Biowulf High Performance Computing at the NIH
VarScan: Variant calling and somatic mutation/CNV detection for next-generation sequencing data

VarScan is an open source tool for variant detection that is compatible with several short read aligners. It is capable of detecting SNPs and indels with high sensitivity and specificity, in both Roche/454 sequencing of individuals and deep Illumina/Solexa sequencing of pooled samples. VarScan2 detects somatic mutations and copy number alterations (CNAs) in exome data from tumor–normal pairs.


Important Notes

Interactive job
Interactive jobs should be used for debugging, graphics, or applications that cannot be run as batch jobs.

Allocate an interactive session and run the program. Sample session:

[user@biowulf]$ sinteractive
[user@cn3316 ~]$ module load VarScan
Download/prepare sample input data:
[user@cn3316 ~]$ URL=""
[user@cn3316 ~]$ wget $URL/wgEncodeUwRepliSeqBg02esG1bAlnRep1.bam -O myData1.bam
[user@cn3316 ~]$ wget $URL/wgEncodeUwRepliSeqBg02esG2AlnRep1.bam -O myData2.bam
[user@cn3316 ~]$ samtools sort  myData1.bam > myData1_sorted.bam 
[user@cn3316 ~]$ samtools sort  myData2.bam > myData2_sorted.bam 
[user@cn3316 ~]$ ln -s /fdb/igenomes/Homo_sapiens/UCSC/hg19/Sequence/WholeGenomeFasta/genome.fa hg19.fa
[user@cn3316 ~]$ samtools mpileup -B -f hg19.fa  myData1_sorted.bam  > myData1.pileup
[user@cn3316 ~]$ samtools mpileup -B -f hg19.fa  myData2_sorted.bam  > myData2.pileup
Run a VarScan executable on the inputs:
[user@cn3316 ~]$varscan somatic myData1.pileup myData2.pileup --output-snp snp --output-indel indel
Normal Pileup: myData1.pileup
Tumor Pileup: myData2.pileup
NOTICE: While dual input files are still supported, using a single mpileup file (normal-tumor) with the --mpileup 1 setting is strongly recommended.
Min coverage:   8x for Normal, 6x for Tumor
Min reads2:     2
Min strands2:   1
Min var freq:   0.2
Min freq for hom:       0.75
Normal purity:  1.0
Tumor purity:   1.0
Min avg qual:   15
P-value thresh: 0.99
Somatic p-value:        0.05
52831015 positions in tumor
52831015 positions shared in normal
225836 had sufficient coverage for comparison
0 were called Reference
0 were mixed SNP-indel calls and filtered
225836 were called Germline
0 were called LOH
0 were called Somatic
0 were called Unknown
0 were called Variant
[user@cn3144 ~]$ exit
salloc.exe: Relinquishing job allocation 46116226
[user@biowulf ~]$

Batch job
Most jobs should be run as batch jobs.

Create a batch input file (e.g. For example:

module load VarScan
varscan somatic myData1.pileup myData2.pileup --output-snp snp12 --output-indel indel12
varscan somatic myData3.pileup myData4.pileup --output-snp snp34 --output-indel indel34

Submit this job using the Slurm sbatch command.

sbatch [--cpus-per-task=#] [--mem=#]