No peaks in CUT&RUN
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5 months ago
Johanna • 0

Hi!

I am new to bioinformatics and am doing it for the first time for my master's thesis. I've performed CUT&RUN between two conditions with H3K27ac and IgG as a control. I crosslinked the histone mod to DNA with PFA which is known to reduce signal.

Everything was looking fine between the two conditions, nothing strange during alignment (Bowtie2) or clean-up (remove MAPQ <10, chrM/Un/N), both conditions show very similar alignment rates, number of reads dropped, and size distributions. Even after peak calling (SEACR) the FRiP scores are very similar. However, the IGV tracks are almost completely peakless for one of the conditions, and correspondingly DiffBind showed 11 differential peaks in this condition and >5000 in the other.

Example tracks of the two conditions

What I can't seem to figure out is why the data processing doesn't give any indication of a failed experiment? Or does it, and I've just been looking in the wrong places?

Let me know if there is any relevant information missing. Thanks!

bowtie2 ngs chip-seq SEACR • 325 views
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Are these tracks scaled or normalized? I would say in the lower one you do not see anything as the scale is too large for the present signal. Is this the BAM files or bigwigs?

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Thank you for the reply! The tracks are scaled. These are bedgraph files, the output from https://github.com/Henikoff/Cut-and-Run/blob/master/spike_in_calibration.csh. I agree that the scale is too large, but I set it with regards to the tracks for condition 1.

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