ENCODE ATAC-seq Pipeline

When to Use

• User wants to run an ATAC-seq processing pipeline from FASTQ to peaks and signal tracks
• User asks about "ATAC-seq pipeline", "Tn5 shift", "chromatin accessibility pipeline", or "Bowtie2 for ATAC"
• User needs to process ATAC-seq data with proper Tn5 insertion site correction
• Example queries: "process my ATAC-seq FASTQs", "run ENCODE ATAC-seq pipeline", "call accessibility peaks from ATAC-seq"

Execute the ENCODE ATAC-seq processing pipeline from raw FASTQ files through Tn5 offset correction, peak calling, IDR analysis, and signal track generation. This skill provides a complete Nextflow DSL2 implementation following ENCODE uniform analysis standards.

Overview

ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) uses the Tn5 transposase to probe open chromatin regions. The ENCODE pipeline processes ATAC-seq data through quality control, alignment with Bowtie2, Tn5 insertion site correction (+4/-5 bp offset), mitochondrial read removal, nucleosome-free fragment selection, peak calling with MACS2, and IDR-based replicate consistency analysis.

Key differences from ChIP-seq: Bowtie2 aligner (optimized for short fragments), Tn5 transposase shift correction, aggressive mitochondrial read filtering (can be 30-80% of reads), nucleosomal fragment size distribution as a QC metric, and TSS enrichment score as the primary quality indicator.

Key Literature

Pipeline Stages

FASTQ ──> FastQC / Trim Galore ──> Bowtie2 ──> Mito Removal + Tn5 Shift
  │                                                       │
  │           ┌──────────────────────────────────────────┘
  │           v
  │     Picard MarkDup ──> Blacklist Filter ──> Size Selection
  │                                                   │
  │                    ┌─────────────────┬────────────┘
  │                    v                 v
  │             NFR Fragments     Mono-Nucleosome
  │                    │
  │                    v
  │           MACS2 Peak Calling ──> IDR Analysis
  │                    │                    │
  │                    v                    v
  │             Signal Tracks         QC Report (MultiQC + ataqv)
  v
 Raw QC Report

Stage Summary

Input Requirements

Required Files

• ATAC-seq FASTQ: Paired-end reads (strongly recommended; single-end supported)
• Reference genome: Bowtie2-indexed genome (GRCh38 for human, mm10 for mouse)

Sample Sheet Format

sample_id,read1,read2,replicate
SAMPLE1_rep1,atac_R1.fq.gz,atac_R2.fq.gz,1
SAMPLE1_rep2,atac_R1.fq.gz,atac_R2.fq.gz,2

No input control needed: Unlike ChIP-seq, ATAC-seq does not require a separate input or IgG control. MACS2 calls peaks against a local background model.

Tn5 Transposase Offset Correction

The Tn5 transposase inserts sequencing adapters with a 9-bp duplication. To center reads on the actual cut site:

• Forward strand (+): shift +4 bp
• Reverse strand (-): shift -5 bp

This correction is essential for accurate footprinting and motif analysis.

Fragment Size Distribution

ATAC-seq produces a characteristic nucleosomal ladder pattern:

For peak calling, use nucleosome-free reads (<150 bp) only.

QC Thresholds

TSS Enrichment Score

The TSS enrichment score measures the fold enrichment of ATAC-seq signal at transcription start sites compared to flanking regions. It is the single most informative QC metric for ATAC-seq:

Execution

Quick Start (Local Docker)

nextflow run scripts/main.nf \
  -profile local \
  --reads 'fastq/*_R{1,2}.fq.gz' \
  --genome GRCh38 \
  --outdir results/

SLURM HPC

nextflow run scripts/main.nf \
  -profile slurm \
  --reads 'fastq/*_R{1,2}.fq.gz' \
  --genome GRCh38 \
  --outdir results/

Google Cloud

nextflow run scripts/main.nf \
  -profile gcp \
  --reads 'gs://bucket/fastq/*_R{1,2}.fq.gz' \
  --genome GRCh38 \
  --outdir 'gs://bucket/results/'

AWS Batch

nextflow run scripts/main.nf \
  -profile aws \
  --reads 's3://bucket/fastq/*_R{1,2}.fq.gz' \
  --genome GRCh38 \
  --outdir 's3://bucket/results/'

Cloud Cost Estimates

Output Directory Structure

results/
  fastqc/                   # Raw and trimmed QC reports
  trimmed/                  # Trimmed FASTQ files
  aligned/                  # Sorted BAM files (pre-filtering)
  filtered/
    shifted/                # Tn5-corrected BAM files
    nfr/                    # Nucleosome-free fragments (<150 bp)
    mononuc/                # Mono-nucleosome fragments (150-300 bp)
  peaks/
    narrow/                 # MACS2 narrowPeak files
    idr/                    # IDR-filtered reproducible peaks
  signal/                   # bigWig signal tracks
  qc/
    tss_enrichment