Question: VarScan warning and exit after Invalid format for pileup at line 1
0
gravatar for n.tear
13 months ago by
n.tear20
n.tear20 wrote:

I'm trying to variant call using VarScan but after sorting the BAM files and creating mpileup files using samtools I get this error using VarScan

Im a novice so any help would be greatly appreciated

java -jar ~/workspace/bioinformaticstools/varscan/varscan-master/VarScan.v2.4.3.jar mpileup2snp /mnt/z/Nathan/WESsamples/unsolved/X366.mpileup >X366.VarScan.snp

Only SNPs will be reported
Warning: No p-value threshold provided, so p-values will not be calculated
Min coverage:   8
Min reads2:     2
Min var freq:   0.2
Min avg qual:   15
P-value thresh: 0.01

Reading input from /mnt/z/Nathan/WESsamples/unsolved/X366.mpileup

Warning: Line ignored: Invalid format for pileup at line 1

╝     �═ BC╗ �╔�B�BCF╝�╗  1 10 11 12 13 14 15 16 17 18 19 2 20 21 22 3 4 5 6 7 8 9 MT X Y GL000192.1 GL000225.1 GL000194.1 GL000193.1 GL000200.1 GL000222.1 GL000212.1 GL000195.1 GL000223.1 GL000224. GL000219.1 GL000205.1 GL000215.1 GL000216.1 GL000217.1 GL000199.1 GL000211.1 GL000213.1 GL000220.1 GL000218.1 GL
000209.1 GL000221.1 GL000214.1 GL000228.1 GL000227.1 GL000191.1 GL000208.1 GL000198.1 GL000204.1 GL000233.1 GL000237.1 GL000230.1 GL000242.1 GL000243.1 GL000241.1 GL000236.1 GL000240.1 GL000206.1 GL000232.1 GL000234.1 GL000202.1 GL000238.1 GL000244.1 GL000248.1 GL000196.1 GL000249.1 GL000246.1 GL000203.1 GL000197.1 GL000245.1 GL000247.1 GL000201.1 GL000235.1 GL000239.1 GL000210.1 GL000231. GL000229.1 GL000226.1 GL000207.1 A   /mnt/z/NathanHaffordTear/WESsamples/PPCDunsolved/X366.sorted.ba �
GL000219.1 GL000205.1 GL000215.1 GL000216.1 GL000217.1 GL000199.1 GL000211.1 GL000213.1 GL000220.1 GL                                                                                  ##samtoolsVersion=0.1.19-96b5f2294a
ADD COMMENTlink modified 12 months ago by Biostar ♦♦ 20 • written 13 months ago by n.tear20
1

The pileup is corrupted. Please post the entire command that was used to generate it. Did you use nohup?

ADD REPLYlink written 13 months ago by ATpoint35k

Hi Thanks for the response. This was the command I used to generate the mpile up file:

samtools mpileup -E -uf /mnt/z/Nathan/ReferenceGenome/Homo_sapiens.GRCh37.dna.primary_assembly.fa /mnt/z/NathanHaffordTear/WESsamples/unsolved/X366.sorted.bam > X366.mpileup

I was following instruction from this paper: https://www.biorxiv.org/content/biorxiv/early/2017/10/11/201145.full.pdf

ADD REPLYlink modified 13 months ago • written 13 months ago by n.tear20

Is that the full command? No nohup or anything, or the command embedded in a wrapper function?

ADD REPLYlink written 13 months ago by ATpoint35k

Yes thats the full command. Sorry im not familiar with nohup or embedding in a wrapper

ADD REPLYlink written 13 months ago by n.tear20

The thing is that your pileup looks like it was contaminated by some binary information, maybe a mix of stderr and stdout, that is why I was asking. Can you post the output of

samtools mpileup -E -uf /mnt/z/Nathan/ReferenceGenome/Homo_sapiens.GRCh37.dna.primary_assembly.fa /mnt/z/NathanHaffordTear/WESsamples/unsolved/X366.sorted.bam | head -n 20
ADD REPLYlink written 13 months ago by ATpoint35k
     head -20 X366.mpileup
╝     ═ BC╗ ╔BBCF╝╗  1 10 11 12 13 14 15 16 17 18 19 2 20 21 22 3 4 5 6 7 8 9 MT X Y GL000192.1 GL000225.1 GL000194.1 GL000193.1 GL000200.1 GL000222.1 GL000212.1 GL000195.1 GL000223.1 GL000224.1 GL000219.1 GL000205.1 GL000215.1 GL000216.1 GL000217.1 GL000199.1 GL000211.1 GL000213.1 GL000220.1 GL000218.1 GL000209.1 GL000221.1 GL000214.1 GL000228.1 GL000227.1 GL000191.1 GL000208.1 GL000198.1 GL000204.1 GL000233.1 GL000237.1 GL000230.1 GL000242.1 GL000243.1 GL000241.1 GL000236.1 GL000240.1 GL000206.1 GL000232.1 GL000234.1 GL000202.1 GL000238.1 GL000244.1 GL000248.1 GL000196.1 GL000249.1 GL000246.1 GL000203.1 GL000197.1 GL000245.1 GL000247.1 GL000201.1 GL000235.1 GL000239.1 GL000210.1 GL000231.1 GL000229.1 GL000226.1 GL000207.1 A   /mnt/z/Nathan/WESsamples/unsolved/X366.sorted.bam                                                                                                                                                                                   ##samtoolsVersion=0.1.19-96b5f2294a##reference=file:///mnt/z/NathanHaffordTear/ReferenceGenome/Homo_sapiens.GRCh37.dna.primary_assembly.fa##contig=<ID=1,length=249250621>##contig=<ID=10,length=135534747>
    ##contig=<ID=11,length=135006516>
    ##contig=<ID=12,length=133851895>
    ##contig=<ID=13,length=115169878>
    ##contig=<ID=14,length=107349540>
    ##contig=<ID=15,length=102531392>
    ##contig=<ID=16,length=90354753>
    ##contig=<ID=17,length=81195210>
    ##contig=<ID=18,length=78077248>
    ##contig=<ID=19,length=59128983>
    ##contig=<ID=2,length=243199373>
    ##contig=<ID=20,length=63025520>
    ##contig=<ID=21,length=48129895>
    ##contig=<ID=22,length=51304566>
    ##contig=<ID=3,length=198022430>
    ##contig=<ID=4,length=191154276>
    ##contig=<ID=5,length=180915260>

I also get this warning when running mpileup with samtools. should I be using bcftools?

[warning] samtools mpileup option `u` is functional, but deprecated. Please switch to using bcftools mpileup in future.
ADD REPLYlink modified 13 months ago • written 13 months ago by n.tear20

I do not know what this is. A mpileup file looks like this:

chr1    3000238 a   1   ^K. A
chr1    3000239 t   1   .   A
chr1    3000240 t   1   .   A
chr1    3000241 t   1   .   A
chr1    3000242 c   1   .   A
chr1    3000243 a   1   .   E
chr1    3000244 t   1   .   E
chr1    3000245 c   1   .   E
chr1    3000246 c   1   .   E
chr1    3000247 a   1   .   E

Do you run this directly via the command line?

ADD REPLYlink written 13 months ago by ATpoint35k

okay thanks yes I am running from a ubuntu bash shell on windows. perhaps I need to QC my bam files to check they are correct

ADD REPLYlink written 13 months ago by n.tear20

Looks like a partial VCF file. Maybe a BCF file?

ADD REPLYlink written 13 months ago by RamRS27k
1

You should also add the samtools command that you used to generate the pileup, because from the error message it seems like the problem lies there.

ADD REPLYlink written 13 months ago by RamRS27k

Please use the formatting bar (especially the code option) to present your post better. You can use backticks for inline code (`text` becomes text), or select a chunk of text and use the highlighted button to format it as a code block. I've done it for you this time.
code_formatting

ADD REPLYlink written 13 months ago by RamRS27k

Hi RamRS. Sorry, will do thanks

ADD REPLYlink written 13 months ago by n.tear20
1

I made the changes already :)

ADD REPLYlink written 13 months ago by ATpoint35k
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