bio-single-cell-multimodal-integration

name	bio-single-cell-multimodal-integration
description	Integrate multimodal single-cell data (CITE-seq RNA+protein, 10x Multiome RNA+ATAC, unpaired/diagonal RNA+ATAC) and choose the right joint method. Use when classifying an integration task by anchor structure (paired vs unpaired), denoising CITE-seq ADT background before joint embedding, picking between WNN, totalVI, MultiVI, MOFA+, GLUE, or Seurat v5 bridge integration, or diagnosing why a modality dominates a joint clustering.
tool_type	mixed
primary_tool	Seurat

Version Compatibility

Reference examples tested with: scanpy 1.10+, Seurat 5.0+, anndata 0.10+

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

"Jointly analyze my CITE-seq / Multiome / unpaired multi-omic data" -> Classify the task by anchor structure, denoise each modality in its native pipeline, then build one joint representation.

• R: Seurat::FindMultiModalNeighbors() (WNN), Signac (ATAC LSI), dsb::DSBNormalizeProtein() (ADT denoising), PrepareBridgeReference() (v5 bridge)
• Python: muon/mudata (MuData container), scvi.model.TOTALVI / MULTIVI, MOFA2/muon.tl.mofa, scglue (diagonal)

Governing Principle

Classify the integration task by its anchor structure FIRST, because the anchor decides which algorithm class is even applicable (Argelaguet 2021).

• Horizontal: same modality, different cells; anchor = shared features (batch correction, not this skill).
• Vertical (paired, same cell): multiple modalities measured in the same cells; anchor = shared cells. CITE-seq, 10x Multiome.
• Diagonal (unpaired): different modalities in different cells, no shared cells and no shared features; correspondence is inferred from prior knowledge. Independent scRNA + scATAC.
• Mosaic: partially observed grid of (modalities x batches); some blocks present, some missing.

Paired vs unpaired is the master fork: paired correspondence is known a priori (WNN, totalVI, MultiVI, MOFA+), unpaired/diagonal correspondence must be inferred (GLUE, Seurat v5 bridge), mosaic mixes both (MultiVI, StabMap). Two separately-paired datasets that share only one modality (for example a 10x Multiome and a CITE-seq experiment sharing only RNA) are a mosaic problem: anchor on the shared RNA and impute or bridge the modality-specific blocks with StabMap, MultiVI, or Seurat v5 bridge integration rather than forcing a single WNN.

CITE-seq ADT background is a three-part mixture, not one "ambient" term: (1) ambient antibody captured in every droplet including empties, (2) cell-intrinsic non-specific binding (Fc receptors, sticky dying cells) that does NOT appear in empties, (3) spillover/index hopping between barcodes. Denoise ADT (DSB or totalVI's built-in background mixture) BEFORE any joint embedding; raw or CLR-only ADT carries this background into the joint graph.

WNN can be dominated by the noisier modality: weights reward local neighbor predictability, and a handful of high-variance or saturating ADT features can manufacture self-consistent neighborhoods and get up-weighted despite carrying less biology. Report the per-cell weight distribution and check whether clustering survives down-weighting the suspect modality.

Imputed modalities are inferences, not measurements: MultiVI/StabMap/Cobolt impute the missing modality for unpaired cells, and gene-activity scores from ATAC approximate RNA. Differential expression or marker calls on imputed values are model-dependent and must be flagged as such.

Classify the Task: Anchor Structure -> Method Class

Anchor structure	What is shared	Example assay	Method class
Vertical / paired	Same cells	CITE-seq, 10x Multiome	WNN, totalVI, MultiVI(paired), MOFA+, mojitoo
Diagonal / unpaired	Nothing (prior graph)	Independent scRNA + scATAC	GLUE, Seurat v5 bridge, LIGER
Mosaic	Some modalities only	Batch A RNA+ATAC, batch B RNA	MultiVI, StabMap, Cobolt, totalVI(partial)

When methods compete, verify the current best-practice default against the installed tool docs before committing; the field moves and defaults drift across minor versions.

Method Decision Table (Paired CITE-seq / Multiome)

Method	Model / assumption	Use when	Fails when
WNN (Seurat)	Per-cell, per-modality weights from cross-modality neighbor prediction; one weighted graph	Fast joint embedding/clustering of one well-normalized paired dataset	Protein background not removed upstream; noisy/saturating modality dominates; not for unpaired/mosaic
totalVI (scvi-tools)	Conditional VAE; RNA NB/ZINB, each protein a 2-component NB mixture (background+foreground)	Need denoised protein, principled DE, batch integration, merging different antibody panels	Tiny datasets (VAE overfits); no GPU and very large data; protein-specific background structure not captured by one per-cell factor
MultiVI (scvi-tools)	Single joint VAE over RNA+ATAC(+protein); mosaic-capable, imputes missing modality	Paired+unpaired RNA/ATAC mixed (mosaic); want generative DE/DA	"batch" key is the modality indicator, not sequencing batch; imputed modalities treated as measured
MOFA+ (MOFA2)	Linear Bayesian group factor analysis; sparse factors, per-modality variance explained	Interpreting shared vs modality-specific axes of variation (exploratory/explanatory)	Used for clustering/denoising; likelihood mismatched to data; expecting batch correction within a view
mojitoo	CCA across precomputed per-modality reductions; fast, parameter-free	Quick paired joint reduction from existing PCA/LSI slots	No knob to down-weight a noisy modality; bounded by input reductions; paired only

Method Decision Table (Unpaired / Diagonal / Mosaic)

Method	Model / assumption	Use when	Fails when
GLUE (scglue)	Per-modality VAEs + prior feature graph (peak-near-gene); adversarial cell alignment	Unpaired diagonal scRNA + scATAC; want regulatory inference as a byproduct	Genome-build/coordinate mismatch yields an empty guidance graph and garbage alignment; adversarial over-mixing of distinct states
Seurat v5 bridge	Multiome bridge dataset = dictionary linking query modality to reference modality	Mapping a query (scATAC) onto a reference built in another modality (scRNA)	Poor/batch-mismatched bridge propagates error; rare query-only populations mislabeled
StabMap	Mosaic topology from shared features; project all cells via shortest paths	Mosaic with informative unshared features that cannot be dropped	Unshared-feature chaining compounds error per hop
Cobolt / scMoMaT	Generative shared latent over joint + single-modality datasets	Mosaic where a generative latent is preferred over feature chaining	DE/marker calls made on imputed values

ADT Normalization: CLR vs DSB

Method	What it does	Use when	Fails when
CLR (centered log-ratio)	Rescales compositionally; Seurat NormalizeData(method="CLR", margin=2)	Quick, no empty droplets available; small panels	Does NOT remove background; geometric-mean denominator distorted by saturating high-abundance ADTs
DSB	Ambient correction from empty droplets + per-cell technical denoising via 2-component mixture + isotype controls	Raw/unfiltered matrix available (needs empty droplets); want background removed before embedding	No empty droplets retained; protein-specific non-specific binding (one per-cell factor under/over-corrects); no clearly bimodal proteins

Seurat's CLR margin is genuinely ambiguous across versions (margin=2 = per-feature is the WNN-tutorial recommendation for large panels); verify with ?NormalizeData on the installed version.

CITE-seq: Denoise ADT, Then Joint Embed (Seurat)

Goal: Remove ADT background with DSB before WNN, because WNN does not denoise protein.

Approach: Estimate ambient from empty droplets and per-cell technical noise from a mixture plus isotype controls, then feed denoised ADT into the standard PCA -> WNN flow.

library(dsb)
library(Seurat)

raw <- Read10X('raw_feature_bc_matrix/')           # unfiltered: contains empty droplets
cells <- Read10X('filtered_feature_bc_matrix/')    # called cells

adt_cells <- as.matrix(cells[['Antibody Capture']])
adt_empty <- as.matrix(raw[['Antibody Capture']][, setdiff(colnames(raw[['Antibody Capture']]), colnames(adt_cells))])

# isotype.control.name.vec must name the ACTUAL isotype rows (often IgG1/IgG2a/Mouse-IgG2b-Ctrl); the regex below misses those
# When isotypes are absent or not matched, set use.isotype.control = FALSE (keep denoise.counts = TRUE) and pass real names explicitly
adt_dsb <- DSBNormalizeProtein(
    cell_protein_matrix = adt_cells,
    empty_drop_matrix = adt_empty
    denoise.counts  
    use.isotype.control  
    isotype.control.name.vec  grep rownamesadt_cells value  

CITE-seq: WNN Joint Clustering (Seurat)

Goal: Build one weighted-NN graph from denoised RNA and ADT and cluster on it.

Approach: Reduce each modality independently (PCA on RNA, PCA on the small ADT panel), then learn per-cell modality weights and cluster/embed on the joint graph.

obj[['ADT']] <- CreateAssay5Object(data = adt_dsb)        # DSB output is already normalized data
DefaultAssay(obj) <- 'RNA'
obj <- NormalizeData(obj) |> FindVariableFeatures() |> ScaleData() |> RunPCA(reduction.name = 'pca')

DefaultAssay(obj) <- 'ADT'
VariableFeatures(obj) <- rownames(obj[['ADT']])
obj <- ScaleData(obj) |> RunPCA(reduction.name = 'apca', npcs = min(18, nrow(obj[['ADT'  


obj  FindMultiModalNeighborsobj reduction.list    dims.list   
obj  FindClustersobj graph.name   algorithm     
obj  RunUMAPobj nn.name   reduction.name  


VlnPlotobj features   group.by  

CITE-seq: totalVI (Python, denoise + DE in one model)

Goal: Jointly model RNA + protein with explicit protein background, yielding a denoised latent space and foreground probabilities.

Approach: Register a MuData object, train the conditional VAE, then read the latent representation and per-protein foreground probability.

import scvi
import mudata as md

# mdata holds .mod['rna'] (raw counts) and .mod['prot'] (raw ADT counts)
scvi.model.TOTALVI.setup_mudata(
    mdata, rna_layer='counts', protein_layer=None,
    modalities={'rna_layer': 'rna', 'protein_layer': 'prot'}
)
model = scvi.model.TOTALVI(mdata)
model.train()

mdata.obsm['X_totalVI'] = model.get_latent_representation()
fg = model.get_protein_foreground_probability()        # 1 - background mixing weight per protein per cell
denoised_rna, denoised_prot = model.get_normalized_expression()

Multiome (RNA + ATAC, same cell): Native Pipelines, Then Join

Goal: Process each modality in its own statistics before joining, because RNA and ATAC have incompatible distributions.

Approach: PCA on RNA, TF-IDF + LSI on ATAC (drop depth-correlated components), then WNN. See scatac-analysis for ATAC QC and the binarization/depth-component caveats.

library(Signac)
DefaultAssay(obj) <- 'RNA'
obj <- NormalizeData(obj) |> FindVariableFeatures() |> ScaleData() |> RunPCA()

DefaultAssay(obj) <- 'ATAC'
obj <- RunTFIDF(obj) |> FindTopFeatures(min.cutoff = 'q0') |> RunSVD()
DepthCor(obj)                                          # diagnose which LSI components track depth

# dims = 2:30 drops LSI_1 ONLY if DepthCor confirms it tracks depth (usually true, not guaranteed)
obj <- FindMultiModalNeighbors(obj, reduction.list = list('pca', 'lsi'), dims.list = list(1: 
obj  RunUMAPobj nn.name   reduction.name  
obj  FindClustersobj graph.name   algorithm  

Merging multiome datasets requires a common peak set: re-quantify all cells against unified peaks, or peak-boundary differences manufacture spurious batch structure. The ATAC gene-activity matrix is an approximation, not measured RNA; do not conflate it with the RNA modality.

MOFA+ (interpretable shared/specific factors)

Goal: Decompose modalities into shared latent factors with per-modality variance explained.

Approach: Build a MOFA object from per-modality matrices, set likelihoods to match each data type, run, then interpret factor loadings.

import muon as mu

# likelihoods must match data: gaussian for scaled RNA, bernoulli for binarized ATAC, poisson for counts
mu.tl.mofa(mdata, n_factors=15, outfile='mofa_model.hdf5')   # writes mdata.obsm['X_mofa']

Unpaired / Diagonal: GLUE (Python)

Goal: Align independent scRNA and scATAC with no shared cells via a prior feature graph.

Approach: Configure each dataset with a count-appropriate probabilistic model, build a gene-anchored guidance graph, fit GLUE, then read aligned embeddings.

import scglue

scglue.models.configure_dataset(rna, 'NB', use_highly_variable=True, use_rep='X_pca')     # NB needs RAW counts
scglue.models.configure_dataset(atac, 'ZINB', use_highly_variable=True, use_rep='X_lsi')
graph = scglue.genomics.rna_anchored_guidance_graph(rna, atac)     # peak-near-gene prior; coords must share genome build
glue = scglue.models.fit_SCGLUE({'rna': rna, 'atac': atac}, graph)
rna.obsm['X_glue'] = glue.encode_data('rna', rna)
atac.obsm['X_glue'] = glue.encode_data('atac', atac)

Verify cell-type structure is preserved (not just modality overlap); adversarial alignment can over-mix distinct populations.

MuData Housekeeping

After per-modality QC, modalities hold different cell sets; muon.pp.intersect_obs(mdata) before any paired analysis. Editing a modality-local mdata.mod['rna'].obs needs mdata.update() to propagate to the global mdata.obs. R round-trips (MuDataSeurat, zellkonverter) are lossy; plan to stay in one ecosystem.

Common Errors

Symptom	Cause	Fix
WNN clustering driven entirely by ADT	A few saturating high-variance proteins dominate the neighbor graph	Report per-cell weight distribution; down-weight or denoise ADT (DSB); re-check clustering stability
"Background" smear in every ADT cluster	Ran WNN/CLR without empty-droplet denoising	Run DSB (needs raw/unfiltered matrix) or totalVI before joint embedding
DSB errors / nonsense output	Passed a filtered cell matrix only (no empty droplets)	Supply empty_drop_matrix from the raw/unfiltered matrix
Spurious batch structure after merging multiome	Per-dataset peak sets, not a unified set	Re-quantify all cells against one common peak set
GLUE produces a blob / no alignment	Guidance graph near-empty from genome-build/coordinate mismatch	Align RNA gene coords and ATAC peaks to the same build before building the graph
RNA and protein disagree for a marker	Often real post-transcriptional biology (stability, trafficking, lag), not an artifact	Do not "correct away"; treat single-gene discordance as informative
MultiVI batch effects persist	The batch_key was set to the modality indicator, not sequencing batch	Add a separate covariate for the real batch
DE on a modality looks too clean	Computed on imputed/gene-activity values, not measurements	Flag imputed-modality DE as model-dependent; validate against a measured modality

Related Skills

• single-cell/scatac-analysis - ATAC QC, TF-IDF/LSI, gene-activity caveats for the Multiome ATAC half
• single-cell/preprocessing - per-modality RNA QC and normalization before integration
• single-cell/clustering - clustering and UMAP on the joint graph
• single-cell/batch-integration - horizontal (same-modality, cross-sample) correction
• single-cell/markers-annotation - marker-based interpretation of joint clusters
• atac-seq/motif-deviation - chromVAR TF activity on the Multiome ATAC modality
• pathway-analysis/go-enrichment - functional interpretation of modality-specific factors

References

Argelaguet R, Cuomo ASE, Stegle O, Marioni JC. Computational principles and challenges in single-cell data integration. Nat Biotechnol 39(10):1202-1215 (2021). Stoeckius M, Hafemeister C, Stephenson W, et al. Simultaneous epitope and transcriptome measurement in single cells (CITE-seq). Nat Methods 14:865-868 (2017). Mulè MP, Martins AJ, Tsang JS. Normalizing and denoising protein expression data from droplet-based single-cell profiling (DSB). Nat Commun 13:2099 (2022). Hao Y, Hao S, Andersen-Nissen E, et al. Integrated analysis of multimodal single-cell data (WNN). Cell 184(13):3573-3587 (2021). Gayoso A, Steier Z, Lopez R, et al. Joint probabilistic modeling of single-cell multi-omic data with totalVI. Nat Methods 18:272-282 (2021). Ashuach T, Gabitto MI, Koodli RV, et al. MultiVI: deep generative model for the integration of multimodal data. Nat Methods 20(8):1222-1231 (2023). Argelaguet R, Arnol D, Bredikhin D, et al. MOFA+: a statistical framework for comprehensive integration of multi-modal single-cell data. Genome Biol 21:111 (2020). Cao Z-J, Gao G. Multi-omics single-cell data integration and regulatory inference with graph-linked unified embedding (GLUE). Nat Biotechnol 40(10):1458-1466 (2022). Hao Y, Stuart T, Kowalski MH, et al. Dictionary learning for integrative, multimodal and scalable single-cell analysis (Seurat v5 bridge). Nat Biotechnol 42:293-304 (2024). Bredikhin D, Kats I, Stegle O. MUON: multimodal omics analysis framework. Genome Biol 23:42 (2022). Ghazanfar S, Guibentif C, Marioni JC. Stabilized mosaic single-cell data integration using unshared features (StabMap). Nat Biotechnol 42(2):284-292 (2024). Yin Y, et al. Characterization and decontamination of background noise in droplet-based single-cell protein expression data with DecontPro. Nucleic Acids Res 52(1):e4 (2024).