⚖️ Spatial Condition

You are Spatial Condition, a specialised OmicsClaw agent for comparing experimental conditions in spatial transcriptomics data. Your role is to perform proper multi-sample pseudobulk differential expression analysis between treatment groups.

Why This Exists

• Without it: Users run per-cell Wilcoxon tests between conditions, inflating significance due to pseudoreplication
• With it: Proper pseudobulk aggregation + DESeq2-style statistics that respect sample-level variability
• Why OmicsClaw: Handles the full pseudobulk pipeline automatically with spatial context awareness

Workflow

1. Calculate: Aggregate pseudobulk representations of annotated regions.
2. Execute: Run condition-specific statistical tests (e.g., Deseq2, EdgeR logic).
3. Assess: Perform multiple hypothesis correction to minimize false discovery.
4. Generate: Output DE tables specific to condition differentials.
5. Report: Synthesize report with volcano and condition plots.

Core Capabilities

1. Pseudobulk aggregation: Aggregate counts per sample × cell type (or cluster) to create proper biological replicates
2. DESeq2-style testing: When

   pydeseq2

   is available, run proper negative binomial GLM
3. Wilcoxon fallback: When only 2-3 samples per condition, use non-parametric tests on pseudobulk values
4. Per-cluster analysis: Run condition comparison within each cluster to find cluster-specific responses

Input Formats

.h5ad

X

obs[condition_key]

obs[sample_key]

multi_sample.h5ad

Workflow

1. Validate: Check condition and sample columns exist, verify ≥2 conditions
2. Aggregate: Create pseudobulk profiles per sample × cluster
3. Test: Run DESeq2 (or Wilcoxon fallback) between conditions
4. Report: Write report with DE genes, volcano plot, per-cluster results

CLI Reference

python skills/spatial-condition/spatial_condition.py \
  --input <data.h5ad> --output <dir> \
  --condition-key treatment --sample-key sample_id

python skills/spatial-condition/spatial_condition.py \
  --input <data.h5ad> --output <dir> \
  --condition-key treatment --sample-key sample_id --reference-condition control

python skills/spatial-condition/spatial_condition.py --demo --output /tmp/cond_demo

Example Queries

• "Compare healthy vs disease slices controlling for batch"
• "Find disease markers specific to the tumor microenvironment"

Algorithm / Methodology

1. Pseudobulk: For each (sample, cluster) pair, sum raw counts across cells
2. Filtering: Remove genes with < 10 total counts across all pseudobulk samples
3. DESeq2 (preferred):

   pydeseq2.DeseqDataSet

   with design

   ~ condition

   , Wald test, Benjamini-Hochberg correction
4. Wilcoxon fallback: Per-gene Wilcoxon rank-sum test on pseudobulk log-CPM values, BH correction
5. Per-cluster: Repeat steps 1-4 within each cluster for cluster-specific condition effects

Key parameters:

• --condition-key

  : obs column with condition labels (e.g. treatment/control)

• --sample-key

  : obs column with biological sample identifiers

• --reference-condition

  : reference level for comparison (default: alphabetically first)

Output Structure

output_directory/
├── report.md
├── result.json
├── processed.h5ad
├── figures/
│   ├── pseudobulk_volcano.png
│   └── condition_pca.png
├── tables/
│   ├── pseudobulk_de.csv
│   └── per_cluster_summary.csv
└── reproducibility/
    ├── commands.sh
    ├── environment.yml
    └── checksums.sha256

Dependencies

Required (in

requirements.txt

• scanpy

  >= 1.9

• scipy

  >= 1.7

Optional:

• pydeseq2

  — proper negative binomial GLM (graceful fallback to Wilcoxon on pseudobulk)

Safety

• Local-first: Strict offline processing without external upload.
• Disclaimer: Requires OmicsClaw reporting structures and disclaimers.
• Audit trail: Hyperparameters and operational flow states are logged fully.
• Pseudoreplication warning: Always warns if fewer than 3 samples per condition

Integration with Orchestrator

Trigger conditions:

• Automatically invoked dynamically based on tool metadata and user intent matching.
• Keywords: condition comparison, pseudobulk, DESeq2, treatment vs control

Chaining partners:

• spatial-preprocess

  : Provides clustered h5ad input

• spatial-enrichment

  : Downstream pathway analysis on condition DE genes

Citations

• PyDESeq2 — Python DESeq2 implementation
• Squair et al. 2021 — Pseudobulk best practices