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Extract Fragment Lengths

Multiple studies have used fragment lengths (count distributions) to detect cancer [REFS].

Base command​

cfdna lengths --help

cfdna lengths \
  --bam <sample>.bam \
  --output-dir <sample_directory>/lengths \
  --output-prefix <sample_id> \
  --n-threads 12 \
  --blacklist <path>/hg38-blacklist.v2.bed \
  --blacklist <path>/<another_blacklist>.bed \
  --by-size 1000000

Use length bins​

...

The default length bins are specified as --length-bins 30:1001:1, which gives us the full single-bp resolution fragment length distribution between 30bp and 1000bp.

cfdna lengths \
  ... \
  # Bin every 10bp from 30-500bp
  --length-bins 30:500:10
  # OR Specify edges directly (last end is exclusive)
  --length-bins 100 151 221

GC-bias correction example​

cfdna lengths \
  --bam <sample>.bam \
  --output-dir <sample_directory>/lengths \
  --output-prefix <sample_id> \
  --n-threads 12 \
  --blacklist <path>/hg38-blacklist.v2.bed \
  --by-size 1000000 \
  --gc-file <sample_directory>/gc_bias/gc_bias_correction.npz \
  --ref-2bit <path>/hg38.2bit

Genomic smoothing example​

cfdna lengths \
  --bam <sample>.bam \
  --output-dir <sample_directory>/lengths \
  --output-prefix <sample_id> \
  --n-threads 12 \
  --blacklist <path>/hg38-blacklist.v2.bed \
  --by-size 1000000 \
  --scaling-factors <sample_directory>/coverage_weights/<sample_id>.scaling_factors.tsv

GC-bias correction + genomic smoothing​

cfdna lengths \
  --bam <sample>.bam \
  --output-dir <sample_directory>/lengths \
  --output-prefix <sample_id> \
  --n-threads 12 \
  --blacklist <path>/hg38-blacklist.v2.bed \
  --by-size 1000000 \
  --gc-file <sample_directory>/gc_bias/gc_bias_correction.npz \
  --ref-2bit <path>/hg38.2bit \
  --scaling-factors <sample_directory>/coverage_weights/<sample_id>.scaling_factors.tsv

Adjusting for indels​

The default lengths behavior is to use the fragment span on the reference genome and ignore whether insertions or deletions (InDels) are present in the reads.

By specifying --indel-mode adjust, the fragment lengths are adjusted for indels, which should be closer to the size of the original DNA molecule.

cfdna lengths \
  ... \
  --indel-mode adjust

This is an analysis choice, not a baseline requirement. If you are unsure, start without it.