bam-to-fragment-file-conversion

BAM-to-fragment-file conversion

Summary

Convert coordinate-sorted BAM files into compressed fragment files (BED.gz or .zst format) containing fragment coordinates, cell barcodes, and quality metrics. This preprocessing step is essential for downstream single-cell ATAC-seq analysis in SnapATAC2.

When to use

When you have coordinate-sorted BAM files from single-cell ATAC-seq experiments (e.g., 10X Genomics scATAC-seq) and need to generate a compressed fragment file for efficient downstream analysis. Use this step before matrix generation, clustering, or peak calling in SnapATAC2.

When NOT to use

• Input BAM file is not coordinate-sorted; use samtools sort -c to validate or re-sort first.
• Fragment file already exists and has been validated; re-running is redundant unless BAM has been re-aligned.
• Working with RNA-seq or other modalities that do not generate fragment-level data (use pp.import_fragments for pre-existing fragment files instead).

Inputs

• coordinate-sorted BAM file
• BAM file with cell barcode information in header (for 10X or tagged BAM)

Outputs

• compressed fragment file (BED.gz or .zst format)
• fragment coordinates with cell barcodes and quality metrics

How to apply

Load the coordinate-sorted BAM file and invoke pp.make_fragment_file with source='10x' if the input is a 10X BAM file, or with default settings for standard BAM inputs. The function processes alignments, extracts fragment coordinates (chromosome, start, end), assigns cell barcodes from the BAM header, and computes quality metrics including duplication rate. Output is generated in compressed format (BED.gz or zst) with standard BED fields plus barcode and count columns. Validate output by confirming the file is non-empty, contains properly formatted BED columns, and that QC metrics (duplication rate, read counts per cell) are accessible for downstream QC decisions.

Related tools

• SnapATAC2 (Python/Rust framework providing pp.make_fragment_file function for BAM-to-fragment conversion with built-in quality metrics and barcode extraction) — https://github.com/scverse/SnapATAC2
• precellar (Alternative universal preprocessing package that automates BAM-to-fragment conversion for multiple single-cell genomics platforms via alignment and fragment file generation) — https://github.com/regulatory-genomics/precellar
• samtools (Utility for BAM file validation, sorting, and inspection before fragment file conversion)