Question: No mutations in BAM (IGV) but a mutation in final VCF?
gravatar for mhmtgenc85
2.2 years ago by
mhmtgenc8540 wrote:

I use GATK 4.0 for the variant calling pipeline. my steps involve MarkDuplicates, BaseRecalibration, ApplyBaseRecalibration and HaplotypeCaller. When I check in a loci there is no mutation in the original BAM file in IGV, but there is a mutation in final VCF and when I check the bamout of the HaplotypeCaller there seems to be a mutation. Then I tried Sanger sequencing and see that there is actually no mutation. So the original Bam file is the right one and bamout is the wrong mutation.

So how could I overcome this problem? This is a serious issue and occurs several times. Thanks in advance.

ADD COMMENTlink modified 2.2 years ago by WouterDeCoster43k • written 2.2 years ago by mhmtgenc8540


could you please post the line of the variant in your vcf file, a screenshot of the original bam in IGV and one with the bamout?

fin swimmer

ADD REPLYlink written 2.2 years ago by finswimmer13k

I am trying but having problems on uploading images :(

ADD REPLYlink written 2.2 years ago by mhmtgenc8540

Just google for "free image hoster" and choose one. In the meantime you can post the vcf entry.

ADD REPLYlink written 2.2 years ago by finswimmer13k

Here are two ways you can add an image to your post - using Markdown syntax, and with inline HTML. Markdown syntax looks better, while Inline HTML gives you more control and is easier. I'll demonstrate both methods here, and you can pick the one most appropriate to you.

Upload your image:

  1. Pick a free image upload site. I'd recommend imgbb (accessible in most countries) or cubeupload (easier to use)
  2. Upload your image on the site
  3. Use either of the methods below to add the image to your post:

a. Markdown method

  • Get the direct URL to the image. To do this, you'll need to right click on the uploaded image and choose "Copy Image Address" or an equivalent option.

    On cubeupload, simply copy the first link under the uploaded image

  • Click on the image option on the toolbar, and paste the copied URL in the dialog box.

b. Inline HTML method:

This option is not available on cubeupload. You'll need an HTML embed code for this method.

  • On imgbb, scroll down and look for a tab that says Embed codes.
  • Click on this Embed codes tab. Copy the code in the HTML full image box.
  • Paste the HTML embed code as part of the text in your post
ADD REPLYlink modified 2.2 years ago by RamRS27k • written 2.2 years ago by genomax84k

Dear genomax and Ram thanks for the detailed expanation,

And finswimmer, here is the IGV image the top one is the original Bam and the bottom one is the bamout produced by Haplotypecaller. Ekran_Al_nt_s

And this is the VCF secreenshot; Ekran_Al_nt_s_VCF

ADD REPLYlink written 2.1 years ago by mhmtgenc8540

Hello mhmtgenc85,

thanks for posting the screenshots. I didn't have a solution for you until now. Could you please post the whole entry of the INFO column from your vcf. (That's the column after PASS in your screenshot). Because the values there are the most interested part to decide whether the variant is correct.

The allel frequency for the variants is only about 25%. That normaly indicates that there is a pseudogen/homologies regions. But I couldn't found one :(

fin swimmer

ADD REPLYlink written 2.1 years ago by finswimmer13k

Here is another screenshot with detatiled INFO column :)


ADD REPLYlink written 2.1 years ago by mhmtgenc8540

Hello mhmtgenc85!

It appears that your post has been cross-posted to another site:

This is typically not recommended as it runs the risk of annoying people in both communities.

ADD REPLYlink written 2.2 years ago by WouterDeCoster43k
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