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HaplotypeCaller misses a true variant

BolognaMember

Hi,
I'm updating my pipeline for exome sequencing analysis, so I'm experiencing the HaplotypeCaller capabilities! I have analyzed the same sample with the UnifiedGenotyper walker and the HC one and I have examined the differences between the two output vcf files and I had a very bad finding... HC failed to find a true novel variant!! I know that this is a true variants because I validated that with Sanger sequencing after the first calling with UG.

I have run UG using GATK version 1.6-11-g3b2fab9. This is the VCF line of the variant:

I have run HC using GATK version 2.7-4-g6f46d11 both in a single- and in a multi-sample manner but not the shadow of this variant in the VCF output..
I also noticed that together with this novel variant, HC lost other two variants upstream the first; these are the VCF lines:

How is it possible?

Best, Flavia

Tagged:

• BolognaMember

Hi Geraldine,

I've just uploaded my data in the FTP server in a file named flapa_data.tar.gz; I created a BAM file for the whole chr7 in which the non-called variants fall.

I hope this can be helpful!

Flavia

I was able to reproduce your issue, so I'm now passing this on to the devs for in-depth debugging.

Hi Flavia,

I've taken a look at your example and would like to explain what's happening. If you look carefully at the HC call in that region you'll notice that it assembles it into a very large (120bp) deletion (with 90% of your reads supporting that call). The HC believes that those "SNPs" aren't real, but rather are artifacts from a misalignment around the deletion.

I've attached a screenshot of your data that illustrates it quite nicely. The upper half shows the nice clean HC re-alignments around the deletion. The lower half shows the original reads; notice that the coverage drops dramatically over the deletion and that those "SNPs" occur near the breakpoints. These are classic signs of mis-alignments.

Is it possible that the Sanger sequencing validation could be interpreted in this way too?

• BolognaMember

Hi Eric,

The gene sequence is very repetitive; so, after your explanation, I think that also the Sanger sequencing could be interpreted in this way.
Now I'm trying to perform a more specific PCR and I'll let you know if I'll reply the validation.

Thanks for yor help!
Flavia