Hi,

I have ATAC-seq data for 8 time points of 4 conditions --> 32 samples in total. The only replicates I had were technical which were pooled early in the pipeline. There are no biological replicates. I have completed MACS2 peak calling and now want the differential binding (DB). I would like to use HTseq to create the counts table and then DEseq2 to analyze for DB.

I have read a number of posts all replied with good advice from Micheal Love (DEseq2 extended to open chromatin anlaysis: normalization, dispersion fit, and "too many" differences, Replicate for DESeq2 and A: GFOLD file as input for DESeq2) regarding DEseq2, ATAC-seq and replicates....but none that address what to do when there are no biological replicates. Can HTseq and DEseq2 run with no replicates?

Also I'm a bit confused as to what needs to be counted. I have the BAM files and the output of MACS2 I have filtered to remove overlapping peaks (as per guidance). So do I simply count the number of reads in the original data that align to the remaining non-overlapping uniquely named peaks (separately for each sample) - is that what the count table (of each individual sample) should consist of? For example:

peak_name     read count

peak1                  452

peak2                  34

peak3                  458

I will be monitoring this post so let me know if I can clarify question(s) or provide additional information.

Thank you all for your help,

Kenneth

Actually the approach of calling peaks separately in each sample would lead to overestimation of significance in a DB analysis even if you did have replicates. See

https://doi.org/10.1093/nar/gku351

for an explanation of why this is so.

Honestly, I don't think it's much use to run DESeq2 without replicates. The software allows for this analysis with large caveats (see the relevant section in ?results) that such an analysis is only vaguely exploratory. But you could just as well plot the log ratios of peak counts across conditions and skip running DESeq2.