1
$\begingroup$

I am analyzing Sanger sequencing data to verify whether or not the correct protein coding sequence was inserted during cloning.

For each sample, I have both forward and reverse Sanger sequencing reads that cover the coding sequence.

Would it be better if I first align the forward and reverse reads to get a consensus, and then align the consensus to the reference? Or should I just align each read directly to the reference?

$\endgroup$
2
$\begingroup$

Align all reads from a sample at the same time IMO. This will give you all of the information at once, and it should not be difficult. It should be very obvious whenever there's a disagreement between reads or between reads and reference.

I would also recommend inspecting chromatograms directly at all sites of interest (such disagreements, insertion sites, possible errors or mutations).

$\endgroup$

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.