Diffbind for ATAC-seq data with no controls

Question

[daniele.avancini]

Hi all, I'm trying to use Diffbind to identify differentially open regions (identified using ATAC-seq) between three different cell types. I have three biological replicates for each cell type. I'm building now the sample sheet to import the data into Diffbind, but I don't know what I should indicate as a control since ATAC do not have controls. Is there a way to avoid this problem?

Answer 1

[Rory Stark]

Control reads, such as those from an Input sample, are optional in DiffBind, so you can just leave out the control column in the sample sheet.

[Rory Stark]

Just to expand on this answer, controls in DiffBind are used in two possible ways:

1. Generation of greylists. Used to exclude peaks in regions that appear to have anomalous levels of enrichment in the control. This is the default usage of controls if specified.
2. Read subtraction. Control read counts may be subtracted from the associated sample read counts. In this case, control reads are scaled based on the ratio of library size between the sample and control.

In general, control reads are most useful in modelling the "background" signal in the peak calling step, which occurs outside of DiffBind.