CatsCradle Spatial Vignette
Introduction
Here we describe the tools that CatsCradle offers for exploiting the spatial relationships in spatial transcriptomics data.
We will be using a subset of a Xenium data set that profiles the mouse hippocampus available from 10x genomics (https://www.10xgenomics.com/datasets/fresh-frozen-mouse-brain-for-xenium-explorer-demo-1-standard)
Here we visualise this subset coloured by cell type. Here cell clusters (Louvain cluster) have been assigned cell type identities using RCTD along with a reference dataset (https://www.dropbox.com/s/cuowvm4vrf65pvq/allen_cortex.rds?dl=1) followed by minimal manual curation. Please note these assignments are not definitive and are for illustratory purporses only.
library(Seurat,quietly=TRUE)
library(CatsCradle,quietly=TRUE)
getExample = make.getExample()
smallXenium = getExample('smallXenium')
ImageDimPlot(smallXenium, cols = "polychrome", size = 1)
We will want to answer questions like the following:
Is the expression of a particular gene localised? Who are the immediate neighbours of a given cell? Do certain types of cells tend to co-localise? Is the spatial association of two cell types, (say, glia and macrophages) statistically significant? For any pair of neighbouring cells, are they engaged in ligand-repceptor interactions? Can we classify different types of tissue neighbourhoods? Are different neighbourhood types characterised by the cell types found in them? The genes expressed in them? The ligand-receptor interactions taking place in them?
Neighbourhoods
A key concept here is that of a neighbourhood. A neighbourhood is a spatially contiguous set of cells. In every case we will consider, a neighbourhood is a set of cells centred on a particular cell.
The simplest sort of neighbourhood consists of a cell together with its immediate neighbours. We compute these as a Delaunay triangulation using the centroids of the cells:
centroids = GetTissueCoordinates(smallXenium)
rownames(centroids) = centroids$cell
clusters = smallXenium@active.ident
delaunayNeighbours = computeNeighboursDelaunay(centroids)
head(delaunayNeighbours)
#> nodeA nodeB
#> 1 16307 16316
#> 2 16314 16317
#> 3 16296 16299
#> 4 16299 16307
#> 5 16309 16316
#> 6 16309 16314Two cells are neighbours if they appear on the same row. The neighbourhood of the cell 16307 consists of the following 7 cells:
idx = (delaunayNeighbours$nodeA == 16307 |
delaunayNeighbours$nodeB == 16307)
nbhd = unique(c(delaunayNeighbours$nodeA[idx],
delaunayNeighbours$nodeB[idx]))
nbhd
#> [1] "16307" "16299" "16303" "16298" "16296" "16316" "16317"We can compute extended neighbourhoods by considering (say) each cell’s neighbours, their neighbours, their neighbours and their neighbours. Here we have such an extended neighbourhood as a list and again as a data frame with each row giving a pair of cells in a common extended neighbourhood. In this case we are building an extended neighbourhood of combinatorial radius 4.
extendedNeighboursList = getExtendedNBHDs(delaunayNeighbours, 4)
#> radius 2
#> radius 3
#> radius 4
extendedNeighbours = collapseExtendedNBHDs(extendedNeighboursList, 4)Now the extended neighbourhood of cell 16307 consists of 92 cells:
idx = (extendedNeighbours$nodeA == 16307 |
extendedNeighbours$nodeB == 16307)
nbhd = unique(c(extendedNeighbours$nodeA[idx],
extendedNeighbours$nodeB[idx]))
length(nbhd)
#> [1] 92We can also create neighbours based on Euclidean distance in the tissue.
We see two reasons for looking at these extended neighbourhoods. One is that while the Delaunay triangulation might be more appropriate for looking at cell to cell interactions involving direct contact, extended neighbourhoods might be more appropriate for studying interactions based on diffusible ligands. The second is that when we go on to characterise types of neighbourhoods, these extended neighbourhoods exhibit less noise.
We offer two viewpoints as to what is “going on” in a neighbourhood. One is that a neighbourhood is characterised by the cell types that are found in it. In this view, just as a cell expresses genes, a neighbourhood “expresses” cell types. A second viewpoint is that a neighbourhood also expresses genes. That is, for each neighbourhood, we can compute the total gene expression across all the cells in that neighbourhood.
In this vignette we will focus on the first viewpoint. This is not because we think the first viewpoint is more important or because we necessarily expect it to be more fruitful. It’s because the second viewpoint so directly parallels standard Seurat single cell transcriptomics analyses that it requires less explanation. So before we move on to the neighbourhoods / cell types viewpoint, we give an overview of the neighbourhoods / aggregate gene expression viewpoint.
Neighbourhoods as characterised by gene expression
Here we create a Seurat object based on the aggregate gene expression in each of our extended neighbourhoods and display the clustering of the neighbourhoods (called aggregation_clusters) both on the tissue plot and on the resulting UMAP.
agg = aggregateGeneExpression(smallXenium,extendedNeighbours,
verbose=FALSE)
#> Warning: Data is of class matrix. Coercing to dgCMatrix.
smallXenium$aggregateNBHDClusters = agg@active.ident
ImageDimPlot(smallXenium,group.by='aggregateNBHDClusters',cols='polychrome')
Since neighbourhoods here are indexed by individual cells, the neighbourhood gene aggregation Seurat object is formally identical to a standard Seurat object. In particular, this allows the standard sorts of analyses including clustering of neighbourhoods into types; dimension reduction using PCA, UMAP, tSNE; discovery of marker genes for each neighbourhood type; plotting of aggregate gene expression on neighbourhood UMAP; use of CatsCradle to discover novel clustering of genes based on their expression across the different neighbourhoods.
Neighbourhoods as characterised by cell types
Calculation of neighbourhood celltype composition
Given any of the spatial graphs describing neighbourhoods calculated above, we can now calculate the cell type composition of neighbourhoods.
In the resulting matrix neighbourhoods are rows and cell types are columns. The values in the matrix indicate the number of cells of a given type within a neighbourhood.
Let’s do the same for our extended neighbourhoods.
Analysis of contact based interactions between cell types
We can go on to calculate a matrix which gives the fraction of contacts cells of a given type make with cells of another cell type.
Rows and columns both correspond to cell types, but they are playing different roles. For a given row (say, cell type A) the entry in column B represents the fraction of cells in neighbourhoods of cells of type A that are of type B.
We can then display this as a force directed graph. Here we choose only to display contact based interactions that constitute at least 5% of a cell type’s interactions. Of note, this graph is directed as, for example, 50% of cell type A’s interactions might be with cell type B, but only 5% of cell type B’s interactions might be with cell type A.
colours = DiscretePalette(length(levels(clusters)), palette = "polychrome")
names(colours) = levels(clusters)
cellTypesPerCellTypeGraphFromCellMatrix(cellTypesPerCellTypeMatrix,
minWeight = 0.05, colours = colours)
#> IGRAPH 4d779b1 DNW- 16 55 --
#> + attr: coords (g/n), name (v/c), color (v/c), weight (e/n), width
#> | (e/n)
#> + edges from 4d779b1 (vertex names):
#> [1] 0_Oligo->3_Endo 0_Oligo->5_Astro 0_Oligo->10_Astro
#> [4] 0_Oligo->11_Oligo 0_Oligo->13_Microglia 0_Oligo->14_Ependymal
#> [7] 3_Endo ->0_Oligo 3_Endo ->5_Astro 3_Endo ->10_Astro
#> [10] 3_Endo ->11_Oligo 4_Astro->0_Oligo 4_Astro->3_Endo
#> [13] 4_Astro->5_Astro 4_Astro->6_VLMC 4_Astro->7_Granule
#> [16] 4_Astro->10_Astro 4_Astro->11_Oligo 4_Astro->14_Ependymal
#> [19] 4_Astro->17_Lamp5 4_Astro->20_VLMC 5_Astro->3_Endo
#> + ... omitted several edgesHere arrows are directed from rows to columns. Thus, we see an arrow from 12_Pvalb to 18_pyramidal because neighbourhoods of cells of type 12_Pvalb are composed (across the dataset) of ~15% cells of type 18_pyramidal. We do not see an arrow from 18_pyramidal to 12_Pvalb because neighbourhoods of cells of type 18_pyramidal have only 3% cells of type 12_Pvalb, which falls below the chosen cutoff.
It’s worth pointing out the following. The number of edges from cells of type A to cells of type B is the same as the number of edges from cells of type B to cells of type A. Thus the matrix of counts of these edges is symmetric. However, numbers of cells of types A and B are not necessarily equal, and this accounts for the assymmetry in the fractions.
Let’s do the same for our extended nighbourhoods.
cellTypesPerCellTypeMatrixExtended = computeCellTypesPerCellTypeMatrix(NBHDByCTMatrixExtended,clusters)
cellTypesPerCellTypeGraphFromCellMatrix(cellTypesPerCellTypeMatrixExtended,
minWeight = 0.05, colours = colours)
#> IGRAPH 6b00f4b DNW- 16 62 --
#> + attr: coords (g/n), name (v/c), color (v/c), weight (e/n), width
#> | (e/n)
#> + edges from 6b00f4b (vertex names):
#> [1] 0_Oligo->3_Endo 0_Oligo->10_Astro 0_Oligo->11_Oligo
#> [4] 0_Oligo->14_Ependymal 3_Endo ->0_Oligo 3_Endo ->5_Astro
#> [7] 3_Endo ->6_VLMC 3_Endo ->10_Astro 3_Endo ->11_Oligo
#> [10] 3_Endo ->14_Ependymal 4_Astro->0_Oligo 4_Astro->5_Astro
#> [13] 4_Astro->6_VLMC 4_Astro->7_Granule 4_Astro->10_Astro
#> [16] 4_Astro->11_Oligo 4_Astro->14_Ependymal 4_Astro->20_VLMC
#> [19] 5_Astro->6_VLMC 5_Astro->11_Oligo 6_VLMC ->10_Astro
#> + ... omitted several edgesWe can also calculate p values (upper tail, one-sided) for whether cell types are more commonly neighbours than expected by chance. To do this we compare the actual spatial neighbour graph to randomised spatial neighbour graphs where edges are randomised but the degree of each vertice is preserved. As this is carried out on the level of counts of undirected edges between celltypes the p value matrix is symmetric.
cellTypesPerCellTypePValues = computeNeighbourEnrichment(delaunayNeighbours,
clusters, nSim=100,verbose = FALSE)
#> Warning in simGraph$nodeB[selfEdge] <- nodeB[(selfEdge + 1)%%n]: number of
#> items to replace is not a multiple of replacement length
#> Warning in simGraph$nodeB[(selfEdge + 1)%%n] <- nodeB[selfEdge]: number of
#> items to replace is not a multiple of replacement lengthLet’s plot -log10(pvalue)
cellTypesPerCellTypePValuesNegLog = -log10(cellTypesPerCellTypePValues)
pheatmap(cellTypesPerCellTypePValuesNegLog)
Analysis of neighbourhoods based on cell type composition
As mentioned above we can create Seurat objects of neighbourhoods where the underlying counts matrix gives the the number of cells of each type in a given neighbourhood. We can now perform dimensionality reduction and clustering based on neighbourhood composition. As the dimensionality of the feature space is relatively low (number of cell types) we calculate the UMAP using features rather than PCs.
NBHDByCTSeurat = computeNBHDVsCTObject(NBHDByCTMatrix,verbose=FALSE)
#> Warning: Feature names cannot have underscores ('_'), replacing with dashes
#> ('-')
#> Warning: Data is of class matrix. Coercing to dgCMatrix.
#> Warning in irlba(A = t(x = object), nv = npcs, ...): You're computing too large
#> a percentage of total singular values, use a standard svd instead.
#> Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
#>
#> Number of nodes: 4261
#> Number of edges: 165647
#>
#> Running Louvain algorithm...
#> Maximum modularity in 10 random starts: 0.9825
#> Number of communities: 17
#> Elapsed time: 0 secondsAdd cell type information to the neighbourhoodSeurat object.
Visualise neighbourhood clusters.
DimPlot(NBHDByCTSeurat, group.by = c("neighbourhood_clusters"), cols = "polychrome", reduction = "umap")
We can now add information on neighbourhood clusters to our original Xenium object and visualise these on the tissue.
smallXenium$NBHDCluster = NBHDByCTSeurat@active.ident
ImageDimPlot(smallXenium, group.by = "NBHDCluster", size = 1, cols = "polychrome")
Let’s try the same thing with our extended neighbourhoods up to degree 4.
NBHDByCTSeuratExtended = computeNBHDVsCTObject(NBHDByCTMatrixExtended,
verbose=FALSE)
#> Warning: Feature names cannot have underscores ('_'), replacing with dashes
#> ('-')
#> Warning: Data is of class matrix. Coercing to dgCMatrix.
#> Warning in irlba(A = t(x = object), nv = npcs, ...): You're computing too large
#> a percentage of total singular values, use a standard svd instead.
#> Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
#>
#> Number of nodes: 4261
#> Number of edges: 105168
#>
#> Running Louvain algorithm...
#> Maximum modularity in 10 random starts: 0.9694
#> Number of communities: 8
#> Elapsed time: 0 secondsAdd cell type information to the NBHDByCTSeuratExtended object.
Visualise extended neighbourhood clusters.
DimPlot(NBHDByCTSeuratExtended, group.by = c("neighbourhood_clusters"), cols = "polychrome", reduction = "umap")
We can now add information on extended neighbourhood clusters to our original Xenium object and visualise these on the tissue.
smallXenium$NBHDClusterExtended=
NBHDByCTSeuratExtended@active.ident
ImageDimPlot(smallXenium, group.by = c("NBHDClusterExtended"),
size = 1, cols = "polychrome")
Here we retrieve fewer clusters, and these describe tissue architecture rather than small variations in cellular niche.
We leave it to the user to decide which approach is most applicable to the biological question at hand.
Relating cell type clusters to neighbourhood clusters
We can now ask how clusters defined transcriptomically (cell type) relate to those defined based on neighbourhoods (cell niche). Put differently, each cell has its (transcriptomic) cell type and sits at the center of a neighbourhood with a given neighbourhood type. For each cell type A and each neighbourhood type B, we can ask what percentage of the time a cell of type A sits at the center of a neighbourhood of type B.
CTByNBHDCluster = table(NBHDByCTSeurat$cellType,NBHDByCTSeurat@active.ident)
CTByNBHDCluster = CTByNBHDCluster/rowSums(CTByNBHDCluster)
rownames(CTByNBHDCluster) = paste0("CellType",rownames(CTByNBHDCluster))
colnames(CTByNBHDCluster) = paste0("NBHDCluster",colnames(CTByNBHDCluster))
pheatmap(CTByNBHDCluster,
fontsize_row=8,
fontsize_col=8,
cellheight=10,
cellwidth=10)
This allows us to see which cell types share the same niche (neighbourhood clusters). Let’s also perform this analysis using the extended neighbourhoods.
CTByNBHDClusterExtended = table(NBHDByCTSeuratExtended$cellType,NBHDByCTSeuratExtended@active.ident)
CTByNBHDClusterExtended = CTByNBHDClusterExtended/rowSums(CTByNBHDClusterExtended)
rownames(CTByNBHDClusterExtended) = paste0("CellType",rownames(CTByNBHDClusterExtended))
colnames(CTByNBHDClusterExtended) = paste0("NBHDCluster",colnames(CTByNBHDClusterExtended))
pheatmap(CTByNBHDClusterExtended,
fontsize_row=8,
fontsize_col=8,
cellheight=10,
cellwidth=10)
Analysing cell types based on their neighbourhoods
Now we perform dimensionality reduction and clustering of cell types, based on the neighbourhoods they are found in. Note that this is based on the transpose of the matrix used in the previous section.
First we create a Seurat object for a cell type by neighbourhood matrix (the transposed NBHDByCTMatrix). Here we have a large feature space (number of cells) however a low number of observations (number of cell types). Therefore we compute the UMAP using PCs, however due to the low number of observations we need to set a lower value for n.neighbours.
CTByNBHDSeurat =
computeNBHDVsCTObject(t(NBHDByCTMatrix), npcs = 10,
transpose = TRUE, resolution = 1, n.neighbors = 5,
verbose=FALSE)
#> Warning: Data is of class matrix. Coercing to dgCMatrix.
#> Warning in irlba(A = t(x = object), nv = npcs, ...): You're computing too large
#> a percentage of total singular values, use a standard svd instead.
#> Warning: k.param set larger than number of cells. Setting k.param to number of
#> cells - 1.
#> Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
#>
#> Number of nodes: 16
#> Number of edges: 120
#>
#> Running Louvain algorithm...
#> Maximum modularity in 10 random starts: 0.0000
#> Number of communities: 1
#> Elapsed time: 0 seconds
CTByNBHDSeurat$cellType = colnames(CTByNBHDSeurat)
DimPlot(CTByNBHDSeurat, group.by = "cellType", cols = "polychrome",
reduction = "umap", label = TRUE)
Note how Pyramidal, Pvalb and Lamp5 neurons are located close together in UMAP space indicating they are often found in the same neighbourhoods.
We can also compute a force-directed graph embedding.
CTByNBHDSeurat= computeGraphEmbedding(CTByNBHDSeurat)
#> Warning: Keys should be one or more alphanumeric characters followed by an
#> underscore, setting key from graph to graph_
DimPlot(CTByNBHDSeurat,group.by = "cellType", cols = "alphabet", reduction = "graph", label = TRUE)
From this 11_oligo and 5_Astro appear to be in neighbourhoods distinct from the other cell types. Looking at cell type localisation in the tissue shows that they colocalise.
Clustering cell types using the Louvain algorithm acheives only one cluster. Let’s try hierachical clustering.
Here we decide to cut the tree at the correct level to form 11 clusters.
Let’s look at the celltype composition of these clusters.
CTComposition = table(CTByNBHDSeurat$cellType, CTByNBHDSeurat$neighbourhood_clusters)
pheatmap(CTComposition)
Although most clusters here are formed of a single cell type, cluster 2 contains several cell types. We anticipate this analysis may be more informative when performed on a larger tissue area, as some cell types are poorly represented in this subset. To this end we demonstrate how to explore the relationship between neighbourhood clusters and cell type clusters.
averageExpMatrix = getAverageExpressionMatrix(NBHDByCTSeurat,
CTByNBHDSeurat,
clusteringName='neighbourhood_clusters')
averageExpMatrix = tagRowAndColNames(averageExpMatrix,
ccTag='neighbourhoodClusters_',
gcTag='cellTypeClusters_')
pheatmap(averageExpMatrix,
fontsize_row=8,
fontsize_col=8,
cellheight=10,
cellwidth=10)
Detection of genes with spatially variable expression.
Given a neighbour graph describing the spatial relationships between cells, we can compute spatial autocorrelation (here we use Moran’s I). This describes how clustered gene expression values are in space. We derive an upper tail p value based on permutation testing where expression values for genes are permuted. N.B in this implementation equal weights are given to all neighbours of a cell. Minimum values are limited by nSim, the number of permutations used.
moransI = runMoransI(smallXenium, delaunayNeighbours, assay = "SCT",
layer = "data", nSim = 20, verbose = FALSE)Look at most spatially autocorrelated genes.
head(moransI)
#> moransI pValues
#> Nwd2 0.8759895 0.05
#> Neurod6 0.8592572 0.05
#> 2010300C02Rik 0.8232980 0.05
#> Cabp7 0.8084018 0.05
#> Epha4 0.8058667 0.05
#> Bhlhe22 0.8050729 0.05Look at least spatially autocorrelated genes.
tail(moransI)
#> moransI pValues
#> Arhgap25 0.1602768 0.05
#> Myl4 0.1519870 0.25
#> Gm19410 0.1517466 0.35
#> Rxfp1 0.1509323 0.20
#> Opn3 0.1497122 0.25
#> Trbc2 0.1294289 0.80Here we visualise the most spatially autocorrelated gene.
Here we visualise the least spatially autocorrelated gene.
Ligand receptor analysis
We can perform an analysis of ligand receptor interactions in order to infer communication between cells. Here we calculate whether interactions between ligand receptor pairs occur on edges of the spatial graph, i.e. between neighbouring cells, where cell A expresses a ligand and cell B expresses a receptor. Note that the distance between cell A and cell B will vary depending on how the graph has been constructed, larger distances may be desired for analyses involving diffusable ligands. We also analyse the number of ligand receptor interactions that occur between cells from the same cluster and between cells from different clusters. Note that clusters may represent cell type, or another property of the cells such as their neighbourhood type. We leave this for the user to decide. We then calculate how frequently the number of ligand-receptor interactions observed between/within clusters is higher than in simulated data - this allows us to assign a pvalue for the enrichment of these interactions.
## Running this example takes a while:
## ligandReceptorResults = performLigandReceptorAnalysis(smallXenium, delaunayNeighbours,
## "mouse", clusters,verbose=FALSE)
## Accordingly, we retrieve precomputed results:
ligandReceptorResults = getExample('ligandReceptorResults')We look at interactions on edges. For concision, we display the first six of our 28 ligand-receptor pairs. Since ligand-receptor interactions are directed, each undirected edge will show up both as cell A - cell B and as cell B - cell A.
head(ligandReceptorResults$interactionsOnEdges[,1:10],10)
#> clusterA clusterB nodeA nodeB Pecam1_Pecam1 Cdh4_Cdh4 Col1a1_Cd44
#> 1 6_VLMC 11_Oligo 16307 16316 TRUE FALSE FALSE
#> 2 5_Astro 5_Astro 16314 16317 FALSE FALSE FALSE
#> 3 6_VLMC 6_VLMC 16296 16299 FALSE FALSE FALSE
#> 4 6_VLMC 6_VLMC 16299 16307 TRUE FALSE FALSE
#> 5 5_Astro 11_Oligo 16309 16316 FALSE TRUE FALSE
#> 6 5_Astro 5_Astro 16309 16314 FALSE FALSE FALSE
#> 7 18_Pyramidal 18_Pyramidal 12028 12032 FALSE FALSE FALSE
#> 8 18_Pyramidal 7_Granule 12032 16914 FALSE FALSE FALSE
#> 9 18_Pyramidal 7_Granule 12032 16257 FALSE FALSE FALSE
#> 10 4_Astro 7_Granule 2975 3339 FALSE FALSE FALSE
#> Fn1_Cd44 Spp1_Cd44 Cort_Npy2r
#> 1 FALSE FALSE FALSE
#> 2 FALSE FALSE FALSE
#> 3 FALSE FALSE FALSE
#> 4 FALSE FALSE FALSE
#> 5 FALSE FALSE FALSE
#> 6 FALSE FALSE FALSE
#> 7 FALSE FALSE FALSE
#> 8 FALSE FALSE FALSE
#> 9 FALSE FALSE FALSE
#> 10 FALSE FALSE FALSELook at total interactions between/within clusters:
head(ligandReceptorResults$totalInteractionsByCluster[,1:10],10)
#> clusterPair totalEdges Pecam1_Pecam1 Cdh4_Cdh4
#> 0_Oligo-0_Oligo 0_Oligo-0_Oligo 310 2 2
#> 0_Oligo-10_Astro 0_Oligo-10_Astro 44 1 5
#> 0_Oligo-11_Oligo 0_Oligo-11_Oligo 44 1 0
#> 0_Oligo-12_Pvalb 0_Oligo-12_Pvalb 9 0 3
#> 0_Oligo-13_Microglia 0_Oligo-13_Microglia 45 4 0
#> 0_Oligo-14_Ependymal 0_Oligo-14_Ependymal 103 2 1
#> 0_Oligo-15_Oligo 0_Oligo-15_Oligo 29 0 0
#> 0_Oligo-16_Astro 0_Oligo-16_Astro 2 0 0
#> 0_Oligo-17_Lamp5 0_Oligo-17_Lamp5 11 0 0
#> 0_Oligo-18_Pyramidal 0_Oligo-18_Pyramidal 4 0 0
#> Col1a1_Cd44 Fn1_Cd44 Spp1_Cd44 Cort_Npy2r Cort_Htr1f
#> 0_Oligo-0_Oligo 2 3 0 0 0
#> 0_Oligo-10_Astro 0 1 0 0 0
#> 0_Oligo-11_Oligo 0 0 0 0 0
#> 0_Oligo-12_Pvalb 0 0 0 0 0
#> 0_Oligo-13_Microglia 0 0 1 0 0
#> 0_Oligo-14_Ependymal 0 6 0 0 0
#> 0_Oligo-15_Oligo 0 0 0 0 0
#> 0_Oligo-16_Astro 0 0 0 0 0
#> 0_Oligo-17_Lamp5 0 0 0 0 0
#> 0_Oligo-18_Pyramidal 0 0 0 0 0
#> Cort_Chrm2
#> 0_Oligo-0_Oligo 3
#> 0_Oligo-10_Astro 2
#> 0_Oligo-11_Oligo 1
#> 0_Oligo-12_Pvalb 0
#> 0_Oligo-13_Microglia 0
#> 0_Oligo-14_Ependymal 2
#> 0_Oligo-15_Oligo 1
#> 0_Oligo-16_Astro 0
#> 0_Oligo-17_Lamp5 0
#> 0_Oligo-18_Pyramidal 0Look at mean interactions per edge between/within clusters (sum of ligand receptor interactions on A-B edges)/(total A-B edges):
head(ligandReceptorResults$meanInteractionsByCluster[,1:10],10)
#> clusterPair totalEdges Pecam1_Pecam1 Cdh4_Cdh4
#> 0_Oligo-0_Oligo 0_Oligo-0_Oligo 310 0.006451613 0.006451613
#> 0_Oligo-10_Astro 0_Oligo-10_Astro 44 0.022727273 0.113636364
#> 0_Oligo-11_Oligo 0_Oligo-11_Oligo 44 0.022727273 0.000000000
#> 0_Oligo-12_Pvalb 0_Oligo-12_Pvalb 9 0.000000000 0.333333333
#> 0_Oligo-13_Microglia 0_Oligo-13_Microglia 45 0.088888889 0.000000000
#> 0_Oligo-14_Ependymal 0_Oligo-14_Ependymal 103 0.019417476 0.009708738
#> 0_Oligo-15_Oligo 0_Oligo-15_Oligo 29 0.000000000 0.000000000
#> 0_Oligo-16_Astro 0_Oligo-16_Astro 2 0.000000000 0.000000000
#> 0_Oligo-17_Lamp5 0_Oligo-17_Lamp5 11 0.000000000 0.000000000
#> 0_Oligo-18_Pyramidal 0_Oligo-18_Pyramidal 4 0.000000000 0.000000000
#> Col1a1_Cd44 Fn1_Cd44 Spp1_Cd44 Cort_Npy2r Cort_Htr1f
#> 0_Oligo-0_Oligo 0.006451613 0.009677419 0.00000000 0 0
#> 0_Oligo-10_Astro 0.000000000 0.022727273 0.00000000 0 0
#> 0_Oligo-11_Oligo 0.000000000 0.000000000 0.00000000 0 0
#> 0_Oligo-12_Pvalb 0.000000000 0.000000000 0.00000000 0 0
#> 0_Oligo-13_Microglia 0.000000000 0.000000000 0.02222222 0 0
#> 0_Oligo-14_Ependymal 0.000000000 0.058252427 0.00000000 0 0
#> 0_Oligo-15_Oligo 0.000000000 0.000000000 0.00000000 0 0
#> 0_Oligo-16_Astro 0.000000000 0.000000000 0.00000000 0 0
#> 0_Oligo-17_Lamp5 0.000000000 0.000000000 0.00000000 0 0
#> 0_Oligo-18_Pyramidal 0.000000000 0.000000000 0.00000000 0 0
#> Cort_Chrm2
#> 0_Oligo-0_Oligo 0.009677419
#> 0_Oligo-10_Astro 0.045454545
#> 0_Oligo-11_Oligo 0.022727273
#> 0_Oligo-12_Pvalb 0.000000000
#> 0_Oligo-13_Microglia 0.000000000
#> 0_Oligo-14_Ependymal 0.019417476
#> 0_Oligo-15_Oligo 0.034482759
#> 0_Oligo-16_Astro 0.000000000
#> 0_Oligo-17_Lamp5 0.000000000
#> 0_Oligo-18_Pyramidal 0.000000000Look at number of times observed interactions are more frequent than expected:
head(ligandReceptorResults$simResults[,1:10],10)
#> Pecam1_Pecam1 Cdh4_Cdh4 Col1a1_Cd44 Fn1_Cd44 Spp1_Cd44
#> 0_Oligo-0_Oligo 16 380 229 235 0
#> 0_Oligo-10_Astro 254 1000 0 572 0
#> 0_Oligo-11_Oligo 264 0 0 0 0
#> 0_Oligo-12_Pvalb 0 1000 0 0 0
#> 0_Oligo-13_Microglia 906 0 0 0 802
#> 0_Oligo-14_Ependymal 202 510 0 985 0
#> 0_Oligo-15_Oligo 0 0 0 0 0
#> 0_Oligo-16_Astro 0 0 0 0 0
#> 0_Oligo-17_Lamp5 0 0 0 0 0
#> 0_Oligo-18_Pyramidal 0 0 0 0 0
#> Cort_Npy2r Cort_Htr1f Cort_Chrm2 Cort_Gpr17 Sst_Npy2r
#> 0_Oligo-0_Oligo 0 0 689 897 0
#> 0_Oligo-10_Astro 0 0 950 863 0
#> 0_Oligo-11_Oligo 0 0 797 0 0
#> 0_Oligo-12_Pvalb 0 0 0 0 0
#> 0_Oligo-13_Microglia 0 0 0 0 0
#> 0_Oligo-14_Ependymal 0 0 843 722 0
#> 0_Oligo-15_Oligo 0 0 821 995 0
#> 0_Oligo-16_Astro 0 0 0 0 0
#> 0_Oligo-17_Lamp5 0 0 0 0 0
#> 0_Oligo-18_Pyramidal 0 0 0 0 0Look at pvalues (upper tail) for the enrichment of interactions.
head(ligandReceptorResults$pValues,10)
#> Pecam1_Pecam1 Cdh4_Cdh4 Col1a1_Cd44 Fn1_Cd44 Spp1_Cd44
#> 0_Oligo-0_Oligo 0.984 0.620 0.771 0.765 1.000
#> 0_Oligo-10_Astro 0.746 0.001 1.000 0.428 1.000
#> 0_Oligo-11_Oligo 0.736 1.000 1.000 1.000 1.000
#> 0_Oligo-12_Pvalb 1.000 0.001 1.000 1.000 1.000
#> 0_Oligo-13_Microglia 0.094 1.000 1.000 1.000 0.198
#> 0_Oligo-14_Ependymal 0.798 0.490 1.000 0.015 1.000
#> 0_Oligo-15_Oligo 1.000 1.000 1.000 1.000 1.000
#> 0_Oligo-16_Astro 1.000 1.000 1.000 1.000 1.000
#> 0_Oligo-17_Lamp5 1.000 1.000 1.000 1.000 1.000
#> 0_Oligo-18_Pyramidal 1.000 1.000 1.000 1.000 1.000
#> Cort_Npy2r Cort_Htr1f Cort_Chrm2 Cort_Gpr17 Sst_Npy2r
#> 0_Oligo-0_Oligo 1 1 0.311 0.103 1
#> 0_Oligo-10_Astro 1 1 0.050 0.137 1
#> 0_Oligo-11_Oligo 1 1 0.203 1.000 1
#> 0_Oligo-12_Pvalb 1 1 1.000 1.000 1
#> 0_Oligo-13_Microglia 1 1 1.000 1.000 1
#> 0_Oligo-14_Ependymal 1 1 0.157 0.278 1
#> 0_Oligo-15_Oligo 1 1 0.179 0.005 1
#> 0_Oligo-16_Astro 1 1 1.000 1.000 1
#> 0_Oligo-17_Lamp5 1 1 1.000 1.000 1
#> 0_Oligo-18_Pyramidal 1 1 1.000 1.000 1
#> Sst_Htr1f Sst_Chrm2 Sst_Gpr17 Nts_Ntsr2 Nts_Tacr1
#> 0_Oligo-0_Oligo 1.000 0.001 0.001 0.051 1.000
#> 0_Oligo-10_Astro 1.000 0.058 1.000 1.000 1.000
#> 0_Oligo-11_Oligo 1.000 1.000 1.000 1.000 0.005
#> 0_Oligo-12_Pvalb 1.000 1.000 1.000 1.000 1.000
#> 0_Oligo-13_Microglia 0.038 0.077 0.044 0.055 1.000
#> 0_Oligo-14_Ependymal 1.000 1.000 1.000 0.020 1.000
#> 0_Oligo-15_Oligo 1.000 1.000 0.018 0.027 1.000
#> 0_Oligo-16_Astro 1.000 1.000 1.000 1.000 1.000
#> 0_Oligo-17_Lamp5 1.000 1.000 1.000 1.000 1.000
#> 0_Oligo-18_Pyramidal 1.000 1.000 1.000 1.000 1.000
#> Nts_Gpr17 Pthlh_Rxfp1 Crh_Rxfp1 Penk_Npy2r Penk_Htr1f
#> 0_Oligo-0_Oligo 0.005 1 1 1 0.374
#> 0_Oligo-10_Astro 1.000 1 1 1 0.256
#> 0_Oligo-11_Oligo 0.076 1 1 1 1.000
#> 0_Oligo-12_Pvalb 1.000 1 1 1 1.000
#> 0_Oligo-13_Microglia 1.000 1 1 1 1.000
#> 0_Oligo-14_Ependymal 0.130 1 1 1 1.000
#> 0_Oligo-15_Oligo 0.001 1 1 1 1.000
#> 0_Oligo-16_Astro 1.000 1 1 1 1.000
#> 0_Oligo-17_Lamp5 1.000 1 1 1 1.000
#> 0_Oligo-18_Pyramidal 1.000 1 1 1 1.000
#> Penk_Chrm2 Penk_Gpr17 Pdyn_Npy2r Pdyn_Htr1f Pdyn_Chrm2
#> 0_Oligo-0_Oligo 0.001 0.001 1 1 0.789
#> 0_Oligo-10_Astro 0.015 0.293 1 1 1.000
#> 0_Oligo-11_Oligo 1.000 1.000 1 1 1.000
#> 0_Oligo-12_Pvalb 0.002 1.000 1 1 1.000
#> 0_Oligo-13_Microglia 0.181 1.000 1 1 0.452
#> 0_Oligo-14_Ependymal 0.284 0.234 1 1 1.000
#> 0_Oligo-15_Oligo 0.028 0.001 1 1 1.000
#> 0_Oligo-16_Astro 1.000 1.000 1 1 1.000
#> 0_Oligo-17_Lamp5 1.000 1.000 1 1 1.000
#> 0_Oligo-18_Pyramidal 1.000 1.000 1 1 1.000
#> Pdyn_Gpr17 Vip_Rxfp1 Cdh6_Cdh9
#> 0_Oligo-0_Oligo 0.869 0.014 1.000
#> 0_Oligo-10_Astro 1.000 1.000 1.000
#> 0_Oligo-11_Oligo 1.000 1.000 0.570
#> 0_Oligo-12_Pvalb 1.000 1.000 1.000
#> 0_Oligo-13_Microglia 1.000 1.000 1.000
#> 0_Oligo-14_Ependymal 1.000 0.029 0.889
#> 0_Oligo-15_Oligo 1.000 1.000 0.477
#> 0_Oligo-16_Astro 1.000 1.000 1.000
#> 0_Oligo-17_Lamp5 1.000 1.000 1.000
#> 0_Oligo-18_Pyramidal 1.000 1.000 1.000Plot a heatmap showing -log10(pvalues) for the enrichment of ligand-receptor interactions between pairs of clusters.
ligRecMatrix = makeLRInteractionHeatmap(ligandReceptorResults, clusters, colours = colours, labelClusterPairs = FALSE)
Make a heatmap of total ligand-receptor interactions between clusters.
cellTypePerCellTypeLigRecMatrix = makeSummedLRInteractionHeatmap(ligandReceptorResults, clusters, "total")
Make a heatmap of summed mean ligand/receptor interactions between clusters i.e., (sum of all ligand receptor interactions on A-B edges)/(total A-B edges)
cellTypePerCellTypeLigRecMatrix = makeSummedLRInteractionHeatmap(ligandReceptorResults, clusters, "mean", logScale = TRUE)
Look at a histogram of values for summed mean ligand/receptor interactions between clusters.
Visualise in graph format.
cellTypesPerCellTypeGraphFromCellMatrix(cellTypePerCellTypeLigRecMatrix,
minWeight = 0.4, colours = colours)
#> IGRAPH 23112b4 DNW- 16 54 --
#> + attr: coords (g/n), name (v/c), color (v/c), weight (e/n), width
#> | (e/n)
#> + edges from 23112b4 (vertex names):
#> [1] 0_Oligo ->3_Endo 0_Oligo ->7_Granule 0_Oligo ->12_Pvalb
#> [4] 0_Oligo ->15_Oligo 3_Endo ->6_VLMC 3_Endo ->10_Astro
#> [7] 3_Endo ->15_Oligo 3_Endo ->17_Lamp5 4_Astro ->0_Oligo
#> [10] 4_Astro ->3_Endo 4_Astro ->7_Granule 4_Astro ->14_Ependymal
#> [13] 5_Astro ->10_Astro 6_VLMC ->3_Endo 6_VLMC ->5_Astro
#> [16] 6_VLMC ->10_Astro 6_VLMC ->11_Oligo 6_VLMC ->13_Microglia
#> [19] 7_Granule->5_Astro 7_Granule->10_Astro 7_Granule->14_Ependymal
#> + ... omitted several edgesHere the arrows are very thick - we can scale by a constant factor to improve the presentation.
scaleFactor = 3
cellTypesPerCellTypeGraphFromCellMatrix(cellTypePerCellTypeLigRecMatrix/scaleFactor,
minWeight = 0.4/scaleFactor, colours = colours)
#> IGRAPH d9fc19b DNW- 16 54 --
#> + attr: coords (g/n), name (v/c), color (v/c), weight (e/n), width
#> | (e/n)
#> + edges from d9fc19b (vertex names):
#> [1] 0_Oligo ->3_Endo 0_Oligo ->7_Granule 0_Oligo ->12_Pvalb
#> [4] 0_Oligo ->15_Oligo 3_Endo ->6_VLMC 3_Endo ->10_Astro
#> [7] 3_Endo ->15_Oligo 3_Endo ->17_Lamp5 4_Astro ->0_Oligo
#> [10] 4_Astro ->3_Endo 4_Astro ->7_Granule 4_Astro ->14_Ependymal
#> [13] 5_Astro ->10_Astro 6_VLMC ->3_Endo 6_VLMC ->5_Astro
#> [16] 6_VLMC ->10_Astro 6_VLMC ->11_Oligo 6_VLMC ->13_Microglia
#> [19] 7_Granule->5_Astro 7_Granule->10_Astro 7_Granule->14_Ependymal
#> + ... omitted several edgesVisualising ligand-receptor interactions: Seurat objects of edges
We wish to create a Seurat object where the points are edges between cells as a way of visualising ligand-receptor interactions. Since these interactions are asymmetric we need to consider directed edges. We will place the spatial coordinates of the edges A-B and B-A slightly separate but both between the centroids of cells A and B. The “expression matrix” is the binarised presence/absence of an interaction (ligand receptor pair) on an edge. This is useful for visualising where ligand receptor interactions occur spatially.
edgeSeurat = computeEdgeObject(ligandReceptorResults, centroids)
#> Warning: Feature names cannot have underscores ('_'), replacing with dashes
#> ('-')
#> Warning: Data is of class matrix. Coercing to dgCMatrix.For example we can visualise the Pdyn-Npy2r interaction.
ImageFeaturePlot(edgeSeurat, features = "Pdyn-Npy2r")
#> Warning: No layers found matching search pattern provided
#> Warning in FetchData.Assay5(object = object[[DefaultAssay(object = object)]], :
#> data layer is not found and counts layer is used
In principle, the edge Seurat object allows for the computation of an edge UMAP and Louvain clustering of the edges based on ligand-receptor interaction. However some of these analyses are computationally expensive due to large number of edges.
Spatial autocorrelation of ligand-receptor interactions
We can compute a spatial graph where edges in the original delaunayNeighbours become nodes and A-B edges (in the original graph) become connected to all A- edges and all B- edges. This allows us to perfom graph-based analyses associated with the spatial localisation of ligand receptor pairs on edges.
Compute Moran’s I for the spatial autocorrelation of ligand-receptor interactions.
moransILigandReceptor = runMoransI(edgeSeurat, edgeNeighbours, assay = "RNA",
layer = "counts", nSim = 100)View most spatially autocorrelated ligand-receptor interactions.
moransILigandReceptor = getExample('moransILigandReceptor')
head(moransILigandReceptor)
#> moransI pValues
#> Penk-Htr1f 0.8392801 0.01
#> Vip-Rxfp1 0.7866141 0.02
#> Pthlh-Rxfp1 0.7812148 0.21
#> Pdyn-Npy2r 0.7593318 0.01
#> Nts-Ntsr2 0.7582268 0.01
#> Pecam1-Pecam1 0.7397425 0.01View least spatially autocorrelated ligand-receptor interactions.
tail(moransILigandReceptor)
#> moransI pValues
#> Spp1-Cd44 0.6559306 0.01
#> Nts-Tacr1 0.6388787 0.13
#> Nts-Gpr17 0.5903279 0.51
#> Sst-Npy2r 0.5636993 0.58
#> Sst-Htr1f 0.5442672 0.71
#> Sst-Gpr17 0.5305249 0.75View ligand-receptor interaction with the highest spatial autocorrelation (Moran’s I).
ImageFeaturePlot(edgeSeurat, "Penk-Htr1f")
#> Warning: No layers found matching search pattern provided
#> Warning in FetchData.Assay5(object = object[[DefaultAssay(object = object)]], :
#> data layer is not found and counts layer is used
View ligand-receptor interaction with the lowest spatial autocorrelation (Moran’s I).
ImageFeaturePlot(edgeSeurat, "Sst-Gpr17")
#> Warning: No layers found matching search pattern provided
#> Warning in FetchData.Assay5(object = object[[DefaultAssay(object = object)]], :
#> data layer is not found and counts layer is used
Quality control of Delaunay neighbours
We use Delaunay triangulation to estimate neighbour to neighbour contact between cells. However, a cell which happens to sit next to a tissue void is likely to have one or more Delaunay neighbours which are cells that sit on the other side of this void. We hope to detect these spurious edges based on their lengths. However, the distances between neighbouring cells’ centroids tend to depend on the types of the neighbouring cells. Accordingly, we have supplied functions to annotate edges with their distances and cell types; compute estimates for edge-length cutoffs based on cell type pair; plot the distributions of lengths together with proposed cutoffs; and subset the edges based on cutoffs.
edgeLengthsAndCellTypePairs
This functions annotates edges with their lengths and the cell types of the two cells in question.
annEdges =
edgeLengthsAndCellTypePairs(delaunayNeighbours,clusters,centroids)
head(annEdges)
#> nodeA nodeB length cellTypePair
#> 1 16307 16316 19.883354 6_VLMC_11_Oligo
#> 2 16314 16317 35.634141 5_Astro_5_Astro
#> 3 16296 16299 8.127726 6_VLMC_6_VLMC
#> 4 16299 16307 8.957429 6_VLMC_6_VLMC
#> 5 16309 16316 25.730502 5_Astro_11_Oligo
#> 6 16309 16314 18.029393 5_Astro_5_AstroVisualising the edge lengths
Here we visualise the distribution of edge lengths together with proposed cutoffs. We will say more in a moment about how these proposed cutoffs are calculated.
cutoffDF = edgeCutoffsByPercentile(annEdges,percentileCutof=95)
g = edgeLengthPlot(annEdges,cutoffDF,whichPairs=60)
print(g)
#> Warning: Removed 23 rows containing non-finite outside the scale range
#> (`stat_density()`).
In this case, there are 111 types of edges based on the cell types of the cells they connect. By setting whichPairs=60, we are restricting to those types for which there are at least 60 such edges. Here we are also using a default xlim of 100. Passing the value NULL for cutoffDF will produce a plot without these cutoffs.
Estimating edge length cutoffs
We offer several methods for estimating edge length cutoffs. We recommend visual inspection before settling on any specific set of results. Each of these returns a data frame with two columns, cellTypePair and cutoff. As we have just seen this can be used for the plotting function and it can also be used for culling the edges. Since these results are returned as a data frame, they can be tuned “by hand” by modifying the cutoff value for any individual cell type pair.
edgeCutoffsByClustering
This method works well in a small percentage of cases. For each cell type pair it performs k-means clustering of the lengths with k=2 and produces a cutoff halfway between the two clusters. This works well when there really are two clusters of edges, those between truly neighbouring cells and those across tissue voids. However, in the case where all the edges are between neighbouring cells, this will produce a spurious cutoff.
edgeCutoffsByPercentile
This method produces cutoffs based on a chosen percentile applied across all cell type pairs. It has the advantages that its results are easily comprehensible in terms of the chosen parameter. The user my want to “mix and match” using different percentiles for different cell type pairs.
edgeCutoffsByZScore
Very similar to the previous method, using z-score instead of percentile.
edgeCutoffsByWatershed
For each cell type pair, this function first computes a histogram of the distances. It then uses watershed segmentation to discover the “humps” of this histogram. The most important of these will be the one containing the median of the distances. This reports as a cutoff the midpoint of the first histogram bin which is past this main hump. The parameter nbins specifies the number of bins for the histogram. The parameter tolerance controls the sensitivity of the watershed algorithm.
cullEdges
cutoffDF = edgeCutoffsByWatershed(annEdges,nbins=15,tolerance=10)
culledEdges = cullEdges(annEdges,cutoffDF)
nrow(annEdges)
#> [1] 12759
nrow(culledEdges)
#> [1] 12512This subsets the edges according to the parameter cutoffSpec. This can be a cutoff data frame as produced by any of the edgeCutoff functions or can be a single number in which case all the cell type pairs will be trimmed to the same cutoff.
sessionInfo()
#> R version 4.4.2 (2024-10-31)
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#> attached base packages:
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#> [3] SummarizedExperiment_1.37.0 Biobase_2.67.0
#> [5] GenomicRanges_1.59.1 GenomeInfoDb_1.43.2
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#> [15] foreign_0.8-87 maps_3.4.2.1
#> [17] tictoc_1.2.1 pheatmap_1.0.12
#> [19] ggplot2_3.5.1 Seurat_5.1.0
#> [21] SeuratObject_5.0.2 sp_2.1-4
#> [23] CatsCradle_1.1.0 rmarkdown_2.29
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#> [22] cowplot_1.1.3 pbapply_1.7-2 buildtools_1.0.0
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- Neighbourhoods as characterised by cell types
- Analysis of neighbourhoods based on cell type composition
- Relating cell type clusters to neighbourhood clusters
- Analysing cell types based on their neighbourhoods
- Detection of genes with spatially variable expression.
- Ligand receptor analysis
- Quality control of Delaunay neighbours