markdup 0.0.3

Comprehensive BAM file deduplication that automatically handles multiple sequencing conditions and edge cases

MarkDup

A comprehensive Python tool for deduplicating BAM files that automatically handles multiple sequencing conditions and edge cases with intelligent UMI clustering and biological positioning.

⚠️ Early Development Stage: This tool is currently in alpha development. While functional, it may have bugs and the API may change. Please report any issues you encounter.

🆕 Version 0.0.2: Enhanced error handling, better input validation, improved memory management, and more detailed progress reporting.

🎯 Key Differentiators

Unlike other deduplication tools, MarkDup automatically handles multiple sequencing conditions and edge cases:

• 🔬 Multi-condition Support: Works with or without UMIs, single-end or paired-end reads
• 🧬 Biological Positioning: Automatically handles strand-aware positioning (start-only, end-only, or full fragment)
• 🎯 Intelligent Clustering: Frequency-aware UMI clustering prevents unrealistic merging
• ⚡ Edge Case Handling: Automatically detects and handles various sequencing artifacts
• 🔧 Adaptive Processing: Automatically adjusts algorithms based on input data characteristics

🚀 Features

Core Capabilities

• 🔬 UMI-based deduplication with quality-based read selection
• 📍 Coordinate-based deduplication for files without UMIs
• 🧬 Biological positioning for strand-aware clustering
• ⚡ Process-based parallelism for multi-core performance
• 🎯 Advanced clustering with edit distance and frequency-aware algorithms
• 📊 Comprehensive statistics and progress tracking

Automatic Edge Case Handling

• 🔄 UMI Detection: Automatically detects UMI presence and format
• 🧬 Strand Awareness: Automatically handles forward/reverse strand reads
• 📏 CIGAR Handling: Properly processes reads with indels and complex CIGAR strings
• 🎯 Position Grouping: Intelligent grouping based on biological vs. reference coordinates
• ⚖️ Frequency Balancing: Prevents over-clustering of high-frequency UMIs
• 🔧 Quality Selection: Multiple quality metrics with automatic fallback

📦 Installation

From PyPI (Recommended)

pip install markdup

From Source

git clone https://github.com/y9c/markdup.git
cd markdup
pip install .

Using uv (Development)

git clone https://github.com/y9c/markdup.git
cd markdup
uv sync

🚀 Quick Start

Automatic UMI Detection and Processing

# Tool automatically detects UMIs and chooses appropriate method
markdup input.bam output.bam

# With multiple threads
markdup input.bam output.bam --threads 8

# Keep duplicates and mark them
markdup input.bam output.bam --keep-duplicates

Explicit Method Selection

# Force UMI-based deduplication
markdup input.bam output.bam --method umi

# Force coordinate-based deduplication (no UMIs)
markdup input.bam output.bam --method coordinate

Advanced Positioning Options

# Start-only positioning (e.g., for ChIP-seq)
markdup input.bam output.bam --start-only

# End-only positioning (e.g., for reverse-complemented reads)
markdup input.bam output.bam --end-only

# Full fragment positioning (default, handles both start and end)
markdup input.bam output.bam

UMI Clustering Tuning

# Custom edit distance threshold
markdup input.bam output.bam --min-edit-dist-frac 0.17

# Frequency-aware clustering to prevent over-merging
markdup input.bam output.bam --min-frequency-ratio 0.1

# Custom UMI separator
markdup input.bam output.bam --umi-separator ":"

📋 Command Line Interface

Global Options

Option	Description	Default
--help	Show help message	-
--version	Show version information	-

Input/Output Options

Option	Description	Default
INPUT_BAM	Input BAM file path	Required
OUTPUT_BAM	Output BAM file path	Required
--force	Overwrite output file if it exists	False

Deduplication Method

Option	Description	Default
--method	Deduplication method: umi or coordinate	umi

UMI Options

Option	Description	Default
--umi-separator	Separator for extracting UMIs from read names	_
--min-edit-dist-frac	Minimum UMI edit distance as fraction of UMI length	0.1
--min-frequency-ratio	Minimum frequency ratio for UMI clustering	0.1

Positioning Options

Option	Description	Default
--start-only	Group reads by start position only	False
--end-only	Group reads by end position only	False

Quality Selection

Option	Description	Default
--best-read-by	Select best read by: mapq, avg_base_q	avg_base_q

Processing Options

Option	Description	Default
--threads	Number of threads for parallel processing	1
--window-size	Size of genomic windows for processing	100000
--keep-duplicates	Keep duplicate reads and mark them	False

🧬 Algorithm Details

Automatic Condition Detection

The tool automatically detects and handles:

1. UMI Presence: Scans read names for UMI patterns
2. Read Type: Single-end vs. paired-end detection
3. Strand Orientation: Forward vs. reverse strand handling
4. CIGAR Complexity: Indel and complex alignment handling
5. Quality Metrics: Available quality scores and selection criteria

UMI-based Deduplication

1. Fragment Creation: Reads are grouped into fragments (single-end or paired-end)
2. Biological Positioning: Fragments are positioned using strand-aware coordinates
3. Position Grouping: Fragments are grouped by biological position and strand
4. UMI Clustering: Within each position group, UMIs are clustered using:
   • Exact matching for identical UMIs
   • Edit distance clustering for similar UMIs
   • Frequency-aware clustering to prevent unrealistic merging
5. Quality Selection: The highest quality read from each cluster is selected
6. Output Generation: Selected reads are written with comprehensive cluster information

Coordinate-based Deduplication

1. Fragment Creation: Reads are grouped into fragments
2. Position Grouping: Fragments are grouped by genomic coordinates
3. Quality Selection: The highest quality read from each group is selected
4. Output Generation: Selected reads are written

Biological Positioning

• Forward strand: Biological start = reference start, Biological end = reference end
• Reverse strand: Biological start = reference end, Biological end = reference start
• Strand-aware clustering: Ensures proper grouping regardless of strand orientation
• CIGAR-aware positioning: Properly handles indels and complex alignments

📊 Output Format

BAM Tags

Tag	Description
cn	Cluster name with genomic coordinates and UMI (format: chr:start-end:strand:UMI)
cs	Cluster size (number of reads in cluster)

Example Output

read1_UMI123    0    chr1    1001    60    50M    *    0    0    ATGC...    IIII...    cn:Z:chr1:1001-1050:+:UMI123    cs:i:3
read2_UMI123    1024  chr1    1001    50    50M    *    0    0    ATGC...    IIII...    cn:Z:chr1:1001-1050:+:UMI123    cs:i:3
read3_UMI123    1024  chr1    1001    45    50M    *    0    0    ATGC...    IIII...    cn:Z:chr1:1001-1050:+:UMI123    cs:i:3

📚 Documentation

• Installation Guide
• Usage Guide
• Algorithm Details
• FAQ
• Contributing