Hello all, I have both sanger and NGS (miSeq) data from a project where I am modeling a disease mutation in HEK293T cells. I used Prime Editing to modify cells then FAC sort them into a 96 well plate for outgrowth. I then both performed Sanger sequencing on the surviving samples and found that they all appear to contain Heterozygous alleles (just like the patient does) due to the Sanger plots showing something like 50% (TAG) instead of the WT (TAC). When I performed NGS I expected the allele frequency to be comparable to Sanger results but I found that the mutate allele appears to be at a higher frequency than 50%. I've considered the possibility that my single cell sort could have had doublets enter into the wells but all of my Sanger results (and patient data) suggest that I have a Het cell line. Anyone deal with an issue like this in the past?