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sc-perturb-prep

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Load when attaching cell-barcode → sgRNA assignments from a mapping TSV/CSV onto a Perturb-seq expression AnnData, producing standardised perturbation / sgRNA / target-gene obs columns. Skip when the AnnData already has perturbation labels (go straight to sc-perturb) or for raw guide-calling from FASTQ (use upstream demuxlet / cellranger guide pipelines).

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sc-perturb-prep

When to use

The user has Perturb-seq expression data (10x h5 / matrix dir / h5ad) and an upstream barcode-to-sgRNA mapping table (TSV / CSV from demultiplex / cellranger / cellbender output) and needs them merged into a single AnnData with:

  • obs[--pert-key] (default perturbation) — canonical perturbation label
  • obs[--sgrna-key] (default sgRNA) — guide identifier
  • obs[--target-key] (default target_gene) — inferred target gene
  • obs["assignment_status"] — single_guide / multi_guide / unassigned
  • obs["n_sgrnas"] — count per cell

Single backend: mapping_tsv. Then chain to sc-perturb for Mixscape classification. This skill does NOT infer guide identities from FASTQ — bring an upstream assignment table.

Inputs & Outputs

InputFormatRequired
Expression.h5ad / 10x .h5 / 10x diryes (unless --demo)
Barcode → sgRNA mapping.tsv / .csv (--mapping-file)yes (unless --demo)
Column overrides--barcode-column / --sgrna-column / --target-column / --sepoptional (auto-detect by default)
OutputPathNotes
Standardised AnnDataprocessed.h5adadds obs["perturbation"] / obs["sgRNA"] / obs["target_gene"] / obs["assignment_status"] / obs["n_sgrnas"]; non-gene features removed from var
Per-cell assignmentstables/perturbation_assignments.csvalways
Status countstables/assignment_status_counts.csvsingle_guide / multi_guide / unassigned tally
Perturbation tallytables/perturbation_counts.csvcells per perturbation label
Dropped multi-guidetables/dropped_multi_guide_cells.csvwhen multi-guide cells were dropped
Feature summarytables/feature_type_summary.csvgene vs non-gene feature breakdown
Figurefigures/perturbation_counts.pngalways
Reportreport.md + result.jsonalways

Flow

  1. Load expression input via smart_load; load mapping via load_sgrna_mapping (auto-detect --sep if unset).
  2. Strip non-gene features from var (e.g., 10x guide / antibody capture rows).
  3. Collapse mapping rows per cell: tag single_guide / multi_guide / unassigned based on row count.
  4. Drop multi_guide cells unless --keep-multi-guide is set.
  5. Match each sgRNA against --control-patterns (,-separated, includes default NT-style patterns); rewrite matches to --control-label (default NT).
  6. Infer target gene from sgRNA ID via --delimiter + --gene-position, OR use --target-column from the mapping if provided.
  7. Save standardised AnnData, tables, figure, report.md, result.json.

Gotchas

  • All preflight failures raise SystemExit, not ValueError. sc_perturb_prep.py:205 raises SystemExit("Provide --input or use --demo"); :207 raises SystemExit("Perturbation preparation requires --mapping-file for real inputs. Generate barcode-to-guide assignments upstream first.") when --mapping-file is missing on a real (non-demo) run. Wrappers expecting standard ValueError need to catch SystemExit.
  • multi_guide cells are DROPPED by default. Step 4 in the flow filters them out unless --keep-multi-guide is passed. result.json["n_cells_multi_guide_dropped"] (line 267 / 362) records the count. If your screen has high MOI on purpose (combinatorial perturbations), --keep-multi-guide is mandatory.
  • Target gene is inferred by default, not read from mapping. Without --target-column, the script splits the sgRNA ID by --delimiter (default _) and takes token at --gene-position (default 0). For sgRNA IDs like EGFR_sg1 this gives EGFR; for non-standard formats (sg-EGFR-1, EGFR.sg1) you must pass --delimiter accordingly or supply --target-column.
  • Control matching is pattern-based, not exact. --control-patterns (default from DEFAULT_CONTROL_PATTERNS) is a comma-separated list — any sgRNA whose ID contains one of the patterns is rewritten to --control-label (default NT). False positives are possible if a real guide's ID contains a control-pattern substring; review tables/perturbation_assignments.csv after the run.
  • Non-gene features in var are silently removed only when var["feature_types"] exists. sc_perturb_prep.py:222 calls keep_gene_expression_features(adata); the helper early-returns the unchanged AnnData if feature_types isn't a var column (typical for user-loaded h5ads). When it IS present (e.g., 10x cellranger output), antibody-capture / guide-capture rows are stripped silently and result.json["n_non_gene_features_removed"] records the count. A 0 value means either the column was absent or there were no non-gene rows to remove.

Key CLI

# Demo (synthetic expression + mapping)
python omicsclaw.py run sc-perturb-prep --demo --output /tmp/sc_perturb_prep_demo

# Real run with auto-detected mapping columns + delimiter
python omicsclaw.py run sc-perturb-prep \
  --input cellranger/raw_feature_bc_matrix.h5 \
  --mapping-file guide_assignments.tsv \
  --output results/

# Custom column names + non-default delimiter
python omicsclaw.py run sc-perturb-prep \
  --input expression.h5ad \
  --mapping-file mapping.csv \
  --barcode-column cell_id --sgrna-column guide_id --target-column gene \
  --sep ',' --delimiter '-' --gene-position 1 \
  --output results/

# Keep combinatorial multi-guide cells (high-MOI screens)
python omicsclaw.py run sc-perturb-prep \
  --input expression.h5ad --mapping-file mapping.tsv \
  --keep-multi-guide --output results/

See also

  • references/parameters.md — every CLI flag, mapping-file conventions
  • references/methodology.md — assignment status semantics; control-pattern matching
  • references/output_contract.md — obs["perturbation"] / obs["sgRNA"] / obs["target_gene"] / obs["assignment_status"] schema
  • Adjacent skills: sc-count / sc-multi-count (upstream — produces the expression matrix; the mapping comes from cellranger / demuxlet output), sc-perturb (downstream — Mixscape classification on the standardised AnnData), sc-de (alternative downstream — direct DE between perturbed and control without Mixscape)