# Errors with Varscan Somatic and GATK pipline **URL:** https://help.galaxyproject.org/t/errors-with-varscan-somatic-and-gatk-pipline/451 **Category:** Uncategorized **Tags:** variant-analysis **Created:** [January 15, 2019, 9:01am UTC](https://help.galaxyproject.org/t/errors-with-varscan-somatic-and-gatk-pipline/451 "2019-01-15T09:01:22Z") **Posts on this page:** 14 **Page:** 1
### Author: ![Tarek\_zakaria](https://avatars.discourse-cdn.com/v4/letter/t/3d9bf3/32.png) [@Tarek\_zakaria](https://help.galaxyproject.org/u/Tarek_zakaria) #### Post date: [January 15, 2019, 9:01am UTC](https://help.galaxyproject.org/t/errors-with-varscan-somatic-and-gatk-pipline/451/1 "2019-01-15T09:01:22Z")
Hey everyone, i have been having some difficulties using the GATK tools and Varscan Somatic in Galaxy to analyse tumor vs normal tissue samples. Basically after adding the bam files of the tumor/normal pair Varscan somatic tool runs for a couple of hours then crashes. This happened three times now. i don’t have the same problem running sam mileup then Varscan but i would have likes to use the Varscan Somatic tool. Also in using GATK Indel Realigner as an example. isn’t it possible to use a bulit in genome refrence as in BWA-MEM? or do i have to upload mine because i would have liked to use the exact same refrence files used in mapping provided within galaxy? Thanks in advance ---
### Author: ![wm75](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/wm75/32/56_2.png) [@wm75](https://help.galaxyproject.org/u/wm75) #### Post date: [January 15, 2019, 9:48am UTC](https://help.galaxyproject.org/t/errors-with-varscan-somatic-and-gatk-pipline/451/2 "2019-01-15T09:48:40Z")
Hmm, can you be a bit more specific? - Where are you trying this? AFAIK, [usegalaxy.org](http://usegalaxy.org) doesn’t currently have the _Varscan somatic_ tool installed, so are you using [usegalaxy.eu](http://usegalaxy.eu), or something else? - Which version of the tool are you using? - What is the error message when the tool “crashes”? The Varscan somatic tool has undergone a relatively big update recently and, as the person responsible for that update, I’d be really interested to learn if there is a problem with it, so I can fix it. ---
### Author: ![Tarek\_zakaria](https://avatars.discourse-cdn.com/v4/letter/t/3d9bf3/32.png) [@Tarek\_zakaria](https://help.galaxyproject.org/u/Tarek_zakaria) #### Post date: [January 15, 2019, 10:21am UTC](https://help.galaxyproject.org/t/errors-with-varscan-somatic-and-gatk-pipline/451/3 "2019-01-15T10:21:07Z")
Hi, thanks for your feedback, yeah i am using the European Galaxy/Galaxy uni Freiburg. The tool version stated is varscan\_somatic 2.4.3.2. Apparently it always crashes around calling the variants in CH12. i’m not sure exactly where the error exactly is after viewing the report, but here is the code generated if it would help? ln -s -f ‘/data/7/galaxy\_db/files/008/154/dataset\_8154422.dat’ normal.bam && ln -s -f ‘/data/7/galaxy\_db/files/008/154/dataset\_8154424.dat’ tumor.bam && ln -s -f ‘/data/7/galaxy\_db/files/\_metadata\_files/000/149/metadata\_149016.dat’ normal.bam.bai && ln -s -f ‘/data/7/galaxy\_db/files/\_metadata\_files/000/149/metadata\_149013.dat’ tumor.bam.bai && python3 /opt/galaxy/shed\_tools/toolshed.g2.bx.psu.edu/repos/iuc/varscan\_somatic/d37adcc2ec03/varscan\_somatic/varscan.py --normal ‘normal.bam’ --tumor ‘tumor.bam’ --normal-purity 1.0 --tumor-purity 1.0 --ofile variants\_out --split-output --threads ${GALAXY\_SLOTS:-2} --verbose ‘/data/db/reference\_genomes/mm10/seq/mm10.fa’ ---
### Author: ![wm75](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/wm75/32/56_2.png) [@wm75](https://help.galaxyproject.org/u/wm75) #### Post date: [January 15, 2019, 10:43am UTC](https://help.galaxyproject.org/t/errors-with-varscan-somatic-and-gatk-pipline/451/4 "2019-01-15T10:43:34Z")
I see. This is indeed the latest version of the tool so I may have to fix something here. You should see the exact error message when you click the little bug icon attached to the failed dataset (right next to the info button). Can you post the complete contents of that please? ---
### Author: ![Tarek\_zakaria](https://avatars.discourse-cdn.com/v4/letter/t/3d9bf3/32.png) [@Tarek\_zakaria](https://help.galaxyproject.org/u/Tarek_zakaria) #### Post date: [January 15, 2019, 11:51am UTC](https://help.galaxyproject.org/t/errors-with-varscan-somatic-and-gatk-pipline/451/5 "2019-01-15T11:51:40Z")
Hey, here is the complete report generated by the tool: Fatal error: Exit code 1 () STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Input stream not ready, waiting for 5 seconds… Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 66783358 positions in mpileup file 7033032 had sufficient coverage for comparison 7029951 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 2600 were called Germline 15 were called LOH 466 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Input stream not ready, waiting for 5 seconds… Input stream not ready, waiting for 5 seconds… Input stream not ready, waiting for 5 seconds… Input stream not ready, waiting for 5 seconds… Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 45047113 positions in mpileup file 4947460 had sufficient coverage for comparison 4946536 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 747 were called Germline 6 were called LOH 171 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 49655798 positions in mpileup file 7536260 had sufficient coverage for comparison 7534896 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 990 were called Germline 9 were called LOH 365 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 41549526 positions in mpileup file 4138840 had sufficient coverage for comparison 4138070 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 627 were called Germline 2 were called LOH 141 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 41566134 positions in mpileup file 4179204 had sufficient coverage for comparison 4178045 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 939 were called Germline 4 were called LOH 216 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 42608322 positions in mpileup file 5241621 had sufficient coverage for comparison 5240440 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 854 were called Germline 2 were called LOH 325 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 36121558 positions in mpileup file 4026104 had sufficient coverage for comparison 4025330 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 567 were called Germline 11 were called LOH 196 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 32354208 positions in mpileup file 3313437 had sufficient coverage for comparison 3312612 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 653 were called Germline 3 were called LOH 169 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 35216264 positions in mpileup file 5003693 had sufficient coverage for comparison 5001855 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 1577 were called Germline 6 were called LOH 254 were called Somatic 1 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 30029133 positions in mpileup file 2806005 had sufficient coverage for comparison 2805414 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 465 were called Germline 4 were called LOH 122 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 22762761 positions in mpileup file 3327674 had sufficient coverage for comparison 3327007 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 478 were called Germline 3 were called LOH 186 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 114548 positions in mpileup file 30084 had sufficient coverage for comparison 29799 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 272 were called Germline 3 were called LOH 10 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 167022 positions in mpileup file 38614 had sufficient coverage for comparison 38181 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 411 were called Germline 3 were called LOH 19 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 114415 positions in mpileup file 25673 had sufficient coverage for comparison 25354 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 303 were called Germline 0 were called LOH 16 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 11264 positions in mpileup file 299 had sufficient coverage for comparison 299 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 143854 positions in mpileup file 34366 had sufficient coverage for comparison 34071 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 276 were called Germline 1 were called LOH 18 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 66689831 positions in mpileup file 8919088 had sufficient coverage for comparison 8917373 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 1238 were called Germline 5 were called LOH 472 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 51158002 positions in mpileup file 5137528 had sufficient coverage for comparison 5136422 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 893 were called Germline 3 were called LOH 210 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 56412510 positions in mpileup file 6725410 had sufficient coverage for comparison 6712791 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 12044 were called Germline 46 were called LOH 528 were called Somatic 1 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 16449 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 10960 positions in mpileup file 7711 had sufficient coverage for comparison 7359 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 349 were called Germline 0 were called LOH 3 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 92551 positions in mpileup file 37842 had sufficient coverage for comparison 37842 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 89743 positions in mpileup file 35219 had sufficient coverage for comparison 35218 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 1 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input f … nput files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 32 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 689 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 25075 positions in mpileup file 585 had sufficient coverage for comparison 570 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 9 were called Germline 1 were called LOH 5 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 12048 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 15006 positions in mpileup file 1888 had sufficient coverage for comparison 1819 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 67 were called Germline 0 were called LOH 2 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 5410 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 4655 positions in mpileup file 873 had sufficient coverage for comparison 870 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 2 were called Germline 0 were called LOH 1 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 6412 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 8639 positions in mpileup file 744 had sufficient coverage for comparison 740 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 4 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 6241 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 5408 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 11460 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 6274 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 11796 positions in mpileup file 4366 had sufficient coverage for comparison 4242 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 120 were called Germline 0 were called LOH 4 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 10330 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 5435 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 14362 positions in mpileup file 4294 had sufficient coverage for comparison 4252 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 37 were called Germline 1 were called LOH 4 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 7155 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 4909 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 3409 positions in mpileup file 325 had sufficient coverage for comparison 295 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 20 were called Germline 0 were called LOH 10 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 17803 positions in mpileup file 2534 had sufficient coverage for comparison 2523 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 11 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 15418 positions in mpileup file 1185 had sufficient coverage for comparison 1184 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 1 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 7395 positions in mpileup file 526 had sufficient coverage for comparison 526 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 101268 positions in mpileup file 34242 had sufficient coverage for comparison 33871 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 298 were called Germline 1 were called LOH 72 were called Somatic 0 were called Unknown 0 were called Variant Traceback (most recent call last): File “/opt/galaxy/shed\_tools/toolshed.g2.bx.psu.edu/repos/iuc/varscan\_somatic/d37adcc2ec03/varscan\_somatic/varscan.py”, line 1178, in varscan\_call(\*\*args) ---
### Author: ![Tarek\_zakaria](https://avatars.discourse-cdn.com/v4/letter/t/3d9bf3/32.png) [@Tarek\_zakaria](https://help.galaxyproject.org/u/Tarek_zakaria) #### Post date: [January 15, 2019, 11:52am UTC](https://help.galaxyproject.org/t/errors-with-varscan-somatic-and-gatk-pipline/451/6 "2019-01-15T11:52:12Z")
File “/opt/galaxy/shed\_tools/toolshed.g2.bx.psu.edu/repos/iuc/varscan\_somatic/d37adcc2ec03/varscan\_somatic/varscan.py”, line 884, in varscan\_call v.merge\_and\_postprocess(\*out, \*\*args) File “/opt/galaxy/shed\_tools/toolshed.g2.bx.psu.edu/repos/iuc/varscan\_somatic/d37adcc2ec03/varscan\_somatic/varscan.py”, line 814, in merge\_and\_postprocess invcf, \*\*filter\_args File “/opt/galaxy/shed\_tools/toolshed.g2.bx.psu.edu/repos/iuc/varscan\_somatic/d37adcc2ec03/varscan\_somatic/varscan.py”, line 609, in \_postprocess\_variant\_records for allele in record.alleles File “/opt/galaxy/shed\_tools/toolshed.g2.bx.psu.edu/repos/iuc/varscan\_somatic/d37adcc2ec03/varscan\_somatic/varscan.py”, line 609, in for allele in record.alleles File “/opt/galaxy/shed\_tools/toolshed.g2.bx.psu.edu/repos/iuc/varscan\_somatic/d37adcc2ec03/varscan\_somatic/varscan.py”, line 437, in get\_allele\_specific\_pileup\_column\_stats for base in pile\_column.get\_query\_sequences(): File “pysam/libcalignedsegment.pyx”, line 2990, in pysam.libcalignedsegment.PileupColumn.get\_query\_sequences AssertionError ---
### Author: ![wm75](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/wm75/32/56_2.png) [@wm75](https://help.galaxyproject.org/u/wm75) #### Post date: [January 15, 2019, 2:08pm UTC](https://help.galaxyproject.org/t/errors-with-varscan-somatic-and-gatk-pipline/451/7 "2019-01-15T14:08:24Z")
Thanks for posting the error message - it really helped. So the issue seems to be that there is at least one genomic region to which you have more than 10,000 reads aligning, and the tool isn’t prepared to handle this (though arguably it should). The fact that you have such regions of extremely high coverage though, is most likely due to some of your reads mapping to more than one place in the genome. This can happen for sequences with highly similar multiple copies in the genome that make it hard/impossible for a read alignment tool to decide to which of these copies it should map a given read. If the strategy it uses is to report _all_ possible mappings, this can lead to _a lot_ of reads piling up in such multi-copy regions. Now, if you are not interested in these ambiguous mappings, an easy way to reduce coverage in the problematic regions is to discard all, but the primary read mappings in your bam input. You can use this tool: [https://usegalaxy.eu/root?tool\_id=toolshed.g2.bx.psu.edu/repos/devteam/samtool\_filter2/samtool\_filter2/](https://usegalaxy.eu/root?tool_id=toolshed.g2.bx.psu.edu/repos/devteam/samtool_filter2/samtool_filter2/) to filter your BAM files and exclude non-primary mappings of reads. To do so select: `Filter on bitwise flag` then, under `Skip alignments with any of these flag bits set`: `The alignment or this read is not primary` If your data is paired-end sequencing data, you may consider being a bit stricter and may want to select, under `Only output alignments with all of these flag bits set`: `Read is mapped in a proper pair` You can do this for your GATK tools-treated bam files, but it would have saved some runtime to filter your reads before. Anyway, chances are that with the filtered datasets you can get _Varscan somatic_ to run successfully. ---
### Author: ![Tarek\_zakaria](https://avatars.discourse-cdn.com/v4/letter/t/3d9bf3/32.png) [@Tarek\_zakaria](https://help.galaxyproject.org/u/Tarek_zakaria) #### Post date: [January 15, 2019, 5:06pm UTC](https://help.galaxyproject.org/t/errors-with-varscan-somatic-and-gatk-pipline/451/8 "2019-01-15T17:06:20Z")
Thanks a lot for your reply i would try that out and get back to you. It would be amazing if you can help me out with the GATK tools issue regarding the reference genome. Can’t we use the Galaxy built in mouse mm10 reference genome? ---
### Author: ![wm75](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/wm75/32/56_2.png) [@wm75](https://help.galaxyproject.org/u/wm75) #### Post date: [January 16, 2019, 8:15am UTC](https://help.galaxyproject.org/t/errors-with-varscan-somatic-and-gatk-pipline/451/9 "2019-01-16T08:15:46Z")
> [@Tarek\_zakaria](#): > > It would be amazing if you can help me out with the GATK tools issue regarding the reference genome. Can’t we use the Galaxy built in mouse mm10 reference genome? This is not quite as simple as it sounds. Offering a built in genome doesn’t just mean providing access to a fasta file on the server, but also freeing the tool from building any index files it may require by hosting prebuilt ones. @bjoern.gruening do you want to comment more on GATK tools and builtin genomes for them? Human hg19 seems to be the only cached genome choice available for them on [usegalaxy.eu](http://usegalaxy.eu), but the OP is looking for mm10. ---
### Author: ![bjoern.gruening](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/bjoern.gruening/32/10_2.png) [@bjoern.gruening](https://help.galaxyproject.org/u/bjoern.gruening) #### Post date: [January 16, 2019, 8:34am UTC](https://help.galaxyproject.org/t/errors-with-varscan-somatic-and-gatk-pipline/451/10 "2019-01-16T08:34:14Z")
Which GATK tool are you using? GATK-2 used to use a nonstandard sorting. But GATK2 is very old and we do not recommend to use it anymore. ---
### Author: ![Tarek\_zakaria](https://avatars.discourse-cdn.com/v4/letter/t/3d9bf3/32.png) [@Tarek\_zakaria](https://help.galaxyproject.org/u/Tarek_zakaria) #### Post date: [January 21, 2019, 11:22am UTC](https://help.galaxyproject.org/t/errors-with-varscan-somatic-and-gatk-pipline/451/11 "2019-01-21T11:22:03Z")
Thanks for your feedback, i was trying to use the **Indel Realigner** - (Galaxy Version 2.8.0) and Base Recalibrator (Galaxy Version 2.8.0). my target genome is mm10. ---
### Author: ![Tarek\_zakaria](https://avatars.discourse-cdn.com/v4/letter/t/3d9bf3/32.png) [@Tarek\_zakaria](https://help.galaxyproject.org/u/Tarek_zakaria) #### Post date: [January 22, 2019, 5:39am UTC](https://help.galaxyproject.org/t/errors-with-varscan-somatic-and-gatk-pipline/451/12 "2019-01-22T05:39:59Z")
Hi, i followed your recommendation and ran Varscan somatic after filtering the non primary alignments as you suggested but unfortunately it crashed again. I will paste the full error message again. Would be great if you can help. Varscan somatic is the only tool in galaxy that is suitable for my samples and would be great to use it. Thanks in advance. Fatal error: Exit code 1 () STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 50836856 positions in mpileup file 7267074 had sufficient coverage for comparison 7264082 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 2704 were called Germline 30 were called LOH 258 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 34586836 positions in mpileup file 5109210 had sufficient coverage for comparison 5108285 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 787 were called Germline 7 were called LOH 131 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 39836230 positions in mpileup file 7773168 had sufficient coverage for comparison 7771917 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 1022 were called Germline 9 were called LOH 220 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 31518825 positions in mpileup file 4280804 had sufficient coverage for comparison 4280017 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 671 were called Germline 5 were called LOH 110 were called Somatic 1 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 31547523 positions in mpileup file 4322878 had sufficient coverage for comparison 4321789 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 952 were called Germline 12 were called LOH 125 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 32543046 positions in mpileup file 5381370 had sufficient coverage for comparison 5380378 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 834 were called Germline 8 were called LOH 150 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 27833499 positions in mpileup file 4155010 had sufficient coverage for comparison 4154326 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 578 were called Germline 4 were called LOH 102 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 24605730 positions in mpileup file 3420820 had sufficient coverage for comparison 3420114 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 632 were called Germline 5 were called LOH 69 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 27699588 positions in mpileup file 5146375 had sufficient coverage for comparison 5144549 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 1637 were called Germline 5 were called LOH 184 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 22707947 positions in mpileup file 2893450 had sufficient coverage for comparison 2892904 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 463 were called Germline 3 were called LOH 80 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 18095380 positions in mpileup file 3424012 had sufficient coverage for comparison 3423474 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 467 were called Germline 3 were called LOH 68 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 102473 positions in mpileup file 30757 had sufficient coverage for comparison 30453 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 287 were called Germline 2 were called LOH 15 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 144644 positions in mpileup file 39848 had sufficient coverage for comparison 39383 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 447 were called Germline 0 were called LOH 18 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 96426 positions in mpileup file 26011 had sufficient coverage for comparison 25693 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 300 were called Germline 1 were called LOH 17 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 6877 positions in mpileup file 354 had sufficient coverage for comparison 354 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 127604 positions in mpileup file 34884 had sufficient coverage for comparison 34605 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 265 were called Germline 5 were called LOH 9 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 52136699 positions in mpileup file 9200436 had sufficient coverage for comparison 9198964 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 1274 were called Germline 12 were called LOH 186 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 38065039 positions in mpileup file 5303165 had sufficient coverage for comparison 5302098 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 936 were called Germline 7 were called LOH 124 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Input stream not ready, waiting for 5 seconds… Reading input from STDIN Reading mpileup input… 43560014 positions in mpileup file 6902682 had sufficient coverage for comparison 6887399 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 14832 were called Germline 57 were called LOH 394 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 9773 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 9947 positions in mpileup file 7747 had sufficient coverage for comparison 7390 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 347 were called Germline 0 were called LOH 10 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 69959 positions in mpileup file 36036 had sufficient coverage for comparison 36035 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 1 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 68250 positions in mpileup file 33845 had sufficient coverage for comparison 33843 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 2 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 62134 positions in mpileup file 21648 had sufficient coverage for comparison 21648 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 1178 positions in mpi … OM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 94 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 959 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 17728 positions in mpileup file 261 had sufficient coverage for comparison 257 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 4 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 7144 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 9542 positions in mpileup file 1503 had sufficient coverage for comparison 1422 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 81 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 1489 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 3288 positions in mpileup file 884 had sufficient coverage for comparison 879 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 5 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 1528 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 5541 positions in mpileup file 781 had sufficient coverage for comparison 769 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 12 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 2797 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 4049 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 5663 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 2381 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 10432 positions in mpileup file 4486 had sufficient coverage for comparison 4360 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 123 were called Germline 0 were called LOH 3 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 4396 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 3157 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 15042 positions in mpileup file 4203 had sufficient coverage for comparison 4171 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 30 were called Germline 1 were called LOH 1 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 3813 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 2907 positions in mpileup file 0 had sufficient coverage for comparison 0 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 0 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 1921 positions in mpileup file 152 had sufficient coverage for comparison 141 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 7 were called Germline 0 were called LOH 4 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 14443 positions in mpileup file 2368 had sufficient coverage for comparison 2356 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 12 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant ---
### Author: ![Tarek\_zakaria](https://avatars.discourse-cdn.com/v4/letter/t/3d9bf3/32.png) [@Tarek\_zakaria](https://help.galaxyproject.org/u/Tarek_zakaria) #### Post date: [January 22, 2019, 5:40am UTC](https://help.galaxyproject.org/t/errors-with-varscan-somatic-and-gatk-pipline/451/13 "2019-01-22T05:40:40Z")
STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 8647 positions in mpileup file 1108 had sufficient coverage for comparison 1107 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 1 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 6604 positions in mpileup file 545 had sufficient coverage for comparison 540 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 5 were called Germline 0 were called LOH 0 were called Somatic 0 were called Unknown 0 were called Variant STDERR OUTPUT FROM SAMTOOLS MPILEUP/BCFTOOLS: [mpileup] 2 samples in 2 input files Min coverage: 8x for Normal, 8x for Tumor Min reads2: 2 Min strands2: 1 Min var freq: 0.1 Min freq for hom: 0.75 Normal purity: 1.0 Tumor purity: 1.0 Min avg qual: 13 P-value thresh: 0.99 Somatic p-value: 0.05 Reading input from STDIN Reading mpileup input… 92206 positions in mpileup file 30297 had sufficient coverage for comparison 30120 were called Reference 0 were mixed SNP-indel calls and filtered 0 were removed by the strand filter 158 were called Germline 0 were called LOH 19 were called Somatic 0 were called Unknown 0 were called Variant Traceback (most recent call last): File “/opt/galaxy/shed\_tools/toolshed.g2.bx.psu.edu/repos/iuc/varscan\_somatic/d37adcc2ec03/varscan\_somatic/varscan.py”, line 1178, in varscan\_call(\*\*args) File “/opt/galaxy/shed\_tools/toolshed.g2.bx.psu.edu/repos/iuc/varscan\_somatic/d37adcc2ec03/varscan\_somatic/varscan.py”, line 884, in varscan\_call v.merge\_and\_postprocess(\*out, \*\*args) File “/opt/galaxy/shed\_tools/toolshed.g2.bx.psu.edu/repos/iuc/varscan\_somatic/d37adcc2ec03/varscan\_somatic/varscan.py”, line 814, in merge\_and\_postprocess invcf, \*\*filter\_args File “/opt/galaxy/shed\_tools/toolshed.g2.bx.psu.edu/repos/iuc/varscan\_somatic/d37adcc2ec03/varscan\_somatic/varscan.py”, line 609, in \_postprocess\_variant\_records for allele in record.alleles File “/opt/galaxy/shed\_tools/toolshed.g2.bx.psu.edu/repos/iuc/varscan\_somatic/d37adcc2ec03/varscan\_somatic/varscan.py”, line 609, in for allele in record.alleles File “/opt/galaxy/shed\_tools/toolshed.g2.bx.psu.edu/repos/iuc/varscan\_somatic/d37adcc2ec03/varscan\_somatic/varscan.py”, line 437, in get\_allele\_specific\_pileup\_column\_stats for base in pile\_column.get\_query\_sequences(): File “pysam/libcalignedsegment.pyx”, line 3041, in pysam.libcalignedsegment.PileupColumn.get\_query\_sequences AssertionError ---
### Author: ![wm75](https://sea2.discourse-cdn.com/flex020/user_avatar/help.galaxyproject.org/wm75/32/56_2.png) [@wm75](https://help.galaxyproject.org/u/wm75) #### Post date: [January 22, 2019, 9:31am UTC](https://help.galaxyproject.org/t/errors-with-varscan-somatic-and-gatk-pipline/451/14 "2019-01-22T09:31:51Z")
Hmm, that’s still the same error. Would you mind sharing the problematic history and datasets with us? For sharing you can follow the instructions for [Workflow doesn't execute all steps after successfully being invoked.](https://help.galaxyproject.org/t/workflow-doesnt-execute-all-steps-after-successfully-being-invoked/271/5?u=wm75). Just make sure that, after making the history and its datasets available, you send the link and a reference to this discussion here to [contact@usegalaxy.eu](mailto:contact@usegalaxy.eu). Thanks!