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Preparing overlapping bisufite sequences for methylation analysis - (Mar/16/2014 )

Hi

 

Wondering if you could help.

 

I am analysing methylation in a transgene using bisulfite treatment. I had to use five primer pairs to get the a.1500bp section I needed and I have 10 clones of each primer set from each transgenic line. The sequences from each primer set do overlap and so my question is do I cut out the sequences so that there are no overlapping regions before analysis? I was just wondering because some of these overlapping regions are very large and I'm worried they may skew the results.

 

The program I am using is Kismeth in case that is important.

 

Many thanks in advance

-Kate Warner-

 

 

my question is do I cut out the sequences so that there are no overlapping regions before analysis?

I understand that you meant the analysis phase, right? You don't need to cut out the overlapping regions. actually by overlapping regions, you can obtain more representative data, i.e., an overlapping region is represented by 20 clones instead of just 10. So I think the data should be included and presented. 

-pcrman-

Thanks pcrman that makes a lot of sense and that's my question solved smile.png .

-Kate Warner-