Parameter selection (VeraFISH Mouse Hippocampus)
8 June 2025
Contents
Here, we demonstrate a grid search of clustering parameters with a mouse
hippocampus VeraFISH dataset. BANKSY currently provides four algorithms for
clustering the BANKSY matrix with clusterBanksy: Leiden (default), Louvain,
k-means, and model-based clustering. In this vignette, we run only Leiden
clustering. See ?clusterBanksy for more details on the parameters for
different clustering methods.
1 Loading the data
The dataset comprises gene expression for 10,944 cells and 120 genes in 2
spatial dimensions. See ?Banksy::hippocampus for more details.
# Load libs
library(Banksy)
library(SummarizedExperiment)
library(SpatialExperiment)
library(scuttle)
library(scater)
library(cowplot)
library(ggplot2)
# Load data
data(hippocampus)
gcm <- hippocampus$expression
locs <- as.matrix(hippocampus$locations)Here, gcm is a gene by cell matrix, and locs is a matrix specifying the
coordinates of the centroid for each cell.
head(gcm[,1:5])
#> cell_1276 cell_8890 cell_691 cell_396 cell_9818
#> Sparcl1 45 0 11 22 0
#> Slc1a2 17 0 6 5 0
#> Map 10 0 12 16 0
#> Sqstm1 26 0 0 2 0
#> Atp1a2 0 0 4 3 0
#> Tnc 0 0 0 0 0
head(locs)
#> sdimx sdimy
#> cell_1276 -13372.899 15776.37
#> cell_8890 8941.101 15866.37
#> cell_691 -14882.899 15896.37
#> cell_396 -15492.899 15835.37
#> cell_9818 11308.101 15846.37
#> cell_11310 14894.101 15810.37Initialize a SpatialExperiment object and perform basic quality control. We keep cells with total transcript count within the 5th and 98th percentile:
se <- SpatialExperiment(assay = list(counts = gcm), spatialCoords = locs)
colData(se) <- cbind(colData(se), spatialCoords(se))
# QC based on total counts
qcstats <- perCellQCMetrics(se)
thres <- quantile(qcstats$total, c(0.05, 0.98))
keep <- (qcstats$total > thres[1]) & (qcstats$total < thres[2])
se <- se[, keep]Next, perform normalization of the data.
# Normalization to mean library size
se <- computeLibraryFactors(se)
aname <- "normcounts"
assay(se, aname) <- normalizeCounts(se, log = FALSE)2 Parameters
BANKSY has a few key parameters. We describe these below.
2.1 AGF usage
For characterising neighborhoods, BANKSY computes the weighted neighborhood
mean (H_0) and the azimuthal Gabor filter (H_1), which estimates gene
expression gradients. Setting compute_agf=TRUE computes both H_0 and H_1.
2.2 k-geometric
k_geom specifies the number of neighbors used to compute each H_m for
m=0,1. If a single value is specified, the same k_geom will be used
for each feature matrix. Alternatively, multiple values of k_geom can be
provided for each feature matrix. Here, we use k_geom[1]=15 and
k_geom[2]=30 for H_0 and H_1 respectively. More neighbors are used to
compute gradients.
For datasets generated using Visium v1/v2, use
k_geom=18(ork_geom <- c(18, 18)ifcompute_agf = TRUE), since that corresponds to taking as neighbourhood two concentric rings of spots around each spot.
We compute the neighborhood feature matrices using normalized expression
(normcounts in the se object).
k_geom <- c(15, 30)
se <- computeBanksy(se, assay_name = aname, compute_agf = TRUE, k_geom = k_geom)
#> Computing neighbors...
#> Spatial mode is kNN_median
#> Parameters: k_geom=15
#> Done
#> Computing neighbors...
#> Spatial mode is kNN_median
#> Parameters: k_geom=30
#> Done
#> Done
#> Centering
#> DonecomputeBanksy populates the assays slot with H_0 and H_1 in this
instance:
se
#> class: SpatialExperiment
#> dim: 120 10205
#> metadata(1): BANKSY_params
#> assays(4): counts normcounts H0 H1
#> rownames(120): Sparcl1 Slc1a2 ... Notch3 Egfr
#> rowData names(0):
#> colnames(10205): cell_1276 cell_691 ... cell_11635 cell_10849
#> colData names(4): sample_id sdimx sdimy sizeFactor
#> reducedDimNames(0):
#> mainExpName: NULL
#> altExpNames(0):
#> spatialCoords names(2) : sdimx sdimy
#> imgData names(1): sample_id2.3 lambda
The lambda parameter is a mixing parameter in [0,1] which
determines how much spatial information is incorporated for downstream analysis.
With smaller values of lambda, BANKY operates in cell-typing mode, while at
higher levels of lambda, BANKSY operates in domain-finding mode. As a
starting point, we recommend lambda=0.2 for cell-typing and lambda=0.8 for
zone-finding, except for datasets generated using the Visium v1/v2 technology, for which
we recommend
lambda=0.2 for domain finding. See the note in the tutorial on the
main page for more info.
Here, we run lambda=0 which corresponds to non-spatial
clustering, and lambda=0.2 for spatially-informed cell-typing. We compute PCs
with and without the AGF (H_1).
lambda <- c(0, 0.2)
se <- runBanksyPCA(se, use_agf = c(FALSE, TRUE), lambda = lambda, seed = 1000)
#> Using seed=1000
#> Using seed=1000
#> Using seed=1000
#> Using seed=1000runBanksyPCA populates the reducedDims slot, with each combination of
use_agf and lambda provided.
reducedDimNames(se)
#> [1] "PCA_M0_lam0" "PCA_M0_lam0.2" "PCA_M1_lam0" "PCA_M1_lam0.2"2.4 Clustering parameters
Next, we cluster the BANKSY embedding with Leiden graph-based clustering. This
admits two parameters: k_neighbors and resolution. k_neighbors determines
the number of k nearest neighbors used to construct the shared nearest
neighbors graph. Leiden clustering is then performed on the resultant graph
with resolution resolution. For reproducibiltiy we set a seed for each
parameter combination.
k <- 50
res <- 1
se <- clusterBanksy(se, use_agf = c(FALSE, TRUE), lambda = lambda, k_neighbors = k, resolution = res, seed = 1000)
#> Using seed=1000
#> Using seed=1000
#> Using seed=1000
#> Using seed=1000clusterBanksy populates colData(se) with cluster labels:
colnames(colData(se))
#> [1] "sample_id" "sdimx"
#> [3] "sdimy" "sizeFactor"
#> [5] "clust_M0_lam0_k50_res1" "clust_M0_lam0.2_k50_res1"
#> [7] "clust_M1_lam0_k50_res1" "clust_M1_lam0.2_k50_res1"3 Comparing cluster results
To compare clustering runs visually, different runs can be relabeled to
minimise their differences with connectClusters:
se <- connectClusters(se)
#> clust_M1_lam0_k50_res1 --> clust_M0_lam0_k50_res1
#> clust_M0_lam0.2_k50_res1 --> clust_M1_lam0_k50_res1
#> clust_M1_lam0.2_k50_res1 --> clust_M0_lam0.2_k50_res1Visualise spatial coordinates with cluster labels.
cnames <- colnames(colData(se))
cnames <- cnames[grep("^clust", cnames)]
cplots <- lapply(cnames, function(cnm) {
plotColData(se, x = "sdimx", y = "sdimy", point_size = 0.1, colour_by = cnm) +
coord_equal() +
labs(title = cnm) +
theme(legend.title = element_blank()) +
guides(colour = guide_legend(override.aes = list(size = 2)))
})
plot_grid(plotlist = cplots, ncol = 2)
Compare all cluster outputs with compareClusters. This function computes
pairwise cluster comparison metrics between the clusters in colData(se) based
on adjusted Rand index (ARI):
compareClusters(se, func = "ARI")
#> clust_M0_lam0_k50_res1 clust_M0_lam0.2_k50_res1
#> clust_M0_lam0_k50_res1 1.000 0.67
#> clust_M0_lam0.2_k50_res1 0.670 1.00
#> clust_M1_lam0_k50_res1 1.000 0.67
#> clust_M1_lam0.2_k50_res1 0.747 0.87
#> clust_M1_lam0_k50_res1 clust_M1_lam0.2_k50_res1
#> clust_M0_lam0_k50_res1 1.000 0.747
#> clust_M0_lam0.2_k50_res1 0.670 0.870
#> clust_M1_lam0_k50_res1 1.000 0.747
#> clust_M1_lam0.2_k50_res1 0.747 1.000or normalized mutual information (NMI):
compareClusters(se, func = "NMI")
#> clust_M0_lam0_k50_res1 clust_M0_lam0.2_k50_res1
#> clust_M0_lam0_k50_res1 1.000 0.741
#> clust_M0_lam0.2_k50_res1 0.741 1.000
#> clust_M1_lam0_k50_res1 1.000 0.741
#> clust_M1_lam0.2_k50_res1 0.782 0.915
#> clust_M1_lam0_k50_res1 clust_M1_lam0.2_k50_res1
#> clust_M0_lam0_k50_res1 1.000 0.782
#> clust_M0_lam0.2_k50_res1 0.741 0.915
#> clust_M1_lam0_k50_res1 1.000 0.782
#> clust_M1_lam0.2_k50_res1 0.782 1.000See ?compareClusters for the full list of comparison measures.
4 Session information
Vignette runtime:
#> Time difference of 50.63552 secssessionInfo()
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