Bam file is empty

[Tirza Doniger]

Hi,

I have tried running qualimap v 2.1.1 . I keep getting the following error message:

"Failed to analyze RNA-seq data. bam file is empty."

The bam file is not empty. What could be causing this error message?

Much Thanks!

Tirza Doniger

[Konstantin Okonechnikov]

Hi!

Thanks a lot for report!

Did you try running Qualimap BAM QC or samtools flagstat command to check how many reads are mapped? Which tool was used to create the BAM file? Also, was the BAM file already sorted by name?

It would be great if you could share the BAM file or a small subsample from it (http://okko73313.blogspot.de/2013/03/random-subsample-from-bam-file.html), so I can try checking this issue in detail. Additionally the link to GTF file that was used for analysis will be helpful.

--

  Konstantin

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[Tirza Doniger]

Hi,

Thanks for the prompt reply.

1) I ran Qualimap BamQC - that runs fine

2) just a note- I was unable to get Repitools to install - could that be the reason for the  failure?

3) STAR was used to create the bam file. It was then sorted using samtools- i dont think by name. i think by coordinates.

4) gtf: http://ftp.ensembl.org/pub/release-74/gtf/mus_musculus/Mus_musculus.GRCm38.74.gtf.gz (mm10)

5) link to sample.bam : https://drive.google.com/file/d/0B31nVw8m2-c3R2daSi0wMUk2ZXc/view?usp=sharing

Thanks so much!

Tirza

[Tirza Doniger]

Also-

I installed qualimap on another machine - which does have repitools installed and tried running RNAQC and Count on these files- and I still get the same error that the bam file is empty.

Thanks!

Tirza

[Konstantin Okonechnikov]

Hi Tirza,

thanks a lot for sharing the data!

The problem actually occurs due to the difference of chromosome names in the BAM file and in the annotation file. This is also reported by Qualimap in the log.

The chromosome names in the BAM file start with "chr": chr1, chr2, chr3, etc, while chromosome names in the GTF file are without "chr" prefix: 1,2,3, etc. Additionally "chrM" in the BAM file called "MT" in the GTF file.

This is a well-known problem due to differences in chromosome names between Ensembl and UCSC databases.

Here are some links with more information:
http://plindenbaum.blogspot.de/2011/10/reference-genome-with-or-without-chr.html
https://usegalaxy.org/u/jeremy/p/transcriptome-analysis-faq#faq5

I converted the chromosome names in the GTF file and launched with Qualimap - everything worked fine.

Would be great if you also run the analysis with this annotation file again.

Please find the converted annotation here:
https://drive.google.com/file/d/0B616vf21NOpPSFVzVXYxbmNRNVk/view?usp=sharing

Let me know if there any additional questions.

--
  Konstantin











I converted