Question about mutation results

[shanyfr]

Hi,

I ran this pipeline on some samples and got a confusing result in one of them. Please help me figure this out.
In this sample when looking at the mutation results I can see that the same mutation was counted three times. When looking at the neat files for the sample and its undiluted matched library in IGV, I don't see this mutation in the undiluted sample but I do see it in almost all the fragments of the diluted nanoseq sample (more than three fragments). Why is this mutation then counted? why three times?
Another question for this sample I got a duplicate rate of 0.78 and an efficiency of 0.056 which I think are good but I got a big error bar for the mutation burden which I don't understand why. Do you have any idea why this might have happened?
I attached a photo of the three mutations from the mutation results vcf file and a photo from the IGV (the top sample is the undiluted matched library)
Thanks!

[fa8sanger]

Hi,
It's strange that the mutation is so frequent in your nanoseq sample but not in the matched normal. How were each of them sequenced (platform)? Could it be possible that you have DNA contamination or a sample swap? Which burdens are you getting? Seeing a mutation called multiple times is not an error, NanoSeq calls mutations at single molecules. Have you looked at the burdens pre vs post-masking?

Regarding your large confindence intervals... Your efficiency is very good, but still you can have large CIs if you don't have enough mutations/coverage. CIs are poisson confidence intervals, very simple to calculate. If you provide more details I may be able to help more
I hope this helps