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  • what to do about uneven reads between ATACseq samples?

    I'm trying to look for differential open chromatin between 2 groups with two replicates each. After bowtie2 alignment, Group A has 70-76million paired reads while Group B had 80-95million paired reads.

    Peak calling with Genrich using replicate mode called out 20k peaks in Group A, 30k peaks in Group B.

    I'm guessing there's more identified peaks in Group B mainly because the two replicates in Group B had more reads to begin with. Because there's more called peaks in Group B than A, it's affecting my differential peak analysis.

    I used DiffBind for differential analysis and 95% of the differential peaks were unique to Group B. I had to input both the peak files along with the bam files so shouldn't Diffbind's normalization account for the uneven reads between the groups?

    Is there a critical normalization step I should have taken early on? Maybe downsampling the fastq files to equal number of reads? This is my first time doing ATACseq and I haven't been able to find any info on read normalization for atacseq.

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