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Preparing overlapping bisufite sequences for methylation analysis


Best Answer pcrman, 08 April 2014 - 09:30 AM

 

 

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. 

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#1 Kate Warner

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Posted 16 March 2014 - 08:21 AM

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



#2 pcrman

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Posted 08 April 2014 - 09:30 AM   Best Answer

 

 

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. 



#3 Kate Warner

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Posted 11 April 2014 - 06:24 AM

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






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