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 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

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Table of Contents

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

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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.

• Flexible Installation:

  • Supports installation via pip using setup.py.
  • Provides Conda environment setup for easy dependency management.

• Subcommands:

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

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Installation

VNtyper 2.0 can be installed using either pip with setup.py or via Conda environments for streamlined dependency management.

Using setup.py and pip

1. Clone the Repository:

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

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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 on paired-end FASTQ files or BAM files:

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

Alternatively, using a BAM file:

vntyper pipeline \
    --config-path /path/to/config.json \
    --bam /path/to/sample.bam \
    --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 pipeline \
    --config-path /path/to/config.json \
    --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.

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

docker pull saei/vntyper:2.0.0-alpha.52

docker run -w /opt/vntyper --rm \
    -v /local/input/folder/:/opt/vntyper/input \
    -v /local/output/folder/:/opt/vntyper/output \
    vntyper:2.0.0-alpha.52 \
    vntyper pipeline --bam /local/input/folder/filename.bam \
    -o /local/output/folder/filename/

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

apptainer pull docker://saei/vntyper:2.0.0-alpha.52

apptainer run --pwd /opt/vntyper \
    -B /local/input/folder/:/opt/vntyper/input \
    -B /local/output/folder/:/opt/vntyper/output \
    vntyper_2.0.0-alpha.52.sif vntyper pipeline \
    --bam /opt/vntyper/input/filename.bam \
    -o /opt/vntyper/output/filename/

2. Installing References

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

3. Generating Reports

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

Process raw FASTQ files to prepare them for genotyping:

vntyper fastq \
    --fastq1 /path/to/sample_R1.fastq.gz \
    --fastq2 /path/to/sample_R2.fastq.gz \
    --output-dir /path/to/output/dir

vntyper bam \
    --alignment /path/to/sample.bam \
    --output-dir /path/to/output/dir \
    --threads 4

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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.

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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.

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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

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Results

Once the pipeline completes, you will have:

• BAM or FASTQ slices containing MUC1-specific reads.
• VCF files with genotyping results (for Kestrel and optional adVNTR).
• HTML summary report detailing coverage stats, genotyping calls, and relevant logs.

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Notes

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

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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.

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Contributing

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

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License

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

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Contact

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