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Using Germline Blood for PON creation


I'm running ~80 tumour/normal (peripheral blood) pairs at 100x/30x through the GATK4/mutect2 best practices and I have a quick question about PON creation.

I've been looking at the advice here:


"As a result, the most important selection criteria for choosing normals to include in any PON are the technical properties of how the data was generated. It's very important to use normals that are as technically similar as possible to the tumor (same exome or genome preparation methods, sequencing technology and so on). Additionally, the samples should come from subjects that were young and healthy to minimize the chance of using as normal a sample from someone who has an undiagnosed tumor. Normals are typically derived from blood samples."

The only samples I have done on the analysis platform are the Tumour and matched normals. Can you advise on whether it would be beneficial to construct a PON from the 80 germline samples themselves? Or would it be better to just use the matched pairs and possibly gnomad frequencies? I tried to see if a similar question had been asked before and it's not clear to me if it's worth doing:



"Typically the PON is made from normals not comprising the matched normals. One of the rules for accounting for presence of a variant in the PON looks at how many samples carry the variant, so the inclusion of your matched normal would indeed bias the result a little bit."

In my case, the PON would contain the matched normal.


"...PON was created with 125 whole genome samples derived using 2012 technology. The sample libraries were from the blood normal tissue of cancer patients. We do NOT use matched normal tissue samples, as matched normal tissue samples can be contaminated with tumor/pre-tumor tissue, as they are typically derived from tissue"

I'm not sure in this example whether the cancer germline samples used were from the same study (or whether that matters).

Thanks a lot




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