I am testing a methylation-specific ddPCR assay and observed two positive droplet layers on the FAM channel. Based on the 2D group it does not seem to be affected by the signal from HEX channel. What would be the possible reasons to have double positive clouds?
Hi, there are several options for this phenotype.
What method did you use to detect methylation? Bisulfite conversion, MSRE, enzymatic?
It looks like the lower cloud has less efficient amplification compared to the upper cloud. This could be due to:
- a longer (or other) amplicon
- a slight variation in sequence. If you have multiple methylation sites in the amplicon, it might be 1 vs 2 sites that are methylated (and converted) or it could be incomplete conversion for example.
Depending on the method, do you have a 100% converted positive control (just a gblock or ultramer sequence) ? Do you see the same phenotype there?
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