bio-workflows-atacseq-pipeline

name	bio-workflows-atacseq-pipeline
description	End-to-end ATAC-seq workflow from FASTQ files to differential accessibility and TF footprinting. Covers alignment, peak calling with MACS3, QC metrics, and optional TOBIAS footprinting. Use when running end-to-end ATAC-seq analysis from FASTQ to differential accessibility.
tool_type	mixed
primary_tool	MACS3
workflow	true
depends_on	["read-qc/fastp-workflow","read-alignment/bowtie2-alignment","alignment-files/duplicate-handling","atac-seq/atac-peak-calling","atac-seq/atac-qc","atac-seq/differential-accessibility","atac-seq/footprinting"]
qc_checkpoints	[{"after_qc":"Q30 >85%, adapter content <5%"},{"after_alignment":"Mapping rate >80%, mitochondrial <20%"},{"after_peaks":"FRiP >20%, TSS enrichment >5"},{"after_footprinting":"Motif enrichment validates TF activity"}]

ATAC-seq Pipeline

Complete workflow from raw ATAC-seq FASTQ files to accessibility peaks, differential analysis, and TF footprinting.

Workflow Overview

FASTQ files
    |
    v
[1. QC & Trimming] -----> fastp (Nextera adapters)
    |
    v
[2. Alignment] ---------> Bowtie2
    |
    v
[3. BAM Processing] ----> filter, shift, dedup
    |
    v
[4. Peak Calling] ------> MACS3
    |
    v
[5. QC] ----------------> TSS enrichment, FRiP, fragment size
    |
    v
[6. Differential] ------> DiffBind (optional)
    |
    v
[7. Footprinting] ------> TOBIAS (optional)
    |
    v
Accessibility peaks + TF activity

Primary Path: Bowtie2 + MACS3

Step 1: Quality Control with fastp

# ATAC-seq uses Nextera adapters
NEXTERA_R1="CTGTCTCTTATACACATCT"
NEXTERA_R2="CTGTCTCTTATACACATCT"

for sample in sample1 sample2 sample3; do
    fastp -i ${sample}_R1.fastq.gz -I ${sample}_R2.fastq.gz \
        -o trimmed/${sample}_R1.fq.gz -O trimmed/${sample}_R2.fq.gz \
        --adapter_sequence ${NEXTERA_R1} \
        --adapter_sequence_r2 ${NEXTERA_R2} \
        --qualified_quality_phred 20 \
        --length_required 25 \
        --html qc/${sample}_fastp.html
done

Step 2: Alignment with Bowtie2

# Build index (once)
bowtie2-build genome.fa bt2_index/genome

# Align with ATAC-seq specific settings
for sample in sample1 sample2 sample3; do
    bowtie2 -p 8 -x bt2_index/genome \
        -1 trimmed/${sample}_R1.fq.gz \
        -2 trimmed/${sample}_R2.fq.gz \
        --very-sensitive \
        --no-mixed --no-discordant \
        -X 2000 \
        2> aligned/${sample}.log | \
    samtools view -@ 4 -bS -q 30 -f 2 - | \
    samtools sort -@ 4 -o aligned/${sample}.bam
done