Call Peaks Command¶

Call quantile-based peaks from bigWig files.

Usage¶

quantnado call-peaks [OPTIONS]

Description¶

The call-peaks command identifies peaks in bigWig files using quantile-based thresholding. It's useful for determining enriched regions from ChIP-seq, ATAC-seq, and similar assays.

Options¶

BigWig Directory (`--bigwig-dir`)¶

Required. Directory containing bigWig (.bw) files:

quantnado call-peaks --bigwig-dir ./bigwigs/ --output-dir ./peaks/ --chromsizes hg38.chrom.sizes

All .bw files in the directory will be processed.

Output Directory (`--output-dir`)¶

Required. Directory to save peak files in BED format:

quantnado call-peaks \
  --bigwig-dir ./bigwigs/ \
  --output-dir ./peaks/ \
  --chromsizes hg38.chrom.sizes

Output BED files will be named: <bigwig_name>.peaks.bed

Chromsizes (`--chromsizes`)¶

Required. Path to chromsizes file:

quantnado call-peaks \
  --bigwig-dir ./bigwigs/ \
  --output-dir ./peaks/ \
  --chromsizes /reference/hg38.chrom.sizes

Quantile (`--quantile`)¶

Quantile threshold for peak calling (default: 0.98):

# Stricter threshold - fewer, higher-confidence peaks
quantnado call-peaks --bigwig-dir ./bigwigs/ --output-dir ./peaks/ \
  --chromsizes hg38.chrom.sizes --quantile 0.99

# More relaxed threshold - more peaks
quantnado call-peaks --bigwig-dir ./bigwigs/ --output-dir ./peaks/ \
  --chromsizes hg38.chrom.sizes --quantile 0.95

Higher quantile = fewer peaks (more stringent)

Quantile	Interpretation
0.90	90^th percentile - relaxed
0.95	95^th percentile - standard
0.98	98^th percentile - stringent
0.99	99^th percentile - very stringent

Tile Size (`--tilesize`)¶

Size of genomic tiles for peak calling (default: 128 bp):

quantnado call-peaks --bigwig-dir ./bigwigs/ --output-dir ./peaks/ \
  --chromsizes hg38.chrom.sizes --tilesize 50

Smaller tiles = more sensitive to local changes Larger tiles = more robust to noise

Blacklist (`--blacklist`)¶

Path to BED file with regions to exclude:

quantnado call-peaks \
  --bigwig-dir ./bigwigs/ \
  --output-dir ./peaks/ \
  --chromsizes hg38.chrom.sizes \
  --blacklist /reference/hg38-blacklist.bed

Blacklist format (tab-separated):

chr1    1000    2000
chr2    5000    6000

Merge (`--merge` / `--no-merge`)¶

Merge overlapping peaks (default: disabled with --no-merge):

# Enable merging
quantnado call-peaks \
  --bigwig-dir ./bigwigs/ \
  --output-dir ./peaks/ \
  --chromsizes hg38.chrom.sizes \
  --merge

# Disable merging (default)
quantnado call-peaks \
  --bigwig-dir ./bigwigs/ \
  --output-dir ./peaks/ \
  --chromsizes hg38.chrom.sizes \
  --no-merge

Temporary Directory (`--tmp-dir`)¶

Temporary directory for intermediate files (default: tmp):

quantnado call-peaks \
  --bigwig-dir ./bigwigs/ \
  --output-dir ./peaks/ \
  --chromsizes hg38.chrom.sizes \
  --tmp-dir /scratch/tmp

Log File (`--log-file`)¶

Save logs (default: quantnado_peaks.log):

quantnado call-peaks \
  --bigwig-dir ./bigwigs/ \
  --output-dir ./peaks/ \
  --chromsizes hg38.chrom.sizes \
  --log-file peak_calling.log

Verbose (`--verbose`, `-v`)¶

Enable debug logging:

quantnado call-peaks \
  --bigwig-dir ./bigwigs/ \
  --output-dir ./peaks/ \
  --chromsizes hg38.chrom.sizes \
  --verbose

Examples¶

Basic Peak Calling¶

Call peaks from bigWig files:

quantnado call-peaks \
  --bigwig-dir ./bigwigs/ \
  --output-dir ./peaks/ \
  --chromsizes hg38.chrom.sizes

Stringent Thresholding¶

Call only high-confidence peaks:

quantnado call-peaks \
  --bigwig-dir ./bigwigs/ \
  --output-dir ./peaks/ \
  --chromsizes hg38.chrom.sizes \
  --quantile 0.99 \
  --merge

With Blacklist¶

Exclude problematic regions:

quantnado call-peaks \
  --bigwig-dir ./bigwigs/ \
  --output-dir ./peaks/ \
  --chromsizes hg38.chrom.sizes \
  --blacklist /reference/hg38-blacklist.bed \
  --quantile 0.98

Fine-Tuned Tile Size¶

Use smaller tiles for better resolution:

quantnado call-peaks \
  --bigwig-dir ./bigwigs/ \
  --output-dir ./peaks/ \
  --chromsizes hg38.chrom.sizes \
  --tilesize 50 \
  --quantile 0.98

Full Pipeline¶

Comprehensive peak calling with all options:

quantnado call-peaks \
  --bigwig-dir ./bigwigs/ \
  --output-dir ./peaks/ \
  --chromsizes /reference/hg38.chrom.sizes \
  --blacklist /reference/hg38-blacklist.bed \
  --quantile 0.98 \
  --tilesize 128 \
  --merge \
  --tmp-dir /scratch/tmp \
  --log-file peak_calling.log \
  --verbose

Output¶

Output BED files contain called peaks:

chr1    1000    2000    peak_1    100    .
chr1    5000    6500    peak_2    150    .
chr2    10000   11000   peak_3    120    .

Columns: 1. Chromosome 2. Start position 3. End position 4. Peak name 5. Score (signal intensity) 6. Strand (always . for this implementation)

Performance¶

Typical Run Times¶

Peak calling time depends on bigWig file size:

Genome Coverage	Time
Low coverage	1-5 minutes
Medium coverage	5-15 minutes
High coverage	15-30 minutes

Resource Requirements¶

Memory: 2-8 GB (depends on tile size and coverage)
Disk: ~2-3× bigWig file size (for temporary files)

Troubleshooting¶

No bigWig files found¶

Problem: Directory is empty or wrong format

Solution: Check directory and file extensions:

ls -l ./bigwigs/*.bw

Missing chromsizes¶

Problem: FileNotFoundError: chromsizes file not found

Solution: Download or provide chromsizes:

# Download from UCSC
wget http://hgdownload.cse.ucsc.edu/goldenpath/hg38/bigZips/hg38.chrom.sizes

Out of memory¶

Problem: Memory error during peak calling

Solution: Use larger tile size (fewer tiles):

quantnado call-peaks \
  --bigwig-dir ./bigwigs/ \
  --output-dir ./peaks/ \
  --chromsizes hg38.chrom.sizes \
  --tilesize 256

No peaks detected¶

Problem: Output peak files are empty

Likely causes: - Quantile threshold too stringent - BigWig files have low signal - Incorrect chromsizes

Solution: Lower quantile threshold:

quantnado call-peaks \
  --bigwig-dir ./bigwigs/ \
  --output-dir ./peaks/ \
  --chromsizes hg38.chrom.sizes \
  --quantile 0.90

Preparing BigWig Files¶

Convert BAM to bigWig if needed:

# Create bedGraph
bedtools genomecov -ibam sample.bam -bg -o sample.bedgraph

# Convert to bigWig
bedGraphToBigWig sample.bedgraph hg38.chrom.sizes sample.bw

Python Equivalent¶

There's currently no direct Python API for peak calling. Use the CLI command.