bio-workflows-atacseq-pipeline

Version Compatibility

Reference examples tested with: Bowtie2 2.5.3+, MACS3 3.0+, bedtools 2.31+, deepTools 3.5+, fastp 0.23+, samtools 1.19+

Before using code patterns, verify installed versions match. If versions differ:

• R: packageVersion('<pkg>') then ?function_name to verify parameters
• CLI: <tool> --version then <tool> --help to confirm flags

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

ATAC-seq Pipeline

"Run end-to-end ATAC-seq analysis from FASTQ to differential accessibility" → Orchestrate QC, Bowtie2 alignment, MACS3 peak calling, FRiP/TSS enrichment QC, differential accessibility, and optional TOBIAS footprinting.

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