how to look at sequence traces? - (Dec/14/2008 )
I am a beginner at looking at dna sequence traces. I have some questions. I am looking at traces in a sequencing software and I'd like to know what it means if there are more than one peak or overlapping peaks?? I attached a small image with a sequencing trace. The peaks circled in red are either slightly overlapping peaks or more than one peak. In the software I am using, these are scored as perfectly good traces. I was always taught that good traces are peaks that are separate and resolved very well. So I do not understand these overlapping or double/quadruple peaks. Is it just because the background is so low? If someone can help me understand the nuances of sequencing traces, it would be very helpful.
Low peaks are mostly background noise. Overlapping peaks mean that the DNA at that location has more than one allele, usually caused by polymorphism, partial methylation (in bisulfite sequencing), and DNA contamination (for example your PCR product is a mix of two close located bands).
Quickest reply to your question. Nobody ever said that peaks are baseline-resolved. In the first pair of peaks you circled, it's just that there are two different bases, whereas the other two examples are with the same base repeated.
What part of the sequence did your image come from? The further through the run you go, the smaller the relative difference between molecules, so the harder it becomes to completely resolve them.
If all of my traces came back as clean as yours, I would be a very happy camper!