I encountered 2 interesting sequencing problems:)
First- In case of one PCR product, forward and reverse primers give me a different nucleotide in certain position. I mean that sequencing with forward primer gives for example A and sequencing with reverse primer gives for example G in the same position. The rest of the sequence is identical. It is not an accident, I repeated PCR and sequencing several times and it was the same all the time. All forwards say A and all reverses say G. The chromatogram looks good, no mixed peaks.
The second mysterious error is quite similar.
The sequence from reverse primer looks like that: TTT
and the sequence from forward looks like that: -TT
Peaks surrounding that region are of the good quality and they look good and clear in that region too. And again…I repeated PCR and sequencing and the error persisted. How can I know if the true sequence is TTT or just TT?
Do you have any suggestions? I do not know how to decide about the true sequence in these two cases. Thank you.
Without seeing the traces it is hard to comment.
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