Question: ChIPseq - Identical peaks in IP and input samples
gravatar for dorota.komar
22 months ago by
dorota.komar10 wrote:

Hi guys, I have obtained some strange results from my ChIP-seq analysis: the ChIP sample looks exactly the same as Input sample, bacisally covering uniformly the entire genome. I have already performed other ChIP-seq experiment with the same line and antibody - that time it worked nicely (but we got some unexpected results, so wanted to make sure they are not an artefact, but a real protein behaviour). I have also checked the immunoprecipited DNA by q-PCR and could see clear difference in DNA abundance of positive and negative regions.

We sent 4 samples for sequencing: 2 ChIP and 2 inputs. The library preparation of one of ChIP samples did not work, so we have finally only sequenced 1 ChIP and 1 input. The very simple explanation could be that the sequencing service made a mistake and sequenced both inputs, or that I mislabelled the samples, but since one momber of the lab got the same results some time in the past, I am thinking there might be more difficult answer to that.

I was thinking since the binding of my protein is pretty low, the peaks were not wery high and with the high background, after amplification during library preparation and very deep sequencing it could be the expected failed results. But maybe there is another explanation that could save me from that in teh future. Any thoughts?

chip-seq • 697 views
ADD COMMENTlink modified 22 months ago by Devon Ryan93k • written 22 months ago by dorota.komar10

So, you have just aligned the sequenced files and visualized using a browser like IGV?

ADD REPLYlink written 22 months ago by arup1.9k
gravatar for Devon Ryan
22 months ago by
Devon Ryan93k
Freiburg, Germany
Devon Ryan93k wrote:

The likely explanations are:

  1. Mislabelled samples
  2. Poor IP (have a look at this with plotFingerprint in deepTools).

There's no good way to differentiate post hoc whether you have a poor IP or the signal was lost during PCR. I mean, you could look at your duplication levels. If they're high then presumably you over amplified and perhaps that's causing the problem. It's also usually useful to look at a control region in your samples (whatever you're using for your qPCR).

ADD COMMENTlink written 22 months ago by Devon Ryan93k
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