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Frequently Asked Questions

What input coverage do I need?

A minimum of approximately 100x coverage over the MUC1 VNTR region is recommended for reliable variant detection. Whole-genome sequencing at 30x or higher typically provides sufficient local coverage, while whole-exome sequencing may fall short depending on the capture kit's coverage of the MUC1 VNTR.

BAM vs FASTQ -- which is faster?

BAM input is faster. When you provide a BAM or CRAM file, VNtyper 2 extracts only the reads overlapping the MUC1 region before processing. Starting from FASTQ files requires an additional alignment step, which adds runtime.

Do I need adVNTR?

No. adVNTR is an optional module that provides independent, alignment-based validation of Kestrel calls. Enabling it adds approximately 9 minutes to the runtime. It can be useful for confirmation in research settings but is not required for routine genotyping.

What does Low_Precision mean?

A variant classified as Low_Precision (also called Low Confidence) was detected but has marginal depth support -- the depth score falls between the low and high thresholds defined in kestrel_config.json. These calls may benefit from manual review or independent validation. See Scoring & Confidence for detailed threshold definitions.

Can I use GRCh38?

Yes. VNtyper 2 supports both UCSC and NCBI naming conventions. Use the --reference-assembly flag with any of the following values:

  • hg19 or GRCh37
  • hg38 or GRCh38

Example:

vntyper pipeline --bam input.bam --reference-assembly hg38 -o output/

Docker vs local install?

Docker bundles all external dependencies (BWA, samtools, fastp, Java 11, Kestrel JAR) into a single image, so you do not need to install them yourself. This is the easiest way to get started.

A local install gives you more control and avoids container overhead, but you must ensure that BWA, samtools, fastp, and Java 11 are available on your PATH.

How do I interpret the HTML report?

The HTML report includes an embedded IGV viewer for visual inspection of variants and a summary table of detected mutations with confidence levels. For a detailed walkthrough, see Output Files.

SHARK fails with BAM input

SHARK requires FASTQ input. If you want to use the SHARK module, provide reads with the --fastq1 and --fastq2 flags along with --extra-modules shark:

vntyper pipeline --fastq1 R1.fastq.gz --fastq2 R2.fastq.gz \
    --extra-modules shark -o output/

How do I run multiple samples?

There are two approaches:

  1. Cohort command -- VNtyper 2's built-in cohort subcommand processes a directory of samples and produces an aggregated summary. See Cohort Analysis.
  2. Snakemake workflow -- For large-scale batch processing with parallelization and cluster support, use the provided Snakemake workflow. See Snakemake.

Where can I get help?

Open an issue on GitHub. Please search existing issues first to avoid duplicates.