Errors with Varscan Somatic and GATK pipline

varscan

#1

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


#2

Hmm, can you be a bit more specific?

  • Where are you trying this? AFAIK, usegalaxy.org doesn’t currently have the Varscan somatic tool installed, so are you using 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.


#3

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’


#4

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?


#5

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)


#6

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


#7

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/

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.


#8

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?


#9

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, but the OP is looking for mm10.


#10

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.


#11

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.


#12

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


#13

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


#14

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.. 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.
Thanks!