Version Compatibility

Reference examples tested with: anndata 0.10+, matplotlib 3.8+, numpy 1.26+, pandas 2.2+, scanpy 1.10+

Before using code patterns, verify installed versions match. If versions differ:

• Python:

  pip show <package>

  then

  help(module.function)

  to check signatures

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Spatial Deconvolution

Estimate cell type composition in spatial spots using scRNA-seq references.

Required Imports

import scanpy as sc
import anndata as ad
import numpy as np
import pandas as pd
import matplotlib.pyplot as plt

Overview

Deconvolution estimates cell type proportions in each spatial spot using a reference single-cell dataset. Essential for Visium data where spots contain multiple cells.

Using cell2location

Goal: Estimate cell type abundances per spatial spot using a probabilistic model trained on scRNA-seq reference signatures.

Approach: Train a regression model on reference scRNA-seq to extract cell type signatures, then decompose spatial spots using those signatures.

"Deconvolve my Visium spots into cell types" -> Train a reference signature model on scRNA-seq, then map cell type abundances to spatial locations using cell2location.

import cell2location
from cell2location.utils.filtering import filter_genes
from cell2location.models import RegressionModel

# Load reference scRNA-seq
adata_ref = sc.read_h5ad('reference_scrna.h5ad')
adata_ref.obs['cell_type'] = adata_ref.obs['cell_type'].astype('category')

# Load spatial data
adata_vis = sc.read_h5ad('spatial_data.h5ad')

# Find shared genes
intersect = np.intersect1d(adata_vis.var_names, adata_ref.var_names)
adata_ref = adata_ref[:, intersect].copy()
adata_vis = adata_vis[:, intersect].copy()

Train Reference Signature Model

Goal: Learn cell type gene expression signatures from annotated single-cell reference data.

Approach: Filter genes, set up a regression model on the scRNA-seq reference, train it, and export per-cell-type mean expression signatures.

# Select genes for deconvolution
selected = filter_genes(adata_ref, cell_count_cutoff=5, cell_percentage_cutoff2=0.03,
                        nonz_mean_cutoff=1.12)
adata_ref = adata_ref[:, selected].copy()

# Prepare reference
cell2location.models.RegressionModel.setup_anndata(
    adata_ref,
    labels_key='cell_type',
)

# Train reference model
mod = RegressionModel(adata_ref)
mod.train(max_epochs=250, use_gpu=True)

# Export reference signatures
adata_ref = mod.export_posterior(adata_ref, sample_kwargs={'num_samples': 1000})
ref_sig = adata_ref.varm['means_per_cluster_mu_fg']

Run Spatial Deconvolution

Goal: Decompose each spatial spot into cell type abundances using trained reference signatures.

Approach: Set up the Cell2location model with reference signatures and expected cells per spot, then train on the spatial data.

# Ensure spatial data has same genes
adata_vis = adata_vis[:, adata_ref.var_names].copy()

# Setup spatial data
cell2location.models.Cell2location.setup_anndata(adata_vis)

# Train deconvolution model
mod_spatial = cell2location.models.Cell2location(
    adata_vis,
    cell_state_df=ref_sig,
    N_cells_per_location=10,  # Expected cells per spot
    detection_alpha=20,
)
mod_spatial.train(max_epochs=30000, use_gpu=True)

# Export results
adata_vis = mod_spatial.export_posterior(adata_vis, sample_kwargs={'num_samples': 1000})

Access Deconvolution Results

# Cell type abundances stored in obsm
abundances = adata_vis.obsm['q05_cell_abundance_w_sf']
print(f'Cell types: {abundances.shape[1]}')

# Convert to proportions
proportions = abundances / abundances.sum(axis=1, keepdims=True)
adata_vis.obsm['cell_type_proportions'] = proportions

# Add dominant cell type
cell_types = adata_ref.obs['cell_type'].cat.categories
adata_vis.obs['dominant_cell_type'] = cell_types[proportions.argmax(axis=1)]

Using Tangram (Alternative)

Goal: Map single-cell reference data to spatial locations using optimal transport.

Approach: Find marker genes from the reference, align single cells to spatial spots using Tangram's mapping algorithm, then project cell type annotations.

import tangram as tg

# Load data
adata_sc = sc.read_h5ad('reference_scrna.h5ad')
adata_sp = sc.read_h5ad('spatial_data.h5ad')

# Preprocess
sc.pp.normalize_total(adata_sc)
sc.pp.log1p(adata_sc)

# Find marker genes
sc.tl.rank_genes_groups(adata_sc, groupby='cell_type', method='wilcoxon')
markers = sc.get.rank_genes_groups_df(adata_sc, group=None)
markers = markers[markers['pvals_adj'] < 0.01].groupby('group').head(100)
marker_genes = markers['names'].unique().tolist()

# Prepare for Tangram
tg.pp_adatas(adata_sc, adata_sp, genes=marker_genes)

# Map single cells to spatial locations
ad_map = tg.map_cells_to_space(
    adata_sc,
    adata_sp,
    mode='clusters',
    cluster_label='cell_type',
    device='cuda:0',
)

# Get cell type proportions
tg.project_cell_annotations(ad_map, adata_sp, annotation='cell_type')
# Results in adata_sp.obsm['tangram_ct_pred']

Using RCTD (via R)

# RCTD runs in R; use rpy2 for integration
import rpy2.robjects as ro
from rpy2.robjects import pandas2ri
pandas2ri.activate()

# Save data for R
adata_vis.write_h5ad('spatial_for_rctd.h5ad')
adata_ref.write_h5ad('reference_for_rctd.h5ad')

# R code for RCTD
r_code = '''
library(spacexr)
library(Seurat)

# Load data (convert from h5ad first)
# ... R-specific loading code ...

# Create RCTD object
rctd <- create.RCTD(puck, reference, max_cores=4)
rctd <- run_RCTD(rctd, doublet_mode='full')

# Get results
results <- rctd@results
weights <- normalize_weights(results$weights)
'''

Visualize Cell Type Proportions

Goal: Display estimated cell type abundances as spatial heatmaps across the tissue.

Approach: Plot each cell type's proportion as a separate spatial panel using Scanpy's spatial plot.

# Plot cell type abundances spatially
cell_types_to_plot = ['T_cell', 'Macrophage', 'Epithelial', 'Fibroblast']

fig, axes = plt.subplots(2, 2, figsize=(12, 12))
for ax, ct in zip(axes.flatten(), cell_types_to_plot):
    ct_idx = list(adata_ref.obs['cell_type'].cat.categories).index(ct)
    adata_vis.obs[f'{ct}_proportion'] = proportions[:, ct_idx]
    sc.pl.spatial(adata_vis, color=f'{ct}_proportion', ax=ax, show=False,
                  title=ct, cmap='Reds', vmin=0, vmax=1)
plt.tight_layout()
plt.savefig('cell_type_proportions.png', dpi=150)

Pie Chart Per Spot (Advanced)

from matplotlib.patches import Wedge

def plot_pie_spatial(adata, proportions, cell_types, spot_size=0.5):
    fig, ax = plt.subplots(figsize=(12, 12))
    colors = plt.cm.tab20(np.linspace(0, 1, len(cell_types)))

    coords = adata.obsm['spatial']
    for i in range(adata.n_obs):
        x, y = coords[i]
        props = proportions[i]
        start_angle = 0
        for j, prop in enumerate(props):
            if prop > 0.01:  # Skip tiny proportions
                wedge = Wedge((x, y), spot_size * 50, start_angle,
                             start_angle + prop * 360, color=colors[j])
                ax.add_patch(wedge)
                start_angle += prop * 360

    ax.set_xlim(coords[:, 0].min() - 100, coords[:, 0].max() + 100)
    ax.set_ylim(coords[:, 1].min() - 100, coords[:, 1].max() + 100)
    ax.set_aspect('equal')
    ax.invert_yaxis()

    # Legend
    handles = [plt.Rectangle((0, 0), 1, 1, color=colors[i]) for i in range(len(cell_types))]
    ax.legend(handles, cell_types, loc='upper right')
    plt.savefig('pie_chart_spatial.png', dpi=150)

Evaluate Deconvolution Quality

Goal: Validate deconvolution results by correlating estimated proportions with known marker gene expression.

Approach: For each cell type, compute correlation between its estimated proportion and mean expression of canonical marker genes.

# Check correlation between expected and observed cell counts
# (if you have known cell type markers)

marker_genes = {
    'T_cell': ['CD3D', 'CD3E', 'CD4', 'CD8A'],
    'Macrophage': ['CD68', 'CD14', 'CSF1R'],
    'Epithelial': ['EPCAM', 'KRT8', 'KRT18'],
}

for ct, markers in marker_genes.items():
    available_markers = [m for m in markers if m in adata_vis.var_names]
    if available_markers:
        marker_expr = adata_vis[:, available_markers].X.mean(axis=1)
        ct_idx = list(cell_types).index(ct)
        ct_prop = proportions[:, ct_idx]
        corr = np.corrcoef(marker_expr.flatten(), ct_prop)[0, 1]
        print(f'{ct}: marker-proportion correlation = {corr:.3f}')

Compare Deconvolution Methods

# Store results from different methods
adata_vis.obsm['cell2location'] = cell2location_proportions
adata_vis.obsm['tangram'] = tangram_proportions

# Correlation between methods
for ct_idx, ct in enumerate(cell_types):
    c2l = adata_vis.obsm['cell2location'][:, ct_idx]
    tg = adata_vis.obsm['tangram'][:, ct_idx]
    corr = np.corrcoef(c2l, tg)[0, 1]
    print(f'{ct}: cell2location vs tangram = {corr:.3f}')

Export Results

# Save proportions as CSV
prop_df = pd.DataFrame(
    proportions,
    index=adata_vis.obs_names,
    columns=cell_types
)
prop_df.to_csv('cell_type_proportions.csv')

# Save annotated AnnData
adata_vis.write_h5ad('spatial_deconvolved.h5ad')

Related Skills

• spatial-data-io - Load spatial data
• single-cell/data-io - Load scRNA-seq reference
• spatial-visualization - Visualize deconvolution results
• single-cell/markers-annotation - Annotate reference cell types