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sc-preprocessing

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Load when normalising QC'd scRNA into a PCA-ready AnnData via scanpy / Seurat / SCTransform / Pearson residuals. Skip when QC thresholds are still undecided (use sc-qc) or for batch correction across samples (use sc-batch-integration).

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sc-preprocessing

When to use

The user has a filtered, QC-annotated AnnData and wants the standard "normalise → HVG → PCA" pipeline before clustering or batch integration. Four interchangeable backends are available: scanpy (default; CP10k log + HVG seurat flavour), seurat (R-backed LogNormalize / CLR / RC), sctransform (R-backed regularised NB), and pearson_residuals (raw-count HVG selection plus Pearson residual transformation). The skill stops at PCA — UMAP / clustering live in sc-clustering, multi-sample correction in sc-batch-integration.

Inputs & Outputs

InputFormatRequired
Filtered AnnData.h5ad (raw-count-like or QC-annotated)yes (unless --demo)
OutputPathNotes
Processed AnnDataprocessed.h5adX = normalised, layers["counts"] = raw, obsm["X_pca"], var["highly_variable"], adata.raw populated
Run summarytables/preprocess_summary.csvalways
HVG tabletables/hvg_summary.csvtop-N HVGs
PCA variancetables/pca_variance_ratio.csvper-PC variance + cumulative
PCA embeddingtables/pca_embedding.csvfirst 5 PCs per cell
Per-cell QCtables/qc_metrics_per_cell.csvretained QC metrics
Reportreport.md + result.jsonalways

Flow

  1. Load AnnData; infer species; canonicalise gene-name / expression layout via the shared single-cell standardiser.
  2. Reuse existing QC if n_genes_by_counts / total_counts / pct_counts_mt are present in obs; otherwise compute them.
  3. Apply shared filtering (--min-genes, --min-cells, --max-mt-pct); drop doublets when predicted_doublet / doublet_score columns are present (opt out via --no-remove-doublets).
  4. Run the chosen normalisation backend (scanpy / seurat / sctransform / pearson_residuals).
  5. Select HVGs (--n-top-hvg) and compute PCA (--n-pcs).
  6. Save processed.h5ad, tables, figures, report.md, result.json.

Gotchas

  • result.json["n_pcs_used"] may be smaller than the requested --n-pcs. sc_preprocess.py:876 reads obsm["X_pca"].shape[1] after PCA — small matrices cap the count below the request. Trust n_pcs_used, not the input flag, when handing off to sc-clustering --n-pcs.
  • R-backed seurat / sctransform need a working Rscript env. sc_preprocess.py:271 raises RuntimeError("Seurat preprocessing returned no overlapping cells or genes") when the R round-trip empties the matrix; sc_preprocess.py:296 raises RuntimeError("Seurat preprocessing returned PCA rows that do not align with exported cells") when the R-side PCA shape disagrees with the cell list. Confirm Seurat, SingleCellExperiment, zellkonverter (and sctransform for that method) are installed before picking these methods.
  • Doublet filter is on-by-default whenever sc-doublet-detection ran. sc_preprocess.py:510-511 passes filter_doublets=True and doublet_score_threshold=0.25 when those columns exist in obs. To keep the called-doublet rows, pass --no-remove-doublets.
  • figure_data/gene_expression.csv write failures are silent. sc_preprocess.py:670-672 catches the exception and only logs a warning — figure_data/manifest.json is the source of truth for which figure-data files actually landed.
  • --input is mandatory unless --demo. sc_preprocess.py:1013 raises ValueError("--input required when not using --demo").

Key CLI

# Demo (built-in synthetic data)
python omicsclaw.py run sc-preprocessing --demo --output /tmp/sc_preprocess_demo

# Default scanpy backend
python omicsclaw.py run sc-preprocessing \
  --input filtered.h5ad --output results/

# R-backed Seurat LogNormalize
python omicsclaw.py run sc-preprocessing \
  --input filtered.h5ad --output results/ \
  --method seurat --seurat-normalize-method LogNormalize

# Pearson residuals (recommended for very sparse / heterogeneous data)
python omicsclaw.py run sc-preprocessing \
  --input filtered.h5ad --output results/ \
  --method pearson_residuals --n-top-hvg 3000

See also

  • references/parameters.md — every CLI flag and per-method tuning hint
  • references/methodology.md — when each backend wins; canonicalisation contract
  • references/output_contract.md — obs / obsm / layers / uns schema + table layouts
  • Adjacent skills: sc-qc / sc-filter (upstream — produce the input), sc-batch-integration (parallel — multi-sample alternative path; consumes obsm["X_pca"]), sc-clustering (downstream — consumes obsm["X_pca"] for neighbour-graph + UMAP + Leiden)