Alterlab Scanpy

Scanpy: Single-Cell Analysis

Overview

Scanpy is a scalable Python toolkit for analyzing single-cell RNA-seq data, built on AnnData. Apply this skill for complete single-cell workflows including quality control, normalization, dimensionality reduction, clustering, marker gene identification, visualization, and trajectory analysis.

When to Use This Skill

This skill should be used when:

• Analyzing single-cell RNA-seq data (.h5ad, 10X, CSV formats)
• Performing quality control on scRNA-seq datasets
• Creating UMAP, t-SNE, or PCA visualizations
• Identifying cell clusters and finding marker genes
• Annotating cell types based on gene expression
• Conducting trajectory inference or pseudotime analysis
• Generating publication-quality single-cell plots

Quick Start

Basic Import and Setup

import scanpy as sc
import pandas as pd
import numpy as np

# Configure settings
sc.settings.verbosity = 3
sc.settings.set_figure_params(dpi=80, facecolor='white')
sc.settings.figdir = './figures/'

Loading Data

# From 10X Genomics
adata = sc.read_10x_mtx('path/to/data/')
adata = sc.read_10x_h5('path/to/data.h5')

# From h5ad (AnnData format)
adata = sc.read_h5ad('path/to/data.h5ad')

# From CSV
adata = sc.read_csv('path/to/data.csv')

Understanding AnnData Structure

The AnnData object is the core data structure in scanpy:

adata.X          # Expression matrix (cells × genes)
adata.obs        # Cell metadata (DataFrame)
adata.var        # Gene metadata (DataFrame)
adata.uns        # Unstructured annotations (dict)
adata.obsm       # Multi-dimensional cell data (PCA, UMAP)
adata.raw        # Raw data backup

# Access cell and gene names
adata.obs_names  # Cell barcodes
adata.var_names  # Gene names

Standard Analysis Workflow

1. Quality Control

Identify and filter low-quality cells and genes:

# Identify mitochondrial genes
adata.var['mt'] = adata.var_names.str.startswith('MT-')

# Calculate QC metrics
sc.pp.calculate_qc_metrics(adata, qc_vars=['mt'], inplace=True)

# Visualize QC metrics
sc.pl.violin(adata, ['n_genes_by_counts', 'total_counts', 'pct_counts_mt'],
             jitter=0.4, multi_panel=True)

# Filter cells and genes
sc.pp.filter_cells(adata, min_genes=200)
sc.pp.filter_genes(adata, min_cells=3)
adata = adata[adata.obs.pct_counts_mt < 5, :]  # Remove high MT% cells

Use the QC script for automated analysis:

python scripts/qc_analysis.py input_file.h5ad --output filtered.h5ad

2. Normalization and Preprocessing

# Normalize to 10,000 counts per cell
sc.pp.normalize_total(adata, target_sum=1e4)

# Log-transform
sc.pp.log1p(adata)

# Save raw counts for later
adata.raw = adata

# Identify highly variable genes
sc.pp.highly_variable_genes(adata, n_top_genes=2000)
sc.pl.highly_variable_genes(adata)

# Subset to highly variable genes
adata = adata[:, adata.var.highly_variable]

# Regress out unwanted variation
sc.pp.regress_out(adata, ['total_counts', 'pct_counts_mt'])

# Scale data
sc.pp.scale(adata, max_value=10)

3. Dimensionality Reduction

# PCA
sc.tl.pca(adata, svd_solver='arpack')
sc.pl.pca_variance_ratio(adata, log=True)  # Check elbow plot

# Compute neighborhood graph
sc.pp.neighbors(adata, n_neighbors=10, n_pcs=40)

# UMAP for visualization
sc.tl.umap(adata)
sc.pl.umap(adata, color='leiden')

# Alternative: t-SNE
sc.tl.tsne(adata)

4. Clustering

# Leiden clustering (recommended). Pass flavor='igraph' (the future default) +
# n_iterations=2 — the bare call emits a FutureWarning and uses the slow backend.
sc.tl.leiden(adata, resolution=0.5, flavor='igraph', n_iterations=2)
sc.pl.umap(adata, color='leiden', legend_loc='on data')

# Try multiple resolutions to find optimal granularity
for res in [0.3, 0.5, 0.8, 1.0]:
    sc.tl.leiden(adata, resolution=res, flavor='igraph', n_iterations=2,
                 key_added=f'leiden_{res}')

5. Marker Gene Identification

# Find marker genes for each cluster
sc.tl.rank_genes_groups(adata, 'leiden', method='wilcoxon')

# Visualize results
sc.pl.rank_genes_groups(adata, n_genes=25, sharey=False)
sc.pl.rank_genes_groups_heatmap(adata, n_genes=10)
sc.pl.rank_genes_groups_dotplot(adata, n_genes=5)

# Get results as DataFrame
markers = sc.get.rank_genes_groups_df(adata, group='0')

6. Cell Type Annotation

# Define marker genes for known cell types
marker_genes = ['CD3D', 'CD14', 'MS4A1', 'NKG7', 'FCGR3A']

# Visualize markers
sc.pl.umap(adata, color=marker_genes, use_raw=True)
sc.pl.dotplot(adata, var_names=marker_genes, groupby='leiden')

# Manual annotation
cluster_to_celltype = {
    '0': 'CD4 T cells',
    '1': 'CD14+ Monocytes',
    '2': 'B cells',
    '3': 'CD8 T cells',
}
adata.obs['cell_type'] = adata.obs['leiden'].map(cluster_to_celltype)

# Visualize annotated types
sc.pl.umap(adata, color='cell_type', legend_loc='on data')

7. Save Results

# Save processed data
adata.write('results/processed_data.h5ad')

# Export metadata
adata.obs.to_csv('results/cell_metadata.csv')
adata.var.to_csv('results/gene_metadata.csv')

Common Tasks

Creating Publication-Quality Plots

# Set high-quality defaults
sc.settings.set_figure_params(dpi=300, frameon=False, figsize=(5, 5))
sc.settings.file_format_figs = 'pdf'

# UMAP with custom styling
sc.pl.umap(adata, color='cell_type',
           palette='Set2',
           legend_loc='on data',
           legend_fontsize=12,
           legend_fontoutline=2,
           frameon=False,
           save='_publication.pdf')

# Heatmap of marker genes
sc.pl.heatmap(adata, var_names=genes, groupby='cell_type',
              swap_axes=True, show_gene_labels=True,
              save='_markers.pdf')

# Dot plot
sc.pl.dotplot(adata, var_names=genes, groupby='cell_type',
              save='_dotplot.pdf')

Refer to references/plotting_guide.md for comprehensive visualization examples.

Trajectory Inference

# PAGA (Partition-based graph abstraction)
sc.tl.paga(adata, groups='leiden')
sc.pl.paga(adata, color='leiden')

# Diffusion pseudotime
adata.uns['iroot'] = np.flatnonzero(adata.obs['leiden'] == '0')[0]
sc.tl.dpt(adata)
sc.pl.umap(adata, color='dpt_pseudotime')

Differential Expression Between Conditions

# Compare treated vs control within cell types
adata_subset = adata[adata.obs['cell_type'] == 'T cells']
sc.tl.rank_genes_groups(adata_subset, groupby='condition',
                         groups=['treated'], reference='control')
sc.pl.rank_genes_groups(adata_subset, groups=['treated'])

Gene Set Scoring

# Score cells for gene set expression
gene_set = ['CD3D', 'CD3E', 'CD3G']
sc.tl.score_genes(adata, gene_set, score_name='T_cell_score')
sc.pl.umap(adata, color='T_cell_score')

Batch Correction

# ComBat batch correction
sc.pp.combat(adata, key='batch')

# Alternative: use Harmony or scVI (separate packages)

Key Parameters to Adjust

Quality Control

• min_genes: Minimum genes per cell (typically 200-500)
• min_cells: Minimum cells per gene (typically 3-10)
• pct_counts_mt: Mitochondrial threshold (typically 5-20%)

Normalization

• target_sum: Target counts per cell (default 1e4)

Feature Selection

• n_top_genes: Number of HVGs (typically 2000-3000)
• min_mean, max_mean, min_disp: HVG selection parameters

Dimensionality Reduction

• n_pcs: Number of principal components (check variance ratio plot)
• n_neighbors: Number of neighbors (typically 10-30)

Clustering

• resolution: Clustering granularity (0.4-1.2, higher = more clusters)

Common Pitfalls and Best Practices

1. Always save raw counts: adata.raw = adata before filtering genes
2. Check QC plots carefully: Adjust thresholds based on dataset quality
3. Use Leiden with flavor='igraph': The future default and far faster than Louvain or the legacy leidenalg backend — pip/uv install the igraph package (not bundled with scanpy)
4. Try multiple clustering resolutions: Find optimal granularity
5. Validate cell type annotations: Use multiple marker genes
6. Use use_raw=True for gene expression plots: Shows original counts
7. Check PCA variance ratio: Determine optimal number of PCs
8. Save intermediate results: Long workflows can fail partway through

Bundled Resources

scripts/qc_analysis.py

Automated quality control script that calculates metrics, generates plots, and filters data:

python scripts/qc_analysis.py input.h5ad --output filtered.h5ad \
    --mt-threshold 5 --min-genes 200 --min-cells 3

references/standard_workflow.md

Complete step-by-step workflow with detailed explanations and code examples for:

• Data loading and setup
• Quality control with visualization
• Normalization and scaling
• Feature selection
• Dimensionality reduction (PCA, UMAP, t-SNE)
• Clustering (Leiden, Louvain)
• Marker gene identification
• Cell type annotation
• Trajectory inference
• Differential expression

Read this reference when performing a complete analysis from scratch.

references/api_reference.md

Quick reference guide for scanpy functions organized by module:

• Reading/writing data (sc.read_*, adata.write_*)
• Preprocessing (sc.pp.*)
• Tools (sc.tl.*)
• Plotting (sc.pl.*)
• AnnData structure and manipulation
• Settings and utilities

Use this for quick lookup of function signatures and common parameters.

references/plotting_guide.md

Comprehensive visualization guide including:

• Quality control plots
• Dimensionality reduction visualizations
• Clustering visualizations
• Marker gene plots (heatmaps, dot plots, violin plots)
• Trajectory and pseudotime plots
• Publication-quality customization
• Multi-panel figures
• Color palettes and styling

Consult this when creating publication-ready figures.

assets/analysis_template.py

Complete analysis template providing a full workflow from data loading through cell type annotation. Copy and customize this template for new analyses:

cp assets/analysis_template.py my_analysis.py
# Edit parameters and run
python my_analysis.py

The template includes all standard steps with configurable parameters and helpful comments.

Additional Resources

• Official scanpy documentation: https://scanpy.readthedocs.io/
• Scanpy tutorials: https://scanpy-tutorials.readthedocs.io/
• scverse ecosystem: https://scverse.org/ (related tools: squidpy, scvi-tools, cellrank)
• Best practices: Luecken & Theis (2019) "Current best practices in single-cell RNA-seq"

Tips for Effective Analysis

1. Start with the template: Use assets/analysis_template.py as a starting point
2. Run QC script first: Use scripts/qc_analysis.py for initial filtering
3. Consult references as needed: Load workflow and API references into context
4. Iterate on clustering: Try multiple resolutions and visualization methods
5. Validate biologically: Check marker genes match expected cell types
6. Document parameters: Record QC thresholds and analysis settings
7. Save checkpoints: Write intermediate results at key steps