Single-Cell Genomics and Expression Matrix Analysis

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

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)

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

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)

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)

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)

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

Reasoning Framework for Result Interpretation

Evidence Grading

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

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