ATAC-seq Peak Calling - Usage Guide

ATAC-seq Peak Calling - Usage Guide

Overview

Call accessible chromatin regions from ATAC-seq data using MACS3 or Genrich, with specialized handling for Tn5 transposase cut sites and nucleosome-free region detection.

Prerequisites

pip install macs3
conda install -c bioconda samtools bedtools genrich

Quick Start

Tell your AI agent what you want to do:

• "Call ATAC-seq peaks from my BAM file"
• "Separate nucleosome-free and mono-nucleosomal reads before peak calling"

Example Prompts

Basic Peak Calling

"Call peaks from my ATAC-seq BAM file using MACS3 with proper Tn5 offset correction"

Fragment-Based Analysis

"Separate my ATAC-seq reads by fragment size and call peaks on nucleosome-free regions only"

Consensus Peaks

"Create a consensus peak set from multiple ATAC-seq samples"

Quality Filtering

"Filter my ATAC-seq peaks by q-value and remove blacklist regions"

What the Agent Will Do

1. Filter BAM for properly paired, high-quality reads and remove mitochondrial reads
2. Optionally separate reads by fragment size (NFR, mono-nucleosomal)
3. Call peaks with MACS3 using ATAC-seq specific parameters (--shift -75 --extsize 150 --keep-dup all)
4. Filter peaks by quality and remove blacklist regions
5. Generate consensus peak set if multiple samples provided

Key Differences from ChIP-seq

Fragment Size Selection

Tips

• Use --shift -75 --extsize 150 for paired-end ATAC-seq to account for Tn5 offset
• Always use --keep-dup all since ATAC-seq has legitimate duplicates
• Filter for NFR reads (<100bp) when looking for TF binding sites
• Remove ENCODE blacklist regions to reduce false positives
• For consensus peaks across samples, require presence in at least 2 samples