saltshaker 1.0.2

Pattern classification and visualization for MitoSAlt

SAltShaker

A Python package for classifying and visualizing mitochondrial structural variants from MitoSAlt pipeline output.

Overview

SAltShaker is a Python port and extension of the original MitoSAlt (Basu et al. PLoS Genetics 2020) delplot.R visualization script. The package provides three modular commands for a flexible analysis workflow:

• Event calling: Direct Python port of the original R script's deletion/duplication classification logic
• Pattern classification: Rule-based decision tree to distinguish single and multiple type of events from background
• Visualization: Circular genome plotting based on the original R script visualization with spatial grouping and annotations

Installation

From source

git clone https://gitlab.com/GenomeDX/annotation/saltshaker.git
cd saltshaker
pip install -e .

Docker

# Build the image
docker build -t saltshaker .

# Run with mounted data directory
docker run -v /path/to/your/data:/data saltshaker  [command] [options]

Package registry

SAltShaker is published to PyPI and GitLab PyPI (https://gitlab.com/api/v4/groups/16758292/-/packages/pypi/simple).

Pip

pip install saltshaker or

pip install saltshaker --index-url https://gitlab.com/api/v4/groups/16758292/-/packages/pypi/simple

Poetry

poetry source add --priority=primary dpipe https://gitlab.com/api/v4/groups/16758292/-/packages/pypi/simple
poetry add saltshaker

uv

uv add --index https://gitlab.com/api/v4/groups/16758292/-/packages/pypi/simple saltshaker

Commands

1. saltshaker call - Event calling (R script port)

Python port of the original MitoSAlt R script event calling logic. Calls detected breakpoint clusters as deletions or duplications based on replication origin overlap, following the exact algorithm from delplot.R.

Usage:

saltshaker call \
    --prefix sample \                       # Sample identifier
    --output-dir results/ \                 # Output directory
    -c sample.cluster \                     # cluster file from MitoSAlt
    -p sample.breakpoint \                  # breakpoint file from MitoSAlt
    -r reference.fasta \                    # mitochondrial reference genome
    -g 16569 \                              # genome length
    --ori-h-start 16081 --ori-h-end 407 \   # heavy strand origin
    --ori-l-start 5730 --ori-l-end 5763 \   # light strand origin
    -H 0.01 \                               # heteroplasmy threshold
    -f 15 \                                 # flanking sequence size (bp)
    --blacklist                             # Optional: enable with default MT blacklist, OR
    --blacklist custom_bl.bed               # Optional: enable with custom BED file

Outputs:

• results/sample.saltshaker_call.tsv - Human-readable results matching original R script output format
• results/sample.saltshaker_call_metadata.tsv - Metadata for downstream processing (classify/plot)

Algorithm (from original R script):

The logic is a direct port of the original MitoSAlt R implementation:

1. Data loading: Parses cluster and breakpoint files, merges data to link clusters with D-loop crossing information
2. Deletion size calculation: Handles circular genome wraparound for events crossing position 1
3. Origin-based event calling: Events overlapping replication origins (OriH or OriL) are called as duplications; non-overlapping events are deletions
4. Coordinate handling: Implements the R script's coordinate swapping logic for D-loop crossing events
5. Flanking sequence analysis: Uses Biostrings-equivalent pattern matching to find microhomology sequences near breakpoints

This approach identifies the arc complementary to the actual structural change, consistent with the biological interpretation that origin-overlapping events represent duplications of the non-deleted arc.

Original R script functionality preserved:

• Exact deletion/duplication calling algorithm
• D-loop crossing detection and coordinate handling
• Flanking sequence extraction and microhomology analysis
• Output TSV format and column names
• Heteroplasmy calculation and filtering

Python enhancements in call step:

• Blacklist region detection and flagging if --blacklist flag is supplied.

2. saltshaker classify - Pattern classification

Extended analysis beyond the original R script. Performs spatial grouping and classifies the overall pattern as Single, Multiple or background based on heteroplasmy levels and spatial event distribution.

Usage:

saltshaker classify \
    --prefix sample1 \              # Sample identifier (matches call output)
    --input-dir results/ \          # Input directory with .saltshaker_call.tsv
    --output-dir results/ \         # Output directory (default: same as input-dir)
    --blacklist                     # Optional: enable with default MT blacklist, OR
    --blacklist custom_bl.bed       # Optional: enable with custom BED file
    --vcf \                         # Optional: also output VCF format
    --chr-format \                  # Optional: specify one of MT or chrM (default: chrM)
    --high-het 10 \                 # Optional: high heteroplasmy threshold % (default: 20)
    --noise 0.3 \                   # Optional: noise threshold % (default: 1.0)
    --radius 600 \                  # Optional: spatial clustering radius bp (default: 600)
    --multiple-threshold 5 \       # Optional: event count for Multiple pattern (default: 10)
    --dominant-fraction 0.5        # Optional: fraction for dominant group (default: 0.70)

Outputs:

• results/sample.saltshaker_classify.txt - Detailed analysis report with classification reasoning
• results/sample.saltshaker_classify_metadata.tsv - Events with spatial group assignments (for plotting)
• results/sample.saltshaker.vcf - events in VCF format (if --vcf specified)

Classification criteria:

Single pattern:

• One or few high-heteroplasmy events (≥high-het)
• Dominant spatial group (≥dominant-fraction of events)
• Few total events (≤multiple-threshold)
• Consistent with pathogenic single deletion/duplication

Multiple pattern:

• Many events (>multiple-threshold)
• Dispersed spatial distribution (no dominant group)
• No high-heteroplasmy events
• Consistent with mtDNA maintenance defects

Spatial grouping: Events within radius bp are grouped together.

3. saltshaker plot - Visualization

Generates circular genome plots based on the original R script visualization with enhanced spatial grouping and other features.

Usage:

saltshaker plot \
    --prefix sample1 \
    --input-dir results/ \
    --output-dir results/plots/ \   # Optional: default is input-dir
    --genes \                       # Optional: enable with default MT genes, OR
    --genes custom_genes.bed \      # Optional: enable with custom BED file
    --blacklist \                   # Optional: enable with default MT blacklist, OR
    --blacklist custom_bl.bed \     # Optional: enable with custom BED file
    --figsize 16 10 \               # Optional: width height (default: 16 10)
    --direction clockwise \         # Optional: clockwise or counterclockwise (default: counterclockwise)
    --del-color red \               # Optional: red or blue (default: blue)
    --dup-color blue \              # Optional: red or blue (default: red)
    --scale fixed                   # Optional: dynamic or fixed (default: dynamic)

Output:

• results/plot/sample.saltshaker.png - Circular genome visualization

Visualization features:

• Circular genome with arc-based event display
• Heteroplasmy gradient coloring for all event types (del/dup/blacklist-crossing events - BL)
• Dynamic (min-max %) or fixed (0-100%) heteroplasmy scale
• Spatial grouping for overlapping events
• Optional gene annotations track and labels
• Optional blacklist region marking (BL-crossing events in lime-green gradient)
• Configurable colors and polar direction

Color scaling considerations

Example sample with 5-25% heteroplasmy:

Dynamic scale:
[████████████████████] ← Colors span full gradient from 5% to 25%
5%                 25%

Fixed scale:
[█████░░░░░░░░░░░░░░░] ← Colors span from 0% to 100%, events appear lighter
0%               100%

Input files

Required (from MitoSAlt pipeline)

Cluster file (.cluster)

• Tab-separated clustered breakpoint data
• Generated by MitoSAlt's clustering step
• Columns: cluster ID, read counts, positions, heteroplasmy levels

Breakpoint file (.breakpoint)

• Tab-separated raw breakpoint data
• Generated by MitoSAlt's breakpoint detection step
• Columns: read names, positions, D-loop crossing flags

Reference genome (.fasta)

• Mitochondrial reference sequence
• Used for flanking sequence extraction and coordinate validation

Optional

Blacklist file (.bed)

• BED format regions to flag (e.g., artifacts, repetitive sequences)
• Format: chromosome, start position, end position, name

Genes file (.bed)

• BED format gene regions to plot around the genomic axis
• Format: chromosome, start position, end position, name, score (0), strand (+), thick start (same as start), think end (same as end), color in rgb code (e.g. 255,255,0)

Output formats

Display TSV ({prefix}.saltshaker_call.tsv)

Human-readable format matching original R script output with columns:

• sample: Sample identifier
• cluster_id: Cluster identifier
• alt_reads, ref_reads: Read counts
• heteroplasmy: Heteroplasmy percentage
• del_start_range, del_end_range: Coordinate ranges
• del_size: Event size in base pairs
• final_event: Event type (del/dup)
• final_start, final_end: Final coordinates
• blacklist_crossing: Flag for blacklist overlap
• seq1, seq2, seq: Flanking sequences and microhomology

Metadata files

Call metadata ({prefix}.saltshaker_call_metadata.tsv): Internal format preserving all columns for downstream processing. Contains metadata header with genome length.

Classify metadata ({prefix}.saltshaker_classify_metadata.tsv): Internal format with additional group column for spatial group assignments. Used by plot command.

Analysis summary ({prefix}.saltshaker_classify.txt)

Human-readable analysis report including:

• Pattern classification (Single/Multiple) with reasoning
• Event statistics and heteroplasmy distribution
• Spatial clustering metrics
• Classification criteria scores

VCF format ({prefix}.saltshaker.vcf)

Standard VCF 4.3 format with structural variant fields:

• SVTYPE: DEL or DUP
• END: Variant end position
• SVLEN: Variant length
• HF: Heteroplasmy fraction (0-1)
• GROUP: Spatial group identifier
• CLUSTER: Original cluster ID
• DLOOP: Flag for D-loop crossing
• BLCROSS: Flag for blacklist crossing

Circular plot ({prefix}.saltshaker.png)

Complete workflow example

Single-sample pipeline:

# Step 1: Call events from MitoSAlt output (R script port)
saltshaker call \
    --prefix sample1 \
    --output-dir results/ \
    -c sample1.cluster -p sample1.breakpoint \
    -r reference.fasta \
    -g 16569 --ori-h-start 16081 --ori-h-end 407 \
    --ori-l-start 5730 --ori-l-end 5763 \
    --blacklist

# Step 2: Classify pattern and perform spatial grouping (extended analysis)
saltshaker classify \
    --prefix sample1 \
    --input-dir results/ \
    --blacklist \
    --vcf

# Step 3: Generate visualization (enhanced R script plotting)
saltshaker plot \
    --prefix sample1 \
    --input-dir results/ \
    --output-dir results/plot/ \
    --blacklist \
    --genes

Batch processing multiple samples:

for sample in sample1 sample2 sample3; do
    mkdir -p results/${sample}
    # Call events
    saltshaker call --prefix ${sample }--output-dir results/${sample} \
        -c ${sample}.cluster -p ${sample}.breakpoint \
        -r reference.fasta
        -g 16569 --ori-h-start 16081 --ori-h-end 407 \
        --ori-l-start 5730 --ori-l-end 5763 \
        --blacklist
    # Classify events
    saltshaker classify --prefix ${sample} \
        --input-dir results/${sample} \
        --blacklist
    # Plot events
    saltshaker plot --prefix ${sample} \
        --input-dir results/${sample} \
        --blacklist \
        --genes
done

Configuration

Default classification thresholds are defined in saltshaker/config.py:

# Heteroplasmy thresholds
HIGH_HET_THRESHOLD = 10.0        # High heteroplasmy threshold (%), --high-het
NOISE_THRESHOLD = 0.3            # Noise threshold (%), --noise

# Spatial clustering
CLUSTER_RADIUS = 600             # Spatial grouping radius (bp), --radius
MIN_CLUSTER_SIZE = 2             # Minimum events per cluster (not configurable via CLI)

# Pattern classification
MULTIPLE_EVENT_THRESHOLD = 5     # Event count for Multiple pattern, --multiple-threshold
DOMINANT_GROUP_FRACTION = 0.5    # Fraction for dominant group (70%), --dominant-fraction

These can be customized by modifying the configuration file or via CLI arguments (see saltshaker classify --help).

Command reference

Global options

All commands support:

• -h, --help: Show help message
• --blacklist [FILE]: Enable blacklist regions (default: built-in MT blacklist, use as a flag; optional: custom BED file)

call command

Required:

• --prefix STR: Sample prefix for output files
• -c, --cluster FILE: Cluster file from MitoSAlt
• -p, --breakpoint FILE: Breakpoint file from MitoSAlt
• -r, --reference FILE: Reference genome FASTA
• -g, --genome-length INT: Mitochondrial genome length
• --ori-h-start INT: Heavy strand origin start
• --ori-h-end INT: Heavy strand origin end
• --ori-l-start INT: Light strand origin start
• --ori-l-end INT: Light strand origin end

Optional:

• --output-dir DIR: Output directory (default: .)
• -H, --het-limit FLOAT: Heteroplasmy threshold (default: 0.01)
• -f, --flank-size INT: Flanking sequence size in bp (default: 15)

classify command

Required:

• --prefix STR: Sample prefix (matches call output)
• --input-dir DIR: Input directory containing saltshaker_call_metadata.tsv from call

Optional:

• --output-dir DIR: Output directory (default: input-dir)
• --vcf: Also output VCF format
• --chr-format: Choose chromosome format for VCF output: MT or chrM (default: chrM)
• --high-het FLOAT: High heteroplasmy threshold % (default: 20)
• --noise FLOAT: Noise threshold % (default: 1)
• --radius INT: Spatial clustering radius bp (default: 600)
• --multiple-threshold INT: Event count for Multiple pattern (default: 10)
• --dominant-fraction FLOAT: Fraction for dominant group (default: 0.70)

plot command

Required:

• --prefix STR: Sample prefix (matches classify output)
• --input-dir DIR: Input directory containing saltshaker_classify_metadata.tsv from classify

Optional:

• --output-dir DIR: Output directory (default: input-dir)
• --genes [FILE]: Enable gene annotations (default: built-in MT genes; optional: custom BED file)
• --figsize WIDTH HEIGHT: Figure dimensions (default: 16 10)
• --direction STR: Plot direction - 'clockwise' or 'counterclockwise' (default: counterclockwise, MitoSAlt original)
• --del-color STR: Deletion color - 'red' or 'blue' (default: blue, MitoSAlt original)
• --dup-color STR: Duplication color - 'red' or 'blue' (default: red, MitoSAlt original)
• --scale STR: Heteroplasmy scale - 'dynamic' (min-max per category) or 'fixed' (0-100%) (default: dynamic)

Dependencies

pandas>=1.3.0
numpy>=1.20.0
matplotlib>=3.3.0
biopython>=1.78

Package structure

saltshaker/
├── __init__.py
├── __main__.py                  # CLI entry point with subcommands
├── config.py                    # Configuration and thresholds
├── types.py                     # Type definitions and data structures
├── event_caller.py              # Event calling (R script port)
├── classifier.py                # Pattern classification (single vs musltiple vs background)
├── spatial.py                   # Spatial grouping
├── visualizer.py                # Circular plotting
├── utils.py                     # Utility functions 
│                 
├── layout/                      # Layout engine 
│   ├── __init__.py
│   ├── engine.py                # LayoutEngine class
│   └── types.py                 # Layout-specific types
├── cli/
│   ├── call.py                  # Call subcommand
│   ├── classify.py              # Classify subcommand
│   └── plot.py                  # Plot subcommand
└── io/
    ├── readers.py               # File input
    ├── writers.py               # TSV and summary output
    └── vcf_writer.py            # VCF format output
└── data/
    ├── __init__.py                              # Default file paths
    ├── gencode.v49.annotation.MT_genes.bed      # Default MT gene annotations
    └── mt_blacklist_regions.bed                 # Default MT blacklist regions
└── tests/
    ├── fixtures/
    │   ├── inputs/
    │   │   ├── human_mt_rCRS.fasta         # Reference FASTA
    │   │   ├── test_breakpoints.breakpoint # Small test dataset raw breakpoints  
    │   │   ├── test_clusters.cluster       # Small test dataset raw clusters 
    │   │   ├── viz_sample_small.tsv        # Small test dataset (15 events)
    │   │   └── viz_sample_large.tsv        # Large test dataset (80 events)
    │   └── expected/
    │       ├── viz_sample_small.tsv        # Small test dataset (15 events)
    │       └── visualizer_layouts.json     # Expected visualization characteristics
    │
    ├── unit/
    │   ├── test_helpers.py                 # Utilities tests
    │   ├── test_layout_engine.py           # Layout engine tests
    │   └── test_label_positioning.py       # Visualization unit tests 
    │
    └── integration/
        ├── test_saltshaker_output.py       # End-to-end event calling tests
        └── test_visualizer.py              # End-to-end visualization tests 
docs/
└── classification_algorithm.md  # Detailed classification algorithm documentation

Documentation

• Classification algorithm - Detailed explanation of the Single vs Multiple pattern classification logic