I am setting up a fragment analysis assay in which I need to calculate the ratio of the two peaks. The protocol I have inherited suggests using Peak Height, and I see this used a lot generally for fragment analysis.
However, I am getting stutter peaks and these aren't always consistent between my alleles (e.g. Allele 1 has a stutter, allele 2 doesn't). In this example, the Peak Height of one of the alleles is reduced by the presence of the stutter, whereas the other allele is not affected, which will affect my ratio calculation. I am considering using Peak Area instead of Peak Height. This way, I would be able to include the area of any stutters in my analysis as follows:
[True Allele 1 Peak Area + Stutter 1 Peak Area] / [True Allele 2 Peak Area + Stutter 2 Peak Area]
I feel this would be more robust against the inconsistent presence/absence of stutter peaks. Is this a valid approach? Am I overlooking anything with this approach?
Many thanks in advance for your input
Peak area should be a bit more robust than peak height, assuming that your peak "edges" are well enough defined. Peak height is affected by peak broadening, whereas peak height is not. Peak width is affected by resolution, the smoothing level you choose (in software like GeneMapper), and the product size. That said, peak heights generally give an accurate-enough estimate of quantity unless you need a high degree of accuracy, or peak heights being compared differ significantly. I'd try both methods and compare. If there is a difference significant to your application, go with peak area.
If you are getting inconsistent stutter amounts from run to run and Allele 1 and Allele 2 are reasonably close in size, you may want to check the age/quality of the formamide you are using and your denaturing protocol.
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