2. Example Amplicon Project Design and Analysis
:
2.5 Important Factors in the Assessment of New Variants
: 2.5.5 Flowgram Evidence
2.5.5
Flowgram Evidence
If you filter the alignment to show only those reads containing your variant of interest, you can dig down into the flowgrams of the individual reads to see how convincing the Variant is.
The flowgram lowest on the page represents the subtraction of the reference flowgram from the read flowgram and shows which bases have been overcalled or undercalled in the read according to the reference. Does your potential Variant cause an appropriate shift in the heights of flowgram bars across the series of flowgrams? Is the intensity value of the shift always on the high end or low end of the expected value, or does it more appropriately form a narrow distribution around the middle of the expected value?
If your variant is an insertion or deletion that impacts a single flowgram bar, you only have the intensity of the bar to guide you. If your variant is a substitution, it will simultaneously impact the heights of at least two bars, making the Variant more believable. The most convincing flowgram evidence will be if your Variant happens to cause a nucleotide flow cycle shift in the flowgram. This will be detectable as some inserted flows in the reference flowgram at the top plot or in the read flowgram in the middle plot, that are highlighted in grey.
