deepTools — NGS Data Analysis Toolkit

Overview

deepTools is a command-line toolkit for processing and visualizing high-throughput sequencing data. It converts BAM alignments to normalized coverage tracks (bigWig), performs quality control (correlation, PCA, fingerprint), and generates publication-quality heatmaps and profile plots around genomic features. Supports ChIP-seq, RNA-seq, ATAC-seq, and MNase-seq.

When to Use

• Converting BAM files to normalized bigWig coverage tracks
• Comparing ChIP-seq treatment vs input control (log2 ratio tracks)
• Assessing sample quality: replicate correlation, PCA, coverage depth
• Evaluating ChIP enrichment strength (fingerprint plots)
• Creating heatmaps and profile plots around TSS, peaks, or other genomic regions
• Analyzing ATAC-seq data with Tn5 offset correction
• Generating strand-specific RNA-seq coverage tracks
• For read alignment, use STAR, BWA, or bowtie2 instead
• For peak calling, use MACS2 or HOMER instead
• For BAM/VCF file manipulation, use pysam instead

Prerequisites

bash
pip install deeptools
# Verify installation
bamCoverage --version

Input requirements: BAM files must be sorted and indexed (.bai file present). Generate index with samtools index input.bam. BED files for genomic regions (genes, peaks) in standard 3+ column format.

Quick Start

bash
# Convert BAM to normalized bigWig
bamCoverage --bam sample.bam --outFileName sample.bw \
    --normalizeUsing RPGC --effectiveGenomeSize 2913022398 \
    --binSize 10 --numberOfProcessors 8

# Create heatmap around TSS
computeMatrix reference-point -S sample.bw -R genes.bed \
    -b 3000 -a 3000 --referencePoint TSS -o matrix.gz
plotHeatmap -m matrix.gz -o heatmap.png --colorMap RdBu

Core API

1. BAM to Coverage Conversion

Convert BAM alignments to normalized coverage tracks (bigWig or bedGraph).

bash
# Basic conversion with RPGC normalization
bamCoverage --bam input.bam --outFileName output.bw \
    --normalizeUsing RPGC --effectiveGenomeSize 2913022398 \
    --binSize 10 --numberOfProcessors 8 \
    --extendReads 200 --ignoreDuplicates

# CPM normalization (simpler, no genome size needed)
bamCoverage --bam input.bam --outFileName output.bw \
    --normalizeUsing CPM --binSize 10 -p 8

# RNA-seq: strand-specific coverage
bamCoverage --bam rnaseq.bam --outFileName forward.bw \
    --filterRNAstrand forward --normalizeUsing CPM -p 8
# IMPORTANT: Never use --extendReads for RNA-seq (spans splice junctions)

2. Sample Comparison

Compare treatment vs control or generate ratio tracks.

bash
# Log2 ratio: treatment / control
bamCompare -b1 treatment.bam -b2 control.bam -o log2ratio.bw \
    --operation log2 --scaleFactorsMethod readCount \
    --extendReads 200 -p 8

# Subtract control from treatment
bamCompare -b1 treatment.bam -b2 control.bam -o subtract.bw \
    --operation subtract --scaleFactorsMethod readCount

3. Quality Control

Assess sample quality, replicate concordance, and enrichment strength.

bash
# Sample correlation heatmap
multiBamSummary bins --bamfiles rep1.bam rep2.bam rep3.bam \
    -o counts.npz --binSize 10000 -p 8
plotCorrelation -in counts.npz --corMethod pearson \
    --whatToShow heatmap -o correlation.png
# Good: replicates cluster with r > 0.9

# PCA of samples
plotPCA -in counts.npz -o pca.png --plotTitle "Sample PCA"

# ChIP enrichment fingerprint
plotFingerprint -b input.bam chip.bam -o fingerprint.png \
    --extendReads 200 --ignoreDuplicates
# Good ChIP: steep rise curve; flat diagonal = poor enrichment

# Coverage depth assessment
plotCoverage -b sample.bam -o coverage.png --ignoreDuplicates -p 8

# Fragment size distribution (paired-end)
bamPEFragmentSize -b sample.bam -o fragsize.png

4. Heatmaps and Profile Plots

Visualize signal around genomic features (TSS, peaks, gene bodies).

bash
# Reference-point mode: signal around TSS
computeMatrix reference-point -S chip.bw -R genes.bed \
    -b 3000 -a 3000 --referencePoint TSS -o matrix.gz -p 8

# Scale-regions mode: signal across gene bodies
computeMatrix scale-regions -S chip.bw -R genes.bed \
    -b 1000 -a 1000 --regionBodyLength 5000 -o matrix.gz -p 8

# Generate heatmap
plotHeatmap -m matrix.gz -o heatmap.png \
    --colorMap RdBu --kmeans 3 --sortUsing mean

# Generate profile plot
plotProfile -m matrix.gz -o profile.png \
    --plotType lines --colors blue red

# Multiple signal files: compare marks
computeMatrix reference-point -S h3k4me3.bw h3k27me3.bw -R genes.bed \
    -b 3000 -a 3000 --referencePoint TSS -o multi_matrix.gz
plotHeatmap -m multi_matrix.gz -o multi_heatmap.png

5. Read Filtering and Processing

Filter reads before analysis or correct for assay-specific biases.

bash
# Filter by mapping quality and fragment size
alignmentSieve --bam input.bam --outFile filtered.bam \
    --minMappingQuality 10 --minFragmentLength 150 \
    --maxFragmentLength 700

# ATAC-seq: apply Tn5 offset correction (+4/-5 bp shift)
alignmentSieve --bam atac.bam --outFile shifted.bam --ATACshift
# Then index: samtools index shifted.bam

# GC bias correction (only if significant bias detected)
computeGCBias -b input.bam --effectiveGenomeSize 2913022398 \
    -g genome.2bit --GCbiasFrequenciesFile gc_freq.txt -p 8
correctGCBias -b input.bam --effectiveGenomeSize 2913022398 \
    --GCbiasFrequenciesFile gc_freq.txt -o corrected.bam

6. Enrichment Analysis

Quantify signal enrichment at specific regions.

bash
# Signal enrichment at peak regions
plotEnrichment -b chip.bam input.bam --BED peaks.bed \
    -o enrichment.png --ignoreDuplicates -p 8

Key Concepts

Normalization Methods

| Method | Formula | When to Use | Requires | |--------|---------|-------------|----------| | RPGC | 1× genome coverage | ChIP-seq, ATAC-seq | --effectiveGenomeSize | | CPM | Counts per million | Any assay, quick comparison | Nothing | | RPKM | Per kb per million | RNA-seq gene-level | Nothing | | BPM | Bins per million | Similar to CPM | Nothing | | None | Raw counts | Not recommended for comparison | Nothing |

Rule: Use RPGC for ChIP-seq/ATAC-seq (accounts for genome size). Use CPM for quick comparisons. Use RPKM for RNA-seq gene-level analysis.

Effective Genome Sizes

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

computeMatrix Modes

| Mode | Use When | Key Params | |------|----------|------------| | reference-point | Signal around a fixed point (TSS, peak summit) | -b, -a, --referencePoint | | scale-regions | Signal across variable-length features (gene bodies) | -b, -a, --regionBodyLength |

Common Workflows

Workflow: ChIP-seq QC and Visualization

bash
#!/bin/bash
# Complete ChIP-seq QC + visualization pipeline
CHIP="chip.bam"
INPUT="input.bam"
GENES="genes.bed"
PEAKS="peaks.bed"
GSIZE=2913022398
THREADS=8

# 1. QC: sample correlation
multiBamSummary bins --bamfiles $INPUT $CHIP -o summary.npz -p $THREADS
plotCorrelation -in summary.npz --corMethod pearson --whatToShow heatmap -o correlation.png

# 2. QC: enrichment fingerprint
plotFingerprint -b $INPUT $CHIP -o fingerprint.png --extendReads 200 --ignoreDuplicates

# 3. Convert to normalized bigWig
bamCoverage --bam $CHIP --outFileName chip.bw --normalizeUsing RPGC \
    --effectiveGenomeSize $GSIZE --extendReads 200 --ignoreDuplicates -p $THREADS

# 4. Log2 ratio track
bamCompare -b1 $CHIP -b2 $INPUT -o log2ratio.bw --operation log2 \
    --scaleFactorsMethod readCount --extendReads 200 -p $THREADS

# 5. Heatmap at TSS
computeMatrix reference-point -S chip.bw log2ratio.bw -R $GENES \
    -b 3000 -a 3000 --referencePoint TSS -o tss_matrix.gz -p $THREADS
plotHeatmap -m tss_matrix.gz -o tss_heatmap.png --colorMap RdBu --kmeans 3

# 6. Profile at peaks
computeMatrix reference-point -S chip.bw -R $PEAKS \
    -b 2000 -a 2000 -o peak_matrix.gz -p $THREADS
plotProfile -m peak_matrix.gz -o peak_profile.png

Workflow: ATAC-seq Analysis

bash
#!/bin/bash
ATAC="atac.bam"
PEAKS="atac_peaks.bed"
GSIZE=2913022398
THREADS=8

# 1. Apply Tn5 offset correction (+4/-5 bp)
alignmentSieve --bam $ATAC --outFile shifted.bam --ATACshift -p $THREADS
samtools index shifted.bam

# 2. Generate RPGC-normalized coverage
bamCoverage --bam shifted.bam --outFileName atac.bw \
    --normalizeUsing RPGC --effectiveGenomeSize $GSIZE \
    --binSize 5 --extendReads -p $THREADS

# 3. Check nucleosome periodicity (expect 200bp/400bp peaks)
bamPEFragmentSize -b shifted.bam -o fragsize.png \
    --maxFragmentLength 1000 --binSize 1

# 4. Heatmap at ATAC peaks
computeMatrix reference-point -S atac.bw -R $PEAKS \
    -b 2000 -a 2000 -o atac_matrix.gz -p $THREADS
plotHeatmap -m atac_matrix.gz -o atac_heatmap.png --colorMap Blues --kmeans 2

Key Parameters

| Parameter | Tool(s) | Default | Range | Effect | |-----------|---------|---------|-------|--------| | --normalizeUsing | bamCoverage, bamCompare | None | RPGC, CPM, RPKM, BPM, None | Coverage normalization method | | --effectiveGenomeSize | bamCoverage, bamCompare | — | See table above | Required for RPGC normalization | | --binSize | bamCoverage, multiBamSummary | 50 | 1–10000 | Resolution in bp; smaller = larger files | | --extendReads | bamCoverage, bamCompare | False | integer (bp) | Extend to fragment length (ChIP: YES, RNA: NO) | | --ignoreDuplicates | Most tools | False | True/False | Remove PCR duplicates | | --numberOfProcessors | Most tools | 1 | 1–N cores | Parallel processing | | --operation | bamCompare | log2 | log2, ratio, subtract, add, mean, reciprocal_ratio | Sample comparison operation | | --referencePoint | computeMatrix | TSS | TSS, TES, center | Anchor point for reference-point mode | | -b / -a | computeMatrix | 500 | 100–10000 bp | Upstream/downstream distance from reference | | --kmeans | plotHeatmap | None | 1–20 | Number of clusters for heatmap rows | | --minMappingQuality | Most tools | None | 0–60 | Minimum alignment quality filter |

Best Practices

1. Always extend reads for ChIP-seq: Use --extendReads 200 (or actual fragment length) — ChIP fragments are longer than reads.

1. Never extend reads for RNA-seq: --extendReads would span splice junctions, creating artifacts.

1. Anti-pattern — comparing with different normalizations: Always use the same normalization method across all samples in a comparison.

1. Use --region for parameter testing: Test on a single chromosome (--region chr1:1-10000000) before running on the full genome — saves hours.

1. Always use --numberOfProcessors: Most tools parallelize well — use all available cores.

1. Anti-pattern — using RPGC without --effectiveGenomeSize: Will silently produce wrong results. Always specify the correct genome size.

1. Run QC before analysis: Check fingerprint and correlation before investing time in heatmaps/profiles. Poor enrichment means downstream visualizations will be noise.

Common Recipes

Recipe: Multi-Sample Correlation Matrix

bash
# Compare 6 samples across the genome
multiBamSummary bins --bamfiles sample{1..6}.bam \
    -o all_samples.npz --binSize 10000 -p 8 \
    --labels S1 S2 S3 S4 S5 S6

# Pearson correlation heatmap
plotCorrelation -in all_samples.npz --corMethod pearson \
    --whatToShow heatmap -o pearson_corr.png --plotNumbers

# Spearman correlation + PCA
plotCorrelation -in all_samples.npz --corMethod spearman \
    --whatToShow heatmap -o spearman_corr.png
plotPCA -in all_samples.npz -o pca.png

Recipe: Gene Body Coverage Profile

bash
# Scale-regions mode for gene body analysis
computeMatrix scale-regions -S sample.bw -R genes.bed \
    -b 1000 -a 1000 --regionBodyLength 5000 -o gene_body.gz -p 8
plotProfile -m gene_body.gz -o gene_body_profile.png \
    --plotType lines --perGroup

Troubleshooting

| Problem | Cause | Solution | |---------|-------|----------| | BAM index not found | Missing .bai file | Run samtools index input.bam | | Out of memory | Large genome, small bin size | Increase --binSize; process with --region chr1 | | Very slow processing | Single-threaded execution | Add -p 8 (or available cores) | | bigWig files very large | Bin size too small | Increase --binSize 50 or larger | | Flat ChIP fingerprint | Poor ChIP enrichment | Biological issue — consider repeating ChIP experiment | | RNA-seq artifacts at exon boundaries | --extendReads used with RNA-seq | Remove --extendReads for RNA-seq data | | ATAC-seq signal offset | Missing Tn5 correction | Apply alignmentSieve --ATACshift before analysis | | Mismatched genome assemblies | BAM and BED use different assemblies | Verify both use same genome build (hg38 vs hg19) |

Related Skills

• pysam-genomic-files — programmatic BAM/VCF manipulation for custom filtering before deepTools
• matplotlib-scientific-plotting — customize deepTools output figures beyond built-in options

References

• deepTools documentation — official user guide and tool reference
• deepTools Galaxy — web-based interface
• Ramirez et al. (2016) "deepTools2: a next generation web server for deep-sequencing data analysis" — Nucleic Acids Research