pywombat 1.0.1

A CLI tool for processing and filtering bcftools tabulated TSV files with pedigree support

PyWombat 🦘

A high-performance CLI tool for processing and filtering bcftools tabulated TSV files with advanced filtering capabilities, pedigree support, and de novo mutation detection.

Features

✨ Fast Processing: Uses Polars for efficient data handling
🔬 Quality Filtering: Configurable depth, quality, and VAF thresholds
👨‍👩‍👧 Pedigree Support: Trio and family analysis with parent genotypes
🧬 De Novo Detection: Sex-chromosome-aware DNM identification
📊 Flexible Output: TSV, compressed TSV, or Parquet formats
🎯 Expression Filters: Complex filtering with logical expressions
🏷️ Boolean Flag Support: INFO field flags (PASS, DB, etc.) extracted as True/False columns
⚡ Streaming Mode: Memory-efficient processing of large files

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Quick Start

One-Time Usage (Recommended for Most Users)

Use uvx to run PyWombat without installation:

# Basic formatting
uvx pywombat input.tsv -o output

# With filtering
uvx pywombat input.tsv -F examples/rare_variants_high_impact.yml -o output

# De novo mutation detection
uvx pywombat input.tsv --pedigree pedigree.tsv \
  -F examples/de_novo_mutations.yml -o denovo

Installation for Development/Repeated Use

# Clone the repository
git clone https://github.com/bourgeron-lab/pywombat.git
cd pywombat

# Install with uv
uv sync

# Run with uv run
uv run wombat input.tsv -o output

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What Does PyWombat Do?

PyWombat transforms bcftools tabulated TSV files into analysis-ready formats by:

1. Expanding the (null) INFO column: Extracts all NAME=value fields (e.g., DP=30;AF=0.5;AC=2) and boolean flags (e.g., PASS, DB) into separate columns
2. Melting sample columns: Converts wide-format sample data into long format with one row per variant-sample combination
3. Extracting genotype data: Parses GT:DP:GQ:AD format into separate columns with calculated VAF
4. Adding parent data: Joins father/mother genotypes when pedigree is provided
5. Applying filters: Quality filters, expression-based filters, or de novo detection

Example Transformation

Input (Wide Format):

#CHROM  POS  REF  ALT  (null)                      Sample1:GT:DP:GQ:AD  Sample2:GT:DP:GQ:AD
chr1    100  A    T    DP=30;AF=0.5;PASS;AC=2      0/1:15:99:5,10      1/1:18:99:0,18

Output (Long Format):

#CHROM  POS  REF  ALT  AC  AF   DP  PASS  sample   sample_gt  sample_dp  sample_gq  sample_ad  sample_vaf
chr1    100  A    T    2   0.5  30  true  Sample1  0/1        15         99         10         0.6667
chr1    100  A    T    2   0.5  30  true  Sample2  1/1        18         99         18         1.0

Generated Columns:

• INFO fields with =: Extracted as separate columns (e.g., DP, AF, AC)
• INFO boolean flags: Extracted as True/False columns (e.g., PASS, DB, SOMATIC)
• sample: Sample identifier
• sample_gt: Genotype (e.g., 0/1, 1/1)
• sample_dp: Read depth (total coverage)
• sample_gq: Genotype quality score
• sample_ad: Alternate allele depth (from second value in AD field)
• sample_vaf: Variant allele frequency (sample_ad / sample_dp)

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Basic Usage

Format Without Filtering

# Output to file
uvx pywombat input.tsv -o output

# Output to stdout (useful for piping)
uvx pywombat input.tsv

# Compressed output
uvx pywombat input.tsv -o output -f tsv.gz

# Parquet format (fastest for large files)
uvx pywombat input.tsv -o output -f parquet

# With verbose output
uvx pywombat input.tsv -o output --verbose

With Pedigree (Trio/Family Analysis)

Add parent genotype information for inheritance analysis:

uvx pywombat input.tsv --pedigree pedigree.tsv -o output

Pedigree File Format (tab-separated):

FID sample_id FatherBarcode MotherBarcode Sex Pheno
FAM1 Child1 Father1 Mother1 1 2
FAM1 Father1 0 0 1 1
FAM1 Mother1 0 0 2 1

• FID: Family ID
• sample_id: Sample name (must match VCF)
• FatherBarcode: Father's sample name (0 = unknown)
• MotherBarcode: Mother's sample name (0 = unknown)
• Sex: 1=male, 2=female (or M/F)
• Pheno: 1=unaffected, 2=affected

Output with pedigree includes additional columns:

• father_gt, father_dp, father_gq, father_ad, father_vaf
• mother_gt, mother_dp, mother_gq, mother_ad, mother_vaf

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Advanced Filtering

PyWombat supports two types of filtering:

1. Expression-based filtering: For rare variants, impact filtering, frequency filtering
2. De novo mutation detection: Specialized logic for identifying DNMs in trios/families

1. Rare Variant Filtering

Filter for ultra-rare, high-impact variants:

uvx pywombat input.tsv \
  -F examples/rare_variants_high_impact.yml \
  -o rare_variants

Configuration (rare_variants_high_impact.yml):

quality:
  sample_dp_min: 10           # Minimum read depth
  sample_gq_min: 19           # Minimum genotype quality
  sample_vaf_het_min: 0.25    # Het VAF range: 25-75%
  sample_vaf_het_max: 0.75
  sample_vaf_homalt_min: 0.85 # Hom alt VAF ≥ 85%
  apply_to_parents: true      # Also filter parent genotypes
  filter_no_alt_allele: true  # Exclude 0/0 genotypes

expression: "VEP_CANONICAL = YES & VEP_IMPACT = HIGH & VEP_LoF = HC & VEP_LoF_flags = . & ( fafmax_faf95_max_genomes = null | fafmax_faf95_max_genomes <= 0.001 )"

What this does:

• Filters for canonical transcripts with HIGH impact
• Loss-of-function (LoF) with high confidence, no flags
• Ultra-rare: ≤0.1% frequency in gnomAD genomes
• Stringent quality: DP≥10, GQ≥19, appropriate VAF

2. De Novo Mutation Detection

Identify de novo mutations in trio data:

uvx pywombat input.tsv \
  --pedigree pedigree.tsv \
  -F examples/de_novo_mutations.yml \
  -o denovo

Configuration (de_novo_mutations.yml):

quality:
  sample_dp_min: 10
  sample_gq_min: 18
  sample_vaf_min: 0.20

dnm:
  enabled: true
  parent_dp_min: 10          # Parent quality thresholds
  parent_gq_min: 18
  parent_vaf_max: 0.02       # Max 2% VAF in parents
  
  sample_vaf_hemizygous_min: 0.85  # For X male, Y, and hom variants
  
  fafmax_faf95_max_genomes_max: 0.001  # Max 0.1% in gnomAD
  genomes_filters_pass_only: true      # Only PASS variants
  
  par_regions:               # Pseudo-autosomal regions (GRCh38)
    grch38:
      PAR1:
        chrom: X
        start: 10000
        end: 2781479
      PAR2:
        chrom: X
        start: 155701383
        end: 156030895

Sex-Chromosome Aware Logic:

• Autosomes & PAR: Both parents must be 0/0 with VAF<2%
• X in males (non-PAR): Mother must be 0/0, father not informative
• Y in males: Father must be 0/0, mother N/A
• Hemizygous variants: Require VAF ≥ 85%

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Expression-Based Filtering

Create custom filters using logical expressions:

Available Operators

• Comparison: =, !=, <, >, <=, >=
• Logical: & (AND), | (OR)
• Grouping: (, )
• Null checks: = null, != null

Example Expressions

# High or moderate impact with low frequency
expression: "(VEP_IMPACT = HIGH | VEP_IMPACT = MODERATE) & gnomad_AF < 0.001"

# Canonical transcripts with CADD score
expression: "VEP_CANONICAL = YES & CADD_PHRED >= 20"

# Specific consequence types
expression: "VEP_Consequence = frameshift_variant | VEP_Consequence = stop_gained"

# Multiple criteria
expression: "VEP_IMPACT = HIGH & VEP_CANONICAL = YES & gnomad_AF < 0.01 & CADD_PHRED >= 25"

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Debug Mode

Inspect specific variants for troubleshooting:

uvx pywombat input.tsv \
  -F config.yml \
  --debug chr11:70486013

Shows:

• All rows matching the position
• VEP_SYMBOL if available
• All columns mentioned in filter expression
• Useful for understanding why variants pass/fail filters

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Output Formats

TSV (Default)

uvx pywombat input.tsv -o output          # Creates output.tsv
uvx pywombat input.tsv -o output -f tsv   # Same as above

Compressed TSV

uvx pywombat input.tsv -o output -f tsv.gz  # Creates output.tsv.gz

Parquet (Fastest for Large Files)

uvx pywombat input.tsv -o output -f parquet  # Creates output.parquet

When to use Parquet:

• Large cohorts (>100 samples)
• Downstream analysis with Polars/Pandas
• Need for fast repeated access
• Storage efficiency

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Example Workflows

1. Rare Disease Gene Discovery

# Step 1: Filter for rare, high-impact variants
uvx pywombat cohort.tsv \
  -F examples/rare_variants_high_impact.yml \
  -o rare_variants

# Step 2: Further filter with gene list (in downstream analysis)
# Use the output TSV with your favorite tools (R, Python, etc.)

2. Autism Trio Analysis

# Identify de novo mutations in autism cohort
uvx pywombat autism_trios.tsv \
  --pedigree autism_pedigree.tsv \
  -F examples/de_novo_mutations.yml \
  -o autism_denovo \
  --verbose

# Review output for genes in autism risk lists

3. Multi-Family Rare Variant Analysis

# Process multiple families together
uvx pywombat families.tsv \
  --pedigree families_pedigree.tsv \
  -F examples/rare_variants_high_impact.yml \
  -o families_rare_variants \
  -f parquet  # Parquet for fast downstream analysis

4. Custom Expression Filter

Create custom_filter.yml:

quality:
  sample_dp_min: 15
  sample_gq_min: 20
  sample_vaf_het_min: 0.30
  sample_vaf_het_max: 0.70

expression: "VEP_IMPACT = HIGH & (gnomad_AF < 0.0001 | gnomad_AF = null)"

Apply:

uvx pywombat input.tsv -F custom_filter.yml -o output

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Input Requirements

bcftools Tabulated Format

PyWombat expects TSV files created with bcftools:

# From VCF to tabulated TSV
bcftools query -HH -f '%CHROM\t%POS\t%REF\t%ALT\t%FILTER\t%INFO[\t%GT:%DP:%GQ:%AD]\n' \
  input.vcf.gz > input.tsv

# With specific INFO fields
bcftools query -HH \
  -f '%CHROM\t%POS\t%REF\t%ALT\t%FILTER\t%INFO/DP;%INFO/AF;%INFO/AC[\t%GT:%DP:%GQ:%AD]\n' \
  input.vcf.gz > input.tsv

Expected format:

• Tab-separated values
• Header row with column names (use -HH for proper headers)
• (null) column containing INFO fields
• Sample columns in GT:DP:GQ:AD format (or similar)
• Optional FILTER column for quality control

VEP Annotations

For expression-based filtering on VEP annotations, a two-step process is required:

Step 1: Split VEP CSQ Field

IMPORTANT: PyWombat requires VEP annotations to be split into individual fields with the VEP_ prefix:

# Split VEP CSQ field - creates one row per transcript/consequence
# This prefixes all CSQ columns with "VEP_" (e.g., VEP_SYMBOL, VEP_IMPACT)
bcftools +split-vep -c - -p VEP_ -O b -o annotated.split.bcf input.vcf.gz

Step 2: Convert to Tabulated Format

# Extract to TSV with genotype information
bcftools query -HH \
  -f '%CHROM\t%POS\t%REF\t%ALT\t%FILTER\t%INFO[\t%GT:%DP:%GQ:%AD]\n' \
  annotated.split.bcf > annotated.tsv

Complete VEP Workflow

# 1. Annotate with VEP
vep -i input.vcf.gz \
  --cache --offline \
  --format vcf \
  --vcf \
  --everything \
  --canonical \
  --plugin LoF \
  -o annotated.vcf.gz

# 2. Split VEP CSQ field (REQUIRED for PyWombat)
bcftools +split-vep -c - -p VEP_ -O b -o annotated.split.bcf annotated.vcf.gz

# 3. Convert to tabulated format
bcftools query -HH \
  -f '%CHROM\t%POS\t%REF\t%ALT\t%FILTER\t%INFO[\t%GT:%DP:%GQ:%AD]\n' \
  annotated.split.bcf > annotated.tsv

# 4. Process with PyWombat
uvx pywombat annotated.tsv -F examples/rare_variants_high_impact.yml -o output

Why split-vep is required:

• Creates one row per transcript/consequence (instead of all in one CSQ field)
• Prefixes VEP fields with VEP_ making them accessible in expressions
• Enables filtering on VEP_CANONICAL, VEP_IMPACT, VEP_LoF, etc.

Pipeline Optimization

For production workflows, these commands can be piped together:

# Efficient pipeline (single pass through data)
bcftools +split-vep -c - -p VEP_ input.vcf.gz | \
  bcftools query -HH -f '%CHROM\t%POS\t%REF\t%ALT\t%FILTER\t%INFO[\t%GT:%DP:%GQ:%AD]\n' | \
  uvx pywombat - -F config.yml -o output

Note: For multiple filter configurations, it's more efficient to save the intermediate TSV file rather than regenerating it each time.

gnomAD Annotations

For frequency filtering, annotate with gnomAD:

# Using bcftools annotate
bcftools annotate -a gnomad.genomes.vcf.gz \
  -c INFO/AF,INFO/fafmax_faf95_max \
  input.vcf.gz -o annotated.vcf.gz

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Configuration Examples

See the examples/ directory for complete configuration files:

• rare_variants_high_impact.yml: Ultra-rare, high-impact variants
• de_novo_mutations.yml: De novo mutation detection with sex-chromosome handling

Each configuration file is fully documented with:

• Parameter descriptions
• Use case recommendations
• Customization tips
• Example command lines

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Performance Tips

1. Use streaming mode (default): Efficient for most workflows
2. Parquet output: Faster for large files and repeated analysis
3. Pre-filter with bcftools: Filter by region/gene before PyWombat
4. Compressed input: PyWombat handles .gz files natively
5. Filter early: Apply quality filters before complex expression filters

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Development

Setup

# Clone repository
git clone https://github.com/bourgeron-lab/pywombat.git
cd pywombat

# Install dependencies
uv sync

# Run tests (if available)
uv run pytest

Project Structure

pywombat/
├── src/pywombat/
│   ├── __init__.py
│   └── cli.py           # Main CLI implementation
├── examples/            # Configuration examples
│   ├── README.md
│   ├── rare_variants_high_impact.yml
│   └── de_novo_mutations.yml
├── tests/               # Test files and data
├── pyproject.toml       # Project metadata
└── README.md

Technology Stack

• Polars: Fast DataFrame operations
• Click: CLI interface
• PyYAML: Configuration parsing
• uv: Package management

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Troubleshooting

Common Issues

Issue: Column '(null)' not found

• Solution: Ensure input is bcftools tabulated format with INFO column

Issue: No parent genotypes found

• Solution: Check pedigree file format and sample name matching

Issue: DNM filter requires pedigree

• Solution: Add --pedigree option when using de novo config

Issue: Variants missing from output

• Solution: Use --debug chr:pos to see why variants are filtered

Issue: Memory errors on large files

• Solution: Files are processed in streaming mode by default; if issues persist, pre-filter with bcftools

Getting Help

1. Check --help for command options: uvx pywombat --help
2. Review example configurations in examples/
3. Use --debug mode to inspect specific variants
4. Use --verbose to see filtering steps

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Citation

If you use PyWombat in your research, please cite:

PyWombat: A tool for processing and filtering bcftools tabulated files
Bourgeron Lab, Institut Pasteur
https://github.com/bourgeron-lab/pywombat

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License

MIT License - see LICENSE file for details

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Contributing

Contributions are welcome! Please:

1. Fork the repository
2. Create a feature branch
3. Make your changes
4. Submit a pull request

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Authors

• Freddy Cliquet - Bourgeron Lab, Institut Pasteur

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Acknowledgments

• Bourgeron Lab for project support
• Institut Pasteur for infrastructure
• Polars team for the excellent DataFrame library