Single Cell Scrna Seq Analysis Scanpy

Why this exists

This skill encodes the current best practices for scRNA-seq:

• Enforces strict tracking of metadata (adata.obs and adata.var).
• Utilizes the more robust Leiden algorithm over Louvain.
• Bundles automated, reproducible scripts for both QC and full-pipeline analysis.
• Provides immediate access to trajectory inference (PAGA/DPT) and gene set scoring.

Usage

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)
sc.tl.leiden(adata, resolution=0.5)
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, 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)

Example Output

Scanpy Single-Cell QC & Clustering
==================================
Input: 12,450 cells × 32,000 genes
After filtering (min_genes=200, MT% < 5): 10,210 cells × 18,500 genes

Variance explained by top 40 PCs: 68.4%

Clustering (Leiden, res=0.5):
  Found 12 distinct clusters.
  Cluster 0: 3,210 cells (Top markers: CD3D, IL7R) -> Annotated: CD4 T cells
  Cluster 1: 2,800 cells (Top markers: CD14, LYZ)  -> Annotated: CD14+ Monocytes

Saved outputs to: results/
  - processed_data.h5ad
  - UMAP_clusters.pdf (300 dpi)
  - marker_genes_dotplot.pdf (300 dpi)

Best practice

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

Bundled Resources

This skill includes pre-configured tools to accelerate your workflow:

• scripts/qc_analysis.py: Automated QC script calculating metrics, generating plots, and filtering data.
• assets/analysis_template.py: A complete, customizable python template from data loading to cell type annotation.
• references/standard_workflow.md: Step-by-step conceptual explanations.
• references/api_reference.md: Quick lookup for Scanpy function signatures.
• references/plotting_guide.md: Customization guide for publication-ready figures.

Requirements

Inputs

Outputs

Citations