Optimizing ATAC-seq Peak Calling with MACS3

ATAC-seq has become the method of choice for profiling chromatin accessibility. With the release of MACS3, the peak calling landscape has shifted significantly. This guide walks through a complete analysis from raw reads to differential peaks.

Preprocessing

Start with adapter trimming using fastp, then align with Bowtie2:

fastp -i R1.fastq.gz -I R2.fastq.gz -o clean_R1.fq.gz -O clean_R2.fq.gz
bowtie2 -x genome -1 clean_R1.fq.gz -2 clean_R2.fq.gz | samtools sort -o aligned.bam

Peak Calling with MACS3

MACS3 introduces several improvements over MACS2, including better handling of paired-end data and improved statistical models. The key parameters for ATAC-seq are the shift and extension sizes that account for the Tn5 transposase insertion.

Differential Analysis

For comparing conditions, we recommend DiffBind or DESeq2 on the consensus peak set. Both approaches give robust results when biological replicates are available.