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Correct GC-Bias

Fragmentomics features are vulnerable to biases from various sample-handling and sequencing processes, such as PCR amplification. cfDNAlab commands allow correcting the commonly observed GC-bias.

This requires only a few steps.

When running many jobs at once on an HPC cluster, use --temp-dir to place temporary files on node-local scratch and significantly reduce shared-filesystem contention.

Step 1. Build reference GC bias once per assembly​

Calculate the expected GC bias in the reference genome assembly (for example hg38). This output can be reused for all samples aligned to that assembly.

cfdna ref-gc-bias --help

# Run once per assembly
cfdna ref-gc-bias \
  --ref-2bit <path>/hg38.2bit \
  --output-dir <ref_gc_directory> \
  --output-prefix hg38 \
  --n-threads 12 \
  --blacklist <path>/hg38-blacklist.v2.bed \
  --blacklist <path>/<another_blacklist>.bed

Step 2. Build sample-specific GC correction​

Calculate the observed GC-bias for given sample and build the correction matrix:

cfdna gc-bias --help

cfdna gc-bias \
  --bam <sample>.bam \
  --output-dir <sample_directory>/gc_bias \
  --n-threads 12 \
  --ref-2bit <path>/hg38.2bit \
  --ref-gc-file <ref_gc_directory>/hg38.ref_gc_package.zarr \
  --blacklist <path>/hg38-blacklist.v2.bed \
  --blacklist <path>/<another_blacklist>.bed

Use the same blacklist inputs as in step 1.

Step 3. Apply correction in feature extraction commands​

When calling the feature extraction commands, pass the produced GC correction package via --gc-file. Note that the same BAM file should generally be used when calculating and correcting the GC-bias.

cfdna fcoverage \
  --bam <sample>.bam \
  ... \
  --gc-file <sample_directory>/gc_bias/gc_bias_correction.zarr \
  --ref-2bit <path>/hg38.2bit

The same pattern works for lengths, ends, and midpoints.

Alternative: read GC weights from BAM aux tags​

If you prefer to use a different / custom GC-bias tool, the feature extraction commands can also read a GC weight from a BAM aux tag. Use --gc-tag and set the name of the aux tag to use.

cfdna fcoverage \
  --bam <sample>.bam \
  ... \
  --gc-tag 'GC'