[med-svn] [varscan] 01/01: Enhancing packaging
Andreas Tille
tille at debian.org
Wed Apr 16 17:34:24 UTC 2014
This is an automated email from the git hooks/post-receive script.
tille pushed a commit to branch master
in repository varscan.
commit 30722343a0a45d073c5f7cddd8a4bd800ebaca73
Author: Andreas Tille <tille at debian.org>
Date: Wed Apr 16 08:01:01 2014 +0200
Enhancing packaging
---
debian/changelog | 2 +-
debian/control | 4 +-
debian/copyright | 231 +++++++++++++-
debian/faq.txt | 268 ++++++++++++++++
debian/get-manual | 21 ++
debian/manifest | 3 +
debian/upstream/metadata | 23 +-
debian/using-varscan.txt | 801 +++++++++++++++++++++++++++++++++++++++++++++++
8 files changed, 1335 insertions(+), 18 deletions(-)
diff --git a/debian/changelog b/debian/changelog
index 961d122..8dbd399 100644
--- a/debian/changelog
+++ b/debian/changelog
@@ -2,4 +2,4 @@ varscan (2.3.6+dfsg-1) UNRELEASED; urgency=low
* Initial release (Closes: #<bug>)
- -- DMPT <debian-med-packaging at lists.alioth.debian.org> Thu, 24 May 2012 14:30:13 +0200
+ -- Andreas Tille <tille at debian.org> Tue, 15 Apr 2014 13:38:37 +0200
diff --git a/debian/control b/debian/control
index 1305617..8b244ee 100644
--- a/debian/control
+++ b/debian/control
@@ -5,8 +5,8 @@ Maintainer: Debian Med Packaging Team <debian-med-packaging at lists.alioth.debian.
Uploaders: Andreas Tille <tille at debian.org>
Build-Depends: debhelper (>= 9)
Standards-Version: 3.9.5
-Vcs-Browser: http://anonscm.debian.org/viewvc/debian-med/trunk/packages/varscan/trunk/
-Vcs-Svn: svn://anonscm.debian.org/debian-med/trunk/packages/varscan/trunk/
+Vcs-Browser: http://anonscm.debian.org/gitweb/?p=debian-med/varscan.git
+Vcs-Git: git://anonscm.debian.org/debian-med/varscan.git
Homepage: http://varscan.sourceforge.net/
Package: varscan
diff --git a/debian/copyright b/debian/copyright
index ba82f3b..ed6cd04 100644
--- a/debian/copyright
+++ b/debian/copyright
@@ -1,14 +1,237 @@
Format: http://www.debian.org/doc/packaging-manuals/copyright-format/1.0/
Upstream-Name: VarScan
+Upstream-Contact: Dan Koboldt http://genome.wustl.edu/people/individual/dan-koboldt/
Source: http://sourceforge.net/projects/varscan/files/
Files-Excluded:
*.class
META-INF
Files: *
-Copyright: © 20xx-20yy <upstream>
-License: <license>
+Copyright: 2009-2014 by Washington University in St. Louis.
+License: NPOSL-3.0
+ The download page
+ http://sourceforge.net/projects/varscan/files/
+ says:
+ VarScan 2 is licensed under the Non-Profit Open Software License 3.0 (NPOSL-3.0)
+ .
+ Non-Profit Open Software License 3.0 (NPOSL-3.0)
+ .
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Files: debian/*
-Copyright: © 2014 maintainername <maintainer at e.mail>
-License: <license>
+Copyright: 2014 Andreas Tille <tille at debian.org>
+License: LGPL-3+
+ On Debian systems the full text of the GNU Lesser General Public License
+ is available at /usr/share/common-licenses/LGPL-3+
+
diff --git a/debian/faq.txt b/debian/faq.txt
new file mode 100644
index 0000000..8f15168
--- /dev/null
+++ b/debian/faq.txt
@@ -0,0 +1,268 @@
+Support FAQ
+===========
+
+This page contains answers to many of the common questions asked about VarScan
+usage, performance, input/output, etc.
+
+
+USAGE QUESTIONS
+
+Which version of VarScan should I use?
+Which VarScan command should I use?
+Can I use VarScan on WGS, exome, or RNA-seq data?
+Does VarScan work for pooled samples?
+How do I use VarScan for validation?
+Can I use VarScan on my model organism or microbial genome?
+
+
+INPUT QUESTIONS
+
+What input format should I give to VarScan?
+Which aligner should I use?
+Do I need Illumina or Phred scale base qualities?
+What about mapping qualities?
+
+
+OUTPUT QUESTIONS
+
+What do the output columns mean?
+How are the p-values calculated?
+Why are all of my p-values 0.98?
+How should I filter the output files?
+Does VarScan provide a confidence score similar to SAMtools?
+
+
+COMMON ISSUES, WARNINGS, AND ERRORS
+
+Warnings about "resetting normal" or "resetting tumor" file
+Read counts from VarScan are different from SAMtools/IGV counts
+
+
+
+USAGE QUESTIONS
+
+Which version of VarScan should I use?
+
+Always use the latest version! Currently, this is v2.3.x. There are differences
+between versions; notably, between v2.2.2 and v2.2.3 we adjusted how reads are
+counted for indels, which had exhibited strange behavior due to some
+peculiarities in how SAMtools represents indels in the pileup files.
+
+
+Which VarScan command should I use?
+
+If you have a single sample and wish to call variants, you can use pileup2snp
+to call SNPs, pileup2indel to call indels, or pileup2cns to call consensus
+genotypes at every position meeting the coverage requirement. To call both SNPs
+and indels simultaneously, use pileup2cns with the --variants parameter set to
+1. If you have tumor-normal pairs, you should use the somatic command to call
+mutations (SNVs/indels) and the copynumber command to call copy number
+alterations (CNAs).
+
+
+Can I use VarScan on WGS, exome, or RNA-seq data?
+
+Yes, you can use VarScan for any of these. The default settings are optimized
+for exome data, where one expects to have at least 10x or 20x coverage across
+targeted exons. By lowering the minimum coverage requirement (to perhaps 6x)
+one can call variants in lower-coverage data. Warning: the output files for WGS
+data may be quite large, and the runtimes longer, since it will call 3+ million
+variants per genome. VarScan works for RNA-seq data as well, though this data
+tends to be noisier for variant calling due to RT errors, allele-specific
+expression, and alignment difficulties.
+
+
+Does VarScan work for pooled samples?
+
+Absolutely. It's simply a matter of setting appropriate input parameters,
+particularly --min-coverage, --min-var-freq, and --p-value. In pooled data, you
+might specify a higher minimum coverage, a lower variant allele frequency
+(conservatively, 0.50 / the number of samples), and a less-stringent p-value to
+detect rare variants.
+
+
+How do I use VarScan for validation?
+
+If validating germline SNPs/indels on an orthogonal sequencing platform, use
+the pileup2cns command, which will call consensus genotypes at all positions
+with sufficient coverage, rather than just the variants. This lets you
+determine sites that are refuted as wild-type. You might start with the
+following parameters:
+
+
+--min-coverage 20
+--min-var-freq 0.08
+--p-value 0.05
+
+If validating somatic SNPs/indels on an orthogonal sequncing platform, use the
+somatic command with --validation set to 1. You might start with the following
+parameters:
+
+
+--min-coverage 20
+--min-var-freq 0.08
+--p-value 0.10
+--somatic-p-value 0.05
+--validation 1
+
+
+
+Can I use VarScan on my model organism or microbial genome?
+
+Of course. All you need is a BAM file of your reads aligned to a reference
+sequence. If you don't have a reference sequence to which you might align
+reads, then no.
+
+
+INPUT QUESTIONS
+
+What input format should I give to VarScan?
+
+VarScan expects its input in SAMtools pileup format, which is obtained from a
+BAM file via the samtools pileup command. For example:
+
+
+samtools pileup -f myReference.fasta myReads.bam >myPileup.pileup
+java -jar VarScan.jar pileup2snp myPileup.pileup
+
+To save on disk space and I/O, you can also use a UNIX "pipe" command to
+forward the pileup output directly into VarScan:
+
+
+samtools pileup -f myRef.fasta myBam.bam | java -jar VarScan.jar pileup2snp
+
+
+
+Which aligner should I use?
+
+Great question! The choice of an aligner is an important one, and entire review
+articles have been written on the topic. For practical purposes, you should use
+an aligner whose output is (or can be converted to) a SAM/BAM file. I have
+heard that popular aligners for include BWA, Bowtie, and Novoalign for Illumina
+data, SHRiMP and BFAST for SOLiD data, and SSAHA2 and BWA-SW for Roche/454
+data.
+
+
+Do I need Illumina or Phred scale base qualities?
+
+VarScan expects Phred-scaled (Phred+33, also called "Sanger") base qualities,
+in which a score of 20 indicates a 1/1000 probability of base error. By
+default, VarScan requires a minimum base quality of 15 or 20 (depending on the
+application), so this value should be adjusted appropriately if alternate base
+qualities are used.
+
+
+What about mapping qualities?
+
+
+Many aligners provide a Phred-scaled quality value (mapping quality) for every
+read's alignment, which is correlated to the probability that the read is
+correctly mapped. A mapping quality of 0 typically indicates that the given
+read has many possible mapping locations of equal probability, and that the
+location given was chosen randomly. Thus, it's best to exclude reads with
+mapping quality of 0 from most downstream analyses. A minimum mapping quality
+of 10 is even better. It's possible to apply this threshold to a BAM file using
+SAMtools, as follows:
+
+samtools view -b -q 10 myBam.bam | samtools pileup -f myRef.fasta -
+| java -jar VarScan.jar pileup2snp
+
+
+
+OUTPUT QUESTIONS
+
+What do the output columns mean?
+
+By default, all VarScan output files should include headers. For detailed
+descriptions of the output columns and their meanings, see the "output"
+sections of the germline or somatic calling pages. You can also specify VCF
+output, which is widely documented.
+
+
+How are the p-values calculated?
+
+P-values are calculated using a Fisher's Exact Test on the read counts
+supporting reference and variant alleles. For details, see the germline or
+somatic calling pages.
+
+
+Why are all of my p-values 0.98?
+
+The p-value calculations are computationally expensive, so if the user doesn't
+specify a p-value threshold, VarScan skips the calculation in pileup2snp,
+pileup2indel, and pileup2cns. Instead, it inserts a dummy value of 0.98. To get
+p-values, set the --p-value parameter to something like 0.10, 0.05, or 0.01.
+
+
+How should I filter the output files?
+
+VarScan's default parameters aim to be sensitive when detecting variants, with
+the trade-off that it will report a lot of candidate variants, many of which
+will be false positives. These should be further filtered by coverage, variant
+allele frequency, strand representation, and p-value to isolate high-confidence
+calls. When performing the initial discovery, it is recommended that you set
+--strand-filter to 1. After discovery, you should run VarScan filter or
+somaticFilter to further refine your variant calls.
+
+
+Does VarScan provide a confidence score similar to SAMtools?
+
+It is possible to calculate a Phred-scaled confidence score from the p-value
+that VarScan provides:
+
+$score = -10 * log10($p_value);
+$score = 255 if($score > 255);
+
+This is done on a per-sample basis when --vcf-output is specified, since VCF
+format expects Phred-scaled confidence scores.
+
+
+COMMON ISSUES, WARNINGS, AND ERRORS
+
+Warnings about "resetting normal" or "resetting tumor" file
+
+If you provide two separate input (pileup) files, VarScan attempts to use the
+chromosome and position to simultaneously parse both files and match them up.
+Doing so while accounting for alphanumeric chromosome names is difficult,
+especially when there are chromosomes for which only one sample had coverage.
+VarScan does its best to ensure that no chromosomes are missed due to a sorting
+issue by resetting one sample's pileup file, to ensure that no chromosomes are
+missed due to sorting. When you have many chromosomes or contigs with coverage
+in just one sample, you'll see a lot of these warnings.
+
+The best way to address this simultaneous parsing issue is quite simple:
+provide VarScan with a two-sample MPILEUP file (normal and tumor) instead of
+two individual pileup files:
+
+samtools mpileup -f reference.fasta -q 1 -B normal.bam tumor.bam >normal-tumor.mpileup
+java -jar VarScan.jar somatic normal-tumor.mpileup normal-tumor.varScan.output --mpileup 1
+
+In the mpileup file, SAMtools already does the chromosome and position
+matching-up, so there's no room for error. You'll also notice that I provided
+the -B parameter. This disables SAMtools BAQ computation, which is turned on by
+default in SAMtools mpileup, but (as the author admits) occasionally too
+stringent for variant calling.
+
+Read counts from VarScan are different from SAMtools/IGV counts
+
+By default, SAMtools and IGV show and count all bases at a given position,
+regardless of base quality. In contrast, VarScan requires that bases meet the
+minimum Phred quality score (default 15 for most commands) to count them for
+things like read counts (reads1, reads2) and to compute variant allele
+frequency. However, when VarScan reports the depth (such as in the DP field of
+VCF output), it reports SAMtools raw depth. To get VarScan read counts to more
+closely match another tool, set use parameter --min-avg-qual 0. And use
+caution! Low-quality bases, with the occasional exception of BAQ penalties,
+should not be trusted.
+
+Also, VarScan reports variants on a biallelic basis. That is, for a given SNP
+call, the "reads1" column is the number of reference-supporting reads (RD), and
+the "reads2" column is the number of variant-supporting reads (AD). There may
+be additional reads at that position showing other bases (SNP or indel
+variants). If these other variants meet the calling criteria, they will be
+reported in their own line. If not, it may look like you have "missing" reads.
+
+
+
+Copyright © 2009-2013 by Washington University in St. Louis. Design by CSS
+Templates
diff --git a/debian/get-manual b/debian/get-manual
new file mode 100755
index 0000000..27d123d
--- /dev/null
+++ b/debian/get-manual
@@ -0,0 +1,21 @@
+#!/bin/sh
+TMP=`mktemp`
+OUT=using-varscan.txt
+w3m -dump http://varscan.sourceforge.net/using-varscan.html > $TMP
+cat >$OUT <<EOT
+VarScan User's Manual
+=====================
+EOT
+sed "1,/^VarScan User's Manual/d" $TMP >> $OUT
+
+
+OUT=faq.txt
+w3m -dump http://varscan.sourceforge.net/support-faq.html > $TMP
+cat >$OUT <<EOT
+Support FAQ
+===========
+EOT
+sed "1,/^Support FAQ/d" $TMP >> $OUT
+
+rm -f $TMP
+
diff --git a/debian/manifest b/debian/manifest
new file mode 100644
index 0000000..416f0ea
--- /dev/null
+++ b/debian/manifest
@@ -0,0 +1,3 @@
+Manifest-Version: 1.0
+Main-Class: net.sf.varscan.VarScan
+
diff --git a/debian/upstream/metadata b/debian/upstream/metadata
index d8b5812..428fd3e 100644
--- a/debian/upstream/metadata
+++ b/debian/upstream/metadata
@@ -1,12 +1,13 @@
Reference:
- Author:
- Title:
- Journal:
- Year:
- Volume:
- Number:
- Pages:
- DOI:
- PMID:
- URL:
- eprint:
+ Author: Daniel C. Koboldt and Qunyuan Zhang and David E. Larson and Dong Shen and Michael D. McLellan and Ling Lin and Christopher A. Miller and Elaine R. Mardis and Li Ding and Richard K. Wilson
+ Title: "VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing"
+ Journal: Genome Res.
+ Year: 2012
+ Volume: 22
+ Number: 3
+ Pages: 568-76
+ DOI: doi: 10.1101/gr.129684.111
+ PMID: 22300766
+ URL: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3290792/
+ eprint: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3290792/pdf/568.pdf
+
diff --git a/debian/using-varscan.txt b/debian/using-varscan.txt
new file mode 100644
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+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 [pileup file]
+ pileup2indel [pileup file]
+ pileup2cns [pileup file]
+
+ Multi-sample Calling:
+ mpileup2snp [mpileup file]
+ mpileup2indel [mpileup file]
+ mpileup2cns [mpileup file]
+
+ Tumor-normal Comparison:
+ somatic [normal pileup] [tumor pileup] or [normal-tumor mpileup]
+ copynumber [normal pileup] [tumor pileup] or [normal-tumor mpileup]
+
+ Variant Filtering:
+ filter [variants file]
+ somaticFilter [mutations file]
+
+ Utility Functions:
+ limit [variants file]
+ readcounts [pileup file]
+ compare [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) using pileup 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
+
+You 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 output
+
+Both 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-variant
+
+Note 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 tumor
+
+
+copynumber
+
+This command calls variants and identifies their somatic status (Germline/LOH/
+Somatic) using pileup 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 output
+
+You 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 output
+
+Both 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 filters
+
+
+
+somaticFilter
+
+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 variants
+
+
+limit
+
+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 variants
+
+
+readcounts
+
+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 [pileup file]
+ pileup2indel [pileup file]
+ pileup2cns [pileup file]
+ somatic [normal pileup] [tumor pileup]
+ filter [variants file]
+ somaticFilter [mutations file]
+ limit [variants file]
+ readcounts [pileup file]
+ compare [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 error
+
+
+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
+ 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 error
+
+
+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
+ 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 error
+
+
+somatic
+
+This command calls variants and identifies their somatic status (Germline/LOH/
+Somatic) using pileup 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 count
+
+
+filter
+
+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 variants
+
+
+limit
+
+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 variants
+
+
+readcounts
+
+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
+
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+Templates
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