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
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,