shiganluo/skill/chipseq-peak-calling-workflow

ChIP-seq Peak Calling Workflow

Overview

Complete ChIP-seq peak calling subworkflow for the Omics Snakemake framework, ported from the nf-core/chipseq Nextflow pipeline.

Pipeline Steps

1. FastQC (raw) - Quality check raw reads
2. Trim Galore - Adapter trimming
3. FastQC (trimmed) - Quality check trimmed reads
4. Bowtie2 - Alignment to reference genome
5. BigWig tracks - Normalized coverage tracks (bamCoverage)
6. MACS3 - Peak calling
7. FRiP Score - Fraction of Reads in Peaks QC metric
8. HOMER - Peak annotation with genomic features

Key Metrics

• FRiP Score: reads_in_peaks / total_mapped_reads
• >= 0.3 for transcription factors (narrow peaks)
• >= 0.2 for histone marks (broad peaks)

File Structure

Omics/
├── modules/
│   ├── frip_score/
│   │   ├── frip_score.smk
│   │   └── frip_score.yaml
│   └── homer/
│       ├── homer.smk
│       └── homer.yaml
└── subworkflow/
    └── PeakCalling.smk

Config Requirements

# Procedure tools
Procedure:
  samtools: "samtools"
  bedtools: "bedtools"
  macs3: "macs3"
  bamCoverage: "bamCoverage"
  annotatePeaks: "annotatePeaks.pl"
 
# MACS3 parameters
Params:
  macs3:
    bw: 200
    pvalue: "1e-5"
    genome_size: "mm"  # hs (human), mm (mouse), etc.
    cutoff_analysis: false  # true → --cutoff-analysis (~30x slower)
 
# Genome references
genome:
  fasta: "/path/to/genome.fa"
  gtf: "/path/to/genes.gtf"
  bowtie2_index_prefix: "/path/to/bowtie2_index"
 
# Samples
ip_samples: ["sample1", "sample2"]
input_samples: ["input1", "input2"]
sample_ip_input_map:
  sample1: "input1"
  sample2: "input2"

Pitfalls

• MACS3 module expects BAM at indir/{sample_id}/{sample_id}.sorted_markdup.bam (markdup BAM from gatk_prepare, NOT raw BAM)
• HOMER annotatePeaks.pl requires GTF with gene annotations
• FRiP score uses bedtools intersect -u (not -c) to count unique reads
• MACS3 cutoff_analysis: when true, the module must append --cutoff-analysis to the cmd list. A common bug is logging the flag but forgetting to append it (see snakemake-omics-workflow pitfall #14)
• After running cutoff_analysis, interpret results with references/cutoff_analysis_guide.md — includes column meanings, elbow point detection, and threshold selection guide