RELEASE NOTES FOR VARSCAN V2.4.2 26-May-2016 VarScan v2.4.2 is the current release VarScan v2.4.1 is the previous release VarScan v2.4.0 was the first release to VarScan's new home at GitHub, http://dkoboldt.github.io/varscan/ VarScan v2.3.9 and prior releases will persist on SourceForge, as will the support forums and other resources. LICENSE VarScan 2 is free for non-commercial use by academic, government, and non-profit/not-for-profit institutions. A commercial version of the software is available, and licensed through the Office of Technology Management at Washington University School of Medicine. For more information, please contact: Paul Carter, Business Development Director paulcarter@wustl.edu +1 314-362-5426 https://otm.wustl.edu/for-industry/tools/ VERSION 2.4.2 CHANGES The primary changes are minor bugfixes for user-reported issues: 1.) Better handling of mal-formed mpileup lines in VarScan copynumber 2.) Addressing missing segregation STATUS codes in VarScan trio and the addition of a new code (0) for "Unknown") 3.) Addressing a VCF format issue for somatic mutation calling, in which multi-allelic sites (classified as "UNKNOWN") were reported in non-standard VCF format (with forward slashes in the ALT column). VarScan 2 somatic should now report multiple variant alleles using the ALT1,ALT2 format and the genotype fields for normal and tumor samples should reflect the appropriate variant allele. REMINDER: PLEASE USE THE FALSE POSITIVE FILTER The scientific basis of this filter is described in the VarScan 2 publication. It will improve the precision of variant and mutation calling by removing artifacts associated with short-read alignment. -For somatic mutations, generate bam-readcounts with the Tumor BAM. For LOH and Germline, generate readcounts with the Normal BAM -For de novo mutations (trio calling), generate readcounts with the child BAM. The filter requires the bam-readcount utility: https://github.com/genome/bam-readcount USAGE: java -jar VarScan.jar fpfilter [variant file] [readcount file] OPTIONS variant file - A file of SNPs or indels in VarScan-native or VCF format readcount file - The output file from bam-readcount for those positions ***For detailed filtering instructions, please visit http://varscan.sourceforge.net*** OPTIONS: --output-file Optional output file for filter-pass variants --filtered-file Optional output file for filter-fail variants --dream3-settings If set to 1, optimizes filter parameters based on TCGA-ICGC DREAM-3 SNV Challenge results --keep-failures If set to 1, includes failures in the output file FILTERING PARAMETERS: --min-var-count Minimum number of variant-supporting reads [4] --min-var-count-lc Minimum number of variant-supporting reads when depth below somaticPdepth [2] --min-var-freq Minimum variant allele frequency [0.05] --max-somatic-p Maximum somatic p-value [0.05] --max-somatic-p-depth Depth required to test max somatic p-value [10] --min-ref-readpos Minimum average read position of ref-supporting reads [0.1] --min-var-readpos Minimum average read position of var-supporting reads [0.1] --min-ref-dist3 Minimum average distance to effective 3' end (ref) [0.1] --min-var-dist3 Minimum average distance to effective 3' end (var) [0.1] --min-strandedness Minimum fraction of variant reads from each strand [0.01] --min-strand-reads Minimum allele depth required to perform the strand tests [5] --min-ref-basequal Minimum average base quality for ref allele [15] --min-var-basequal Minimum average base quality for var allele [15] --min-ref-avgrl Minimum average trimmed read length for ref allele [90] --min-var-avgrl Minimum average trimmed read length for var allele [90] --max-rl-diff Maximum average relative read length difference (ref - var) [0.25] --max-ref-mmqs Maximum mismatch quality sum of reference-supporting reads [100] --max-var-mmqs Maximum mismatch quality sum of variant-supporting reads [100] --max-mmqs-diff Maximum average mismatch quality sum (var - ref) [50] --min-ref-mapqual Minimum average mapping quality for ref allele [15] --min-var-mapqual Minimum average mapping quality for var allele [15] --max-mapqual-diff Maximum average mapping quality (ref - var) [50] DREAM-3 SETTINGS FOR FPFILTER Please note the --dream3-settings parameter for fpfilter, which (if set to 1) will optimize the false positive filter settings based on the fine-tuning we did for the TCGA-ICGC DREAM-3 SNV Challenge. See the "in silico 3" dataset described here: https://www.synapse.org/#!Synapse:syn312572/wiki/62018 This dataset modeled 100% tumor purity, but three subclones at 50%, 33%, and 20% variant allele frequency. Optimal VarScan settings were established as follows: For SAMtools: mpileup -B (disables BAQ) For VarScan somatic: --min-coverage 3 --min-var-freq 0.08 --p-value 0.10 --somatic-p-value 0.05 --strand-filter 0 For VarScan fpfilter: --min-var-count = 3 --min-var-count-lc = 1 --min-strandedness = 0 --min-var-basequal = 30 --min-ref-readpos = 0.20 --min-ref-dist3 = 0.20 --min-var-readpos = 0.15 --min-var-dist3 = 0.15 --max-rl-diff = 0.05 --max-mapqual-diff = 10 --min-ref-mapqual = 20 --min-var-mapqual = 30 --max-var-mmqs = 100 --max-ref-mmqs = 50 CITING VARSCAN If you use VarScan, please note the version number and cite this publication along with the version-appropriate URL: Koboldt DC, Zhang Q, Larson DE, Shen D, McLellan MD, Lin L, Miller CA, Mardis ER, Ding L, Wilson RK. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Res. 2012 Mar;22(3):568-76. doi: 10.1101/gr.129684.111. https://github.com/dkoboldt/varscan (v2.4.0 and beyond) or http://varscan.sourceforge.net (v2.3.9 and before)