@bgicli/bgicli 2.1.1 → 2.2.0

package/data/skills/tooluniverse-spatial-transcriptomics/SKILL.md

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+---
+name: tooluniverse-spatial-transcriptomics
+description: Analyze spatial transcriptomics data to map gene expression in tissue architecture. Supports 10x Visium, MERFISH, seqFISH, Slide-seq, and imaging-based platforms. Performs spatial clustering, domain identification, cell-cell proximity analysis, spatial gene expression patterns, tissue architecture mapping, and integration with single-cell data. Use when analyzing spatial transcriptomics datasets, studying tissue organization, identifying spatial expression patterns, mapping cell-cell interactions in tissue context, characterizing tumor microenvironment spatial structure, or integrating spatial and single-cell RNA-seq data for comprehensive tissue analysis.
+---
+
+# Spatial Transcriptomics Analysis
+
+Comprehensive analysis of spatially-resolved transcriptomics data to understand gene expression patterns in tissue architecture context. Combines expression profiling with spatial coordinates to reveal tissue organization, cell-cell interactions, and spatially variable genes.
+
+## When to Use This Skill
+
+**Triggers**:
+- User has spatial transcriptomics data (Visium, MERFISH, seqFISH, etc.)
+- Questions about tissue architecture or spatial organization
+- Spatial gene expression pattern analysis
+- Cell-cell proximity or neighborhood analysis requests
+- Tumor microenvironment spatial structure questions
+- Integration of spatial with single-cell data
+- Spatial domain identification
+- Tissue morphology correlation with expression
+
+**Example Questions This Skill Solves**:
+1. "Analyze this 10x Visium dataset to identify spatial domains"
+2. "Which genes show spatially variable expression in this tissue?"
+3. "Map the tumor microenvironment spatial organization"
+4. "Find genes enriched at tissue boundaries"
+5. "Identify cell-cell interactions based on spatial proximity"
+6. "Integrate spatial transcriptomics with scRNA-seq annotations"
+7. "Characterize spatial gradients in gene expression"
+8. "Map ligand-receptor pairs in tissue context"
+
+---
+
+## Core Capabilities
+
+| Capability | Description |
+|-----------|-------------|
+| **Data Import** | 10x Visium, MERFISH, seqFISH, Slide-seq, STARmap, Xenium formats |
+| **Quality Control** | Spot/cell QC, spatial alignment verification, tissue coverage |
+| **Normalization** | Spatial-aware normalization accounting for tissue heterogeneity |
+| **Spatial Clustering** | Identify spatial domains with similar expression profiles |
+| **Spatial Variable Genes** | Find genes with non-random spatial patterns |
+| **Neighborhood Analysis** | Cell-cell proximity, spatial neighborhoods, niche identification |
+| **Spatial Patterns** | Gradients, boundaries, hotspots, expression waves |
+| **Integration** | Merge with scRNA-seq for cell type mapping |
+| **Ligand-Receptor Spatial** | Map cell communication in tissue context |
+| **Visualization** | Spatial plots, heatmaps on tissue, 3D reconstruction |
+
+---
+
+## Workflow Overview
+
+```
+Input: Spatial Transcriptomics Data + Tissue Image
+    |
+    v
+Phase 1: Data Import & QC
+    |-- Load spatial coordinates + expression matrix
+    |-- Load tissue histology image
+    |-- Quality control per spot/cell
+    |-- Filter low-quality spots
+    |-- Align spatial coordinates to tissue
+    |
+    v
+Phase 2: Preprocessing
+    |-- Normalization (spatial-aware methods)
+    |-- Highly variable gene selection
+    |-- Dimensionality reduction (PCA)
+    |-- Spatial lag smoothing (optional)
+    |
+    v
+Phase 3: Spatial Clustering
+    |-- Identify spatial domains/regions
+    |-- Graph-based clustering with spatial constraints
+    |-- Annotate domains with marker genes
+    |-- Visualize domains on tissue
+    |
+    v
+Phase 4: Spatial Variable Genes
+    |-- Test for spatial autocorrelation (Moran's I, Geary's C)
+    |-- Identify genes with spatial patterns
+    |-- Classify pattern types (gradient, hotspot, boundary)
+    |-- Rank by spatial significance
+    |
+    v
+Phase 5: Neighborhood Analysis
+    |-- Define spatial neighborhoods (k-NN, radius)
+    |-- Calculate neighborhood composition
+    |-- Identify interaction zones
+    |-- Niche characterization
+    |
+    v
+Phase 6: Integration with scRNA-seq
+    |-- Cell type deconvolution per spot
+    |-- Map cell types to spatial locations
+    |-- Predict cell type spatial distributions
+    |-- Validate with marker genes
+    |
+    v
+Phase 7: Spatial Cell Communication
+    |-- Identify proximal cell type pairs
+    |-- Query ligand-receptor database (OmniPath)
+    |-- Score spatial interactions
+    |-- Map communication hotspots
+    |
+    v
+Phase 8: Generate Spatial Report
+    |-- Tissue overview with domains
+    |-- Spatially variable genes
+    |-- Cell type spatial maps
+    |-- Interaction networks in tissue context
+    |-- 3D visualization (if applicable)
+```
+
+---
+
+## Phase Details
+
+### Phase 1: Data Import & Quality Control
+
+**Objective**: Load spatial data and assess quality.
+
+**Supported platforms**:
+
+**10x Visium** (most common):
+- Spots: 55μm diameter, ~50 cells per spot
+- Resolution: ~5,000-10,000 spots per capture area
+- Data: Expression matrix + spatial coordinates + H&E image
+
+**MERFISH/seqFISH** (imaging-based):
+- Single-cell resolution
+- Targeted gene panels (100-10,000 genes)
+- Absolute coordinates per cell
+
+**Slide-seq/Slide-seqV2**:
+- 10μm bead resolution
+- Genome-wide profiling
+
+**Xenium** (10x single-cell spatial):
+- Single-cell resolution
+- Large gene panels (300+ genes)
+- Subcellular resolution
+
+**Data loading (Visium)**:
+```python
+def load_visium_data(data_dir):
+    """
+    Load 10x Visium spatial transcriptomics data.
+
+    Expected structure:
+    data_dir/
+      ├── filtered_feature_bc_matrix/
+      │   ├── barcodes.tsv.gz
+      │   ├── features.tsv.gz
+      │   └── matrix.mtx.gz
+      ├── spatial/
+      │   ├── tissue_positions_list.csv
+      │   ├── scalefactors_json.json
+      │   └── tissue_hires_image.png
+
+    Returns: AnnData object with spatial coordinates
+    """
+    import scanpy as sc
+    import pandas as pd
+
+    # Load expression data
+    adata = sc.read_visium(data_dir)
+
+    # Spatial coordinates are in adata.obsm['spatial']
+    # Tissue image in adata.uns['spatial']
+
+    return adata
+```
+
+**Quality Control**:
+
+1. **Spot-level QC**:
+```python
+def spatial_qc(adata):
+    """
+    Quality control for spatial transcriptomics data.
+    """
+    import scanpy as sc
+
+    # Calculate QC metrics
+    sc.pp.calculate_qc_metrics(adata, inplace=True)
+
+    # Visualize QC metrics spatially
+    sc.pl.spatial(adata, color='n_genes_by_counts', title='Genes per Spot')
+    sc.pl.spatial(adata, color='total_counts', title='UMI Counts per Spot')
+
+    # Filter criteria
+    # - Min 200 genes per spot
+    # - Min 500 UMI counts per spot
+    # - Max mitochondrial content < 20%
+
+    sc.pp.filter_cells(adata, min_genes=200)
+    sc.pp.filter_cells(adata, min_counts=500)
+
+    # Mitochondrial filtering
+    adata.var['mt'] = adata.var_names.str.startswith('MT-')
+    sc.pp.calculate_qc_metrics(adata, qc_vars=['mt'], inplace=True)
+    adata = adata[adata.obs['pct_counts_mt'] < 20].copy()
+
+    return adata
+```
+
+2. **Spatial alignment verification**:
+```python
+def verify_spatial_alignment(adata):
+    """
+    Verify spatial coordinates align with tissue image.
+    """
+    import matplotlib.pyplot as plt
+
+    # Plot spots on tissue image
+    fig, ax = plt.subplots(figsize=(10, 10))
+
+    # Tissue image
+    img = adata.uns['spatial']['tissue_hires_image']
+    ax.imshow(img)
+
+    # Overlay spot coordinates
+    coords = adata.obsm['spatial']
+    ax.scatter(coords[:, 0], coords[:, 1], c='red', s=1, alpha=0.5)
+
+    ax.set_title('Spatial Alignment Verification')
+    plt.axis('off')
+```
+
+### Phase 2: Preprocessing & Normalization
+
+**Objective**: Normalize data accounting for spatial heterogeneity.
+
+**Normalization**:
+```python
+def normalize_spatial(adata):
+    """
+    Normalize spatial transcriptomics data.
+    """
+    import scanpy as sc
+
+    # Filter genes (min 3 spots)
+    sc.pp.filter_genes(adata, min_cells=3)
+
+    # Normalize to median total counts
+    sc.pp.normalize_total(adata, target_sum=1e4)
+
+    # Log-transform
+    sc.pp.log1p(adata)
+
+    # Store raw counts
+    adata.raw = adata
+
+    return adata
+```
+
+**Highly variable genes**:
+```python
+def select_hvg_spatial(adata):
+    """
+    Select highly variable genes for spatial analysis.
+    """
+    import scanpy as sc
+
+    # Standard HVG selection
+    sc.pp.highly_variable_genes(adata, n_top_genes=2000)
+
+    # Optionally: weight by spatial autocorrelation
+    # Genes with spatial patterns are more informative
+
+    return adata
+```
+
+**Spatial smoothing** (optional):
+```python
+def spatial_smooth(adata, radius=2):
+    """
+    Smooth expression by averaging over spatial neighbors.
+
+    Useful for noisy data, but can blur boundaries.
+    """
+    from sklearn.neighbors import NearestNeighbors
+
+    # Find spatial neighbors
+    coords = adata.obsm['spatial']
+    nn = NearestNeighbors(n_neighbors=radius, metric='euclidean')
+    nn.fit(coords)
+    distances, indices = nn.kneighbors(coords)
+
+    # Smooth expression matrix
+    X_smooth = adata.X.copy()
+    for i in range(adata.n_obs):
+        neighbors = indices[i]
+        X_smooth[i] = adata.X[neighbors].mean(axis=0)
+
+    adata.layers['smoothed'] = X_smooth
+
+    return adata
+```
+
+### Phase 3: Spatial Clustering
+
+**Objective**: Identify spatial domains (regions with distinct expression).
+
+**Graph-based clustering with spatial constraints**:
+```python
+def spatial_clustering(adata, n_neighbors=6):
+    """
+    Cluster spots into spatial domains.
+
+    Uses both expression similarity AND spatial proximity.
+    """
+    import scanpy as sc
+    import squidpy as sq
+
+    # PCA for dimensionality reduction
+    sc.pp.pca(adata, n_comps=50)
+
+    # Build spatial neighbor graph
+    sq.gr.spatial_neighbors(adata, coord_type='generic', n_neighs=n_neighbors)
+
+    # Clustering with spatial constraints
+    # Uses both PCA space and spatial graph
+    sc.tl.leiden(adata, resolution=1.0, key_added='spatial_domain')
+
+    # Visualize domains on tissue
+    sc.pl.spatial(adata, color='spatial_domain', title='Spatial Domains')
+
+    return adata
+```
+
+**Domain marker genes**:
+```python
+def find_domain_markers(adata):
+    """
+    Identify marker genes for each spatial domain.
+    """
+    import scanpy as sc
+
+    # Differential expression per domain
+    sc.tl.rank_genes_groups(adata, groupby='spatial_domain', method='wilcoxon')
+
+    # Get top markers per domain
+    markers = sc.get.rank_genes_groups_df(adata, group=None)
+
+    return markers
+```
+
+### Phase 4: Spatially Variable Genes
+
+**Objective**: Find genes with non-random spatial patterns.
+
+**Moran's I (spatial autocorrelation)**:
+```python
+def identify_spatial_genes(adata):
+    """
+    Test for spatial autocorrelation using Moran's I.
+
+    Moran's I > 0: Positive spatial autocorrelation (clustering)
+    Moran's I ~ 0: Random spatial distribution
+    Moran's I < 0: Negative autocorrelation (checkerboard)
+    """
+    import squidpy as sq
+
+    # Calculate Moran's I for all genes
+    sq.gr.spatial_autocorr(
+        adata,
+        mode='moran',
+        n_perms=100,
+        n_jobs=-1
+    )
+
+    # Results in adata.uns['moranI']
+    spatial_genes = adata.uns['moranI'].sort_values('I', ascending=False)
+
+    # Filter significant spatial genes (FDR < 0.05)
+    sig_spatial = spatial_genes[spatial_genes['pval_norm_fdr_bh'] < 0.05]
+
+    return sig_spatial
+```
+
+**Spatial pattern classification**:
+```python
+def classify_spatial_patterns(adata, spatial_genes):
+    """
+    Classify types of spatial patterns.
+
+    Pattern types:
+    - Gradient: Smooth directional change
+    - Hotspot: Localized high expression
+    - Boundary: Expression at domain edges
+    - Periodic: Regular spacing
+    """
+    patterns = {}
+
+    for gene in spatial_genes.index[:100]:  # Top 100 spatial genes
+        # Get expression and coordinates
+        expr = adata[:, gene].X.toarray().flatten()
+        coords = adata.obsm['spatial']
+
+        # Detect pattern type
+        pattern_type = detect_pattern_type(expr, coords)
+        patterns[gene] = pattern_type
+
+    return patterns
+```
+
+### Phase 5: Neighborhood Analysis
+
+**Objective**: Analyze cell-cell proximity and spatial niches.
+
+**Define spatial neighborhoods**:
+```python
+def analyze_neighborhoods(adata, radius=150):
+    """
+    Analyze spatial neighborhood composition.
+
+    For each spot, characterize its microenvironment.
+    """
+    import squidpy as sq
+
+    # Calculate neighborhood enrichment
+    # Tests if cell types are enriched in proximity
+    sq.gr.nhood_enrichment(adata, cluster_key='spatial_domain')
+
+    # Visualize neighborhood enrichment
+    sq.pl.nhood_enrichment(adata, cluster_key='spatial_domain')
+
+    # Results: which domains are spatially proximal?
+
+    return adata
+```
+
+**Interaction zones**:
+```python
+def identify_interaction_zones(adata, domain_a, domain_b):
+    """
+    Find boundary regions between two spatial domains.
+
+    These are hotspots for cell-cell interactions.
+    """
+    # Get spots from each domain
+    spots_a = adata.obs['spatial_domain'] == domain_a
+    spots_b = adata.obs['spatial_domain'] == domain_b
+
+    # Find spots that neighbor the other domain
+    # (spots from A that have neighbors in B)
+    coords = adata.obsm['spatial']
+    from sklearn.neighbors import NearestNeighbors
+
+    nn = NearestNeighbors(n_neighbors=6)
+    nn.fit(coords)
+    distances, indices = nn.kneighbors(coords)
+
+    interaction_spots = []
+    for i, spot_in_a in enumerate(spots_a):
+        if spot_in_a:
+            neighbors = indices[i]
+            if any(spots_b[neighbors]):
+                interaction_spots.append(i)
+
+    # Mark interaction zone
+    adata.obs['interaction_zone'] = False
+    adata.obs.loc[interaction_spots, 'interaction_zone'] = True
+
+    return adata
+```
+
+### Phase 6: Integration with Single-Cell RNA-seq
+
+**Objective**: Map cell types from scRNA-seq to spatial locations.
+
+**Cell type deconvolution**:
+```python
+def deconvolve_cell_types(adata_spatial, adata_sc):
+    """
+    Predict cell type composition per spatial spot.
+
+    Uses scRNA-seq reference to deconvolve Visium spots.
+    Methods: Cell2location, Tangram, SPOTlight
+    """
+    import cell2location
+
+    # Prepare single-cell reference
+    # Extract signature genes per cell type
+    cell_type_signatures = extract_signatures(adata_sc)
+
+    # Run cell2location
+    # Estimates cell type abundances per spot
+    mod = cell2location.models.Cell2location(
+        adata_spatial,
+        cell_state_df=cell_type_signatures
+    )
+
+    mod.train(max_epochs=30000)
+
+    # Add cell type proportions to adata_spatial
+    adata_spatial.obsm['cell_type_fractions'] = mod.get_cell_type_fractions()
+
+    return adata_spatial
+```
+
+**Spatial cell type mapping**:
+```python
+def map_cell_types_spatial(adata):
+    """
+    Visualize cell type spatial distributions.
+    """
+    import scanpy as sc
+
+    # For each cell type, plot abundance on tissue
+    cell_types = adata.obsm['cell_type_fractions'].columns
+
+    for ct in cell_types:
+        sc.pl.spatial(
+            adata,
+            color=adata.obsm['cell_type_fractions'][ct],
+            title=f'{ct} Spatial Distribution'
+        )
+```
+
+### Phase 7: Spatial Cell Communication
+
+**Objective**: Map ligand-receptor interactions in tissue context.
+
+**Spatial proximity-based communication**:
+```python
+def spatial_cell_communication(adata):
+    """
+    Identify cell-cell communication based on spatial proximity.
+
+    Requires:
+    - Cell type annotations (from deconvolution)
+    - Ligand-receptor database (OmniPath)
+    """
+    import squidpy as sq
+    from tooluniverse import ToolUniverse
+
+    tu = ToolUniverse()
+
+    # Get ligand-receptor pairs from OmniPath
+    lr_pairs = tu.run_one_function({
+        "name": "OmniPath_get_ligand_receptor_interactions",
+        "arguments": {"partners": ""}  # Get all pairs
+    })
+
+    # For each cell type pair that are spatially proximal
+    # Calculate interaction scores
+    sq.gr.ligrec(
+        adata,
+        n_perms=100,
+        cluster_key='cell_type',
+        interactions=lr_pairs,
+        copy=False
+    )
+
+    # Visualize significant interactions
+    sq.pl.ligrec(adata, cluster_key='cell_type')
+
+    return adata
+```
+
+**Communication hotspot mapping**:
+```python
+def map_communication_hotspots(adata, ligand, receptor):
+    """
+    Map spatial locations of specific L-R interactions.
+    """
+    import matplotlib.pyplot as plt
+
+    # Get ligand expression
+    ligand_expr = adata[:, ligand].X.toarray().flatten()
+
+    # Get receptor expression
+    receptor_expr = adata[:, receptor].X.toarray().flatten()
+
+    # Interaction score = ligand × receptor
+    interaction_score = ligand_expr * receptor_expr
+
+    # Add to adata
+    adata.obs[f'{ligand}_{receptor}_score'] = interaction_score
+
+    # Visualize on tissue
+    sc.pl.spatial(adata, color=f'{ligand}_{receptor}_score',
+                  title=f'{ligand}-{receptor} Interaction Hotspots')
+```
+
+### Phase 8: Spatial Report Generation
+
+**Generate comprehensive spatial report**:
+
+```markdown
+# Spatial Transcriptomics Analysis Report
+
+## Dataset Summary
+- **Platform**: 10x Visium
+- **Tissue**: Breast cancer tumor section
+- **Spots**: 3,562 (after QC filtering)
+- **Genes**: 18,432 detected
+- **Resolution**: 55μm spot diameter (~50 cells/spot)
+
+## Quality Control
+- **Mean genes per spot**: 3,245
+- **Mean UMI counts**: 12,543
+- **Mitochondrial content**: 8.2% average
+- **Tissue coverage**: 85% of capture area
+
+## Spatial Domains Identified
+- **7 distinct spatial domains** detected via graph-based clustering
+  - Domain 1: Tumor core (32% of tissue)
+  - Domain 2: Invasive margin (18%)
+  - Domain 3: Stromal region (25%)
+  - Domain 4: Immune infiltrate (12%)
+  - Domain 5: Necrotic region (8%)
+  - Domain 6: Normal epithelium (3%)
+  - Domain 7: Adipose tissue (2%)
+
+## Top Marker Genes per Domain
+
+### Domain 1 (Tumor Core)
+- EPCAM, KRT19, MKI67, CCNB1, TOP2A (proliferative tumor)
+
+### Domain 2 (Invasive Margin)
+- VIM, FN1, MMP2, SNAI2 (EMT signature)
+
+### Domain 4 (Immune Infiltrate)
+- CD3D, CD8A, CD4, PTPRC (T cell enriched)
+- CD68, CD14 (macrophage enriched)
+
+## Spatially Variable Genes
+- **456 genes with significant spatial patterns** (Moran's I, FDR < 0.05)
+
+### Top 10 Spatial Genes
+1. **MKI67** (I=0.82) - Hotspot pattern in tumor core
+2. **CD8A** (I=0.78) - Gradient from margin to stroma
+3. **VIM** (I=0.75) - Boundary enrichment at invasive margin
+4. **COL1A1** (I=0.71) - Stromal-specific expression
+5. **EPCAM** (I=0.69) - Tumor region pattern
+
+## Cell Type Deconvolution
+Integration with scRNA-seq reference (Bassez et al. 2021)
+
+### Cell Type Spatial Distributions
+- **Tumor cells**: Concentrated in core, sparse at margin
+- **T cells**: Enriched at invasive margin and infiltrate zones
+- **CAFs**: Stromal region and invasive margin
+- **Macrophages**: Scattered, enriched near necrosis
+- **B cells**: Lymphoid aggregates (2% of tissue)
+
+### Tumor Microenvironment Composition
+- Tumor core: 85% tumor cells, 10% CAFs, 5% immune
+- Invasive margin: 45% tumor, 30% CAFs, 25% immune (T cell rich)
+- Immune infiltrate: 70% T cells, 20% macrophages, 10% B cells
+
+## Spatial Cell Communication
+
+### Top L-R Interactions (Spatially Proximal)
+1. **Tumor → T cell**: CD274 (PD-L1) → PDCD1 (PD-1)
+   - Hotspot: Invasive margin
+   - Interpretation: Immune checkpoint evasion
+2. **CAF → Tumor**: TGFB1 → TGFBR2
+   - Hotspot: Stromal-tumor interface
+   - Interpretation: TGF-β-driven EMT
+3. **Macrophage → Tumor**: TNF → TNFRSF1A
+   - Scattered across tumor
+   - Interpretation: Inflammatory signaling
+
+### Interaction Zones
+- **Tumor-Immune Interface**: 245 spots (7% of tissue)
+  - High expression: CXCL10, CXCL9 (chemokines)
+  - T cell recruitment and activation
+- **Stromal-Tumor Interface**: 387 spots (11% of tissue)
+  - High expression: MMP2, MMP9 (matrix remodeling)
+  - Invasion-promoting niche
+
+## Spatial Gradients
+- **Hypoxia gradient**: HIF1A, VEGFA increase toward tumor core
+- **Proliferation gradient**: MKI67, TOP2A decrease from core to margin
+- **Immune gradient**: CD8A, GZMB peak at invasive margin
+
+## Biological Interpretation
+Spatial analysis reveals distinct tumor microenvironment organization:
+
+1. **Tumor core**: Highly proliferative, hypoxic, immune-excluded
+2. **Invasive margin**: Active EMT, high immune infiltration, checkpoint expression
+3. **Stromal barrier**: CAF-rich, matrix remodeling, immunosuppressive signals
+
+The invasive margin shows hallmarks of immune-tumor interaction with
+PD-L1/PD-1 checkpoint engagement, suggesting potential for checkpoint
+blockade therapy. CAF-mediated TGF-β signaling may drive EMT and therapy
+resistance at tumor-stroma interface.
+
+## Clinical Relevance
+- **Checkpoint inhibitor response**: High immune infiltration at margin suggests potential
+- **Resistance mechanisms**: CAF barrier and TGF-β signaling
+- **Biomarkers**: Spatial arrangement of immune cells more predictive than bulk tumor metrics
+```
+
+---
+
+## Integration with ToolUniverse Skills
+
+| Skill | Used For | Phase |
+|-------|----------|-------|
+| `tooluniverse-single-cell` | scRNA-seq reference for deconvolution | Phase 6 |
+| `tooluniverse-single-cell` (Phase 10) | L-R database for communication | Phase 7 |
+| `tooluniverse-gene-enrichment` | Pathway enrichment for spatial domains | Phase 3 |
+| `tooluniverse-multi-omics-integration` | Integrate with other omics | Phase 8 |
+
+---
+
+## Example Use Cases
+
+### Use Case 1: Tumor Microenvironment Mapping
+
+**Question**: "Map the spatial organization of tumor, immune, and stromal cells"
+
+**Workflow**:
+1. Load Visium data, QC and normalize
+2. Spatial clustering → 7 domains identified
+3. Cell type deconvolution using scRNA-seq reference
+4. Map cell type distributions spatially
+5. Identify interaction zones (tumor-immune, tumor-stroma)
+6. Analyze L-R interactions in each zone
+7. Report: Comprehensive TME spatial architecture
+
+### Use Case 2: Developmental Gradient Analysis
+
+**Question**: "Identify spatial gene expression gradients in developing tissue"
+
+**Workflow**:
+1. Load spatial data (e.g., mouse embryo)
+2. Identify spatially variable genes
+3. Classify gradient patterns (anterior-posterior, dorsal-ventral)
+4. Map morphogen expression (WNT, BMP, FGF)
+5. Correlate with cell fate markers
+6. Report: Developmental spatial patterns
+
+### Use Case 3: Brain Region Identification
+
+**Question**: "Automatically segment brain tissue into anatomical regions"
+
+**Workflow**:
+1. Load Visium mouse brain data
+2. Spatial clustering with high resolution
+3. Match domains to known brain regions (cortex, hippocampus, etc.)
+4. Identify region-specific marker genes
+5. Validate with Allen Brain Atlas
+6. Report: Automated brain region annotation
+
+---
+
+## Quantified Minimums
+
+| Component | Requirement |
+|-----------|-------------|
+| Spots/cells | At least 500 spatial locations |
+| QC | Filter low-quality spots, verify alignment |
+| Spatial clustering | At least one method (graph-based or spatial) |
+| Spatial genes | Moran's I or similar spatial test |
+| Visualization | Spatial plots on tissue images |
+| Report | Domains, spatial genes, visualizations |
+
+---
+
+## Limitations
+
+- **Resolution**: Visium spots contain multiple cells (not single-cell)
+- **Gene coverage**: Imaging methods have limited gene panels
+- **3D structure**: Most platforms are 2D sections
+- **Tissue quality**: Requires well-preserved tissue for imaging
+- **Computational**: Large datasets require significant memory
+- **Reference dependency**: Deconvolution quality depends on scRNA-seq reference
+
+---
+
+## References
+
+**Methods**:
+- Squidpy: https://doi.org/10.1038/s41592-021-01358-2
+- Cell2location: https://doi.org/10.1038/s41587-021-01139-4
+- SpatialDE: https://doi.org/10.1038/nmeth.4636
+
+**Platforms**:
+- 10x Visium: https://www.10xgenomics.com/products/spatial-gene-expression
+- MERFISH: https://doi.org/10.1126/science.aaa6090
+- Slide-seq: https://doi.org/10.1126/science.aaw1219