/pipeline-dnaseseq

name: pipeline-dnaseseq
description: "Execute ENCODE DNase-seq pipeline from FASTQ to hotspots and footprints. Child of pipeline-guide. Provides Nextflow execution with Docker and cloud deployment. Use when processing DNase-seq data, calling DNase hypersensitive sites, performing footprinting analysis. Trigger on: DNase-seq pipeline, DNase hypersensitive, DHS, Hotspot2, footprinting, DNase I, chromatin accessibility DNase."

ENCODE DNase-seq Pipeline: FASTQ to Hotspots and Footprints

When to Use

• User wants to run a DNase-seq processing pipeline from FASTQ to hotspots and footprints
• User asks about "DNase-seq pipeline", "DNase hypersensitive sites", "Hotspot2", "footprinting", or "DHS"
• User needs to process DNase-seq data for chromatin accessibility and TF footprint analysis
• Example queries: "process my DNase-seq FASTQs", "call DNase hypersensitive sites", "run footprinting analysis on DNase-seq"

Execute the ENCODE DNase-seq pipeline for chromatin accessibility profiling, producing DNase hypersensitive sites (DHSs) via Hotspot2 and transcription factor footprints.

Pipeline Overview

FASTQ -> Trim -> BWA-MEM align -> Filter/dedup -> Hotspot2 -> DHS peaks
                                       |                        |
                                    Signal track         Footprinting (HINT)

ENCODE Repository

• **GitHub**: `ENCODE-DCC/dnase-seq-pipeline`
• **Container**: `encodedcc/dnase-seq-pipeline`
• **WDL**: Available for Cromwell execution
• **This skill**: Nextflow DSL2 reimplementation for portability

Core Tools and Versions

| Tool | Version | Purpose | Citation | |------|---------|---------|----------| | BWA-MEM | 0.7.17 | Alignment | Li & Durbin 2009 | | samtools | 1.19 | BAM operations | Li et al. 2009 | | Picard | 3.1.1 | Duplicate marking | Broad Institute | | Hotspot2 | 2.3.1 | DHS calling (ENCODE standard) | John et al. 2011 | | bedtools | 2.31.0 | Genomic arithmetic | Quinlan & Hall 2010 | | HINT-ATAC | 0.13.2 | TF footprinting | Li et al. 2019 | | FastQC | 0.12.1 | Read quality | Andrews (Babraham) | | MultiQC | 1.21 | Aggregated QC | Ewels et al. 2016 |

Key Literature

1. **John et al. 2011** - "Chromatin accessibility pre-determines glucocorticoid receptor binding patterns" (Nature Genetics, ~600 citations) DOI: 10.1038/ng.759

2. **Thurman et al. 2012** - "The accessible chromatin landscape of the human genome" (Nature, ~3,000 citations) DOI: 10.1038/nature11232

3. **Vierstra et al. 2020** - "Global reference mapping of human transcription factor footprints" (Nature, ~600 citations) DOI: 10.1038/s41586-020-2528-x

4. **Amemiya et al. 2019** - "The ENCODE Blacklist" (Scientific Reports, ~1,372 citations) DOI: 10.1038/s41598-019-45839-z

5. **Li et al. 2019** - "Identification of transcription factor binding sites using ATAC-seq" (Genome Biology) -- HINT-ATAC footprinting DOI: 10.1186/s13059-019-1642-2

Execution

Quick Start (Local)

nextflow run main.nf \
    -profile local \
    --reads '/data/fastq/*_R{1,2}.fastq.gz' \
    --bwa_index '/ref/bwa_index/genome.fa' \
    --chrom_sizes '/ref/hg38.chrom.sizes' \
    --hotspot_index '/ref/hotspot2_index/' \
    --blacklist '/ref/hg38-blacklist.v2.bed' \
    --outdir results/ \
    -resume

SLURM HPC

nextflow run main.nf \
    -profile slurm \
    --reads '/data/fastq/*_R{1,2}.fastq.gz' \
    --bwa_index '/ref/bwa_index/genome.fa' \
    --chrom_sizes '/ref/hg38.chrom.sizes' \
    --hotspot_index '/ref/hotspot2_index/' \
    --blacklist '/ref/hg38-blacklist.v2.bed' \
    --outdir results/ \
    -resume

Cloud (GCP / AWS)

nextflow run main.nf \
    -profile gcp \
    --reads 'gs://bucket/fastq/*_R{1,2}.fastq.gz' \
    --bwa_index 'gs://bucket/ref/genome.fa' \
    --chrom_sizes 'gs://bucket/ref/hg38.chrom.sizes' \
    --hotspot_index 'gs://bucket/ref/hotspot2_index/' \
    --blacklist 'gs://bucket/ref/hg38-blacklist.v2.bed' \
    --outdir 'gs://bucket/results/' \
    -resume

Resource Requirements

| Step | CPUs | RAM | Time (per sample) | |------|------|-----|-------------------| | BWA-MEM align | 8 | 16 GB | 1-2 hours | | Filter/dedup | 4 | 8 GB | 30-60 min | | Hotspot2 | 4 | 8 GB | 30-60 min | | Signal generation | 2 | 4 GB | 15-30 min | | Footprinting | 4 | 8 GB | 1-2 hours | | **Total** | **8** | **16 GB** | **3-6 hours** |

Pipeline Parameters

| Parameter | Default | Description | |-----------|---------|-------------| | `--reads` | required | Glob pattern to paired FASTQ files | | `--bwa_index` | required | Path to BWA genome index (.fa) | | `--chrom_sizes` | required | Chromosome sizes file | | `--hotspot_index` | required | Hotspot2 mappability index directory | | `--blacklist` | required | ENCODE blacklist BED file | | `--outdir` | `./results` | Output directory | | `--fdr` | `0.05` | Hotspot2 FDR threshold | | `--skip_footprint` | `false` | Skip footprinting analysis | | `--motif_db` | `null` | JASPAR motif database for footprinting |

Output Files

results/
  fastqc/                          # Raw read quality
  alignment/
    {sample}.filtered.bam          # Filtered, deduplicated BAM
    {sample}.filtered.bam.bai
  hotspots/
    {sample}.hotspots.fdr0.05.bed  # DHS peaks (primary output)
    {sample}.peaks.narrowPeak      # narrowPeak format
    {sample}.density.bw            # Signal track (bigWig)
    {sample}.allcalls.bed          # All hotspot calls (unfiltered)
    {sample}.SPOT.txt              # SPOT score
  footprints/
    {sample}.footprints.bed        # TF footprints
    {sample}.footprint_scores.txt  # Per-motif footprint scores
  qc/
    {sample}.flagstat.txt
    {sample}.insert_sizes.txt
  multiqc/
    multiqc_report.html

QC Thresholds (ENCODE Standards)

| Metric | Pass | Warning | Fail | |--------|------|---------|------| | SPOT score (Signal Portion of Tags) | >0.4 | 0.2-0.4 | <0.2 | | Hotspot count | >50,000 | 20,000-50,000 | <20,000 | | Mapping