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Using Samtools mpileup with Varscan

Hi all,

I am using samtools mpile up and varscan to find variants.below is my command:

samtools mpileup \
  -f hg19.fa \
  -D 8000 \
  -S \
  -l stat3_jak1.bed \
  -b $input_dir/tcell_bam_list | \
    /bin/java -jar mutation_tools/VarScan.v2.3.7.jar mpileup2cns \
    --vcf-sample-list $input_dir/tcell_bam_list \
    --min-coverage 6 \
    --min-var-freq 0.08 \
    --p-value 0.01 \
    --output-vcf 1 > $output_dir/mutation_analysis_result.vcf

My bed file looks like(for two genes,STAT3 and JAK1)

chr17    40465342    40540513    uc002hzk.1    0    -    40468844    40500534    0    24    "2476,63,43,213,140,95,53,136,99,84,48,94,30,60,93,159,152,95,82,96,99,145,151,217,"    "0,3464,3857,8957,9679,9935,10248,11386,11638,12791,16085,16229,16422,18147,20348,20566,24110,24438,25406,25989,3223
chr1744,40465342954,"   40540513    uc002hzl.1    0    -    40467762    40500534    0    24    "2476,113,43,213,140,95,53,136,99,84,48,94,30,60,93,159,152,95,82,96,99,145,151,217,"    "0,3464,3857,8957,9679,9935,10248,11386,11638,12791,16085,16229,16422,18147,20348,20566,24110,24438,25406,25989,3223
chr1744,40465342954,"   40540513    uc002hzm.1    0    -    40467762    40500534    0    24    "2476,113,43,210,140,95,53,136,99,84,48,94,30,60,93,159,152,95,82,96,99,145,129,217,"    "0,3464,3857,8960,9679,9935,10248,11386,11638,12791,16085,16229,16422,18147,20348,20566,24110,24438,25406,25989,3223
chr1744,40465342954,"   40540513    uc010wgh.1    0    -    40467762    40497654    0    23    "2476,113,43,213,140,95,53,136,99,84,48,94,30,60,93,159,152,95,82,96,99,145,217,"    "0,3464,3857,8957,9679,9935,10248,11386,11638,12791,16085,16229,16422,18147,20348,20566,24110,24438,25406,25989,3223
chr1744,40465342    40540513    uc002hzn.1    0    -    40467762    40500534    0    24    "2476,113,43,213,140,95,53,136,99,84,48,94,30,60,93,159,152,95,82,96,99,145,129,217,"    "0,3464,3857,8957,9679,9935,10248,11386,11638,12791,16085,16229,16422,18147,20348,20566,24110,24438,25406,25989,3223
4,33244,35064,74954,"

tcell_bam_list contains the list of all bam files.

I get this following error:

Min coverage:    6
Min reads2:    2
Min var freq:    0.08
Min avg qual:    15
P-value thresh:    0.01
[mpileup] 24 samples in 24 input files
<mpileup> Set max per-file depth to 333
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...
ERROR: Gave up waiting after 500 seconds...
next-gen rna-seq varscan samtools
• 8,618 views • link
updated by Ram Ram • written by Ron Ron

I can't see any error; Varscan is just waiting for the first variants....

• 0 views • link
written by Pierre Lindenbaum Pierre Lindenbaum

It gives up after a certain time.

• 0 views • link
written by Ron Ron

Try -mpileup instead mpileup2cns

• 0 views • link
written by poisonAlien poisonAlien

2 answers

I had the same problem and figured it out.

That's just VarScan's fancy way of saying, can't find your input.

Very frustrating.

• 0 views • link
written by field654 • field654

This is it!! Registered on BioStars just to upvote you. Thanks!

• 0 views • link
written by freitas.sn • freitas.sn

Thank you, that's so encouraging. As a nonprofessional, it's especially enjoying to help.

• 0 views • link
written by field654 • field654

Which VarScan command are you using? I know that there are problems with using pileup2snp/pileup2indel if you used mpileup (rather than pileup).

I don't think this is the error message that I would expect, but I do have a pipeline that includes variant calling with VarScan (if you are willing to modify some Perl code): https://docs.google.com/file/d/0B1xpw6_kQMKuaElwcTVXcnFmZ1E

• 0 views • link
updated by Ram Ram • written by Charles Warden Charles Warden

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