<!-- README.md is generated from README.Rmd. Please edit that file --> ## Overview BANKSY is a method for clustering spatial omics data by augmenting the features of each cell with both an average of the features of its spatial neighbors along with neighborhood feature gradients. By incorporating neighborhood information for clustering, BANKSY is able to - improve cell-type assignment in noisy data - distinguish subtly different cell-types stratified by microenvironment - identify spatial domains sharing the same microenvironment BANKSY is applicable to a wide array of spatial technologies (e.g. 10x Visium, Slide-seq, MERFISH, CosMX, CODEX) and scales well to large datasets. For more details, check out: - the [paper](https://www.nature.com/articles/s41588-024-01664-3), - the [peer review file](https://static-content.springer.com/esm/art%3A10.1038%2Fs41588-024-01664-3/MediaObjects/41588_2024_1664_MOESM3_ESM.pdf), - a [tweetorial](https://x.com/shyam_lab/status/1762648072360792479?s=20) on BANKSY, - a set of [vignettes](https://prabhakarlab.github.io/Banksy) showing basic usage, - usage compatibility with Seurat ([here](https://github.com/satijalab/seurat-wrappers/blob/master/docs/banksy.md) and [here](https://satijalab.org/seurat/articles/visiumhd_analysis_vignette#identifying-spatially-defined-tissue-domains)), - a [Python version](https://github.com/prabhakarlab/Banksy_py) of this package, - a [Zenodo archive](https://zenodo.org/records/10258795) containing scripts to reproduce the analyses in the paper, and the corresponding [GitHub Pages](https://github.com/jleechung/banksy-zenodo) (and [here](https://github.com/prabhakarlab/Banksy_py/tree/Banksy_manuscript) for analyses done in Python). ## Installation The *Banksy* package can be installed via Bioconductor. This currently requires R `>= 4.4.0`. ``` r BiocManager::install('Banksy') ``` To install directly from GitHub instead, use ``` r remotes::install_github("prabhakarlab/Banksy") ``` To use the legacy version of *Banksy* utilising the `BanksyObject` class, use ``` r remotes::install_github("prabhakarlab/Banksy@legacy") ``` *Banksy* is also interoperable with [*Seurat*](https://satijalab.org/seurat/) via [*SeuratWrappers*](https://github.com/satijalab/seurat-wrappers). Documentation on how to run BANKSY on Seurat objects can be found [here](https://github.com/satijalab/seurat-wrappers/blob/master/docs/banksy.md). For installation of *SeuratWrappers* with BANKSY version `>= 0.1.6`, run ``` r remotes::install_github('satijalab/seurat-wrappers') ``` ## Quick start Load *BANKSY*. We’ll also load *SpatialExperiment* and *SummarizedExperiment* for containing and manipulating the data, *scuttle* for normalization and quality control, and *scater*, *ggplot2* and *cowplot* for visualisation. ``` r library(Banksy) library(SummarizedExperiment) library(SpatialExperiment) library(scuttle) library(scater) library(cowplot) library(ggplot2) ``` Here, we’ll run *BANKSY* on mouse hippocampus data. ``` r data(hippocampus) gcm <- hippocampus$expression locs <- as.matrix(hippocampus$locations) ``` Initialize a SpatialExperiment object and perform basic quality control and normalization. ``` r se <- SpatialExperiment(assay = list(counts = gcm), spatialCoords = locs) # QC based on total counts qcstats <- perCellQCMetrics(se) thres <- quantile(qcstats$total, c(0.05, 0.98)) keep <- (qcstats$total > thres[1]) & (qcstats$total < thres[2]) se <- se[, keep] # Normalization to mean library size se <- computeLibraryFactors(se) aname <- "normcounts" assay(se, aname) <- normalizeCounts(se, log = FALSE) ``` Compute the neighborhood matrices for *BANKSY*. Setting `compute_agf=TRUE` computes both the weighted neighborhood mean ($\mathcal{M}$) and the azimuthal Gabor filter ($\mathcal{G}$). The number of spatial neighbors used to compute $\mathcal{M}$ and $\mathcal{G}$ are `k_geom[1]=15` and `k_geom[2]=30` respectively. We run *BANKSY* at `lambda=0` corresponding to non-spatial clustering, and `lambda=0.2` corresponding to *BANKSY* for cell-typing. > **An important note about choosing the `lambda` parameter for the > older [Visium v1 / v2 55um > datasets](https://doi.org/10.1038/s41593-020-00787-0) or the original > [ST 100um technology](https://doi.org/10.1038/s41596-018-0045-2):** > > For most modern high resolution technologies like Xenium, Visium HD, > StereoSeq, MERFISH, STARmap PLUS, SeqFISH+, SlideSeq v2, and CosMx > (and others), we recommend the usual defults for `lambda`: For cell > typing, use `lambda = 0.2` (as shown below, or in [this > vignette](https://prabhakarlab.github.io/Banksy/articles/parameter-selection.html)) > and for [domain > segmentation](https://prabhakarlab.github.io/Banksy/articles/domain-segment.html), > use `lambda = 0.8`. These technologies are either imaging based, > having true single-cell resolution (e.g., MERFISH), or are sequencing > based, having barcoded spots on the scale of single-cells (e.g., > [Visium > HD](https://www.10xgenomics.com/products/visium-hd-spatial-gene-expression)). > We find that the usual defaults work well at this measurement > resolution. > > However, for the older **Visium v1/v2** or **ST** technologies, with > their much lower resolution spots (55um and 100um diameter, > respectively), we find that `lambda = 0.2` seems to work best for > domain segmentation. This could be because each spot already measures > the average transcriptome of several cells in a neighbourhood. It > seems that `lambda = 0.2` shares enough information between these > neighbourhoods to lead to good domain segmentation performance. For > example, in the [human DLPFC > vignette](https://prabhakarlab.github.io/Banksy/articles/multi-sample.html), > we use `lambda = 0.2` on a Visium v1/v2 dataset. Also note that in > these lower resolution technologies, each spot can have multiple cells > of different types, and as such *cell-typing* is not defined for them. ``` r lambda <- c(0, 0.2) k_geom <- c(15, 30) se <- Banksy::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 #> Computing harmonic m = 0 #> Using 15 neighbors #> Done #> Computing harmonic m = 1 #> Using 30 neighbors #> Centering #> Done ``` Next, run PCA on the BANKSY matrix and perform clustering. Setting `use_agf=TRUE` uses both $\mathcal{M}$ and $\mathcal{G}$ to construct the BANKSY matrix. ``` r set.seed(1000) se <- Banksy::runBanksyPCA(se, use_agf = TRUE, lambda = lambda) se <- Banksy::runBanksyUMAP(se, use_agf = TRUE, lambda = lambda) se <- Banksy::clusterBanksy(se, use_agf = TRUE, lambda = lambda, resolution = 1.2) ``` Different clustering runs can be relabeled to minimise their differences with `connectClusters`: ``` r se <- Banksy::connectClusters(se) #> clust_M1_lam0.2_k50_res1.2 --> clust_M1_lam0_k50_res1.2 ``` Visualise the clustering output for non-spatial clustering (`lambda=0`) and BANKSY clustering (`lambda=0.2`). ``` r cnames <- colnames(colData(se)) cnames <- cnames[grep("^clust", cnames)] colData(se) <- cbind(colData(se), spatialCoords(se)) plot_nsp <- plotColData(se, x = "sdimx", y = "sdimy", point_size = 0.6, colour_by = cnames[1] ) plot_bank <- plotColData(se, x = "sdimx", y = "sdimy", point_size = 0.6, colour_by = cnames[2] ) plot_grid(plot_nsp + coord_equal(), plot_bank + coord_equal(), ncol = 2) ``` <img src="man/figures/README-unnamed-chunk-13-1.png" width="100%" /> For clarity, we can visualise each of the clusters separately: ``` r plot_grid( plot_nsp + facet_wrap(~colour_by), plot_bank + facet_wrap(~colour_by), ncol = 2 ) ``` <img src="man/figures/README-unnamed-chunk-14-1.png" width="100%" /> Visualize UMAPs of the non-spatial and BANKSY embedding: ``` r rdnames <- reducedDimNames(se) umap_nsp <- plotReducedDim(se, dimred = grep("UMAP.*lam0$", rdnames, value = TRUE), colour_by = cnames[1] ) umap_bank <- plotReducedDim(se, dimred = grep("UMAP.*lam0.2$", rdnames, value = TRUE), colour_by = cnames[2] ) plot_grid( umap_nsp, umap_bank, ncol = 2 ) ``` <img src="man/figures/README-unnamed-chunk-15-1.png" width="100%" /> <details> <summary> Runtime for analysis </summary> #> Time difference of 57.44879 secs </details> <details> <summary> Session information </summary> ``` r sessionInfo() #> R version 4.3.2 (2023-10-31) #> Platform: aarch64-apple-darwin20 (64-bit) #> Running under: macOS Sonoma 14.2.1 #> #> Matrix products: default #> BLAS: /Library/Frameworks/R.framework/Versions/4.3-arm64/Resources/lib/libRblas.0.dylib #> LAPACK: /Library/Frameworks/R.framework/Versions/4.3-arm64/Resources/lib/libRlapack.dylib; LAPACK version 3.11.0 #> #> locale: #> [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8 #> #> time zone: America/Detroit #> tzcode source: internal #> #> attached base packages: #> [1] stats4 stats graphics grDevices utils datasets methods #> [8] base #> #> other attached packages: #> [1] cowplot_1.1.3 scater_1.30.1 #> [3] ggplot2_3.4.4 scuttle_1.12.0 #> [5] SpatialExperiment_1.12.0 SingleCellExperiment_1.24.0 #> [7] SummarizedExperiment_1.32.0 Biobase_2.62.0 #> [9] GenomicRanges_1.54.1 GenomeInfoDb_1.38.6 #> [11] IRanges_2.36.0 S4Vectors_0.40.2 #> [13] BiocGenerics_0.48.1 MatrixGenerics_1.14.0 #> [15] matrixStats_1.2.0 Banksy_0.99.12 #> #> loaded via a namespace (and not attached): #> [1] tidyselect_1.2.0 viridisLite_0.4.2 #> [3] farver_2.1.1 dplyr_1.1.4 #> [5] vipor_0.4.7 viridis_0.6.5 #> [7] bitops_1.0-7 fastmap_1.1.1 #> [9] RCurl_1.98-1.14 digest_0.6.34 #> [11] rsvd_1.0.5 lifecycle_1.0.4 #> [13] magrittr_2.0.3 dbscan_1.1-12 #> [15] compiler_4.3.2 rlang_1.1.3 #> [17] tools_4.3.2 igraph_2.0.1.1 #> [19] utf8_1.2.4 yaml_2.3.8 #> [21] data.table_1.15.0 knitr_1.45 #> [23] labeling_0.4.3 S4Arrays_1.2.0 #> [25] mclust_6.0.1 DelayedArray_0.28.0 #> [27] abind_1.4-5 BiocParallel_1.36.0 #> [29] withr_3.0.0 grid_4.3.2 #> [31] fansi_1.0.6 beachmat_2.18.0 #> [33] colorspace_2.1-0 aricode_1.0.3 #> [35] scales_1.3.0 cli_3.6.2 #> [37] rmarkdown_2.25 crayon_1.5.2 #> [39] leidenAlg_1.1.2 generics_0.1.3 #> [41] rstudioapi_0.15.0 rjson_0.2.21 #> [43] DelayedMatrixStats_1.24.0 ggbeeswarm_0.7.2 #> [45] RcppHungarian_0.3 zlibbioc_1.48.0 #> [47] parallel_4.3.2 XVector_0.42.0 #> [49] vctrs_0.6.5 Matrix_1.6-5 #> [51] BiocSingular_1.18.0 BiocNeighbors_1.20.2 #> [53] ggrepel_0.9.5 irlba_2.3.5.1 #> [55] beeswarm_0.4.0 magick_2.8.2 #> [57] glue_1.7.0 codetools_0.2-19 #> [59] uwot_0.1.16 RcppAnnoy_0.0.22 #> [61] gtable_0.3.4 ScaledMatrix_1.10.0 #> [63] munsell_0.5.0 tibble_3.2.1 #> [65] pillar_1.9.0 htmltools_0.5.7 #> [67] GenomeInfoDbData_1.2.11 R6_2.5.1 #> [69] sparseMatrixStats_1.14.0 evaluate_0.23 #> [71] sccore_1.0.4 lattice_0.22-5 #> [73] highr_0.10 Rcpp_1.0.12 #> [75] gridExtra_2.3 SparseArray_1.2.4 #> [77] xfun_0.42 pkgconfig_2.0.3 ``` </details>