Overview of Voyager
Sep 20, 2023
As including a more detailed vignette inside the package makes the package exceed the tarball size, more detailed vignettes are hosted on an external website. This is a simplified vignette.
1 Installation
This package can be installed from Bioconductor:
if (!requireNamespace("BiocManager")) install.packages("BiocManager")
BiocManager::install("Voyager")
# Devel version
# install.packages("remotes")
remotes::install_github("pachterlab/Voyager")2 Introduction
In non-spatial scRNA-seq, the SingleCellExperiment (SCE) package implements a data structure and other packages such as scater implement methods for quality control (QC), basic exploratory data analysis (EDA), and plotting functions, using SCE to organize the data and results. Voyager to SpatialFeatureExperiment (SFE) aims to be analogous scater to SFE, implementing basic exploratory spatial data analysis (ESDA) and plotting. SFE inherits from SCE and SpatialExperiment (SPE), so all methods written for SCE and SPE can be used for SFE as well.
In this first version, ESDA is based on the classic geospatial package spdep, but future versions will incorporate methods from GWmodel, adespatial, and etc.
These are the main functionalities of the Voyager at present:
- Univariate global spatial statistics, such as Moran’s I, Geary’s C, permutation testing of I and C, correlograms, global G, and semivariogram.
- Univariate local spatial statistics, such as local Moran’s I, local Geary’s C, Getis-Ord Gi*, Moran scatter plot, and local spatial heteroscedasticity (LOSH).
- Multivariate spatial statistics, such as MULTISPATI PCA and a multivariate generalization of local Geary’s C.
- Bivariate spatial statistics, such as Lee’s L (global and local) and cross variograms.
- Plotting gene expression and
colDataalong with annotation geometries, with colorblind friendly default palettes. The actual geometries are plotted, not just centroids as inSeurat. The tissue image can be plotted behind the geometries. - Plotting permutation testing results and correlograms, multiple genes in the same plot, can color by gene, sample, or any other attribute.
- Clustering correlograms and Moran’s scatter plots
- Plotting local spatial statistics in space
- Plotting dimension reduction in space
- Plotting spatial neighborhood graphs
- Plotting variograms and variogram maps
Future versions may add user friendly wrappers of some successful spatial transcriptomics data analysis packages for spatially variable genes, cell type deconvolution, and spatial regions on CRAN, Bioconductor, pip, and conda, to provide a uniform syntax and avoid object conversion, as is done in Seurat for some non-spatial scRNA-seq methods.
3 Dataset
Here we use a mouse skeletal muscle Visium dataset from Large-scale integration of single-cell transcriptomic data captures transitional progenitor states in mouse skeletal muscle regeneration. It’s in the SFEData package, as an SFE object, which contains Visium spot polygons, myofiber and nuclei segmentations, and myofiber and nuclei morphological metrics.
library(SFEData)
library(SpatialFeatureExperiment)
library(SpatialExperiment)
library(ggplot2)
library(Voyager)
library(scater)
library(scran)
library(pheatmap)This is the H&E image:
if (!file.exists("tissue_lowres_5a.jpeg")) {
download.file("https://raw.githubusercontent.com/pachterlab/voyager/main/vignettes/tissue_lowres_5a.jpeg",
destfile = "tissue_lowres_5a.jpeg")
}knitr::include_graphics("tissue_lowres_5a.jpeg")
sfe <- McKellarMuscleData()
#> see ?SFEData and browseVignettes('SFEData') for documentation
#> loading from cacheThis dataset was not originally in the standard Space Ranger output format, so we can’t use read10xVisiumSFE(). But the image can be added later for plotting.
sfe <- addImg(sfe, file = "tissue_lowres_5a.jpeg", sample_id = "Vis5A",
image_id = "lowres", scale_fct = 1024/22208)The image needs to be flipped to match the spots, because the origin of the image is at the top left corner while the origin of the spots is at the bottom left.
sfe <- mirrorImg(sfe, sample_id = "Vis5A", image_id = "lowres")# Only use spots in tissue here
sfe <- sfe[,colData(sfe)$in_tissue]
sfe <- logNormCounts(sfe)
sfe
#> class: SpatialFeatureExperiment
#> dim: 15123 932
#> metadata(0):
#> assays(2): counts logcounts
#> rownames(15123): ENSMUSG00000025902 ENSMUSG00000096126 ...
#> ENSMUSG00000064368 ENSMUSG00000064370
#> rowData names(6): Ensembl symbol ... vars cv2
#> colnames(932): AAACATTTCCCGGATT AAACCTAAGCAGCCGG ... TTGTGTTTCCCGAAAG
#> TTGTTGTGTGTCAAGA
#> colData names(13): barcode col ... in_tissue sizeFactor
#> reducedDimNames(0):
#> mainExpName: NULL
#> altExpNames(0):
#> spatialCoords names(2) : imageX imageY
#> imgData names(4): sample_id image_id data scaleFactor
#>
#> unit: full_res_image_pixels
#> Geometries:
#> colGeometries: spotPoly (POLYGON)
#> annotGeometries: tissueBoundary (POLYGON), myofiber_full (POLYGON), myofiber_simplified (POLYGON), nuclei (POLYGON), nuclei_centroid (POINT)
#>
#> Graphs:
#> Vis5A:4 Univariate spatial statistics
A spatial neighborhood graph is required for all spdep analyses.
colGraph(sfe, "visium") <- findVisiumGraph(sfe)All of the numerous univariate methods can be used with runUnivariate(), which stores global results in rowData(sfe) and local results in localResults(sfe). Here we compute Moran’s I for one gene. While Ensembl IDs are used internally, the user can specify more human readable gene symbols. A warning will be given if the gene symbol matches multiple Ensembl IDs.
features_use <- c("Myh1", "Myh2")sfe <- runUnivariate(sfe, type = "moran", features = features_use,
colGraphName = "visium", swap_rownames = "symbol")
# Look at the results
rowData(sfe)[rowData(sfe)$symbol %in% features_use,]
#> DataFrame with 2 rows and 8 columns
#> Ensembl symbol type means
#> <character> <character> <character> <numeric>
#> ENSMUSG00000033196 ENSMUSG00000033196 Myh2 Gene Expression 0.97476
#> ENSMUSG00000056328 ENSMUSG00000056328 Myh1 Gene Expression 4.82572
#> vars cv2 moran_Vis5A K_Vis5A
#> <numeric> <numeric> <numeric> <numeric>
#> ENSMUSG00000033196 24.0374 25.2984 0.625500 2.21641
#> ENSMUSG00000056328 302.2385 12.9785 0.635718 2.68736Since Moran’s I is very commonly used, one can call runMoransI rather than runUnivariate.
Compute a local spatial statistic, Getis-Ord Gi*, which is commonly used to detect hotspots and coldspots. The include_self argument is only for Getis-Ord Gi*; when set to TRUE Gi* is computed as the spatial graph includes self-directing edges, and otherwise Gi is computed.
sfe <- runUnivariate(sfe, type = "localG", features = features_use,
colGraphName = "visium", include_self = TRUE,
swap_rownames = "symbol")
# Look at the results
head(localResults(sfe, name = "localG")[[1]])
#> localG G*i E(G*i) V(G*i) Z(G*i) Pr(z != E(G*i))
#> 1 0.9539649 0.0013011969 0.001072961 5.72403e-08 0.9539649 0.3401014102
#> 2 2.2196433 0.0014737468 0.001072961 3.26030e-08 2.2196433 0.0264429927
#> 3 -1.4500290 0.0008111398 0.001072961 3.26030e-08 -1.4500290 0.1470504457
#> 4 3.5457397 0.0017131908 0.001072961 3.26030e-08 3.5457397 0.0003915127
#> 5 1.1947382 0.0012886869 0.001072961 3.26030e-08 1.1947382 0.2321893509
#> 6 -1.4750237 0.0008066266 0.001072961 3.26030e-08 -1.4750237 0.1402061682
#> -log10p -log10p_adj cluster
#> 1 0.4683916 0.0000000 High
#> 2 1.5776894 0.6745994 High
#> 3 0.8325337 0.0000000 High
#> 4 3.4072541 2.5041641 High
#> 5 0.6341577 0.0000000 High
#> 6 0.8532329 0.0000000 LowSpatial statistics can also be computed for numeric columns of colData(sfe), with colDataUnivariate(), and for numeric attributes of the geometries with colGeometryUnivariate() and annotGeometryUnivariate(), all with very similar arguments.
5 Bivariate spatial statistics
Akin to runUnivariate() and calculateUnivariate(), the uniform user interface for bivariate spatial statistics is runBivariate() and calculateBivariate(). Here we find top highly variable genes (HVGs) and compute a spatially informed correlation matrix, with Lee’s L. Note that global bivariate results can’t be stored in the SFE object in this version of Voyager.
gs <- modelGeneVar(sfe)
hvgs <- getTopHVGs(gs, fdr.threshold = 0.01)res <- calculateBivariate(sfe, "lee", hvgs)pheatmap(res, color = scales::viridis_pal()(256), cellwidth = 1, cellheight = 1,
show_rownames = FALSE, show_colnames = FALSE)
Here we see groups of genes correlated to each other, taking spatial autocorrelation into account. This matrix can be used in WGCNA to find gene coexpression modules. Note that Lee’s L of a gene with itself is not 1, because of a spatial smoothing factor.
6 Multivariate spatial statistics
Multivariate spatial statistics also have a uniform user interface, runMultivariate(). The matrix results will go to reducedDims, while vector and data frame results can go into colData. Here we perform a form of spatially informed PCA, which maximizes the product of Moran’s I and variance explained by each principal component. This method is called MULTISPATI, which is originally implemented in the adespatial package, but a much faster albeit less flexible implementation is used in Voyager. Because of the Moran’s I, MULTISPATI can give negative eigenvalues, signifying negative spatial autocorrelation. The number of the most negative components to compute is specified in the nfnega argument.
hvgs2 <- getTopHVGs(gs, n = 1000)
sfe <- runMultivariate(sfe, "multispati", colGraphName = "visium", subset_row = hvgs2,
nfposi = 10, nfnega = 10)7 Plotting
Plot gene expression and colData(sfe) together with annotation geometry. Here nCounts is the total UMI counts per spot, which is in colData.
plotSpatialFeature(sfe, c("nCounts", "Myh1"), colGeometryName = "spotPoly",
annotGeometryName = "myofiber_simplified",
aes_use = "color", linewidth = 0.5, fill = NA,
annot_aes = list(fill = "area"), swap_rownames = "symbol")
Plot local results, with the image. The maxcell argument is the maximum number of pixels to plot from the image. If the image has more pixels than that, then it will be downsampled to speed up plotting.
plotLocalResult(sfe, "localG", features = features_use,
colGeometryName = "spotPoly", divergent = TRUE,
diverge_center = 0, swap_rownames = "symbol",
image_id = "lowres", maxcell = 5e+4)
Plot the eigenvalues:
ElbowPlot(sfe, ndims = 10, nfnega = 10, reduction = "multispati") + theme_bw()
Plot dimension reduction (projection of each cell’s gene expression profile on each dimension) in space:
spatialReducedDim(sfe, "multispati", ncomponents = 2, image_id = "lowres",
maxcell = 5e+4, divergent = TRUE, diverge_center = 0)
8 Session info
sessionInfo()
#> R version 4.3.1 (2023-06-16)
#> Platform: x86_64-pc-linux-gnu (64-bit)
#> Running under: Ubuntu 22.04.3 LTS
#>
#> Matrix products: default
#> BLAS: /home/biocbuild/bbs-3.17-bioc/R/lib/libRblas.so
#> LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.10.0
#>
#> locale:
#> [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
#> [3] LC_TIME=en_GB LC_COLLATE=C
#> [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
#> [7] LC_PAPER=en_US.UTF-8 LC_NAME=C
#> [9] LC_ADDRESS=C LC_TELEPHONE=C
#> [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
#>
#> time zone: America/New_York
#> tzcode source: system (glibc)
#>
#> attached base packages:
#> [1] stats4 stats graphics grDevices utils datasets methods
#> [8] base
#>
#> other attached packages:
#> [1] pheatmap_1.0.12 scran_1.28.2
#> [3] scater_1.28.0 scuttle_1.10.2
#> [5] Voyager_1.2.7 ggplot2_3.4.3
#> [7] SpatialExperiment_1.10.0 SingleCellExperiment_1.22.0
#> [9] SummarizedExperiment_1.30.2 Biobase_2.60.0
#> [11] GenomicRanges_1.52.0 GenomeInfoDb_1.36.3
#> [13] IRanges_2.34.1 S4Vectors_0.38.1
#> [15] BiocGenerics_0.46.0 MatrixGenerics_1.12.3
#> [17] matrixStats_1.0.0 SpatialFeatureExperiment_1.2.3
#> [19] SFEData_1.2.0 BiocStyle_2.28.1
#>
#> loaded via a namespace (and not attached):
#> [1] later_1.3.1 bitops_1.0-7
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