7. ATAC-Seq Open Chromatin & Regulatory Footprinting
Assay for Transposase-Accessible Chromatin to profile open promoters, distant enhancers, and transcription factor footprints with Tn5 transposase shift corrections (+4 bp/-5 bp).
| Metric / Requirement | Specification | Target Configuration |
|---|---|---|
| Core Engine / Framework | Bowtie2 / Chromap + MACS3 + HOMER + TOBIAS | Nextflow / Snakemake Compatible |
| Benchmark Runtime | ~4 – 6 hours (50M PE reads) | 16 CPU cores / 32 GB RAM |
| Standard Container | quay.io/biocontainers/macs3:3.0.1--py310h5096538_0 | Docker • Singularity • Conda |
| Input → Deliverable | ATAC FASTQ → Shifted Deduplicated BAM, Narrow Peaks BED, TF Footprints | |
Step 1: Ultra-Fast Chromatin Alignment with Bowtie2
Aligns paired-end ATAC reads, allowing wide insert sizes up to 2 kb to capture multi-nucleosomal fragments.
bowtie2 -p 16 -X 2000 --very-sensitive \
-x /references/GRCh38_bowtie2_index \
-1 atac_R1.fastq.gz -2 atac_R2.fastq.gz | \
samtools view -bS -q 30 -F 1804 - | \
samtools sort -@ 8 -o atac_aligned.bam -
samtools index -@ 8 atac_aligned.bam-X 2000 accommodates large di- and tri-nucleosomal DNA fragments; -q 30 retains only uniquely mapped reads.Step 2: Mitochondrial Depletion & PCR Duplicate Marking
Purges abundant mitochondrial read contamination and flags optical/PCR duplicates.
# Remove chrM reads
samtools idxstats atac_aligned.bam | cut -f 1 | grep -v chrM | xargs samtools view -b atac_aligned.bam > atac_noMito.bam
# Mark and remove duplicates
picard MarkDuplicates \
I=atac_noMito.bam O=atac_dedup.bam M=dup_metrics.txt \
REMOVE_DUPLICATES=true
samtools index atac_dedup.bamStep 3: Tn5 Transposase Shift Correction (+4 bp / -5 bp)
Corrects read coordinates for the 9 bp duplication introduced upon Tn5 dimer transposition (+4 bp on forward, -5 bp on reverse).
alignmentSieve --numberOfProcessors 16 \
--ATACshift -b atac_dedup.bam \
-o atac_shifted.bam
samtools sort -@ 8 atac_shifted.bam -o atac_shifted.sorted.bam
samtools index atac_shifted.sorted.bam--ATACshift ensures peak summits center exactly on the accessible DNA cut site.Step 4: Open Chromatin Peak Calling with MACS3 BAMPE Mode
Calls narrow accessible genomic peaks using exact paired-end insert fragments without empirical single-end modeling.
macs3 callpeak -t atac_shifted.sorted.bam \
-f BAMPE -n sample_atac \
-g hs -q 0.01 --keep-dup all \
--outdir macs3_results/-f BAMPE leverages the paired insert span for exact chromatin accessibility boundary estimation.Step 5: Transcription Factor Motif Discovery with HOMER
Identifies enriched known and de novo transcription factor binding motifs within accessible peak summits.
findMotifsGenome.pl macs3_results/sample_atac_peaks.narrowPeak hg38 homer_motifs_out/ -size 200-size 200 scans a ±100 bp window centered directly on the open chromatin peak summit.🚀 Automated Nextflow (nf-core) Production Execution
Execute this complete workflow across any SLURM, AWS Batch, or Google Cloud HPC cluster using the verified community container pipeline:
nextflow run nf-core/atacseq -r 2.1.2 \
--input samplesheet.csv \
--genome GRCh38 \
--outdir ./results_atacseq/ \
-profile docker