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Fragment Size Ratio (FSR)

Command: krewlyzer fsr

Purpose

Computes ratios of fragment size classes per genomic window. Unlike FSC, this metric is self-normalizing (ratio) and focuses on the proportion of fragment sizes, which is a powerful biomarker for tumor fraction estimation.

Biological Context

The ratio of short to long fragments is a key indicator of tumor burden in cfDNA ("fragmentomics"). See DELFI method for details.

  • Short Fragments (65-149bp): Tumor DNA is typically shorter (~145bp) than healthy DNA (~166bp).
  • Ultra-short Fragments (65-100bp): Associated with transcription factor binding sites and open chromatin.
  • Long Fragments (261-399bp): Often di-nucleosomes, representing stable, healthy chromatin (Leukocytes).

Key Biomarkers: - Short/Long Ratio: The primary metric. Higher ratio = higher probability of tumor DNA. - Ultra-short Ratio: Indicates active gene regulation/transcription factor binding. - Nucleosome Footprints: The 10bp precision of the ranges (150bp vs 167bp) helps separate mono-nucleosomes.

Usage

krewlyzer fsr sample.bed.gz -o output_dir/ --sample-name SAMPLE [options]

Options

  • --bin-input, -b: Bin file (default: data/ChormosomeBins/hg19_window_100kb.bed)
  • --windows, -w: Window size (default: 100000)
  • --continue-n, -c: Consecutive window number (default: 50)
  • --gc-correct/--no-gc-correct: Apply GC bias correction using LOESS (default: True)
  • --verbose, -v: Enable verbose logging
  • --threads, -p: Number of threads.

Output Format

Output: {sample}.FSR.tsv

Column Description Biological Relevance
region Genomic region
ultra_short_count Count 65-100bp TF footprints
short_count Count 65-149bp Tumor enriched
inter_count Count 151-259bp Mono-nucleosome
long_count Count 261-399bp Healthy / Di-nucleosome
total_count Count 65-399bp Total fragments
short_ratio Short / Total Tumor fraction proxy
inter_ratio Intermediate / Total
long_ratio Long / Total Healthy fraction proxy
short_long_ratio Short / Long Primary Cancer Biomarker
ultra_short_ratio Ultra-short / Total TF activity indicator

Clinical Interpretation

Healthy vs Cancer

Metric Healthy Plasma Cancer (ctDNA present)
Modal fragment size ~166bp Left-shifted (~145bp)
short_long_ratio Low (baseline) Elevated
Genome-wide ratio variability Minimal Increased aberrations

Interpretation Guide

  • High short_long_ratio: Indicates higher tumor fraction (ctDNA) or open chromatin.
  • High ultra_short_ratio: Indicates regions of high transcription factor activity.
  • High long_ratio: Indicates stable, nucleosomal DNA (usually healthy background).

Clinical Utility

  • Tumor fraction proxy: Compare short_long_ratio to healthy baseline
  • Treatment monitoring: Track ratio changes over time
  • Performance: DELFI achieves 57-99% sensitivity at 98% specificity

Reference: See Citation & Scientific Background for detailed paper summary.