Pipeline Dnaseseq

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

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

Pipeline Parameters

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)

SPOT Score

The SPOT score (Signal Portion of Tags) is the fraction of reads falling within hotspots. It is the DNase-seq equivalent of FRiP for ChIP-seq.

Higher SPOT = more enrichment in accessible regions = better library quality.

Hotspot2 vs MACS2

IMPORTANT: ENCODE uses Hotspot2 for DNase-seq, NOT MACS2.

Hotspot2 accounts for mappability variation across the genome, which is critical for DNase-seq because DNase I cuts accessible chromatin regardless of whether it is uniquely mappable.

Critical Pitfalls

DNase-seq vs ATAC-seq

These are different assays measuring the same biology (chromatin accessibility):

• DNase-seq: Uses DNase I enzyme, requires more input material
• ATAC-seq: Uses Tn5 transposase, works on fewer cells
• Analysis pipelines differ: Hotspot2 for DNase-seq, MACS2 for ATAC-seq
• Data are largely concordant but not identical

Fragment Size Distribution

DNase-seq produces a characteristic fragment size distribution:

• Peak at ~50-100 bp (sub-nucleosomal fragments at DHS)
• Secondary peak at ~150-200 bp (mononucleosomal fragments)
• Long tail of larger fragments
• If distribution is abnormal, check library preparation protocol

Mappability Index

Hotspot2 requires a pre-computed mappability index. These are read-length and genome-build specific:

• hg38 / 36 bp: Use ENCODE-provided index
• hg38 / 76 bp: Use ENCODE-provided index
• hg38 / 150 bp: May need to generate custom index
• Wrong mappability index = incorrect peak calls

Blacklist Filtering

Always filter peaks against the ENCODE blacklist (Amemiya et al. 2019):

bedtools intersect -a hotspots.bed -b hg38-blacklist.v2.bed -v > hotspots_filtered.bed

Blacklist regions produce artifactual signal in accessibility assays.

Footprinting Analysis

Transcription factor footprinting detects bound TFs from DNase-seq signal:

HINT-ATAC Footprinting

rgt-hint footprinting \
    --atac-seq \
    --paired-end \
    --organism hg38 \
    --output-location footprints/ \
    sample.filtered.bam \
    hotspots.narrowPeak

Interpretation

• Footprints are depressions in the DNase signal where a bound TF protects DNA
• Requires deep sequencing (>100M reads) for reliable footprints
• Sensitivity varies by TF: pioneer factors have shallow footprints
• Vierstra et al. 2020 provides a global reference map for comparison

Provenance Integration

After pipeline completion, log all outputs:

encode_log_derived_file(
    file_path="/results/hotspots/sample1.hotspots.fdr0.05.bed",
    source_accessions=["ENCSR...", "ENCFF..."],
    description="DNase hypersensitive sites from ENCODE DNase-seq pipeline",
    file_type="DHS_peaks",
    tool_used="BWA 0.7.17 + Hotspot2 2.3.1",
    parameters="FDR 0.05, blacklist filtered, ENCODE hg38 mappability index"
)

Reference Files

Detailed step-by-step documentation is provided in the references/ directory:

1. 01-qc-trimming.md -- Read QC and adapter trimming
2. 02-alignment.md -- BWA-MEM alignment for DNase-seq
3. 03-filtering.md -- BAM filtering, deduplication, blacklist removal
4. 04-hotspot-calling.md -- Hotspot2 DHS detection and signal generation
5. 05-footprinting.md -- TF footprint detection with HINT-ATAC

Walkthrough: Processing ENCODE DNase-seq from FASTQ to Hypersensitive Sites

Goal: Process raw DNase-seq FASTQ files through the ENCODE pipeline to generate DNase I hypersensitive site (DHS) peak calls. Context: DNase-seq identifies open chromatin via DNase I enzyme digestion. The pipeline uses BWA alignment and Hotspot2 for DHS identification.

Step 1: Find DNase-seq experiment

encode_search_experiments(assay_title="DNase-seq", biosample_term_name="K562", organism="Homo sapiens")

Expected output:

{
  "total": 8,
  "results": [
    {"accession": "ENCSR000DNS", "assay_title": "DNase-seq", "biosample_summary": "K562", "status": "released"}
  ]
}

Step 2: List and download FASTQ files

encode_list_files(accession="ENCSR000DNS", file_format="fastq")

Step 3: Run the DNase-seq pipeline

nextflow run pipeline-dnaseseq/main.nf \
  --fastq_r1 ENCFF700DN1.fastq.gz \
  --genome GRCh38 \
  --blacklist encode_blacklist_v2.bed \
  -profile docker

Key pipeline steps:

1. Quality trimming
2. BWA-MEM alignment
3. Duplicate removal
4. Hotspot2 DHS calling
5. Signal track generation
6. Footprint analysis (HINT-ATAC)

Step 4: Validate output quality

Step 5: Compare with ATAC-seq

encode_search_experiments(assay_title="ATAC-seq", biosample_term_name="K562", organism="Homo sapiens")

Interpretation: DNase-seq and ATAC-seq both measure accessibility but with different biases. Compare peaks from both assays -- concordant peaks are high confidence.

Integration with downstream skills

• DHS peaks feed into -> accessibility-aggregation alongside ATAC-seq peaks
• Footprint data feeds into -> motif-analysis for TF binding prediction
• Signal tracks feed into -> visualization-workflow
• Peaks integrate with -> regulatory-elements for cCRE classification

Code Examples

1. Survey DNase-seq availability

encode_get_facets(assay_title="DNase-seq", facet_field="organ", organism="Homo sapiens")

Expected output:

{
  "facets": {
    "organ": {"blood": 45, "brain": 30, "liver": 20, "heart": 15, "lung": 12}
  }
}

2. Check for existing DHS peaks

encode_list_files(accession="ENCSR000DNS", file_format="bed", output_type="peaks", assembly="GRCh38")

Expected output:

{
  "files": [
    {"accession": "ENCFF800DHS", "output_type": "peaks", "file_format": "bed narrowPeak", "file_size_mb": 1.5}
  ]
}

3. Track DNase-seq experiments

encode_track_experiment(accession="ENCSR000DNS", notes="K562 DNase-seq for accessibility comparison with ATAC-seq")

Expected output:

{
  "status": "tracked",
  "accession": "ENCSR000DNS",
  "notes": "K562 DNase-seq for accessibility comparison with ATAC-seq"
}

Integration

Related Skills

• pipeline-guide -- Parent skill with compute resource assessment and cloud setup
• accessibility-aggregation -- Aggregate DHS data across samples/tissues
• quality-assessment -- Evaluate pipeline output quality metrics
• data-provenance -- Track all pipeline inputs, outputs, and parameters
• download-encode -- Download ENCODE DNase-seq FASTQ files for pipeline input
• publication-trust -- Verify literature claims backing analytical decisions

Presenting Results

When reporting DNase-seq pipeline results:

• Hotspot counts: Report total Hotspot2 DHS calls at the specified FDR threshold and the number remaining after blacklist filtering
• Signal-to-noise (SPOT score): Report the SPOT score prominently (>0.4 pass, 0.2-0.4 warning, <0.2 fail). This is the DNase-seq equivalent of FRiP
• Footprint depth: If footprinting was performed, report the number of TF footprints detected and note the sequencing depth (>100M reads recommended for reliable footprints)
• FRiP equivalent: Report the fraction of reads in hotspots as a complementary enrichment metric
• Key QC metrics: Present mapping rate (>80%), duplication rate (<30%), NRF (>0.8), PBC1 (>0.9), and insert size peak in a summary table
• Output paths: Provide paths to hotspot BED files, narrowPeak files, signal bigWig tracks, and footprint results
• Mappability note: Confirm which Hotspot2 mappability index was used and that it matches the read length
• Next steps: Suggest motif-analysis for TF motif enrichment in DHS peaks, or accessibility-aggregation for merging DHS data across samples

For the request: "$ARGUMENTS"