low FRiP(Fraction of Reads in Peaks) score in ATAC-seq


I’m doing ATAC-seq analysis of colon tissue.

I analyzed 1)QC -> 2)Mapping -> 3)Post alignment processing(remove mt reads, duplicated reads, multi-mapped reads) -> 4)Peak calling order.

However, as a result of calculating FRiP after peak calling using MACS2, the FRiP score was too low.

No major problems were found in the results of each process until peak calling.
After post alignment processing, a total of 50,212,020 reads were mapped to the bam file(paired-end sequencing).

Below is the code I used for peak calling and FRiP calculation.

macs2 callpeak -t Input.bam --outdir Peak -f BAM -n ID -g hs -q 0.05 --nomodel --shift -100 --extsize 200 --keep-dup all 
awk 'OFS="t" {print $1"-"$2+1"-"$3, $1, $2+1, $3, "."}' Peak/ID_peaks.narrowPeak > ID_peaks.saf
featureCounts -p -a ID_peaks.saf -F SAF -o ID_fcount.txt Input.bam

My FRiP score is 0.037(3.7%)
The result was the same when I calculated the FRiP with samtools rather than featureCounts.

Can anyone tell me what the problem is?


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