Alignment to verify plasmid from forward and reverse Sanger sequencing reads

Question

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?

Answer 1

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).