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Usage Guide

Command Summary

Command Description
motif Motif-based feature extraction
fsc Fragment size coverage (.FSC.tsv)
fsr Fragment size ratio (.FSR.tsv)
fsd Fragment size distribution (.FSD.tsv)
wps Windowed protection score (.WPS.tsv.gz)
ocf Orientation-aware fragmentation (.OCF.tsv)
uxm Fragment-level methylation (.UXM.tsv)
mfsd Mutant fragment size distribution (.mFSD.tsv)
run-all Run all features for a BAM

Reference Data

  • Reference Genome (FASTA):
  • Download GRCh37/hg19 from UCSC
  • BAMs must be sorted, indexed, and aligned to the same build
  • Bin/Region/Marker Files:
  • Provided in krewlyzer/data/ (see options for each feature)

Typical Workflow

The recommended way to run krewlyzer is using the Unified Pipeline via run-all, which processes the BAM file in a single pass for maximum efficiency.

# Optimized Unified Pipeline
krewlyzer run-all sample.bam --reference hg19.fa --output output_dir \
    --variants variants.maf --bin-input targets.bed --threads 4

Alternatively, you can run tools individually. Note that most tools require a fragment BED file (.bed.gz) produced by the extract command.

# 1. Extract fragments (BAM -> BED.gz)
krewlyzer extract sample.bam -g hg19.fa -o output_dir

# 2. Run feature tools using the BED file
# 2. Run feature tools using the BED file
krewlyzer fsc output_dir/sample.bed.gz --output output_dir/
krewlyzer wps output_dir/sample.bed.gz --output output_dir/
# ... (fsd, ocf, etc.)

# 3. Motif analysis (Independent of BED, uses BAM directly)
krewlyzer motif sample.bam -g hg19.fa -o output_dir

Targeted Panel Usage (ACCESS, etc.)

For targeted sequencing panels (e.g., MSK-ACCESS), FSC/FSR require a custom regions BED file instead of the default genome-wide 100kb bins:

# Using run-all with custom target regions
krewlyzer run-all sample.bam --reference hg19.fa --output out/ \
  --bin-input /path/to/MSK-ACCESS-v2_canonicaltargets.bed

# Or run FSC/FSR individually with target regions
# Or run FSC/FSR individually with target regions
krewlyzer fsc motif_out/sample.bed.gz -b targets.bed -w 1 -c 1 --output out_dir/
krewlyzer fsr motif_out/sample.bed.gz -b targets.bed -w 1 -c 1 --output out_dir/

Note: Without --bin-input, FSC/FSR will produce zeros for targeted panels since data only covers specific gene regions, not genome-wide bins. The --output argument for individual tools specifies the output directory, not a filename.