VNtyper 2 - A Pipeline to genotype the MUC1-VNTR

VNtyper 2 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 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.

Features • Installation • Usage • Architecture • Outputs • Dependencies • Citations • License

Features

• Multi-Method Genotyping: Mapping-free k-mer genotyping via Kestrel (local haplotype assembly), optional code-adVNTR profile-HMM repeat-unit validation, and optional SHARK read fishing for FASTQ data.
• Evidence-Based Confidence & QC: First-match, evidence-ranked confidence grades (A–E), automated caller cross-match concordance, and dedicated VNTR coverage QC statistics.
• Interactive & Standard Outputs: Self-contained interactive HTML report featuring an embedded offline IGV.js alignment browser, flattened tabular summaries (pipeline_summary.csv / .tsv), and standard VCF/TSV call tables.
• Cohort Aggregation: vntyper cohort aggregates multiple sample outputs into cohort summaries with allele call frequency metrics and configurable rarity thresholds.
• Production Resilience: Fault-tolerant resumption (--resume), atomic output publishing, and comprehensive audit logs (pipeline.log) with MD5 file checksums.

Installation

VNtyper 2 uses modern Python packaging with pyproject.toml and can be installed via Conda/Mamba, container images, or pip.

Conda / Mamba (Recommended)

Installing via Conda or Mamba provides the complete environment including external bioinformatics binaries (bwa, samtools, fastp, bcftools, and Java 11 for Kestrel):

git clone https://github.com/hassansaei/VNtyper.git
cd VNtyper
mamba env create -f conda/environment_vntyper.yml
conda activate vntyper
pip install -e .

For development and test suites: pip install -e .[dev]

Docker & Apptainer (Zero-Setup)

Prebuilt images with all dependencies and bundled reference data are published to GitHub Packages (GHCR). See Running with Docker & Apptainer.

Python Package (pip)

pip install vntyper installs the complete Python package, entry point scripts, and packaged assets directly from PyPI:

pip install vntyper

Reference Data Setup

Before running the pipeline locally, install the required human reference genomes and MUC1 target models:

vntyper install-references --output-dir reference   # run from the repository root

This downloads and verifies the pre-built reference bundle with SHA-256 checksums.

Run vntyper pipeline from the repository root too. Every tool and reference path in vntyper/config.json is relative to the working directory, so the defaults expect ./reference/ and ./vntyper/dependencies/kestrel/. A job script should cd into the checkout first. If a file is missing, the pipeline stops before reading any input and names the path it tried. See Troubleshooting.

Usage

VNtyper 2 offers multiple subcommands for end-to-end analysis:

• pipeline — Run the full genotyping pipeline on BAM, CRAM, or paired FASTQ files.
• report — Regenerate HTML and TSV reports from an existing run directory.
• cohort — Aggregate multiple run directories into a cohort summary with call frequencies.
• install-references — Download or build reference files and motif databases.
• online — Extract a MUC1 BAM slice and submit it to VNtyper-Online.
• calibrate — Extract, fit, or evaluate opt-in calibration profiles.

1. Running the Genotyping Pipeline

BAM Input

vntyper pipeline \
    --bam /data/inputs/sample.bam \
    --output-dir /data/results/sample/ \
    --threads 4 \
    --fast-mode

CRAM Input

Provide the matching reference FASTA for CRAM decompression:

vntyper pipeline \
    --cram /data/inputs/sample.cram \
    --reference-fasta /data/reference/genome.fa \
    --output-dir /data/results/sample/ \
    --threads 4 \
    --fast-mode

Paired-End FASTQ Input

vntyper pipeline \
    --fastq1 /data/inputs/sample_R1.fastq.gz \
    --fastq2 /data/inputs/sample_R2.fastq.gz \
    --output-dir /data/results/sample/ \
    --threads 4 \
    --fast-mode

Resuming an Interrupted Run

Use --resume to reuse completed stages from an earlier run directory:

vntyper pipeline --bam /data/inputs/sample.bam --output-dir /data/results/sample/ --threads 4 --resume

Optional Modules (adVNTR & SHARK)

• adVNTR: Enable profile-HMM genotyping with --extra-modules advntr.
• SHARK: Enable raw FASTQ k-mer filtering with --extra-modules shark (FASTQ inputs only).

2. Running with Docker & Apptainer

Docker image for VNtyper 2 is provided and can be pulled and used as follows. Released images are published to GHCR as latest (the newest release), the immutable vX.Y.Z and X.Y.Z tags naming one exact release, and the moving X and X.Y series tags. Pin vX.Y.Z for a reproducible run. The main tag is rolling and tracks the default branch. Docker Hub artifacts are legacy, frozen, and unsupported.

Docker

docker pull ghcr.io/hassansaei/vntyper:latest

docker run --user $(id -u):$(id -g) -w /opt/vntyper --rm \
    -v /local/inputs/:/opt/vntyper/input \
    -v /local/results/:/opt/vntyper/output \
    ghcr.io/hassansaei/vntyper:latest \
    vntyper pipeline \
    --bam /opt/vntyper/input/sample.bam \
    -o /opt/vntyper/output/sample/

Note: Input and output must be separate host directories. Mounting the same directory to both input and output is rejected to prevent in-tree side effects.

Apptainer / Singularity

apptainer pull docker://ghcr.io/hassansaei/vntyper:latest

apptainer run --pwd /opt/vntyper \
    -B /local/inputs/:/opt/vntyper/input \
    -B /local/results/:/opt/vntyper/output \
    vntyper_latest.sif vntyper pipeline \
    --bam /opt/vntyper/input/sample.bam \
    -o /opt/vntyper/output/sample/

3. Additional Subcommands

• Regenerate Reports:

  vntyper report --output-dir /data/results/sample/
  

• Cohort Analysis:

  vntyper cohort \
      --input-dirs /data/results/sample1 /data/results/sample2 /data/results/sample3 \
      --output-dir /data/cohort_summary/ \
      --rare-allele-max-frequency 0.05
  

• Online Submission:

  vntyper online --bam /data/inputs/sample.bam --output-dir /data/results/online_submission/
  

Pipeline Architecture

VNtyper 2 integrates multiple steps into a streamlined pipeline. The following is an overview of the steps involved:

flowchart TD
    subgraph Inputs
        A1[FASTQ Pairs]
        A2[BAM / CRAM Alignment]
    end

    subgraph Preprocessing
        B1[fastp QC]
        B2[Optional: SHARK Filtering]
        B3a[BWA-MEM Alignment]
        B3b[Samtools Slicing]
    end

    subgraph Genotyping
        C1[Kestrel Genotyping<br/>k-mer frequency & local assembly]
        C2[Optional: code-adVNTR<br/>profile-HMM repeat counting]
    end

    subgraph Quality & Confidence
        D1[MUC1 Nomenclature & Flagging]
        D2[Caller Cross-Match]
        D3[Confidence Grading A–E]
    end

    subgraph Outputs
        E1[Interactive HTML Report<br/>with embedded IGV.js]
        E2[Flattened Tables<br/>pipeline_summary.csv / .tsv]
        E3[VCF & TSV Call Tables]
        E4[MUC1 Slice BAM]
    end

    A1 --> B1 --> B2 --> B3a --> B3b
    A2 --> B3b
    B3b --> C1
    B3b -.-> C2
    C1 --> D1
    C2 -.-> D1
    D1 --> D2 --> D3
    D3 --> E1 & E2 & E3 & E4

1. Pre-processing & QC: Quality filtering (fastp), optional SHARK k-mer filtering, and targeted MUC1 alignment slicing.
2. Kestrel Genotyping: Mapping-free k-mer frequency counting and local haplotype assembly over the MUC1 VNTR.
3. Optional adVNTR Genotyping: Profile-HMM genotyping for independent repeat-unit validation.
4. Nomenclature & Confidence Grading: Canonical MUC1 nomenclature, evidence flags, caller cross-match concordance, and confidence grades (A–E).
5. Report Generation: Interactive HTML reports (with offline IGV.js), flattened CSV/TSV summaries, and standard VCF/TSV calls.

Outputs & Results

After pipeline completion, --output-dir contains:

• Reports & Summaries:
  • summary_report.html — Interactive HTML report with sample QC, VNTR coverage statistics, confidence grade chip, caller results, and embedded IGV.js browser.
  • pipeline_summary.json — Machine-readable summary with complete parameter and execution provenance.
  • pipeline_summary.csv / .tsv — Flattened, single-row summary per run (when requested via --summary-formats).
  • pipeline_summary_rows.tsv — Flattened table with one row per detected variant call.
• Variant Calls:
  • kestrel/output_indel.vcf.gz (or .vcf) — Standard VCF containing Kestrel variant calls.
  • kestrel/kestrel_result.tsv — Detailed Kestrel calls and k-mer path depths.
  • advntr/output_adVNTR.tsv — adVNTR repeat-unit calls (when enabled).
• Alignments & Logs:
  • fastq_bam_processing/output_sliced.bam & .bai — MUC1-region BAM slice and index.
  • pipeline.log — Full audit log with timestamps and MD5 checksums.

Dependencies

VNtyper 2 relies on several tools and Python libraries. Ensure that the following dependencies are available in your environment:

• Python: >= 3.10 (tested on 3.10–3.13; Docker image uses Python 3.12)
• External Binaries: bwa (≥ 0.7.17), samtools (≥ 1.15), fastp (≥ 0.23), bcftools (≥ 1.15), Java (JRE ≥ 11)
• Key Python Libraries: pandas, numpy, biopython, pysam, regex, jinja2, igv-reports, plotly, rfc8785

All dependencies are pre-packaged in conda/environment_vntyper.yml and in our Docker containers.

Citations

If you use VNtyper 2 in your research, please cite:

• VNtyper 2 & VNtyper-Online (Preprint):
  Popp B, Saei H, Teltsh O, et al. VNtyper 2 enables open-access short-read genotyping of MUC1 VNTR variants in ADTKD at high-speed. medRxiv (2026). doi:10.64898/2026.05.27.26352937

  BibTeX

  @article{popp2026vntyper2,
    title={VNtyper 2 enables open-access short-read genotyping of MUC1 VNTR variants in ADTKD at high-speed},
    author={Popp, Bernt and Saei, Hassan and Teltsh, Omri and Janou{\v{s}}ek, V{\'a}clav and P{\v{r}}istoupilov{\'a}, Anna and Vrback{\'a}, Alena and Hartmannov{\'a}, Hana and Kidd, Kendrah and Helmuth, Johannes and Bleyer, Anthony J and Wiesener, Michael and Fausch, Kathrin and Rowan, Colm and El Hassan, Elhussein and Clince, Michelle and Cavalleri, Gianpiero and Locher, Maurus and Eckardt, Kai-Uwe and Richter-Pechanska, Paulina and {ADTKD-Net Consortium} and Kmoch, Stanislav and Antignac, Corinne and Conlon, Peter and Dorval, Guillaume and {\v{Z}}ivn{\'a}, Martina},
    journal={medRxiv},
    pages={2026.05.27.26352937},
    year={2026},
    publisher={Cold Spring Harbor Laboratory Press},
    doi={10.64898/2026.05.27.26352937}
  }
  

• Original Method (VNtyper v1):
  Saei H, Morinière V, Heidet L, et al. VNtyper enables accurate alignment-free genotyping of MUC1 coding VNTR using short-read sequencing data. iScience 26(7):107171 (2023). doi:10.1016/j.isci.2023.107171

  BibTeX

  @article{saei2023vntyper,
    title={VNtyper enables accurate alignment-free genotyping of MUC1 coding VNTR using short-read sequencing data},
    author={Saei, Hassan and Morini{\`e}re, Vincent and Heidet, Laurence and others},
    journal={iScience},
    volume={26},
    number={7},
    pages={107171},
    year={2023},
    publisher={Elsevier},
    doi={10.1016/j.isci.2023.107171}
  }
  

• Underlying Tools & Archive:

  • Zenodo Archive: Concept DOI 10.5281/zenodo.19744166
  • Kestrel: Audano PA, et al. Bioinformatics 34(10):1659–1665 (2018). doi:10.1093/bioinformatics/btx753
  • code-adVNTR: Park J, et al. iScience 25(8):104785 (2022). doi:10.1016/j.isci.2022.104785
  • SHARK: Denti L, et al. Bioinformatics 37(4):464–472 (2021). doi:10.1093/bioinformatics/btaa779

License & Contributing

VNtyper is licensed under the BSD 3-Clause License. Contributions are welcome; please see CONTRIBUTING.md and AGENTS.md. For questions or issues, please open an issue on GitHub.