Question: Varscan on multiple samples generates error
gravatar for jan
8 weeks ago by
jan10 wrote:


I am using "samtools mpileup" to generate a pileup file from my .bam dataset and then pass it to VarScan2 for variant calling.

Everything works fine until I try to generate a file with multiple samples. The pileup process works as expected and generates a file with more columns to adjust for the second sample. But when I pass this pileup file to VarScan2 it always throws an error.

My Pileup file looks like that (excerpt):

1   2   3   4   5   6   7   8   9   10  11
chr1    183619  G   1   ^?. ?   ?   0   *   *   *
chr1    774701  G   0   *   *   *   2   ^3.^2.  EE  32
chr1    890319  c   2   .,  nG  EI  3   .,, nGG E71
chr1    890320  c   2   .,  mG  EI  3   .,, mGG E71
chr1    890321  g   2   .,  jH  EI  3   .,, iHG E71
chr1    890322  g   2   .,  jH  EI  3   .,, gHE E71

I get the following error when running VarScan2 (I supplied two sample names separeted with a comma)

Fatal error: Tool exception
Got the following sample list: 
C5  F1
Only SNPs will be reported
Min coverage:   8
Min reads2: 10
Min var freq:   0.25
Min avg qual:   15
P-value thresh: 0.99
Reading input from /home/galaxy/galaxy/database/files/000/dataset_782.dat
Parsing Exception on line:
chr1    183619  G   1   ^?. ?   ?   0   *   *   *
For input string: "?"

Galaxy outputs the following commandline command:

echo C5,F1 | awk -F ',' '{ for (i = 1; i <= NF; i++) { print $i; } }' > samples_list.txt &&  varscan mpileup2snp /home/galaxy/galaxy/database/files/000/dataset_782.dat --min-coverage 8 --min-reads2 10 --min-avg-qual 15 --min-var-freq 0.25 --min-freq-for-hom 0.75 --p-value 0.99   --output-vcf 1 > /home/galaxy/galaxy/database/files/000/dataset_788.dat  --vcf-sample-list samples_list.txt

I need two samples in one pileup/varscan file, because I want to compare the samples directly and see if a mutation is "unique" in just one sample or is present in both.

How can I get rid of this error? What am I doing wrong?

Thank you for your support


varscan galaxy mpileup • 110 views
ADD COMMENTlink modified 7 weeks ago by Wolfgang Maier600 • written 8 weeks ago by jan10
gravatar for Wolfgang Maier
7 weeks ago by
Wolfgang Maier600 wrote:

I'm not sure, but it looks as if, in your samtools mpileup run, you had selected the Output mapping quality option. This leads to insertion of columns 7 & 11 in the Pileup file and I don't know whether Varscan is prepared to handle this. Just a guess, but maybe it's worth trying.

ADD COMMENTlink written 7 weeks ago by Wolfgang Maier600

Thank you so much. It works now.

ADD REPLYlink written 7 weeks ago by jan10
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