Using Samtools mpileup with Varscan
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5.7 years ago
Ron ★ 1.0k

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...

RNA-Seq varscan samtools next-gen • 4.2k views
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I can't see any error; Varscan is just waiting for the first variants....

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It gives up after a certain time.

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Try -mpileup instead mpileup2cns

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5.7 years ago

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

 

 

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8 months ago
field654 ▴ 10

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.

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