/pipeline-cutandrun

name: pipeline-cutandrun
description: "Execute CUT&RUN processing pipeline from FASTQ to peaks and signal tracks. Child of pipeline-guide. Provides Nextflow execution with Docker and cloud deployment. Use when processing CUT&RUN or CUT&Tag data, an alternative to ChIP-seq with lower background. Trigger on: CUT&RUN pipeline, CUT&Tag, SEACR, Henikoff, targeted chromatin, pA-MNase, process CUT&RUN."

ENCODE CUT&RUN Pipeline: FASTQ to Peaks and Signal Tracks

When to Use

• User wants to run a CUT&RUN or CUT&Tag processing pipeline from FASTQ to peaks
• User asks about "CUT&RUN pipeline", "CUT&Tag", "SEACR", "spike-in normalization", or "targeted chromatin"
• User needs to process CUT&RUN/CUT&Tag data with spike-in calibration and SEACR peak calling
• Example queries: "process my CUT&RUN FASTQs", "run SEACR on CUT&Tag data", "normalize CUT&RUN with spike-in controls"

Execute the CUT&RUN/CUT&Tag processing pipeline for targeted chromatin profiling, producing peak calls with SEACR and spike-in normalized signal tracks.

Pipeline Overview

FASTQ -> Trim -> Bowtie2 align (genome) -> Filter/dedup -> SEACR peaks
                     |                          |              |
              Bowtie2 align (spike-in)   Spike-in normalize  Signal tracks
                     |
              Scale factor calculation

ENCODE Repository

• **GitHub**: `ENCODE-DCC/cutandrun-pipeline`
• **Container**: `encodedcc/cutandrun-pipeline`
• **This skill**: Nextflow DSL2 reimplementation for portability

Core Tools and Versions

| Tool | Version | Purpose | Citation | |------|---------|---------|----------| | Bowtie2 | 2.5.3 | Alignment (genome + spike-in) | Langmead & Salzberg 2012 | | SEACR | 1.3 | Peak calling (CUT&RUN-specific) | Meers et al. 2019 | | MACS2 | 2.2.9.1 | Alternative peak caller | Zhang et al. 2008 | | Picard | 3.1.1 | Duplicate marking | Broad Institute | | samtools | 1.19 | BAM operations | Li et al. 2009 | | bedtools | 2.31.0 | Genomic arithmetic | Quinlan & Hall 2010 | | deepTools | 3.5.4 | Signal track generation | Ramirez et al. 2016 | | FastQC | 0.12.1 | Read quality | Andrews (Babraham) | | MultiQC | 1.21 | Aggregated QC | Ewels et al. 2016 |

Key Literature

1. **Skene & Henikoff 2017** - "An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites" (eLife, ~1,500 citations) DOI: 10.7554/eLife.21856

2. **Meers et al. 2019** - "Peak calling by Sparse Enrichment Analysis for CUT&RUN chromatin profiling" (Epigenetics & Chromatin, ~800 citations) DOI: 10.1186/s13072-019-0287-4

3. **Kaya-Okur et al. 2019** - "CUT&Tag for efficient epigenomic profiling of small samples and single cells" (Nature Communications, ~1,200 citations) DOI: 10.1038/s41467-019-09982-5

4. **Nordin et al. 2023** - "The CUT&RUN suspect list of problematic regions" (Genome Biology) DOI: 10.1186/s13059-023-02960-3

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

Execution

Quick Start (Local)

nextflow run main.nf \
    -profile local \
    --reads '/data/fastq/*_R{1,2}.fastq.gz' \
    --bowtie2_index '/ref/bowtie2_index/genome' \
    --spikein_index '/ref/bowtie2_ecoli/ecoli' \
    --chrom_sizes '/ref/hg38.chrom.sizes' \
    --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' \
    --bowtie2_index '/ref/bowtie2_index/genome' \
    --spikein_index '/ref/bowtie2_ecoli/ecoli' \
    --chrom_sizes '/ref/hg38.chrom.sizes' \
    --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' \
    --bowtie2_index 'gs://bucket/ref/bowtie2_index/genome' \
    --spikein_index 'gs://bucket/ref/bowtie2_ecoli/ecoli' \
    --chrom_sizes 'gs://bucket/ref/hg38.chrom.sizes' \
    --blacklist 'gs://bucket/ref/hg38-blacklist.v2.bed' \
    --outdir 'gs://bucket/results/' \
    -resume

Resource Requirements

| Step | CPUs | RAM | Time (per sample) | |------|------|-----|-------------------| | Bowtie2 align (genome) | 8 | 8 GB | 30-60 min | | Bowtie2 align (spike-in) | 4 | 4 GB | 10-20 min | | Filter/dedup | 4 | 8 GB | 15-30 min | | SEACR peaks | 2 | 4 GB | 10-20 min | | Signal tracks | 4 | 8 GB | 15-30 min | | **Total** | **8** | **8 GB** | **1.5-3 hours** |

Pipeline Parameters

| Parameter | Default | Description | |-----------|---------|-------------| | `--reads` | required | Glob pattern to paired FASTQ files | | `--bowtie2_index` | required | Bowtie2 genome index prefix | | `--spikein_index` | required | Bowtie2 E. coli spike-in index prefix | | `--chrom_sizes` | required | Chromosome sizes file | | `--blacklist` | required | ENCODE blacklist BED file | | `--outdir` | `./results` | Output directory | | `--seacr_mode` | `stringent` | SEACR mode: `stringent` or `relaxed` | | `--seacr_norm` | `norm` | SEACR normalization: `norm` or `non` | | `--control` | `null` | IgG control BAM (if available) | | `--peak_caller` | `seacr` | Peak caller: `seacr` or `macs2` or `both` | | `--skip_spikein` | `false` | Skip spike-in normalization |

Output Files

results/
  fastqc/                           # Raw read quality
  alignment/
    {sample}.filtered.bam           # Filtered, deduplicated BAM
    {sample}.filtered.bam.bai
  spikein/
    {sample}.spikein_counts.txt     # Spike-in read counts
    {sample}.scale_factor.txt       # Computed scale factor
  peaks/
    {sample}.seacr.stringent.bed    # SEACR stringent peaks
    {sample}.seacr.relaxed.bed      # SEACR relaxed peaks
    {sample}.macs2_peaks.narrowPeak # MACS2 peaks (if requested)
  signal/
    {sample}.normalized.bw          # Spike-in normalized signal
    {sample}.fragments.bed          # Fragment BED file
  qc/