tooluniverse-single-cell

# Single-Cell Genomics and Expression Matrix Analysis

Comprehensive single-cell RNA-seq analysis and expression matrix processing using scanpy, anndata, scipy, and ToolUniverse.

---

## LOOK UP, DON'T GUESS
When uncertain about any scientific fact, SEARCH databases first (PubMed, UniProt, ChEMBL, ClinVar, etc.) rather than reasoning from memory. A database-verified answer is always more reliable than a guess.

---

## When to Use This Skill

Apply when users:
- Have scRNA-seq data (h5ad, 10X, CSV count matrices) and want analysis
- Ask about cell type identification, clustering, or annotation
- Need differential expression analysis by cell type or condition
- Want gene-expression correlation analysis (e.g., gene length vs expression by cell type)
- Ask about PCA, UMAP, t-SNE for expression data
- Need Leiden/Louvain clustering on expression matrices
- Want statistical comparisons between cell types (t-test, ANOVA, fold change)
- Ask about marker genes, batch correction, trajectory, or cell-cell communication

**BixBench Coverage**: 18+ questions across 5 projects (bix-22, bix-27, bix-31, bix-33, bix-36)

**NOT for** (use other skills instead):
- Bulk RNA-seq DESeq2 only -> `tooluniverse-rnaseq-deseq2`
- Gene enrichment only -> `tooluniverse-gene-enrichment`
- VCF/variant analysis -> `tooluniverse-variant-analysis`

---

## Core Principles

1. **Data-first** - Load, inspect, validate before analysis
2. **AnnData-centric** - All data flows through anndata objects
3. **Cell type awareness** - Per-cell-type subsetting when needed
4. **Statistical rigor** - Normalization, multiple testing correction, effect sizes
5. **Question-driven** - Parse what the user is actually asking

---

## Required Packages

```python
import scanpy as sc, anndata as ad, pandas as pd, numpy as np
from scipy import stats
from scipy.cluster.hierarchy import linkage, fcluster
from sklearn.decomposition import PCA
from statsmodels.stats.multitest import multipletests
import gseapy as gp  # enrichment
import harmonypy     # batch correction (optional)
```

Install: `pip install scanpy anndata leidenalg umap-learn harmonypy gseapy pandas numpy scipy scikit-learn statsmodels`

---

## Workflow Decision Tree

```
START: User question about scRNA-seq data
|
+-- FULL PIPELINE (raw counts -> annotated clusters)
|   Workflow: QC -> Normalize -> HVG -> PCA -> Cluster -> Annotate -> DE
|   See: references/scanpy_workflow.md
|
+-- DIFFERENTIAL EXPRESSION (per-cell-type comparison)
|   Most common BixBench pattern (bix-33)
|   See: analysis_patterns.md "Pattern 1"
|
+-- CORRELATION ANALYSIS (gene property vs expression)
|   Pattern: Gene length vs expression (bix-22)
|   See: analysis_patterns.md "Pattern 2"
|
+-- CLUSTERING & PCA (expression matrix analysis)
|   See: references/clustering_guide.md
|
+-- CELL COMMUNICATION (ligand-receptor interactions)
|   See: references/cell_communication.md
|
+-- TRAJECTORY ANALYSIS (pseudotime)
    See: references/trajectory_analysis.md
```

**Data format handling**:
- h5ad -> `sc.read_h5ad()`
- 10X -> `sc.read_10x_mtx()` or `sc.read_10x_h5()`
- CSV/TSV -> `pd.read_csv()` -> Convert to AnnData (check orientation!)

---

## Data Loading

AnnData expects: **cells/samples as rows (obs), genes as columns (var)**

```python
adata = sc.read_h5ad("data.h5ad")  # h5ad already oriented

# CSV/TSV: check orientation
df = pd.read_csv("counts.csv", index_col=0)
if df.shape[0] > df.shape[1] * 5:  # genes > samples by 5x => transpose
    df = df.T
adata = ad.AnnData(df)

# Load metadata
meta = pd.read_csv("metadata.csv", index_col=0)
common = adata.obs_names.intersection(meta.index)
adata = adata[common].copy()
for col in meta.columns:
    adata.obs[col] = meta.loc[common, col]
```

---

## Quality Control

```python
adata.var['mt'] = adata.var_names.str.startswith(('MT-', 'mt-'))
sc.pp.calculate_qc_metrics(adata, qc_vars=['mt'], inplace=True)
sc.pp.filter_cells(adata, min_genes=200)
adata = adata[adata.obs['pct_counts_mt'] < 20].copy()
sc.pp.filter_genes(adata, min_cells=3)
```

See: references/scanpy_workflow.md for details

---

## Complete Pipeline (Quick Reference)

```python
import scanpy as sc

adata = sc.read_10x_h5("filtered_feature_bc_matrix.h5")

# QC
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()
sc.pp.filter_cells(adata, min_genes=200)
sc.pp.filter_genes(adata, min_cells=3)

# Normalize + HVG + PCA
sc.pp.normalize_total(adata, target_sum=1e4)
sc.pp.log1p(adata)
adata.raw = adata.copy()
sc.pp.highly_variable_genes(adata, n_top_genes=2000)
sc.tl.pca(adata, n_comps=50)

# Cluster + UMAP
sc.pp.neighbors(adata, n_pcs=30)
sc.tl.leiden(adata, resolution=0.5)
sc.tl.umap(adata)

# Find markers + Annotate + Per-cell-type DE
sc.tl.rank_genes_groups(adata, groupby='leiden', method='wilcoxon')
```

---

## Differential Expression Decision Tree

```
Single-Cell DE (many cells per condition):
  Use: sc.tl.rank_genes_groups(), methods: wilcoxon, t-test, logreg
  Best for: Per-cell-type DE, marker gene finding

Pseudo-Bulk DE (aggregate counts by sample):
  Use: DESeq2 via PyDESeq2
  Best for: Sample-level comparisons with replicates

Statistical Tests Only:
  Use: scipy.stats (ttest_ind, f_oneway, pearsonr)
  Best for: Correlation, ANOVA, t-tests on summaries
```

---

## Statistical Tests (Quick Reference)

```python
from scipy import stats
from statsmodels.stats.multitest import multipletests

# Pearson/Spearman correlation
r, p = stats.pearsonr(gene_lengths, mean_expression)

# Welch's t-test
t_stat, p_val = stats.ttest_ind(group1, group2, equal_var=False)

# ANOVA
f_stat, p_val = stats.f_oneway(group1, group2, group3)

# Multiple testing correction (BH)
reject, pvals_adj, _, _ = multipletests(pvals, method='fdr_bh')
```

---

## Batch Correction (Harmony)

```python
import harmonypy
sc.tl.pca(adata, n_comps=50)
ho = harmonypy.run_harmony(adata.obsm['X_pca'][:, :30], adata.obs, 'batch', random_state=0)
adata.obsm['X_pca_harmony'] = ho.Z_corr.T
sc.pp.neighbors(adata, use_rep='X_pca_harmony')
sc.tl.leiden(adata, resolution=0.5)
sc.tl.umap(adata)
```

---

## ToolUniverse Integration

### Data Discovery (before analysis)
- **CxGDisc_search_datasets**: Search CELLxGENE Discover for scRNA-seq datasets by disease, tissue, organism. Use broad disease terms (e.g., "breast cancer" not "triple-negative").
- **GEO_search_rnaseq_datasets** / **geo_search_datasets**: Search GEO for scRNA-seq studies
- **NCBI_SRA_search_runs**: Search SRA for sequencing runs (query="single cell RNA-seq [disease]")
- **OmicsDI_search_datasets**: Cross-repository dataset search

### Cell Type Markers
- **CellMarker_search_by_cell_type**: Tissue-specific cell markers (use `CellMarker_list_cell_types` first — exact names required, e.g., "Regulatory T(Treg) cell" not "Regulatory T cell")
- **CellMarker_search_cancer_markers**: Cancer-context markers with experimental evidence
- **CellMarker_search_by_gene**: Reverse lookup — which cell types express a gene?
- **HPA_search_genes_by_query**: Cell-type marker gene search

### Gene Annotation
- **MyGene_query_genes** / **MyGene_batch_query**: Gene ID conversion
- **ensembl_lookup_gene**: Ensembl gene details
- **UniProt_get_function_by_accession**: Protein function

### Cell-Cell Communication
- **OmniPath_get_ligand_receptor_interactions**: L-R pairs (CellPhoneDB, CellChatDB)
- **OmniPath_get_signaling_interactions**: Downstream signaling
- **OmniPath_get_complexes**: Multi-subunit receptors

### Enrichment (Post-DE)
- **PANTHER_enrichment**: GO enrichment (BP, MF, CC)
- **STRING_functional_enrichment**: Network-based enrichment
- **ReactomeAnalysis_pathway_enrichment**: Reactome pathways

### Clinical Context (for tumor immunology)
- **DGIdb_get_drug_gene_interactions**: Drug interactions for immune checkpoint targets (genes=["CD274"] for PD-L1)
- **civic_search_evidence_items**: Clinical evidence for mutations/biomarkers
- **TIMER2_immune_estimation**: TCGA immune infiltration correlation
- **search_clinical_trials**: Clinical trial matching
- **GTEx_get_expression_summary**: Normal tissue baseline expression
- **PubMed_search_articles**: Literature context

---

## Scanpy vs Seurat Equivalents

| Operation | Seurat (R) | Scanpy (Python) |
|-----------|------------|-----------------|
| Load data | `Read10X()` | `sc.read_10x_mtx()` |
| Normalize | `NormalizeData()` | `sc.pp.normalize_total() + sc.pp.log1p()` |
| Find HVGs | `FindVariableFeatures()` | `sc.pp.highly_variable_genes()` |
| PCA | `RunPCA()` | `sc.tl.pca()` |
| Cluster | `FindClusters()` | `sc.tl.leiden()` |
| UMAP | `RunUMAP()` | `sc.tl.umap()` |
| Find markers | `FindMarkers()` | `sc.tl.rank_genes_groups()` |
| Batch correction | `RunHarmony()` | `harmonypy.run_harmony()` |

---

## Reasoning Framework for Result Interpretation

### Evidence Grading

| Grade | Criteria | Example |
|-------|----------|---------|
| **High confidence** | Marker padj < 0.01, log2FC > 1, expressed in > 25% of cluster cells | CD3D as T-cell marker with padj = 1e-50, log2FC = 3.2, pct = 0.85 |
| **Moderate confidence** | padj < 0.05, log2FC > 0.5, or expressed in 10-25% of cluster | FOXP3 in Treg cluster with padj = 0.001, pct = 0.18 |
| **Low confidence** | padj < 0.05 but log2FC < 0.5 or low pct_diff between clusters | Ubiquitously expressed gene with marginal enrichment |
| **Unreliable** | Fewer than 20 cells in cluster, or QC metrics suggest doublets | Cluster with mean nGenes > 6000 and high doublet score |

### Interpretation Guidance

- **QC metric thresholds**: Standard filters are nGenes > 200 (remove empty droplets), nGenes < 5000-6000 (remove doublets), pct_counts_mt < 20% (remove dying cells). These thresholds are tissue-dependent: immune cells tolerate stricter nGene filters; neurons may have higher mitochondrial content naturally. Always visualize distributions before setting cutoffs.
- **Cluster resolution guidance**: Leiden resolution 0.3-0.5 yields broad cell types (T cells, B cells, myeloid). Resolution 0.8-1.2 resolves subtypes (CD4 naive, CD4 memory, Treg). Resolution > 2.0 risks over-clustering (splitting biologically homogeneous populations). Validate by checking that each cluster has distinct marker genes.
- **Marker gene confidence levels**: A strong marker is highly specific (high pct_diff between cluster and rest) and highly expressed (high log2FC). Genes expressed in many clusters with small fold changes are poor markers. Cross-reference with known markers from CellMarker or HPA databases.
- **Pseudo-bulk vs single-cell DE**: For comparing conditions (treatment vs control), pseudo-bulk DE (aggregate by sample, then DESeq2) is more statistically valid than single-cell DE, which inflates significance due to non-independence of cells from the same sample.
- **Batch effects**: If samples cluster by batch rather than biology on UMAP, apply Harmony or other correction before biological interpretation.

### Synthesis Questions

1. Do the identified clusters correspond to known cell types based on canonical markers, or do some clusters lack clear biological identity (potentially doublets or low-quality cells)?
2. At the chosen clustering resolution, are there clusters that merge when resolution is lowered, suggesting they may be a single cell type split by technical noise?
3. For differential expression between conditions, are the results consistent between single-cell and pseudo-bulk approaches, and do the top DE genes have known biological relevance?
4. Do QC-flagged cells (high mito, extreme gene counts) concentrate in specific clusters, and does removing them change the clustering structure?
5. If batch correction was applied, do post-correction clusters still maintain expected cell-type-specific marker expression?

---

## Troubleshooting

| Issue | Solution |
|-------|----------|
| `ModuleNotFoundError: leidenalg` | `pip install leidenalg` |
| Sparse matrix errors | `.toarray()`: `X = adata.X.toarray() if issparse(adata.X) else adata.X` |
| Wrong matrix orientation | More genes than samples? Transpose |
| NaN in correlation | Filter: `valid = ~np.isnan(x) & ~np.isnan(y)` |
| Too few cells for DE | Need >= 3 cells per condition per cell type |
| Memory error | Use `sc.pp.highly_variable_genes()` to reduce features |

---

## Reference Documentation

**Detailed Analysis Patterns**: analysis_patterns.md (per-cell-type DE, correlation, PCA, ANOVA, cell communication)

**Core Workflows**:
- references/scanpy_workflow.md - Complete scanpy pipeline
- references/seurat_workflow.md - Seurat to Scanpy translation
- references/clustering_guide.md - Clustering methods
- references/marker_identification.md - Marker genes, annotation
- references/trajectory_analysis.md - Pseudotime
- references/cell_communication.md - OmniPath/CellPhoneDB workflow
- references/troubleshooting.md - Detailed error solutions