Question

How to Resolve Overlapping Peaks in Sanger Chromatograms?

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Entering edit mode

11 weeks ago

JOSÉ • 0

Hello everyone!

I am trying to sequence a viral genome fragment using the Sanger method, but I am facing an issue. In the agarose gel, I always observe two very close bands, and despite trying several methods, I have not been able to separate them.

When I send the sample for sequencing, the resulting chromatogram shows a lot of noise, indicating the presence of overlapping sequences. Upon analyzing the chromatogram, I notice that in certain positions, there are two peaks, suggesting that two bases were sequenced simultaneously in that region. This makes analysis difficult, as I cannot obtain a clean sequence.

Has anyone encountered this problem before? Is there any software or method that could help separate these sequences and provide a more reliable result?

I appreciate any suggestions!

sequence-analysis sanger-sequencing dna-sequencing chromatogram • 560 views

ADD COMMENT • link 10 weeks ago by JOSÉ • 0

score 2 · Answer 1 · 2025-02-22

The way you finished the post kind of sounds like a bioinformatics question, but to me this is an experimental problem. To answer your question: there is no software that can deconvolute sequences from an overlapping chromatogram.

In the agarose gel, I always observe two very close bands, and despite trying several methods, I have not been able to separate them.

This really shouldn't be that difficult, but you can't be married to doing this using an agarose gel. Try an acrylamide gel, somewhere in the 5-10% range depending on fragment sizes. If you have some knowledge of sequences, doing a restriction digest might split one of your bands into two. If you add terminal adapters and clone them into a plasmid you will be able to sequence them without worrying about overlapping.

Finally, a 2-2.5% agarose gel that you run overnight at a very low voltage (10-20 volts) might be able to separate your bands.