SAMTOOLS mpileup crash

[michalkovac]

Hi,

anyone has an indea why I'm getting this error message?
In-----
samtools mpileup -gf ./Reference_Indexed/Human36.fa ./2007.0214 B.normal.merged.out.bam > normal.test.bcf

It generates sort of 3GB of data when the crash comes in.
Out----
mpileup] 1 samples in 1 input files
<mpileup> Set max per-sample depth to 8000
sh: line 24: 7723 Segmentation fault

It also does not parse the whole bam file when mpileup is piped into bcftools command as is suggested in the manual:
samtools mpileup -ugf ref.fa aln.bam | bcftools view -bvcg - > var.raw.bcf

BTW, pileup does not have any problems with the bam file. It has been generated by SAMTOOLS using SAM output of Stampy-BWA aligners.

Many thanks for any suggestions.

[KNS]

There has been a few issues with the output generated by samtools mpileup - see a couple of recent threads including my post "Unstable output from mpileup/samtools" here in the bioinformatics forum. There do not seem to be good answers to this issue for now, but I am hopeful the developer is having a look.

[lh3]

@michalkovac

try 0.1.11; if that does not help, could you send me the alignment around the point it breaks? Thanks.

@KNS

It is thanksgivings in US. Please give me time. Mpileup has been used for tera bytes of data. It has not segfaulted so far at my hand. There must be something I have not considered in your data. But I can only help if you share me with your data (I cannot reproduce the problem with zee's example). Thank you.

[michalkovac]

Thanks lh3. I've updated samtools to the latest version though is still crashing. As to the point where the crash actually happens: It completes chr13_random, but does not start assembling chr14.

Should it help if I send you the link to my bam file?

Many thanks for you time.
M.

[lh3]

Yes. That would be great. Many thanks.

[michalkovac]

Yes. That would be great. Many thanks.

Link has been posted to your mail. Many thanks.
M.

[lh3]

Please try the latest SVN:

svn co https://samtools.svn.sourceforge.net...trunk/samtools

Your example is really helpful. Thank you very much!

[lh3]

0.1.12 is released, mainly for this bug fix.

[michalkovac]

^^ many thanks. now it seems to work.

[LisaM]

Hi,

I'm having a similar problem with mpileup on the newest revision (397).

In:
samtools mpileup -C50 -gf ref.fasta in.bam > out.bcf

Out:
[mpileup] 1 samples in 1 input files
<mpileup> Set max per-sample depth to 8000
Segmentation fault

The tool has no problem with the bam file if the fasta file is not included (ie. no -f), so the bam file is ok.

However, the default instructions provided in the mpileup manual does work:
samtools mpileup -ugf ref.fasta in.bam | bcftools view -bvcg - > var.raw.bcf

Any help on this would be much appreciated. Please let me know if you need any additional information.

Thanks in advance,
Lisa


-- UPDATE --

I've rerun mpileup a couple of times since, and it looks like it's just the -g or -u tag that's seg faulting. At mpileup -C50 -f, everything seems to run fine. And I also realized that although the default command ran, it did not complete properly, as the resulting .bcf file was not complete.

Hope this helps!

[jmt]

segmentation fault for mpileup

I am having troubles getting 'mpileup' to run correctly w/o a segmentation fault.

I would like to run 'mpileup' on 2 bam files on a set of sites.

I ran the following and got the following error.

samtools mpileup -f $INDEX -l $LIST_OF_SITES $BAM1 $BAM2
[bam_header_read] EOF marker is absent. The input is probably truncated.
[mpileup] 1 samples in 2 input files
<mpileup> Set max per-file depth to 8000
Segmentation fault

However, when I ran on just 1 bam file, it seems to work just fine.

samtools mpileup -f $INDEX -l $LIST_OF_SITES $BAM1
[mpileup] 1 samples in 1 input files
<mpileup> Set max per-file depth to 8000
Y 10645915 T 2 Aa IF
Y 10646057 G 3 Aaa A>,
Y 11726507 T 1 . <
Y 11771361 G 1 a F
Y 11929118 T 34 ..,,,,,,.,.......,..,.,..,,...,... B?IIA?DFFAFI6DFFFFFF8FDEBAGFHI9IFI
Y 19612396 G 1 . 3
Y 19612484 G 1 A 3
Y 19612796 G 1 . I

Could someone tell me what stupid mistake I'm making?

Thanks in advance!

[michalkovac]

samtools mpileup -f $INDEX -l $LIST_OF_SITES $BAM1 $BAM2
[bam_header_read] EOF marker is absent. The input is probably truncated.
[mpileup] 1 samples in 2 input files
<mpileup> Set max per-file depth to 8000
Segmentation fault

I'd suggest you run Picard ValidateSamFile on your BAMs. If it doesn't find EOF, some of your SAM/BAM processing steps have not been fully completed.

[jmt]

Hi michalkovac,

These bam files are the 1000G alignment files from the pilot study. Will running Picard ValidateSamFile correct the EOF error?

At the moment I've resorted to running mpileup on the bam files individually one by one and then writing my own script to combine the pileup output. This way seems to work thus far.

[rna-analys]

samtools mpileup -d8000 will solve the problem

[Idon_Sokolov]

Hello, I´m having the same problem. I´m analysing exome data obtained from exome sequencing. The only step in order to finish my analysis is getting the consensus calls from my samples. This is the command that I´m putting in the console:

samtools mpileup -ugf Homo_sapiens.GRCh38.dna.alt.fa.gz Illumina9.0.0_bwamem.sort.rmdup.bam Illumina9.0.0_bwamem.sort.rmdup.readfiltered.bam | bcftools call -vmO z -o illumina9

But this is my result:

[main] Unrecognized command.
[mpileup] 1 samples in 2 input files
<mpileup> Set max per-file depth to 8000

Please, help me.

Thank you for your time.
Best regard.