools software package.
Generate a mpileup file with the following command:
samtools mpileup -f [reference sequence] [BAM file(s)] >myData.mpileup
Note, to save disk space and file I/O, you can redirect mpileup output directly to VarScan with a "pipe" command. For example:
One sample: samtools mpileup -f reference.fasta myData.bam | java -jar VarScan.v2.2.jar pileup2snp
Multiple samples: samtools mpileup -f reference.fasta sample1.bam sample2.bam | java -jar VarScan.v2.2.jar pileup2snp