Bio Applied Spatial Transcriptomics

Spatial Transcriptomics

When to Use

• Loading or QC-ing 10x Visium, Xenium, MERFISH, seqFISH+, Slide-seq, or Stereo-seq data with spatial coordinates.
• Detecting spatially variable genes (SVGs) or testing spatial autocorrelation (Moran's I, Geary's C).
• Building a spatial neighbor graph from spot/cell coordinates for downstream spatial statistics.
• Visualizing clusters or gene expression overlaid on a tissue histology image.
• Deconvolving multi-cell Visium spots into cell-type proportions using an scRNA-seq reference.

Version Compatibility

• Python: squidpy >=1.4, scanpy >=1.10, anndata >=0.10, Python >=3.10
• R: Seurat >=5.0 (Load10X_Spatial, SCTransform, FindSpatiallyVariableFeatures)

Prerequisites

• pip install squidpy scanpy leidenalg (or install.packages("Seurat") in R)
• Familiarity with the standard scRNA-seq pipeline (normalize -> HVG -> PCA -> neighbors -> Leiden) — see bio-single-cell-preprocessing and bio-single-cell-clustering
• Basic AnnData structure (bio-single-cell-data-io / anndata skill)

Platform Comparison

AnnData spatial slots: adata.obsm["spatial"] (n_spots, 2 pixel coords), adata.uns["spatial"] (tissue image + scale factors), adata.obsp["spatial_connectivities"] (spatial neighbor graph, after sq.gr.spatial_neighbors).

Core Workflow

Goal: Load a Visium dataset, QC-filter spots, and cluster on expression (ignoring spatial coordinates). Approach: reuse the standard scanpy pipeline — spatial coordinates only matter starting at the neighbor-graph step.

import scanpy as sc
import squidpy as sq


def load_and_cluster_visium(min_counts=200, min_cells=5, n_top_genes=2000, resolution=0.5):
    """Load the public Visium mouse-brain dataset, QC-filter, normalize, and Leiden-cluster.

    Returns the processed AnnData with `obs["leiden"]` cluster labels.
    """
    adata = sq.datasets.visium_hne_adata()

    # QC metrics (mitochondrial fraction uses mouse "mt-" prefix; use "MT-" for human)
    adata.var["mt"] = adata.var_names.str.startswith("mt-")
    sc.pp.calculate_qc_metrics(adata, qc_vars=["mt"], inplace=True)
    sc.pp.filter_cells(adata, min_counts=min_counts)
    sc.pp.filter_genes(adata, min_cells=min_cells)

    sc.pp.normalize_total(adata, target_sum=1e4)
    sc.pp.log1p(adata)
    sc.pp.highly_variable_genes(adata, n_top_genes=n_top_genes, subset=False)

    sc.pp.pca(adata, n_comps=50, use_highly_variable=True)
    sc.pp.neighbors(adata, n_pcs=30)
    sc.tl.leiden(adata, resolution=resolution, key_added="leiden")
    return adata

Goal: Build the spatial neighbor graph and detect spatially variable genes (SVGs). Approach: sq.gr.spatial_neighbors connects spots by physical proximity, then sq.gr.spatial_autocorr(mode="moran") computes Moran's I with a permutation p-value per gene.

def detect_spatially_variable_genes(adata, n_genes=500, n_perms=100, n_rings=1):
    """Build a spatial neighbor graph and rank genes by Moran's I spatial autocorrelation.

    n_perms=100 gives approximate p-values for exploration; use n_perms=1000 for
    publication-quality results (permutation testing is required because spatial
    data violates the independence assumption of parametric tests).
    """
    sq.gr.spatial_neighbors(adata, coord_type="visium", n_rings=n_rings)
    sq.gr.spatial_autocorr(
        adata, mode="moran", genes=adata.var_names[:n_genes], n_perms=n_perms
    )
    moran = adata.uns["moranI"].sort_values("I", ascending=False)
    return moran  # columns: I, pval_norm, pval_sim, pval_sim_fdr_bh (top I ~= +1 -> spatial clusters)

Goal: Visualize Leiden clusters and top SVGs overlaid on the tissue image. Approach: sq.pl.spatial_scatter renders points on top of the H&E image stored in adata.uns["spatial"].

def plot_clusters_and_svgs(adata, moran, n_top=6):
    """Plot spatial clusters and the top-N spatially variable genes on the tissue image."""
    sq.pl.spatial_scatter(adata, color="leiden", size=1.4)
    top_svgs = moran.head(n_top).index.tolist()
    sq.pl.spatial_scatter(adata, color=top_svgs, ncols=3, size=1.4, img_alpha=0.4)
    return top_svgs

R Equivalent (Seurat)

Goal: Load a Visium sample, cluster, and find spatially variable features in R. Approach: Seurat's spatial workflow mirrors the Scanpy/Squidpy one, with FindSpatiallyVariableFeatures(method = "moransi") as the SVG-detection step.

library(Seurat)

## Load 10x Space Ranger output (expects filtered_feature_bc_matrix.h5 + spatial/ dir)
brain <- Load10X_Spatial(data.dir = "visium_sample/")

brain <- SCTransform(brain, assay = "Spatial", verbose = FALSE)
brain <- RunPCA(brain, assay = "SCT", verbose = FALSE)
brain <- FindNeighbors(brain, reduction = "pca", dims = 1:30)
brain <- FindClusters(brain, resolution = 0.5)
brain <- RunUMAP(brain, reduction = "pca", dims = 1:30)

SpatialDimPlot(brain, label = TRUE, label.size = 3)

## Moran's I-based spatially variable genes (equivalent to sq.gr.spatial_autocorr)
brain <- FindSpatiallyVariableFeatures(
  brain, assay = "SCT", features = VariableFeatures(brain)[1:500],
  selection.method = "moransi"
)
top_svgs <- head(SpatiallyVariableFeatures(brain, selection.method = "moransi"), 6)
SpatialFeaturePlot(brain, features = top_svgs, ncol = 3)

Cell-type Deconvolution (Visium)

Each Visium spot captures ~10-20 cells; deconvolution estimates per-spot cell-type composition from an scRNA-seq reference.

Result is typically stored as adata.obsm["cell_type_proportions"] (n_spots x n_types), plottable with sq.pl.spatial_scatter(adata, color=proportions.columns).

Pitfalls

• Mitochondrial prefix: use "mt-" for mouse, "MT-" for human when computing pct_counts_mt — wrong case silently zeroes out the QC metric.
• Batch effects: check for batch confounding across sections/slides before interpreting spatial patterns as biological.
• Permutation count: n_perms=100 (Python) gives approximate p-values only; use n_perms=1000+ (or R equivalent) for publication.
• Spot vs. cell resolution: Visium/Slide-seq spots contain multiple cells — cluster labels and SVGs describe local tissue neighborhoods, not single cells, unless deconvolved.
• Deconvolution reference quality: results are highly sensitive to how well the scRNA-seq reference's cell types match the tissue and technology.
• coord_type mismatch: sq.gr.spatial_neighbors(coord_type=...) must match the platform ("visium" for hex-grid spots, "generic" with a radius/n_neighs for imaging-based data) or the graph will be topologically wrong.

See Also

• bio-single-cell-preprocessing — QC/normalization shared with scRNA-seq
• bio-single-cell-clustering — Leiden/Louvain clustering used before spatial analysis
• bio-spatial-transcriptomics-spatial-deconvolution — dedicated cell-type deconvolution methods
• bio-spatial-transcriptomics-spatial-domains — spatial domain detection beyond expression clustering