5 replies to this topic

[claritylight]

Hello,

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.

[mdfenko]

is the sequence before this section clean (like the sequence after this section)?

if so then it would seem that you have two inserts that are mostly similar but have a segment that is different.

[claritylight]

yes, the trace before and after this one segment is all clean. sorry, i don't understand what you mean by having two inserts. can you give an example?  

i also have traces where there are two or three segments like this, but the peaks between them are all clean. and there is no distinct distance between them that i can pick up. for example segment 1 is 250bp away from segment 2, and segment 2 is 140bp away from segment 3, so these segments have overlapped peaks and are not like each 100bp apart or some easy number like that, but different distances from each other.

mdfenko, on 30 August 2010 - 08:46 PM, said:

is the sequence before this section clean (like the sequence after this section)?

if so then it would seem that you have two inserts that are mostly similar but have a segment that is different.

[HomeBrew]

What is it you're sequencing -- a PCR product, an insert in a plasmid vector, something else...?  Is there more than one copy of the insert DNA in the genome from which it came (like a gene encoding a ribosomal RNA)?

[claritylight]

HomeBrew, on 31 August 2010 - 06:12 AM, said:

What is it you're sequencing -- a PCR product, an insert in a plasmid vector, something else...?  Is there more than one copy of the insert DNA in the genome from which it came (like a gene encoding a ribosomal RNA)?

I am sequencing a PCR product. As far as I know, there are no multiple copies of the insert DNA, it is a human gene that encodes a muscle protein.

[HomeBrew]

I'm a bacterial geneticist, but aren't there two alleles, which might be heterozygotes?  Perhaps they're amplifying in close to equal molar amounts, and you're seeing the heterogeneous regions in the sequence trace?  If you clone your PCR product and sequence off the plasmid, the trace will probably be clean -- one variant or the other...