---
description: Investigation of possible contamination of tumor DNA into normal sample
---

# Hotspots In Normals

![](https://2763969089-files.gitbook.io/~/files/v0/b/gitbook-legacy-files/o/assets%2F-M52gq1rRSDQOKMQGEuR%2F-M9Z39Nd7c6cJRknLQBm%2F-M9Z7H_pAeCiMotcoSVV%2FScreen%20Shot%202020-06-11%20at%2012.05.28%20PM.png?alt=media\&token=a092cb6d-f9be-4cb5-8238-ec1b828cf603)

**Theoretical Method**

Extract read counts for mutation hotspots from "normal" sample pileups. Then look into tumor samples to determine whether these mutations may have been due to contamination of tumor into the normal. Unfiltered bams are used for the normal samples to widen the search for hotspots, and duplex bams are then used for tumor samples.&#x20;

**Technical Methods**

* Tool Used:
  * Waltz PileupMetrics
  * BioinfoUtils.jar
  * plots\_module.r
* Input
  * sample\_id-duplex-pileup.txt (for duplex noise calculation)
* Output
  * hotspots-in-normals.txt

**Interpretations**

In the provided example we can see that there was potential contamination of the tumor sample into the normal sample for C-P835W4, as indicated by the 7 unfiltered reads that matched a mutation from the tumor. This may be due to improper separation of tumor and normal sample during extraction, or clonal hematopoiesis.
