Question: VarScan warning and exit after Invalid format for pileup at line 1
0
gravatar for n.tear
8 weeks ago by
n.tear0
n.tear0 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 19 days ago by Biostar ♦♦ 20 • written 8 weeks ago by n.tear0
1

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

ADD REPLYlink written 8 weeks ago by ATpoint18k

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 8 weeks ago • written 8 weeks ago by n.tear0

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

ADD REPLYlink written 8 weeks ago by ATpoint18k

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

ADD REPLYlink written 8 weeks ago by n.tear0

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 8 weeks ago by ATpoint18k
     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 8 weeks ago • written 8 weeks ago by n.tear0

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 8 weeks ago by ATpoint18k

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 8 weeks ago by n.tear0

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

ADD REPLYlink written 8 weeks ago by RamRS22k
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 8 weeks ago by RamRS22k

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 8 weeks ago by RamRS22k

Hi RamRS. Sorry, will do thanks

ADD REPLYlink written 8 weeks ago by n.tear0
1

I made the changes already :)

ADD REPLYlink written 8 weeks ago by ATpoint18k
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 1144 users visited in the last hour