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Pipeline Integration

Run All Features

The run-all command allows you to execute all feature extraction modules for a single BAM file in one go.

Usage

krewlyzer run-all sample.bam --reference hg19.fa --output all_features_out \
    [--variants variants.maf] \
    [--bin-input targets.bed] \
    [--threads N]

Arguments

  • sample.bam: Input BAM file (sorted, indexed).
  • --reference, -g: Reference genome FASTA.
  • --output, -o: Output directory.
  • --variants, -v: (Optional) VCF or MAF file for mfsd analysis.
  • --bin-input, -b: (Optional) Custom bins for FSC/FSR (e.g., targeted panel regions).
  • --threads, -t: Number of threads (default: 0 = all cores).
  • --mapq, -q: Minimum mapping quality (default: 20).
  • --minlen, --maxlen: Fragment length range (default: 65-400).

Nextflow Pipeline

Krewlyzer includes a Nextflow pipeline (krewlyzer.nf) for processing multiple samples in parallel on HPC clusters or local machines.

Usage

nextflow run krewlyzer.nf --samplesheet samplesheet.csv --ref /path/to/reference.fa --outdir results/

Samplesheet Format (CSV)

A CSV file with the following columns: 

sample,bam,meth_bam,vcf,bed,maf,single_sample_maf
sample1,/path/to/sample1.bam,,/path/to/sample1.vcf,,,
sample2,/path/to/sample2.bam,,,,/path/to/sample2.maf,true
sample3,/path/to/sample3.bam,,,,/path/to/cohort.maf,false
sample4,,,,/path/to/pre_extracted.bed.gz,,

Pipeline Logic

The pipeline automatically runs specific modules based on valid input in the samplesheet columns:

  • bam: Triggers the main run-all workflow (Extraction -> Motif -> Features). This is the standard path for WGS BAMs.
  • meth_bam: Triggers uxm (Methylation) analysis. Can be run alongside bam or independently.
  • bed: Triggers fragment-only features (FSC, FSR, WPS, OCF, FSD). Skips extraction/motif steps. Use this for re-running analysis on already extracted fragments.
  • vcf: Used with bam for mFSD (Mutant Fragment Size Distribution) analysis.
  • maf: Multi-sample MAF file for mFSD analysis. The pipeline filters by Tumor_Sample_Barcode matching the sample ID (regex: .*sample_id.*).
  • single_sample_maf: Set to true if the MAF contains only this sample's variants (skips filtering). Set to false or leave empty for multi-sample MAFs.

[!NOTE] If the filtered MAF has zero variants for a sample, MFSD is skipped with a warning.

Note on Column Headers: The samplesheet MUST use these exact headers: sample,bam,meth_bam,vcf,bed,maf,single_sample_maf. Empty fields for optional columns should be left blank (commas required).

Profiles

  • -profile lsf: For LSF clusters.
  • -profile slurm: For SLURM clusters.
  • -profile docker: Run using Docker container.