I have been getting something like the attached image for a segment of the sequencing data, about 100bp long or so. There are double peaks in this segment, but the entire trace is not like this. After this one segment, the rest of the trace is clean. Any suggestions or speculations on the causes of this? Please note that this image is an example taken from another website since I don't have the actual trace with me, but the segment in question looks very similar to this image (overlapped peaks).
My speculation is that it could be that the PCR primers didn't anneal correctly to this region, and thus the double peaks represent a mispriming site. Could it also come from contamination, and what kind? Could it be from a secondary structure, and are these structures created during PCR or sequencing reaction step? I am wondering as it is just a particular segment (or sometimes 2 or 3 'random' segments), not the whole trace that has the mixed trace. Thanks for any help.
mixed-processed.gif 23.5K 10 downloads
Edited by claritylight, 29 August 2010 - 03:09 PM.