Skip to content

Fragment Extraction

Command: krewlyzer extract

Purpose

The extract module serves as the entry point for most analysis workflows. It processes a BAM file to extract valid cell-free DNA (cfDNA) fragments and saves them in a standardized, compressed BED format. It also generates a JSON metadata file containing processing statistics and configuration.

Biological Context

Raw sequencing data (BAM) contains reads that must be paired and filtered to reconstruct physical DNA fragments. This step standardizes the data, removing PCR duplicates and low-quality mappings, ensuring downstream analysis focuses on high-confidence unique molecules.

Usage

krewlyzer extract sample.bam -g hg19.fa -o output_dir/ [options]

Options

  • --genome, -g: Reference genome FASTA (required).
  • --output, -o: Output directory.
  • --mapq, -q: Minimum mapping quality (default: 20).
  • --minlen: Minimum fragment length (default: 65).
  • --maxlen: Maximum fragment length (default: 400).
  • --exclude-regions, -x: BED file of regions to blacklist (e.g., centromeres).

Output Files

1. Fragment File ({sample}.bed.gz)

A block-gzipped, tabix-indexed BED file containing the coordinates of extracted fragments. - Format: BED3+3 (chrom, start, end, name, score, strand) - Note: currently simplified to BED3 or similar depending on implementation. - Coordinates: 0-based, half-open (standard BED).

2. Metadata File ({sample}.metadata.json)

A JSON file capturing run provenance and statistics.

Example Structure: 

{
  "sample_id": "CasePlasma",
  "total_fragments": 8123456,
  "filters": {
    "mapq": 20,
    "min_length": 65,
    "max_length": 400
  },
  "timestamp": "2023-10-27T10:30:00.123456"
}

Fields: - sample_id: Identifier derived from input filename or user argument. - total_fragments: Number of valid fragments extracted after filtering. - filters: Configuration parameters used for the run. - timestamp: Execution time.