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Old 11-20-2019, 11:36 AM   #1
AmitChaurasia
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Location: Delhi, INDIA

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Default Allele Frequency Reproducibility: An interesting observation

Hi All,

I have got an very interesting observation during troubleshooting of an experiment with patient sample as described below (I would like to have your opinion at each point before moving ahead to next point):

The experiment is as follows:

One FFPE and One Germline Sample (In duplicate) total 3 samples libraries have been prepared for targeted BRCA region. Duplicate Germline libraries have been started with same DNA input sample but processed simultaneously for library prep.

The Observation:
Say: FFPE as F1 library and
Germline as G1 and G2 libraries

FFPE Sample got two unique mutations reported:
M1 and M2 for example with allele frequencies (34@ and 17%) respectively

These M1 got 7% Allele frequency in G1 library;
Query1: What do you thing this could be because of? (Please choose your answer before moving ahead to next sentence).

Is it from cfDNA that frequency come into G1 library?

Query2: If YES, but there is no mutation in G2 library although the input DNA was same? How...?

Is there a contamination of G1 from F1 during library processing?

Query3: If Yes, But there is no mutation M2 was found in G1 library. They why it is only M1 mutation is there in G1...?


Guys, please suggest any idea on the hypothesis, what could have gone wrong...? Interesting...!
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Old 11-23-2019, 11:11 PM   #2
chenyudr
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Default

Are these three libraries captured by probes? If yes, they captured in one reaction or individually?
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Old 11-28-2019, 08:04 PM   #3
AmitChaurasia
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Hi Chenyudr,

These libraries are PCR based target amplification but UMI based.
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bioinformatic analysis, library preparation, somatic mutations, troubleshooting routines

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