In IGV is this a heterogeneous mutation or false call?

Question

[Tuck898]

Hi all,

I'm hoping someone might be able to enlighten me on if this could be a true heterogeneous mutation. The count is 22% so a little higher to rule out it not being. How would I go about more investigation to confirm if this mutation is a positive? The read quality are roughly QV 37 average, some 39. I would be interested for any advice. Thanks in advance!

[Tuck898]

I've just displayed clipped bases and this is what it shows. Does this look right?

Answer 1

[Pierre Lindenbaum]

there is a clear shift in the depth just close to this mutation, you should set IGV to display the clipped bases + check if there is a DUP at this place.

[Tuck898]

Hi, I really appreciate you taking the time to reply. I'm still learning with IGV and sorry to sound very inexperienced but how would I check if there is a DUP by displaying the clipped bases please? thanks!

[Tuck898]

Ah I've just found 'show soft clipped bases' and this is what comes up... how come it is all greyed out?

[Pierre Lindenbaum]

yeah.. look at that, there is something wrong in your alignment, many reads were mapped in a location but they are all clipped, so something in 5'/3' is not related to the mapped sequence. Forget about your SNP.

how would I check if there is a DUP by displaying the clipped bases please

look at a clipped sequences, blat it, you might find the location upstream/downstream of the current location

[Tuck898]

Thanks for that! I'll try and have a look and see what is amiss if I can... In your opinion what do you feel is wrong in my alignment from what you can see? I guess the SNP is insignificant :D

Still learning so thanks for all the input.

[Tuck898]

When click the blat it brings me to chr19 and mentions the RYR1 underneath although I was originally looking at the RYR2 gene. Sorry to sound stupid but does this make sense to what I've previously showed about the clipped bases. Image below to where blat sequence took me. many thanks!

[Pierre Lindenbaum]

RYR1 underneath although I was originally looking at the RYR2 gene. Sorry to sound stupid but does this make sense to what I've previously showed about the clipped bases

it's too hard to answer without having a look at the data. It could be a rearrangement between the locus of both genes.

[Tuck898]

No worries, I can completely appreciate that. I am having investigations similar to a condition causes by the RYR2 gene and was wondering if what I am showing you could be a possible cause or not (and if I am wasting my time looking at this region). Is there anything else I can do to double check this exon? thanks again :)

Answer 2

[swbarnes2]

Since all the reads that show the alternate allele have to be clipped to align there, I'd say those reads are misaligned. I would say that is not a true SNP.

[Tuck898]

Thanks for the input :)