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Mutant Fragment Size Distribution (mFSD)

Command: krewlyzer mfsd

Purpose

Compares the size distribution of mutant vs. wild-type fragments at variant sites, with support for all small variant types and 4-way fragment classification.

Biological Context

Mutant ctDNA fragments are typically shorter (~145bp) than wild-type cfDNA (~166bp). This module quantifies this difference using high-depth targeted sequencing data, providing a sensitive marker for ctDNA presence. It performs a Kolmogorov-Smirnov (KS) test to statistically compare the distributions.

Variant Types Supported

Type Example Description
SNV A>T Single nucleotide variant
MNV AT>GC Multi-nucleotide variant
Insertion A>ATG Pure insertion
Deletion ATG>A Pure deletion
Complex ATG>CT Mixed substitution + indel

Fragment Classification

Fragments are classified into 4 categories based on the allele they support:

Category Description Use Case
REF Supports reference allele Healthy cfDNA baseline
ALT Supports alternate allele Tumor signal
NonREF Non-REF, non-ALT, non-N Sequencing errors, subclones
N Contains N at variant position Low quality, uninformative

Usage

krewlyzer mfsd sample.bam --input-file variants.vcf --output output_dir/ [options]

Options

  • --input-file, -i: VCF or MAF file (required)
  • --mapq, -q: Minimum mapping quality (default: 20)
  • --output-distributions, -d: Output per-variant size distributions
  • --sample-name, -s: Sample name for output file
  • --threads, -t: Number of threads (0=all cores)

Output Format

Main Output: {sample}.mFSD.tsv (39 columns)

Variant Info (5)

Column Description
Chrom Chromosome
Pos 1-based position
Ref Reference allele
Alt Alternate allele
VarType SNV, MNV, INS, DEL, or COMPLEX

Counts (5)

Column Description
REF_Count Fragments supporting REF
ALT_Count Fragments supporting ALT
NonREF_Count Fragments with errors
N_Count Fragments with N bases
Total_Count Total fragments

Mean Sizes (4)

Column Description
REF_MeanSize Mean size of REF fragments
ALT_MeanSize Mean size of ALT fragments
NonREF_MeanSize Mean size of error fragments
N_MeanSize Mean size of N fragments

Pairwise Comparisons (18)

For each pair (6 combinations): Delta, KS statistic, KS p-value

  • Delta_ALT_REF, KS_ALT_REF, KS_Pval_ALT_REF (Primary)
  • Delta_ALT_NonREF, KS_ALT_NonREF, KS_Pval_ALT_NonREF
  • Delta_REF_NonREF, KS_REF_NonREF, KS_Pval_REF_NonREF
  • Delta_ALT_N, KS_ALT_N, KS_Pval_ALT_N
  • Delta_REF_N, KS_REF_N, KS_Pval_REF_N
  • Delta_NonREF_N, KS_NonREF_N, KS_Pval_NonREF_N

Derived Metrics (5)

Column Description
VAF_Proxy ALT / (REF + ALT)
Error_Rate NonREF / Total
N_Rate N / Total
Size_Ratio ALT_MeanSize / REF_MeanSize
Quality_Score 1 - N_Rate - Error_Rate

Quality Flags (2)

Column Description
ALT_Confidence HIGH (≥5), LOW (1-4), NONE (0)
KS_Valid TRUE if both REF and ALT ≥2 fragments

Optional: {sample}.mFSD.distributions.tsv

With --output-distributions, outputs per-variant size distributions:

Chrom  Pos  Ref  Alt  Category  Size  Count
chr1   12345  A   T   REF       145   3
chr1   12345  A   T   REF       166   12
chr1   12345  A   T   ALT       142   2

Clinical Interpretation

Healthy vs Cancer

Metric Healthy Cancer (ctDNA)
Delta_ALT_REF ~0 Negative (ALT shorter)
Size_Ratio ~1.0 <1.0
VAF_Proxy 0 >0 (correlates with tumor fraction)

MRD Settings

  • Low fragment counts (1-2) produce NA for statistics
  • ALT_Confidence indicates reliability
  • KS_Valid flags when statistical test is meaningful