Assuming that your disorder is autosomal then the CAG repeats may be long enough to be polymorphic and different lengths on each chromosome. That way sequencing forwards gives homozygous sequence covering the CAT motif but if the 2 chromosomes have 10 CAG and 11 caG repeats then the reverse sequence will produce mixed bases when the CAG and CAT are the same distance from the sequencing primer so in reverse the CAT will be displaced on one chromosome by 3 bases and you will get cat and cag mixed bases from the displaced sequenceThe Simplest way to sequence would be to clone the pcr product and sequence clones which will, at least, be hemizygous and should sequence clean although you will have to sequence more samples. There is software that basecalls the minor sequence and often you can work out the 2 sequences by eye subtracting one sequence from the mixed sequence to leave the other sequence if you have the expected sequence as a reference.

Repetitive sequences like CAG repeats can form secondary structures such as hairpins or loops during the sequencing process. These structures can lead to sequencing errors or discrepancies between the forward and reverse reads. Consider using conditions or reagents that minimize the formation of these secondary structures.

Rakesh Sharma Thanks for answering me. It is definitively one of the possible causes!

Paul Rutland Thank you for your kind reply! I didn’t aware that the difference of length will produce discrepancy results. And according to this logic, I am able to resolve the sequence of two alleles which can explain the discrepancies :)

Excellent Karen Wong well done