Can you post the 2 .abi sequence files so we can see original data please? Tapiwa Nyakauru . Taq does make errors and cloning means that the error is faithfully replicated so it may just be a matter of using different clones until you find one with a fully correct sequence and aligning sequences to get a consensus sequence for the polymorphic base positions as the errors will be quite random

Paul Rutland Thank you for your answer. Taq does make errors, so I then did PCR with high fidelity to get a PCR product without errors. I cloned and sequenced the product. To be frank, I expected the errors to appear as SNPs, but when I aligned the two sequences, the sequence produced by High fidelity had 4 extra bases in the middle of the whole sequence. this confused me.

Tapiwa Nyakauru .When you sequence pcr generated amplimer the errors occur randomly in various cycles so are almost always blotted out by the correct sequence if the error happened late in the pcr and look stronger if generated by the early cycles but the pcr product usually looks normal maybe with slightly stronger underlying base peaks from the changes but on cloning only one amplimer is cloned in each plasmid and colony so you get colonies with many different sequences at 100% incidence because the plasmid is faithfully reproduced in the bacterium. You probably do have correct clones on your plate so it may just be worth screening more colonies.

I cannot explain the 4 extra bases but would still like to see one of each sequencing .abi files in case there is a run of similar bases or dinucleotide bases creating slippage of the enzyme or perhaps a high gc domain looping out of the sequence that the ordinary taq has just reads across at its neck and deleted a loop of 4 bases. Unlikely for so short a sequence but it may be possible