QF-PCR Aneuploidy, Imbalance ratio between allele peaks

[Valerie Tio (2402119161)]

Hi everyone,

I'm seeking some advice on interpreting QF-PCR results for developing aneuploidy testing. I have two NORMAL (control)  samples that I used for evaluation that are puzzling me:

• Sample 1: This sample shows two alleles at 281 bp and 293 bp. However, the ratios between the allele peaks are imbalanced, and there are prominent stutter peaks. 

• Sample 2: This sample has the exact same allele sizes (281 bp and 293 bp) as Sample 1. However, the peak ratios are balanced, and the stutter peaks are less pronounced.

I also encountered the same case with 2 different samples (the same marker, D21S1414), but the two alleles are at 285 bp and 289 bp. One showing normal ratio, while the other shows an imbalance. 

I'm finding it difficult to reconcile these results. Could the imbalance in Sample 1 be due to the high stutter peaks, or is it a true indication of aneuploidy (I am using normal control samples though so I can make a bin)?

Oh, and i tested the UCSC's in silico PCR product sequencing using RepeatMasker to check if the marker is dinucleotide or not, but it's not! 3 different tetranucleotide and that's it. By the way, i'm using 29 cycles of PCR, and multiplex of 4 primer. I then pooled 2 different multiplex amplicon before capillary electrophoresis. So, its 8 primer in total of one capillary run. 

Has anyone encountered a similar situation where the same alleles show different peak ratios and stutter patterns in different samples?

Any insights or suggestions on how to interpret these results or how to avoid/reduce stutter peaks would be greatly appreciated.

Thanks in advance for your help!

Best regards,

Valerie

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