Version Compatibility

Reference examples tested with: numpy 1.26+, 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
• R: packageVersion('<pkg>') then ?function_name to verify parameters

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

Multimodal Integration

"Integrate RNA and protein data from my CITE-seq experiment" → Jointly analyze multiple modalities (RNA + protein, RNA + ATAC) measured in the same cells using weighted nearest neighbor or factor analysis.

• R: Seurat::FindMultiModalNeighbors() for WNN integration
• Python: muon for MuData handling, scanpy + anndata for multimodal objects

Analyze multi-modal single-cell data where multiple measurements are made per cell.

Common Modalities

| Technology | Modalities | Package | |------------|------------|---------| | CITE-seq | RNA + surface proteins (ADT) | Seurat | | 10X Multiome | RNA + ATAC | Seurat, Signac, ArchR | | SHARE-seq | RNA + ATAC | Seurat, Signac | | Spatial (Visium) | RNA + spatial coordinates | Seurat, Squidpy |

CITE-seq Analysis (Seurat)

Load Data

r
library(Seurat)

# Read 10X data with antibody capture
data <- Read10X('filtered_feature_bc_matrix/')

# Separate RNA and ADT
rna_counts <- data$`Gene Expression`
adt_counts <- data$`Antibody Capture`

# Create Seurat object with both assays
obj <- CreateSeuratObject(counts = rna_counts, assay = 'RNA')
obj[['ADT']] <- CreateAssayObject(counts = adt_counts)

QC and Normalization

r
# RNA QC (standard)
obj <- PercentageFeatureSet(obj, pattern = '^MT-', col.name = 'percent.mt')
obj <- subset(obj, nFeature_RNA > 200 & percent.mt < 20)

# Normalize RNA
obj <- NormalizeData(obj, assay = 'RNA')
obj <- FindVariableFeatures(obj, assay = 'RNA')
obj <- ScaleData(obj, assay = 'RNA')

# Normalize ADT (CLR normalization)
obj <- NormalizeData(obj, assay = 'ADT', normalization.method = 'CLR', margin = 2)
obj <- ScaleData(obj, assay = 'ADT')

Weighted Nearest Neighbor (WNN) Clustering

Goal: Jointly cluster cells using both RNA and protein (or ATAC) modalities, weighting each modality's contribution per cell.

Approach: Run PCA separately on each modality, build a weighted nearest neighbor graph that adaptively combines both reductions, then cluster and embed on the combined WNN graph.

r
# Dimensionality reduction for each modality
obj <- RunPCA(obj, assay = 'RNA', reduction.name = 'pca')
obj <- RunPCA(obj, assay = 'ADT', reduction.name = 'apca',
              features = rownames(obj[['ADT']]))

# WNN graph combining both modalities
obj <- FindMultiModalNeighbors(obj,
    reduction.list = list('pca', 'apca'),
    dims.list = list(1:30, 1:18))

# Cluster on WNN graph
obj <- FindClusters(obj, graph.name = 'wsnn', resolution = 0.5)

# UMAP on WNN
obj <- RunUMAP(obj, nn.name = 'weighted.nn', reduction.name = 'wnn.umap')

Visualize

r
# UMAP colored by cluster
DimPlot(obj, reduction = 'wnn.umap', label = TRUE)

# ADT expression on UMAP
FeaturePlot(obj, features = c('adt_CD3', 'adt_CD19', 'adt_CD14'),
            reduction = 'wnn.umap')

# Compare modality weights
VlnPlot(obj, features = 'RNA.weight', group.by = 'seurat_clusters')

10X Multiome (RNA + ATAC)

Load Data

r
library(Seurat)
library(Signac)

# Read RNA counts
rna_counts <- Read10X_h5('filtered_feature_bc_matrix.h5')$`Gene Expression`

# Read ATAC fragments
atac_counts <- Read10X_h5('filtered_feature_bc_matrix.h5')$Peaks
fragments <- CreateFragmentObject('atac_fragments.tsv.gz')

# Create multiome object
obj <- CreateSeuratObject(counts = rna_counts, assay = 'RNA')
obj[['ATAC']] <- CreateChromatinAssay(counts = atac_counts, fragments = fragments,
                                       genome = 'hg38', min.cells = 5)

Process ATAC

r
# ATAC QC
obj <- NucleosomeSignal(obj)
obj <- TSSEnrichment(obj)

# ATAC normalization
obj <- RunTFIDF(obj, assay = 'ATAC')
obj <- FindTopFeatures(obj, assay = 'ATAC', min.cutoff = 'q0')
obj <- RunSVD(obj, assay = 'ATAC')

Joint Analysis

r
# RNA processing
DefaultAssay(obj) <- 'RNA'
obj <- NormalizeData(obj) %>% FindVariableFeatures() %>% ScaleData() %>% RunPCA()

# WNN integration
obj <- FindMultiModalNeighbors(obj, reduction.list = list('pca', 'lsi'),
                                dims.list = list(1:30, 2:30))
obj <- RunUMAP(obj, nn.name = 'weighted.nn', reduction.name = 'wnn.umap')
obj <- FindClusters(obj, graph.name = 'wsnn')

Scanpy/MuData (Python)

CITE-seq with MuData

python
import scanpy as sc
import muon as mu
from muon import prot as pt

# Load multimodal data
mdata = mu.read_10x_h5('filtered_feature_bc_matrix.h5')

# Access modalities
rna = mdata.mod['rna']
prot = mdata.mod['prot']

# Process RNA
sc.pp.filter_cells(rna, min_genes=200)
sc.pp.normalize_total(rna, target_sum=1e4)
sc.pp.log1p(rna)
sc.pp.highly_variable_genes(rna)
sc.tl.pca(rna)

# Process protein (CLR normalization)
pt.pp.clr(prot)

# Multi-omics factor analysis
mu.tl.mofa(mdata, n_factors=20)

# Joint UMAP
mu.tl.umap(mdata)
mu.pl.umap(mdata, color=['rna:leiden', 'prot:CD3'])

Integration Metrics

Modality Weights

r
# Check how much each modality contributes per cell
weights <- obj@reductions$wnn@misc$weights

# Average weight by cluster
aggregate(weights, by = list(obj$seurat_clusters), mean)

Correlation Between Modalities

python
import numpy as np

# Correlate RNA and protein for same genes/proteins
common = set(rna.var_names) & set(prot.var_names)
for gene in common:
    rna_expr = rna[:, gene].X.toarray().flatten()
    prot_expr = prot[:, gene].X.toarray().flatten()
    corr = np.corrcoef(rna_expr, prot_expr)[0, 1]
    print(f'{gene}: r={corr:.3f}')

Marker Discovery

Multi-Modal Markers

r
# Find markers using both modalities
DefaultAssay(obj) <- 'RNA'
rna_markers <- FindAllMarkers(obj, only.pos = TRUE)

DefaultAssay(obj) <- 'ADT'
adt_markers <- FindAllMarkers(obj, only.pos = TRUE)

# Combine
all_markers <- rbind(
    transform(rna_markers, modality = 'RNA'),
    transform(adt_markers, modality = 'ADT')
)

Related Skills

• single-cell/data-io - Loading single-cell data
• single-cell/clustering - Clustering methods
• single-cell/markers-annotation - Cell type annotation
• chip-seq/peak-calling - For ATAC peak calling