Fragment based count matrix generation

fragment-based-count-matrix-generation

Summary

Generate tile-based count matrices from single-cell ATAC-seq fragment data using paired-insertion counting strategy in SnapATAC2. This skill quantifies chromatin accessibility at fixed genomic intervals and produces dense or sparse count matrices suitable for downstream dimensionality reduction and clustering.

When to use

You have a backed AnnData object containing processed fragment data (stored in .obsm['fragment_paired'] or .obsm['fragment_single']) from single-cell ATAC-seq and need to convert raw genomic fragments into a quantitative count matrix indexed by fixed genomic tiles for clustering, embedding, or differential analysis.

When NOT to use

• Input data is already in the form of a peak-by-cell matrix or gene-by-cell matrix; use pp.make_peak_matrix or pp.make_gene_matrix instead.
• Fragment file has not been imported or processed; first convert BAM to fragment file using pp.make_fragment_file or pp.import_fragments.
• You need counts at peak regions rather than fixed tiles; use pp.make_peak_matrix with called or external peak coordinates.

Inputs

• backed AnnData object with fragment data in .obsm['fragment_paired'] or .obsm['fragment_single']
• genomic fragment coordinates (chromosome, start, end, barcode)

Outputs

• count matrix (n_obs × n_vars) indexed by genomic tiles and cell barcodes
• tile coordinate metadata (chromosome, start, end positions for each tile)
• sparse matrix with paired-insertion counts per cell per tile

How to apply

Load the backed AnnData object containing fragment coordinates and invoke pp.add_tile_matrix with counting_strategy='paired_insertion' to aggregate fragment counts across non-overlapping genomic tiles (fixed intervals, typically 5 kb). The function processes paired-end fragments by counting insertions at each genomic position, then sums counts within each tile to generate a sparse count matrix with dimensions n_obs (cells) × n_vars (tiles). Verify the resulting matrix shape matches the number of cells and the expected number of tiles, and confirm that count values are non-zero and reasonably distributed across the tile-cell space (no unexpected sparsity or skew).

Related tools

• SnapATAC2 (Provides pp.add_tile_matrix function to generate tile-based count matrices from fragment data with paired-insertion counting strategy) — https://github.com/scverse/SnapATAC2
• precellar (Upstream preprocessing tool to convert raw fastq files and BAM alignments into fragment files compatible with SnapATAC2 count matrix generation) — https://github.com/regulatory-genomics/precellar
• Python (Host language for SnapATAC2 API and data manipulation)

Examples

import snapatac2 as snap; adata = snap.read('fragments.h5ad', backed='r'); snap.pp.add_tile_matrix(adata, counting_strategy='paired_insertion'); print(adata.X.shape)

Evaluation signals

• Count matrix shape (n_obs × n_vars) matches the number of cells and expected number of genomic tiles (e.g., ~600,000 tiles for 5 kb resolution across human genome).
• Matrix contains non-zero entries distributed across both cells and tiles; median non-zero count per cell is consistent with expected fragment complexity (typically 1,000–100,000 fragments per cell).
• No cells have zero counts across all tiles; no tiles have zero counts across all cells (or if present, they represent genuine biological absence, not data corruption).
• Sparsity (fraction of zero entries) is within expected range for ATAC-seq (typically 95–99% sparse); extremely dense or sparse matrices may indicate counting or filtering errors.
• Count distribution is right-skewed with a long tail, consistent with chromatin accessibility patterns (some tiles have high accessibility, most have low).

Limitations

• Tile-based counting does not account for peak structure; accessibility hotspots may be smoothed across multiple tiles. For peak-aware analysis, use pp.make_peak_matrix instead.
• Fixed tile size (typically 5 kb) is a compromise between resolution and noise; smaller tiles increase sparsity, larger tiles sacrifice resolution. Choice should be motivated by downstream analysis goal and cell-type complexity.
• Paired-insertion counting assumes high-quality paired-end sequencing and correct fragment coordinate assignment; errors in fragment end prediction or barcode assignment will propagate into count matrix.
• SnapATAC2 scales to >10 million cells but matrix generation time and memory scale with both cell count and number of tiles; very large tile matrices may require disk-backed sparse formats.

Evidence

• [other] SnapATAC2 provides a pp.add_tile_matrix function that generates count matrices from fragment data as part of its matrix operation workflow for single-cell ATAC-seq analysis.: "SnapATAC2 provides a pp.add_tile_matrix function that generates count matrices from fragment data as part of its matrix operation workflow for single-cell ATAC-seq analysis."
• [other] Invoke pp.add_tile_matrix with counting_strategy='paired_insertion' to quantify chromatin accessibility at fixed genomic intervals.: "Invoke pp.add_tile_matrix with counting_strategy='paired_insertion' to quantify chromatin accessibility at fixed genomic intervals."
• [other] Load backed AnnData object containing fragment data (stored in .obsm['fragment_paired'] or .obsm['fragment_single']).: "Load backed AnnData object containing fragment data (stored in .obsm['fragment_paired'] or .obsm['fragment_single'])."
• [readme] Blazingly fast preprocessing tools for BAM to fragment files conversion and count matrix generation.: "Blazingly fast preprocessing tools for BAM to fragment files conversion and count matrix generation."
• [methods] Matrix operation including pp.add_tile_matrix, pp.make_peak_matrix, pp.make_gene_matrix: "Matrix operation including pp.add_tile_matrix, pp.make_peak_matrix, pp.make_gene_matrix"