VNtyper 2.0 - A Pipeline to genotype the MUC1-VNTR

VNtyper 2.0 is an advanced pipeline designed to genotype MUC1 coding Variable Number Tandem Repeats (VNTR) in Autosomal Dominant Tubulointerstitial Kidney Disease (ADTKD-MUC1) using Short-Read Sequencing (SRS) data. This version is a refactored version of VNtyper v1 integrates enhanced variant calling algorithms, robust logging mechanisms, and streamlined installation processes to provide researchers with a powerful tool for VNTR analysis.

• We have developed a web server to provide free access to VNtyper, which runs in the background for ease of use.
  Access it through the following link: vntyper-online

---

Table of Contents

1. Features
2. Installation
3. Usage
4. Pipeline Overview
5. Dependencies
6. Linting and Code Formatting
7. Pipeline Logic Diagram
8. Results
9. Notes
10. Citations
11. Contributing
12. License
13. Contact

---

Features

• Variant Calling Algorithms:

  • Kestrel: Mapping-free genotyping using k-mer frequencies.
  • code-adVNTR (optional): Profile-HMM-based method for VNTR genotyping.
  • SHARK (optional, FASTQ-only): Rapid filtering and read extraction for MUC1 region in exome/whole-genome data.

• Comprehensive Logging:

  • Logs both to the console and a dedicated log file.
  • Generates MD5 checksums for all downloaded and processed files.

• Modern Packaging:

  • Uses modern Python packaging with pyproject.toml (PEP 517/518/621).
  • No setup.py required - fully compatible with pip ≥21.3.
  • Provides Conda environment setup for easy dependency management.

• Subcommands:

  • install-references
  • pipeline
  • fastq
  • bam
  • kestrel
  • report
  • cohort
  • online

---

Installation

VNtyper 2.0 uses modern Python packaging with pyproject.toml and can be installed using pip (≥21.3) or via Conda environments for streamlined dependency management.

Using pip

1. Clone the Repository:

   mkdir vntyper
   git clone https://github.com/hassansaei/vntyper.git
   cd vntyper
   pip install .

2. For Development:

   # Install in editable mode with development dependencies
   pip install -e .[dev]

   This installs VNtyper with additional tools for development:

   • Ruff: Fast linter and formatter (replaces flake8 + black)
   • pytest: Testing framework with coverage support

---

Usage

VNtyper 2.0 offers multiple subcommands that can be used depending on your input data and requirements. Below are the main subcommands available:

1. Running the Full Pipeline

To run the entire pipeline using a BAM file:

vntyper --config-path /path/to/config.json pipeline \
    --bam /path/to/sample.bam \
    --output-dir /path/to/output/dir \
    --threads 4 --fast-mode

Alternatively, using paired-end FASTQ files:

vntyper --config-path /path/to/config.json pipeline \
    --fastq1 /path/to/sample_R1.fastq.gz \
    --fastq2 /path/to/sample_R2.fastq.gz \
    --output-dir /path/to/output/dir \
    --threads 4 --fast-mode

The adVNTR genotyping is optional and skipped by default. To enable adVNTR genotyping, use the --extra-modules advntr option.

New: To enable SHARK filtering on FASTQ reads before the usual QC and alignment (for improved MUC1 detection), add shark to the --extra-modules flag (e.g., --extra-modules shark). This can be done as:

vntyper --config-path /path/to/config.json pipeline \
    --fastq1 /path/to/sample_R1.fastq.gz \
    --fastq2 /path/to/sample_R2.fastq.gz \
    --extra-modules shark \
    --threads 4 \
    --output-dir /path/to/output/dir

• SHARK will run first on the raw FASTQ files to extract and filter reads covering the MUC1 VNTR region.
• Important: SHARK is only supported in FASTQ mode. If you try to use --extra-modules shark together with --bam or --cram, VNtyper will exit gracefully with a warning.

2. Running VNtyper with Docker

Docker image for VNtyper 2.0 is provided and can be pulled and used as follows:

# pull the docker image
docker pull saei/vntyper:main

# run the pipeline using the docker image
docker run -w /opt/vntyper --rm \
    -v /local/input/folder/:/opt/vntyper/input \
    -v /local/output/folder/:/opt/vntyper/output \
    saei/vntyper:latest \
    vntyper pipeline \
    --bam /opt/vntyper/input/filename.bam \
    -o /opt/vntyper/output/filename/

Important Host Volume Permissions Note:
When mounting host directories into the container (using the -v flag), please ensure that the host directories (e.g., /local/input/folder/ and /local/output/folder/) have the appropriate permissions so that they are writable by the container's non-root user.

Why Non-Root?
VNtyper runs as a non-root user for enhanced security and to avoid file ownership issues on your host. Running as root may create files owned by root, leading to permission problems later.

There are two ways to ensure proper permissions:

1. Adjust Host Directory Permissions:
   Change the ownership/permissions on the host directories so that the UID and GID match those expected by VNtyper in the container.

2. Use the --user Flag:
   Run the container with the --user flag to specify your current user’s UID and GID. For example:

   docker run --user $(id -u):$(id -g) -w /opt/vntyper --rm \
     -v /local/input/folder/:/opt/vntyper/input \
     -v /local/output/folder/:/opt/vntyper/output \
     saei/vntyper:latest \
     vntyper pipeline \
     --bam /opt/vntyper/input/filename.bam \
     -o /opt/vntyper/output/filename/

Using either method ensures VNtyper can write its log files (e.g., pipeline.log) and other outputs without encountering permission errors.

An Apptainer image can be generated from the Docker image as follows:

# create the apptainer sif image
apptainer pull docker://saei/vntyper:main

# run the pipeline using the apptainer image
apptainer run --pwd /opt/vntyper \
    -B /local/input/folder/:/opt/vntyper/input \
    -B /local/output/folder/:/opt/vntyper/output \
    vntyper_latest.sif vntyper pipeline \
    --bam /opt/vntyper/input/filename.bam \
    -o /opt/vntyper/output/filename/

3. Installing References

vntyper --config-path /path/to/config.json install-references \
    --output-dir /path/to/reference/install \
    --skip-indexing  # Optional: skip BWA indexing if needed

4. Generating Reports

vntyper --config-path /path/to/config.json report \
    --output-dir /path/to/output/dir

---

Pipeline Overview

VNtyper 2.0 integrates multiple steps into a streamlined pipeline. The following is an overview of the steps involved:

1. FASTQ Quality Control: Raw FASTQ files are checked for quality.
2. (Optional) SHARK Filtering: If shark is specified in --extra-modules, raw FASTQ reads are first filtered to extract MUC1-specific reads (especially relevant for exome or large WGS datasets).
3. Alignment: Reads are aligned using BWA (if FASTQ files are provided).
4. Kestrel Genotyping: Mapping-free genotyping of VNTRs.
5. (Optional) adVNTR Genotyping: Profile-HMM-based method for VNTR genotyping (requires additional setup).
6. Summary Report Generation: A final HTML report is generated to summarize the results.

---

Dependencies

VNtyper 2.0 relies on several tools and Python libraries. Ensure that the following dependencies are available in your environment:

• Python >= 3.9
• BWA
• Samtools
• Fastp
• Pandas
• Numpy
• Biopython
• Pysam
• Jinja2
• Matplotlib
• Seaborn
• IGV-Reports

You can easily set up these dependencies via the provided Conda environment file.

---

Linting and Code Formatting

VNtyper adheres to PEP8 style guidelines to ensure clean, readable, and maintainable code. We use Ruff, a modern, extremely fast linter and formatter written in Rust that replaces both flake8 and black.

Quick Start with Makefile

VNtyper provides a Makefile for common development tasks:

# Show all available commands
make help

# Install in development mode
make install-dev

# Run linter
make lint

# Format code (auto-fix issues)
make format

# Check formatting without making changes
make format-check

# Run tests
make test          # All tests
make test-unit     # Unit tests only
make test-cov      # With coverage report

# Clean build artifacts
make clean

Modern Python Packaging

VNtyper uses pyproject.toml for all project configuration (PEP 517/518/621):

• No setup.py required: Modern pip (≥21.3) and setuptools (≥64) fully support pyproject.toml
• Single source of truth: All configuration (build, dependencies, tools) in one file
• Editable installs: Fully supported via PEP 660

---

Pipeline Logic Diagram

Below is a logical overview of the VNtyper pipeline:

graph TD
  A[Input: FASTQ/BAM] -->|Quality Control| B[Alignment BWA]
  B -->|Genotyping| C[Kestrel]
  C --> D[Optional: adVNTR]
  D --> E[Generate Summary Report]
  E --> F[Output: VCF, Summary HTML]

Loading

---

Results

Once the pipeline completes, you will have:

• BAM or FASTQ slices containing MUC1-specific reads.
• VCF files or TSV files with genotyping results (for Kestrel and optional adVNTR).
• HTML summary report including:
  • VNTR Region Coverage Statistics: Detailed coverage metrics specifically for the VNTR region, including mean, median, standard deviation, minimum, and maximum coverage, as well as the percentage of the VNTR region with zero coverage.
  • Genotyping Calls: Results from Kestrel and optional adVNTR analyses.
  • Quality Metrics: When available, includes duplication rate, Q20/Q30 rates, and other quality indicators.
  • Pipeline Log: Comprehensive logging information about the pipeline execution.

---

Notes

1. This tool is for research use only.
2. Ensure high-coverage WES/WGS or targeted data is used to genotype MUC1 VNTR accurately.
3. For questions or issues, refer to the GitHub repository for support.

---

Citations

If you use VNtyper 2.0 in your research, please cite the following:

1. Saei H, Morinière V, Heidet L, et al. VNtyper enables accurate alignment-free genotyping of MUC1 coding VNTR using short-read sequencing data. iScience. 2023.
2. Audano PA, Ravishankar S, et al. Mapping-free variant calling using haplotype reconstruction from k-mer frequencies. Bioinformatics. 2018.
3. Park J, Bakhtiari M, et al. Detecting tandem repeat variants in coding regions using code-adVNTR. iScience. 2022.

---

Contributing

We welcome contributions to VNtyper. Please refer to the CONTRIBUTING.md file for guidelines.

---

License

VNtyper is licensed under the BSD 3-Clause License. See the LICENSE file for more details.

---

Contact

For questions or issues, please open an issue on GitHub or email the corresponding authors listed in the manuscript.