Question: Motif And Down Stream Analysis
gravatar for kanwarjag
7.6 years ago by
United States
kanwarjag1.1k wrote:

This question is taking bioinformatics analysis to down stream analysis.

I have identified 3 Chipseq motifs (obviously I ran chipseq called peaks and identified enrichment regions > identified motifs) which are around 7-9 letters with one mismatch. I took the coordinates of these 3 motif seq's (based on p value <0.001) and intersected them with coordinates from all the enriched regions which I have initially identified in order to find out the selected motif is enriched which of genes in my data set. When I used bed tool I used -Wo option with 50% overlap.

Now when I check the position in the sequence which correspond to motif, I don't find the exact sequence- I should see the exact seq (e.g CMGGATC) with one base mismatch. What I find is 3-4 bases randomly at the same location (e.g AGGGAA)? How can I be sure that I am not designing primers/ EMSA assay for a false motif??? Should I be not having almost all letters except one mismatch in actual motif?

As You will agree it will require lot of effort sand time. If you wonder what I used to do all this I used-MEME suit and has take advice from my previous post discussion. A: motif seq in enriched genes

chipseq sequence motif • 1.7k views
ADD COMMENTlink modified 7.6 years ago by Giovanni M Dall'Olio27k • written 7.6 years ago by kanwarjag1.1k

Did you test the candidate binding regions with qPCR, etc?

ADD REPLYlink written 7.6 years ago by Ian5.7k

That is where the problem is- I am not getting enough confident with these computational approaches. What I am not sure when we see these logos of Chipseq data - Is that complete seq has to be there in the specified gene seq or partial. If partial how partial- 50% ? 90%

ADD REPLYlink written 7.6 years ago by kanwarjag1.1k

I am just wondering whether you have tested that the regions from you ChIP-seq analysis are real, before trying to determine what the motif might be.

ADD REPLYlink written 7.6 years ago by Ian5.7k

Yes we performed qPCR and it seems to be working

ADD REPLYlink written 7.6 years ago by kanwarjag1.1k
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