/pipeline-atacseq

name: pipeline-atacseq
description: "Execute ENCODE ATAC-seq processing pipeline from FASTQ to peaks and signal tracks. Child of pipeline-guide. Provides stage-by-stage Nextflow execution with Docker containers and cloud deployment. Handles Tn5 transposase offset correction, mitochondrial read removal, and nucleosome-free fragment selection. Use when users need to process ATAC-seq data following ENCODE standards. Trigger on: ATAC-seq pipeline, run ATAC-seq, process ATAC-seq, chromatin accessibility, open chromatin, Tn5 shift, TSS enrichment."

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 Nextflow DSL2 implementation following ENCODE uniform analysis standards.

TSS enrichment scoring is a **manual post-processing step**; the workflow does not compute it (see "Manual QC steps" below and `references/05-qc-metrics.md`).

Overview

ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) uses the Tn5 transposase to probe open chromatin regions. This pipeline processes ATAC-seq data through quality control, alignment with Bowtie2, mitochondrial read removal, duplicate removal, Tn5 insertion site correction (+4/-5 bp offset), blacklist filtering, nucleosome-free fragment selection, MACS2 peak calling, FRiP calculation, and an IDR comparison for every pair of replicates.

Key differences from ChIP-seq: Bowtie2 aligner (optimized for short fragments), Tn5 transposase shift correction, mitochondrial read filtering (chrM can be 30-80% of reads), and no input control.

The workflow is **paired-end only**. Passing `--single_end` stops the run with an error, because Tn5 shifting, nucleosome-free selection and BAMPE peak calling all depend on fragment length.

Key Literature

| Reference | Journal | Year | DOI | Relevance | |-----------|---------|------|-----|-----------| | Buenrostro et al. "Transposition of native chromatin (ATAC-seq)" | Nature Methods | 2013 | 10.1038/nmeth.2688 | Original ATAC-seq method (~5,000 citations) | | Corces et al. "An improved ATAC-seq protocol" | Nature Methods | 2017 | 10.1038/nmeth.4396 | Omni-ATAC improvements (~2,500 citations) | | ENCODE Project Consortium "Expanded encyclopaedias" | Nature | 2020 | 10.1038/s41586-020-2493-4 | ENCODE Phase 3 standards | | Amemiya et al. "ENCODE Blacklist" | Scientific Reports | 2019 | 10.1038/s41598-019-45839-z | Artifact regions (~1,372 citations) | | Langmead & Salzberg "Fast gapped-read alignment with Bowtie 2" | Nature Methods | 2012 | 10.1038/nmeth.1923 | Aligner (~30,000 citations) | | Yan et al. "From reads to insight: ATAC-seq analysis" | Genome Biology | 2020 | 10.1186/s13059-020-1929-3 | Analysis best practices |

Pipeline Stages

FASTQ ──> FastQC / Trim Galore ──> Bowtie2 ──> Mito Removal ──> Picard MarkDuplicates
  │                                            (chrM dropped)   (duplicates REMOVED)
  │                                                                       │
  │           ┌───────────────────────────────────────────────────────────┘
  │           v
  │     Tn5 Shift (alignmentSieve --ATACshift) ──> Blacklist Filter ──> Size Selection
  │                                                       │                    │
  │                                                       │        ┌───────────┴────────┐
  │                                                       v        v                    v
  │                                               Signal Track   NFR (<150 bp)   Mono-nucleosome
  │                                                (all frags)      │              (150-300 bp)
  │                                                                 v
  │                                                   MACS2 Peak Calling ──> IDR (every pair)
  │                                                                 │
  │                                                                 v
  │                                                   FRiP (NFR peaks vs final BAM)
  v
 QC reports ────────────────────────────────────────────────────────────────> MultiQC

The Tn5 shift runs **after** duplicate removal, and peaks are called on the nucleosome-free BAM only. The signal track is built from all fragments in the blacklist-filtered BAM, not from the NFR BAM.

Stage Summary

| Stage | Tool | Input | Output | Reference | |-------|------|-------|--------|-----------| | 1. QC & Trimming | FastQC, Trim Galore | Raw FASTQ | Trimmed FASTQ, FastQC reports | references/01-qc-trimming.md | | 2. Alignment | Bowtie2, samtools | Trimmed FASTQ | Sorted BAM, flagstat, bowtie2 log | references/02-alignment.md | | 3. Filtering & Tn5 shift | samtools, Picard, deeptools `alignmentSieve`, bedtools | Sorted BAM | Shifted, filtered, size-selected BAMs | references/03-tn5-filtering.md | | 4. Peak Calling & IDR | MACS2, IDR | NFR BAM | narrowPeak, one `<sampleA>_vs_<sampleB>.idr_peaks.txt` per replicate pair | references/04-peak-calling.md | | 5. Signal, FRiP & QC report | deeptools `bamCoverage`, bedtools, samtools, MultiQC | Filtered BAM, NFR peaks, QC logs | bigWig, `<sample>.frip_mqc.tsv`, multiqc_report.html | references/05-qc-metrics.md |

Input Requirements

Required

• **ATAC-seq FASTQ** (`--reads`): paired-end reads, gzipped. A Nextflow file-pair glob,

e.g. `'fastq/*_R{1,2}.fq.gz'`.

• **Bowtie2 index directory** (`--bowtie2_index`): Bowtie2 is invoked as

`bowtie2 ... -x <dir>/<genome>`, so the directory must hold