Hi,
I have found a denovo mutation in the child:
- coverage: 356
- Ref allele: G
- Alt allele: A
- ref allele: 48% (109+, 63-)
- alt allele: 52% (118+, 66-)
Father:
- coverage: 254
- Ref allele: G
- Alt allele: -
- ref allele: 100% (151+, 103-)
- alt allele: 0%
Mother:
- coverage: 211
- Ref allele: G
- Alt allele: A
- ref allele: 97% (125+, 79-)
- alt allele: 3% (5+, 2-)
this is a link showing some screenshots for father/mother/child in IGV:
questions:
1- Do you think this could be a hint for a low-parental mosaicism in the mother that appeared as denovo in the child?
2- Could you please give me your thoughts on: how to investigate further in this case to make sure if this is/is not real mosaicism?
any hints are much appreciated!
EDIT:
this image shows the 7 reads of the mom carrying the mutation.
So the child is (G, A), the father is (G, G) and the mother is (A, A), but the mother has some low-frequency reference reads at the same position. It doesn't look like a de novo mutation to me.
Sorry, there was a mistake in the AF of the mother, I just corrected it. the mother is being reported as 0/0 by varscan2, because of the low AF of 'A' that is why this mutation is considered denovo in the child
So: child: 0/1, mother: 0/0, father: 0/0
Cool - sorry about the pedantry. Yeah that's pretty interesting - are there any other technical issues that might explain it though: Did you have to PCR the DNA? were the samples sequenced in separate batches?
I am not sure what do you mean by PCR the DNA, I can ask our lab staff though if you give me more details. What I can say is: we always sequence our samples (WGS, illumina 2500) twice in the same run, on 2 different lanes, and then we collect the samples across different lanes. We do this to get a higher coverage.
It might be a low-parental mosaicism.
I would try to confirm this with sanger sequencing. But with this low frequency it is not sure if it's possible. So if you see this mosaic with sanger than it is confirmed, but if you don't see it you cannot exclude it.
fin swimmer
Just out of curiosity / thinking out loud:
Did you check if the Alt reads from the mother are PCR / optical duplicates? Did you check dbSNP for this variant? I am under the impression there is some strand bias, am I wrong?
Thanks h.mon, please think out even louder, those are the sort of things that I need to know: how to investigate more, manually.
Please check this image , it shows the 7 reads of the mother having this mutation.
No duplicates as far as shown, or do consider the 5th and 6th reads as duplicates with opposite strand orientation ?
All 7 reads have a RMSMappingQuality (MQ) = 60.
Are your reads single- or paired-end?
they are paired-end reads