Using ATACseq to do motif analysis in the promoters of a gene set

Question

[dljone02]

Hi,

I have ATACseq data analyzed for my cell of interest. I also have RNAseq from my cells with/without stimulus. After doing differential gene expression from the RNAseq, I want to do motif enrichment in the promoters of this gene set (~4,000 genes). Specifically, I would like to use bedtools to intersect the DNA accessibility regions of the promoters of this gene set, to identify the regions with the highest likelihood of transcription factor binding.

Since RNA is stranded (can be on + or - strand) and ATAC is not, will there be any issue with using a standard bedtools command as follows?

bedtools intersect -a RNAseq_DEG_promoters.bed -b ATAC.bed > result.bed

Any help appreciated

Answer 1

[ATpoint]

Motifs are not stranded, neither is transcription factor binding a strand-specific event. They bind double stranded DNA recognizing a motif. If a sequence is overrepresented in your selection it will be overrepresented no matter if providing the top strand or its reverse complement. So no, you can use intersect as usual, only the coordinates matter. The only thing I would do though is to limit the peak to like 200bp from the peak center. THis is what motif tools such as Homer do by default to avoid motif matches by change and since the actual TF binding (and therefore the motif) is a narrow event. Depending on your peak calling you might get peaks that are notably larger than 200bp and therefore scan way more nucleotides than necessary, increasing the change of false-positives.

[dljone02]

Ok thanks. Was more or less just concerned about using "bedtools intersect" with a dataset that is stranded vs not stranded....this is ok?