alterlab-deeptools

deepTools: NGS Data Analysis Toolkit

Overview

deepTools is a comprehensive suite of Python command-line tools designed for processing and analyzing high-throughput sequencing data. Use deepTools to perform quality control, normalize data, compare samples, and generate publication-quality visualizations for ChIP-seq, RNA-seq, ATAC-seq, MNase-seq, and other NGS experiments.

Core capabilities:

• Convert BAM alignments to normalized coverage tracks (bigWig/bedGraph)
• Quality control assessment (fingerprint, correlation, coverage)
• Sample comparison and correlation analysis
• Heatmap and profile plot generation around genomic features
• Enrichment analysis and peak region visualization

When to Use This Skill

This skill should be used when:

• File conversion: "Convert BAM to bigWig", "generate coverage tracks", "normalize ChIP-seq data"
• Quality control: "check ChIP quality", "compare replicates", "assess sequencing depth", "QC analysis"
• Visualization: "create heatmap around TSS", "plot ChIP signal", "visualize enrichment", "generate profile plot"
• Sample comparison: "compare treatment vs control", "correlate samples", "PCA analysis"
• Analysis workflows: "analyze ChIP-seq data", "RNA-seq coverage", "ATAC-seq analysis", "complete workflow"
• Working with specific file types: BAM files, bigWig files, BED region files in genomics context

Does NOT Trigger

Scenario	Use Instead
Per-read access: CIGAR strings, MAPQ, pileups, read filtering in Python	alterlab-pysam
Interval arithmetic on BED/peak sets (overlaps, Jaccard) or region embeddings	alterlab-gtars, alterlab-geniml
Scanning peaks for transcription-factor motifs	alterlab-jaspar
Counting reads per gene for differential expression	alterlab-rnaseq-quant → alterlab-pydeseq2
Variant calling from the same BAMs	alterlab-nf-core-sarek

Quick Start

For users new to deepTools, start with file validation and common workflows:

1. Validate Input Files

Before running any analysis, validate BAM, bigWig, and BED files using the validation script:

python scripts/validate_files.py --bam sample1.bam sample2.bam --bed regions.bed

This checks file existence, BAM indices, and format correctness.

2. Generate Workflow Template

For standard analyses, use the workflow generator to create customized scripts:

# List available workflows
python scripts/workflow_generator.py --list

# Generate ChIP-seq QC workflow
python scripts/workflow_generator.py chipseq_qc -o qc_workflow.sh \
    --input-bam Input.bam --chip-bams "ChIP1.bam ChIP2.bam" \
    --genome-size 2913022398

# Make executable and run
chmod +x qc_workflow.sh
./qc_workflow.sh

3. Most Common Operations

See assets/quick_reference.md for frequently used commands and parameters.

Installation

uv pip install deeptools          # 4.0.0 as of 2026-09 (needs Python >= 3.12)
# or: conda install -c bioconda deeptools

What changed in deepTools 4.0

bamCoverage, bamCompare, computeMatrix, alignmentSieve and multiBamSummary were rewritten with a Rust core (rayon threading, bigtools bigWig I/O). The old Python implementations remain for now as bamCoverage_old, bamCompare_old, computeMatrix_old, alignmentSieve_old and multiBamSummary_old, and are slated for removal.

Flags that changed, and what to do instead:

Gone in 4.0	Replacement
--ignoreDuplicates on bamCoverage / bamCompare	--samFlagExclude 1024 (the BAM must have duplicates marked, e.g. by samtools markdup). The flag is still valid on alignmentSieve, plotFingerprint, plotCoverage, multiBamSummary and estimateReadFiltering.
--exactScaling	removed — the new backend always scales exactly
--plotFileFormat plotly and the plotly backend	matplotlib output only; --ggplot offers a ggplot-like theme

Also new: gzipped GTF/BED region and blacklist files are accepted by the Rust-backed tools, and plotPCA now uses a scipy/numpy SVD with correct --transpose, --log2/--rowCenter and --ntop handling.

Core Workflows

deepTools workflows typically follow this pattern: QC → Normalization → Comparison/Visualization

ChIP-seq Quality Control Workflow

When users request ChIP-seq QC or quality assessment:

1. Generate workflow script using scripts/workflow_generator.py chipseq_qc
2. Key QC steps:
   • Sample correlation (multiBamSummary + plotCorrelation)
   • PCA analysis (plotPCA)
   • Coverage assessment (plotCoverage)
   • Fragment size validation (bamPEFragmentSize)
   • ChIP enrichment strength (plotFingerprint)

Interpreting results:

• Correlation: Replicates should cluster together with high correlation (>0.9)
• Fingerprint: Strong ChIP shows steep rise; flat diagonal indicates poor enrichment
• Coverage: Assess if sequencing depth is adequate for analysis

Full workflow details in references/workflows.md → "ChIP-seq Quality Control Workflow"

ChIP-seq Complete Analysis Workflow

For full ChIP-seq analysis from BAM to visualizations:

1. Generate coverage tracks with normalization (bamCoverage)
2. Create comparison tracks (bamCompare for log2 ratio)
3. Compute signal matrices around features (computeMatrix)
4. Generate visualizations (plotHeatmap, plotProfile)
5. Enrichment analysis at peaks (plotEnrichment)

Use scripts/workflow_generator.py chipseq_analysis to generate template.

Complete command sequences in references/workflows.md → "ChIP-seq Analysis Workflow"

RNA-seq Coverage Workflow

For strand-specific RNA-seq coverage tracks:

Use bamCoverage with --filterRNAstrand to separate forward and reverse strands.

Important: do not use --extendReads for RNA-seq — extension runs straight over splice junctions and invents coverage in introns.

Use normalization: CPM for fixed bins, RPKM for gene-level analysis.

Template available: scripts/workflow_generator.py rnaseq_coverage

Details in references/workflows.md → "RNA-seq Coverage Workflow"

ATAC-seq Analysis Workflow

ATAC-seq requires Tn5 offset correction:

1. Shift reads using alignmentSieve with --ATACshift
2. Generate coverage with bamCoverage
3. Analyze fragment sizes (expect nucleosome ladder pattern)
4. Visualize at peaks if available

Template: scripts/workflow_generator.py atacseq

Full workflow in references/workflows.md → "ATAC-seq Workflow"

Tool Categories and Common Tasks

deepTools commands group into three categories. Quick command examples for each live in references/usage_playbook.md → "Inline Command Examples by Category"; full parameter documentation is in references/tools_reference.md.

• BAM/bigWig processing — bamCoverage, bamCompare, multiBamSummary, multiBigwigSummary, correctGCBias, alignmentSieve (tools_reference.md → "BAM and bigWig File Processing Tools")
• Quality control — plotFingerprint, plotCoverage, plotCorrelation, plotPCA, bamPEFragmentSize (tools_reference.md → "Quality Control Tools")
• Visualization — computeMatrix, plotHeatmap, plotProfile, plotEnrichment (tools_reference.md → "Visualization Tools")

Normalization Methods

Choosing the correct normalization is critical for valid comparisons. Consult references/normalization_methods.md for comprehensive guidance.

Quick selection guide:

• ChIP-seq coverage: Use RPGC or CPM
• ChIP-seq comparison: Use bamCompare with log2 and readCount
• RNA-seq bins: Use CPM
• RNA-seq genes: Use RPKM (accounts for gene length)
• ATAC-seq: Use RPGC or CPM

Normalization methods:

• RPGC: 1× genome coverage (requires --effectiveGenomeSize)
• CPM: Counts per million mapped reads
• RPKM: Reads per kb per million (accounts for region length)
• BPM: Bins per million
• None: Raw counts (not recommended for comparisons)

Full explanation: references/normalization_methods.md

Effective Genome Sizes

RPGC normalization requires effective genome size. Common values:

Organism	Assembly	Size	Usage
Human	GRCh38/hg38	2,913,022,398	--effectiveGenomeSize 2913022398
Mouse	GRCm38/mm10	2,652,783,500	--effectiveGenomeSize 2652783500
Zebrafish	GRCz11	1,368,780,147	--effectiveGenomeSize 1368780147
Drosophila	dm6	142,573,017	--effectiveGenomeSize 142573017
C. elegans	ce10/ce11	100,286,401	--effectiveGenomeSize 100286401

Complete table with read-length-specific values: references/effective_genome_sizes.md

Common Parameters Across Tools

Many deepTools commands share these options:

Performance:

• --numberOfProcessors, -p: Enable parallel processing (always use available cores)
• --region: Process specific regions for testing (e.g., chr1:1-1000000)

Read Filtering:

• --ignoreDuplicates: skip reads flagged as PCR duplicates. Not available on bamCoverage/bamCompare in 4.0 — use --samFlagExclude 1024 there
• --minMappingQuality: Filter by alignment quality (e.g., --minMappingQuality 10)
• --minFragmentLength / --maxFragmentLength: Fragment length bounds
• --samFlagInclude / --samFlagExclude: SAM flag filtering

Read Processing:

• --extendReads: Extend to fragment length (ChIP-seq: YES, RNA-seq: NO)
• --centerReads: Center at fragment midpoint for sharper signals

Best Practices

File Validation

Always validate files first using scripts/validate_files.py to check:

• File existence and readability
• BAM indices present (.bai files)
• BED format correctness
• File sizes reasonable

Analysis Strategy

1. Start with QC: Run correlation, coverage, and fingerprint analysis before proceeding
2. Test on small regions: Use --region chr1:1-10000000 for parameter testing
3. Document commands: Save full command lines for reproducibility
4. Use consistent normalization: Apply same method across samples in comparisons
5. Verify genome assembly: Ensure BAM and BED files use matching genome builds

ChIP-seq Specific

• Always extend reads for ChIP-seq: --extendReads 200
• Remove duplicates: on bamCoverage/bamCompare use --samFlagExclude 1024 (4.0 dropped --ignoreDuplicates there); --ignoreDuplicates still works on the QC tools
• Check enrichment first: Run plotFingerprint before detailed analysis
• GC correction: Only apply if significant bias is detected, and do not filter duplicates afterwards — correctGCBias adds reads to under-represented regions, so duplicate removal on a corrected BAM discards exactly those synthetic reads and re-introduces the bias

RNA-seq Specific

• Never extend reads for RNA-seq (would span splice junctions)
• Strand-specific: Use --filterRNAstrand forward/reverse for stranded libraries
• Normalization: CPM for bins, RPKM for genes

ATAC-seq Specific

• Apply Tn5 correction: Use alignmentSieve with --ATACshift
• Fragment filtering: Set appropriate min/max fragment lengths
• Check nucleosome pattern: Fragment size plot should show ladder pattern

Performance Optimization

1. Use multiple processors: --numberOfProcessors 8 (or available cores)
2. Increase bin size for faster processing and smaller files
3. Process chromosomes separately for memory-limited systems
4. Pre-filter BAM files using alignmentSieve to create reusable filtered files
5. Use bigWig over bedGraph: Compressed and faster to process

Troubleshooting

Common Issues

BAM index missing:

samtools index input.bam

Out of memory: Process chromosomes individually using --region:

bamCoverage --bam input.bam -o chr1.bw --region chr1

Slow processing: Increase --numberOfProcessors and/or increase --binSize

bigWig files too large: Increase bin size: --binSize 50 or larger

Validation Errors

Run validation script to identify issues:

python scripts/validate_files.py --bam *.bam --bed regions.bed

Common errors and solutions explained in script output.

Reference Documentation

Load the matching reference on demand:

Reference	Load when
references/tools_reference.md	User asks about a specific tool, parameter, or detailed usage. All commands by category (BAM/bigWig 9, QC 6, visualization 3, misc 2) with parameters, examples, and notes.
references/workflows.md	User needs a complete analysis pipeline. ChIP-seq QC, ChIP-seq analysis, RNA-seq coverage, ATAC-seq, multi-sample comparison, peak region analysis, performance tips.
references/normalization_methods.md	User asks about normalization, comparing samples, or which method to use. Per-method detail (RPGC/CPM/RPKM/BPM…), formulas, selection guide, pitfalls.
references/effective_genome_sizes.md	User needs a genome size for RPGC normalization or GC-bias correction. Per-organism and read-length-specific values, custom-genome calculation.
references/usage_playbook.md	Driving the skill: per-request playbooks, example interactions, quick command examples by category, and grep recipes for searching the references above.

Helper Scripts

scripts/validate_files.py

Validates BAM, bigWig, and BED files for deepTools analysis. Checks file existence, indices, and format.

Usage:

python scripts/validate_files.py --bam sample1.bam sample2.bam \
    --bed peaks.bed --bigwig signal.bw

When to use: Before starting any analysis, or when troubleshooting errors.

scripts/workflow_generator.py

Generates customizable bash script templates for common deepTools workflows.

Available workflows:

• chipseq_qc: ChIP-seq quality control
• chipseq_analysis: Complete ChIP-seq analysis
• rnaseq_coverage: Strand-specific RNA-seq coverage
• atacseq: ATAC-seq with Tn5 correction

Usage:

# List workflows
python scripts/workflow_generator.py --list

# Generate workflow
python scripts/workflow_generator.py chipseq_qc -o qc.sh \
    --input-bam Input.bam --chip-bams "ChIP1.bam ChIP2.bam" \
    --genome-size 2913022398 --threads 8

# Run generated workflow
chmod +x qc.sh
./qc.sh

When to use: Users request standard workflows or need template scripts to customize.

Assets

assets/quick_reference.md

Quick reference card with most common commands, effective genome sizes, and typical workflow pattern.

When to use: Users need quick command examples without detailed documentation.

Handling User Requests

Per-request playbooks (new vs experienced users, task-specific responses for "convert BAM to bigWig" / "check ChIP quality" / "create heatmap" / "compare samples"), example interactions, and grep recipes for searching the references all live in references/usage_playbook.md. The common thread: validate files first → pick the right workflow/normalization → generate or run the command → explain the result.

Key Reminders

• deepTools 4.0: --ignoreDuplicates/--exactScaling are gone from bamCoverage and bamCompare — filter duplicates with --samFlagExclude 1024
• Extend reads carefully: --extendReads YES for ChIP-seq, NO for RNA-seq (would span splice junctions)
• Normalization is mutually exclusive in bamCompare: RPGC is a --normalizeUsing value; --scaleFactorsMethod only takes readCount/SES/None
• RPGC requires --effectiveGenomeSize; verify the assembly matches your BAM/BED genome build
• Check QC first (plotFingerprint, correlation) before detailed analysis; test parameters on a --region

Part of the AlterLab Academic Skills suite.