Atac Seq

ATAC Seq

Version Compatibility

Reference examples assume:

• macs3 3.0+
• samtools 1.18+
• deepTools 3.5+

Verify the runtime first:

• CLI: macs3 --version, samtools --version, bamCoverage --version

Overview

Use this skill when the user needs:

• bulk ATAC-seq QC
• peak calling
• accessibility counting
• differential accessibility
• motif deviation or footprint follow-up

When To Use This Skill

• the task is bulk ATAC-seq rather than ChIP-seq
• TSS enrichment, fragment periodicity, or FRiP need review
• the output should include peaks, counts, and downstream accessibility summaries

Quick Route

• paired-end bulk ATAC: use BAMPE
• call peaks without control using ATAC-specific settings
• if TSS enrichment is poor, stop and flag data quality before interpretation

Progressive Disclosure

• Read technical_reference.md for QC gates and assay-specific caveats.
• Read commands_and_thresholds.md for peak-calling commands, thresholds, and output conventions.

Prerequisites

| Check | Guidance |

|---|---:|

| uniquely mapped reads | >= 20M preferred for strong bulk ATAC |

| TSS enrichment | > 7 acceptable, > 10 strong |

| FRiP | > 0.2 often strong for good bulk ATAC |

Expected Inputs

• paired-end ATAC BAM or FASTQ
• reference genome
• sample groups for comparisons

Expected Outputs

• results/peaks/sample_peaks.narrowPeak
• results/matrix/consensus_peak_counts.tsv
• results/diff_accessibility.tsv
• figures/tss_enrichment.pdf
• figures/fragment_size_distribution.pdf

Starter Pattern

macs3 callpeak \
  -t atac.bam \
  -f BAMPE \
  -g hs \
  -n sample \
  --nomodel \
  --shift -100 \
  --extsize 200 \
  -q 0.01 \
  --outdir results/peaks

Key Parameters

| Parameter | Typical value | Notes |

|---|---|---|

| -f | BAMPE | paired-end ATAC should use fragment-aware mode |

| --nomodel | on | standard for ATAC |

| --shift | -100 | common Tn5 offset convention |

| --extsize | 200 | common first-pass extension |

| -q | 0.01 | starting FDR threshold |

Workflow

1. Validate assay QC

Review:

• TSS enrichment
• fragment size periodicity
• duplication
• mapped read depth

2. Call peaks with ATAC-specific settings

Use fragment-aware paired-end mode and Tn5-aware shifting or equivalent settings.

3. Build a consensus peak matrix

Merge peaks across samples, count fragments into consensus intervals, then produce a peak-by-sample matrix.

4. Test differential accessibility

Use replicate-aware statistics and report both effect size and adjusted significance.

5. Run motif or footprint follow-up

Only after peak quality and read depth support it.

Output Artifacts

results/
├── peaks/
│   ├── sample_peaks.narrowPeak
│   └── sample_summits.bed
├── matrix/
│   └── consensus_peak_counts.tsv
└── diff_accessibility.tsv
qc/
├── tss_enrichment.tsv
└── fragment_metrics.tsv
figures/
├── tss_enrichment.pdf
└── fragment_size_distribution.pdf

Quality Review

• TSS enrichment below 7 should trigger caution.
• Strong nucleosome periodicity supports a good bulk ATAC library.
• FRiP below 0.1 is usually weak and needs scrutiny.
• Footprinting should not be trusted on low-depth or poor-quality libraries.

Anti-Patterns

• using generic ChIP peak-calling defaults for ATAC
• running footprinting on weak libraries
• skipping TSS enrichment review
• merging peaks from mixed reference builds

Related Skills

• ChIP Seq
• Gene Regulatory Networks
• Multiome And scATAC

Optional Supplements

• deeptools
• pysam