Voyager

Voyager

Workflows

Standard Workflow

Perform exploratory spatial data analysis (ESDA) on spatial transcriptomics data, including univariate, bivariate, and multivariate spatial statistics, and visualize the results over tissue geometries.

library(SFEData)
library(SpatialFeatureExperiment)
library(SpatialExperiment)
library(ggplot2)
library(Voyager)
library(scater)
library(scran)
library(pheatmap)

# 1. Load required libraries & 2. Read dataset
sfe <- McKellarMuscleData()

# 3. Add and mirror the tissue image
sfe <- addImg(sfe, imageSource = "tissue_lowres_5a.jpeg", sample_id = "Vis5A", image_id = "lowres", scale_fct = 1024/22208)
sfe <- mirrorImg(sfe, sample_id = "Vis5A", image_id = "lowres")

# 4. Filter spots and normalize counts
sfe <- sfe[, colData(sfe)$in_tissue]
sfe <- logNormCounts(sfe)

# 5. Construct a spatial neighborhood graph
colGraph(sfe, "visium") <- findVisiumGraph(sfe)

# 6. Run univariate spatial statistics (Moran's I and Getis-Ord Gi*)
features_use <- c("Myh1", "Myh2")
sfe <- runUnivariate(sfe, type = "moran", features = features_use, colGraphName = "visium", swap_rownames = "symbol")
sfe <- runUnivariate(sfe, type = "localG", features = features_use, colGraphName = "visium", include_self = TRUE, swap_rownames = "symbol")

# 7. Identify highly variable genes and calculate bivariate spatial correlation (Lee's L)
gs <- modelGeneVar(sfe)
hvgs <- getTopHVGs(gs, fdr.threshold = 0.01)
res <- calculateBivariate(sfe, "lee", hvgs)

# 8. Perform spatially-informed dimension reduction (MULTISPATI PCA)
hvgs2 <- getTopHVGs(gs, n = 1000)
sfe <- runMultivariate(sfe, "multispati", colGraphName = "visium", subset_row = hvgs2, nfposi = 10, nfnega = 10)

# 9. Visualize results
plotSpatialFeature(sfe, c("nCounts", "Myh1"), colGeometryName = "spotPoly", annotGeometryName = "myofiber_simplified", aes_use = "color", swap_rownames = "symbol")
plotLocalResult(sfe, "localG", features = features_use, colGeometryName = "spotPoly", divergent = TRUE, swap_rownames = "symbol")
ElbowPlot(sfe, ndims = 10, nfnega = 10, reduction = "multispati")
spatialReducedDim(sfe, "multispati", ncomponents = 2, divergent = TRUE)

Input: A SpatialFeatureExperiment object and a tissue image. Output: Spatial plots of gene expression, local spatial statistics, eigenvalues, and reduced dimensions.

When to Use

• To perform exploratory spatial data analysis (ESDA) on spatial transcriptomics data structured as a SpatialFeatureExperiment (SFE) object.
• To compute univariate global and local spatial statistics such as Moran's I (using runUnivariate with type = "moran") and Getis-Ord Gi* (using type = "localG").
• To calculate bivariate spatial correlation (e.g., Lee's L via calculateBivariate) to identify spatially co-expressed gene modules.
• To run spatially-informed dimension reduction like MULTISPATI PCA (using runMultivariate with type = "multispati").

When NOT to Use

• For non-spatial single-cell RNA-seq data; use standard SingleCellExperiment and scater instead.
• If you need to perform cell type deconvolution or spatial domain segmentation directly; use specialized packages like Seurat or Giotto.

Data Requirements

• A SpatialFeatureExperiment (SFE) object containing spatial coordinates, count matrices, and spot/annotation geometries (e.g., spotPoly).
• A spatial neighborhood graph constructed and stored in the SFE object (e.g., using findVisiumGraph).

Key Parameters

• type ("moran" / "localG" / "lee" / "multispati"): The spatial statistic method to run.
• colGraphName ("visium"): The name of the spatial neighborhood graph stored in the SFE object.
• include_self (TRUE): For Getis-Ord Gi*, determines whether to include self-directing edges in the spatial graph.
• swap_rownames ("symbol"): Column in rowData to swap rownames with (e.g., to use human-readable gene symbols instead of Ensembl IDs).
• nfposi (10): Number of positive spatial autocorrelation components to retain in MULTISPATI PCA.
• nfnega (10): Number of negative spatial autocorrelation components to retain in MULTISPATI PCA.
• divergent (TRUE): Logical indicating whether to use a divergent color palette for local spatial statistics or reduced dimensions.

Best Practices

• Always construct a spatial neighborhood graph using findVisiumGraph before running any spatial statistics.
• Mirror the tissue image using mirrorImg if the origin of the image (top-left) does not align with the origin of the spots (bottom-left).
• Filter spots to keep only those in tissue (colData(sfe)$in_tissue) and normalize counts using logNormCounts before analysis.

Common Pitfalls

• Mismatch between image and spot coordinates: Ensure you call mirrorImg to flip the image if coordinates are inverted.
• Missing spatial graph: Forgetting to assign the output of findVisiumGraph to colGraph(sfe, "visium") will cause downstream spatial functions to fail.

Alternatives

• spdep for classic geospatial analysis.
• adespatial for multivariate spatial analysis (MULTISPATI PCA).
• scater for non-spatial single-cell exploratory data analysis.

Citations

• Moses L, Pachter L (2026). Voyager: Exploratory Spatial Data Analysis for Spatial Bioconductor. R package.

References

• Homepage: bioconductor.org/packages/Voyager
• Vignette: https://bioconductor.org/packages/release/bioc/vignettes/Voyager/inst/doc/voyager.html