tools-only ATAC-seq QC - Usage Guide
Overview
Assess ATAC-seq library quality using TSS enrichment, FRiP scores, fragment size distributions, mitochondrial contamination, and library complexity metrics.
Prerequisites
pip install pysam pyBigWig numpy pandas matplotlib
conda install -c bioconda samtools picard bedtools
Quick Start
Tell your AI agent what you want to do:
- "Run QC on my ATAC-seq BAM file"
- "Calculate TSS enrichment score for my sample"
Example Prompts
Comprehensive QC
"Generate a full QC report for my ATAC-seq sample including TSS enrichment, FRiP, and fragment distribution"
TSS Enrichment
"Calculate the TSS enrichment score for my ATAC-seq data using ENCODE standards"
Fragment Analysis
"Plot the fragment size distribution from my ATAC-seq BAM to check nucleosome periodicity"
Library Complexity
"Calculate library complexity metrics (NRF, PBC1, PBC2) for my ATAC-seq sample"
Batch QC
"Run MultiQC to aggregate QC metrics across all my ATAC-seq samples"
What the Agent Will Do
- Calculate alignment statistics and mitochondrial contamination fraction
- Compute FRiP (Fraction of Reads in Peaks) using peak file
- Generate fragment size distribution plot to verify nucleosome periodicity
- Calculate TSS enrichment score using gene annotations
- Assess library complexity (NRF, PBC1, PBC2)
- Compile metrics into a QC report
Key QC Metrics
| Metric | What It Measures | Target |
|---|---|---|
| TSS Enrichment | Signal at promoters | >7 (ENCODE standard) |
| FRiP | Signal in peaks vs background | >0.2 |
| Fragment Size | Nucleosome periodicity | Clear pattern |
| MT Fraction | Contamination | <20% |
| NRF | Library complexity | >0.9 |
Fragment Size Interpretation
Fragment Size Peak Type
<100 bp Nucleosome-free regions (NFR)
180-247 bp Mono-nucleosome
315-473 bp Di-nucleosome
558-615 bp Tri-nucleosome
Good QC: Clear peaks at ~50bp (NFR), ~200bp (mono), ~400bp (di) Poor QC: Single peak or no periodicity
Tips
- TSS enrichment >7 is the ENCODE standard for high-quality ATAC-seq
- High MT fraction (>20%) indicates incomplete nuclear isolation
- Poor fragment distribution may indicate over-transposition or degraded chromatin
- Use MultiQC to compare QC metrics across multiple samples
- Low correlation between replicates may indicate technical issues or mislabeling