Last updated: 2021-05-05

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Knit directory: STUtility_web_site/

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Region neighbors


Sometimes it can be useful to extract the “neighborhood” of a set of spots. As an example, we show how this can be applied to find all the neighboring spots of any region of interest.

To demonstrate the regional neighbors feature, we will use the Breast Cancer data available on the 10x Genomics website.

The Seurat object below contain 2 breast cancer tissue sections which have already been normalized and clustered.


FeatureOverlay(se, features = "seurat_clusters", sampleids = 1:2, ncols = 2)

Version Author Date
bfe87d1 Ludvig Larsson 2021-05-04
1e9c615 Ludvig Larsson 2021-04-28
4f42429 Ludvig Larsson 2020-06-04


Connected Spatial Network

Once you have defined a region of interest and you want to find all spots neighboring to this region you can use the RegionNeighbours function to automatically detect such spots.

For example, let’s say that we want to select all neighbors to cluster 2. The first step is to make sure that the identity of your seurat object is correct, here we need to set it to “seurat_clusters”.

se <- SetIdent(se, value = "seurat_clusters")
se <- RegionNeighbours(se, id = "2", verbose = TRUE)
Creating Connected Netork using KNN ... 
Found 4763 neighbours for id 2 ... 
Excluding neighbours from the same group ... 
569 neighbours left ... 
Naming all neighbours nbs_2 ... 
Saving neighbour ids to column 'nbs_2' ... 
Finished. 


The default behavior is to find all spots which are neighbors with the selected id but ignoring its label, therefore it will simply be called nbs_2 as in “neighbors to 2”. The output will be stored as a new column in the meta.data slot, and in this example will be called “nbs_2”. The neighborhood detection algorithm is applied to each section separately and can therefore be run on multiple sections at the same time.

FeatureOverlay(se, features = "nbs_2", ncols = 2, sampleids = 1:2, cols = c("red", "lightgray"), pt.size = 2)

Version Author Date
bfe87d1 Ludvig Larsson 2021-05-04
1e9c615 Ludvig Larsson 2021-04-28
4f42429 Ludvig Larsson 2020-06-04


You can also keep all spots from the id group by setting keep.within.id = TRUE.

se <- SetIdent(se, value = "seurat_clusters")
se <- RegionNeighbours(se, id = 2, keep.within.id = T, verbose = TRUE)
Creating Connected Netork using KNN ... 
Found 4763 neighbours for id 2 ... 
Naming all neighbours nbs_2 ... 
Saving neighbour ids to column 'nbs_2' ... 
Finished. 
FeatureOverlay(se, features = "nbs_2", ncols = 2, sampleids = 1:2, cols = c("red", "lightgray"), pt.size = 2)

Version Author Date
bfe87d1 Ludvig Larsson 2021-05-04
1e9c615 Ludvig Larsson 2021-04-28
4f42429 Ludvig Larsson 2020-06-04


Using these two sets of spots, we can run a DE analysis to check what genes are up-regulated outside the cluster border.


library(magrittr)
library(dplyr)

se <- SetIdent(se, value = "nbs_2")
nbs_2.markers <- FindMarkers(se, ident.1 = "2", ident.2 = "nbs_2")
nbs_2.markers$gene <- rownames(nbs_2.markers)
se.subset <- SubsetSTData(se, expression = nbs_2 %in% c("2", "nbs_2"))
sorted.marks <- nbs_2.markers %>% top_n(n = 40, wt = abs(avg_logFC))
sorted.marks <- sorted.marks[order(sorted.marks$avg_logFC, decreasing = T), ]
DoHeatmap(se.subset, features = sorted.marks$gene, group.colors = c("red", "lightgray"), disp.min = -2, disp.max = 2)

Version Author Date
bfe87d1 Ludvig Larsson 2021-05-04
1e9c615 Ludvig Larsson 2021-04-28
34884bd Ludvig Larsson 2020-10-06
4f42429 Ludvig Larsson 2020-06-04


From this DE-test we can for example see that the genes COX6C and FCGR3B genes are up-regulated inside the cluser whereas LGALS1 and CYBA genes are more highly expressed outisde the cluster border.


FeatureOverlay(se.subset, features = c("COX6C", "FCGR3B", "LGALS1", "CYBA"), pt.size = 2,  
               ncols = 2, cols = c("darkblue", "cyan", "yellow", "red", "darkred"))

Version Author Date
bfe87d1 Ludvig Larsson 2021-05-04
1e9c615 Ludvig Larsson 2021-04-28
4f42429 Ludvig Larsson 2020-06-04


And lastly, if you want to keep the labels for the neighbors you can set keep.idents = TRUE and you can keep one label per identity for the neighboring spots, e.g. “label”_nb_to_2


se <- SetIdent(se, value = "seurat_clusters")
se <- RegionNeighbours(se, id = 2, keep.idents = TRUE, verbose = TRUE)
Creating Connected Netork using KNN ... 
Found 4763 neighbours for id 2 ... 
Excluding neighbours from the same group ... 
569 neighbours left ... 
Naming neighbours to id_nb_to* ... 
Saving neighbour ids to column 'nbs_2' ... 
Finished. 
FeatureOverlay(se, features = "nbs_2", ncols = 2, sampleids = 1:2, pt.size = 2)

Version Author Date
bfe87d1 Ludvig Larsson 2021-05-04
1e9c615 Ludvig Larsson 2021-04-28
4f42429 Ludvig Larsson 2020-06-04
 

A work by Joseph Bergenstråhle and Ludvig Larsson

 


sessionInfo()
R version 4.0.5 (2021-03-31)
Platform: x86_64-apple-darwin17.0 (64-bit)
Running under: macOS Mojave 10.14.6

Matrix products: default
BLAS:   /Library/Frameworks/R.framework/Versions/4.0/Resources/lib/libRblas.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/4.0/Resources/lib/libRlapack.dylib

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
[1] dplyr_1.0.5     magrittr_2.0.1  STutility_0.1.0 ggplot2_3.3.3  
[5] Seurat_3.1.5    workflowr_1.6.2

loaded via a namespace (and not attached):
  [1] uuid_0.1-4              backports_1.1.6         systemfonts_0.2.1      
  [4] plyr_1.8.6              igraph_1.2.6            lazyeval_0.2.2         
  [7] sp_1.4-1                splines_4.0.5           crosstalk_1.1.1        
 [10] listenv_0.8.0           digest_0.6.27           foreach_1.5.0          
 [13] htmltools_0.5.1.1       viridis_0.5.1           magick_2.3             
 [16] tiff_0.1-5              gdata_2.18.0            fansi_0.4.2            
 [19] cluster_2.1.1           doParallel_1.0.15       ROCR_1.0-11            
 [22] limma_3.44.1            globals_0.14.0          gmodels_2.18.1         
 [25] jpeg_0.1-8.1            colorspace_2.0-0        blob_1.2.1             
 [28] ggrepel_0.9.1           xfun_0.13               crayon_1.4.1           
 [31] jsonlite_1.7.2          zeallot_0.1.0           survival_3.2-10        
 [34] zoo_1.8-9               iterators_1.0.12        ape_5.4-1              
 [37] glue_1.4.2              gtable_0.3.0            webshot_0.5.2          
 [40] leiden_0.3.7            future.apply_1.7.0      scales_1.1.1           
 [43] DBI_1.1.0               miniUI_0.1.1.1          Rcpp_1.0.6             
 [46] viridisLite_0.3.0       xtable_1.8-4            spData_0.3.5           
 [49] units_0.6-6             reticulate_1.18         spdep_1.1-3            
 [52] rsvd_1.0.3              akima_0.6-2             tsne_0.1-3             
 [55] htmlwidgets_1.5.3       httr_1.4.2              RColorBrewer_1.1-2     
 [58] ellipsis_0.3.1          ica_1.0-2               farver_2.1.0           
 [61] pkgconfig_2.0.3         uwot_0.1.10             deldir_0.1-25          
 [64] utf8_1.2.1              labeling_0.4.2          tidyselect_1.1.0       
 [67] rlang_0.4.10            manipulateWidget_0.10.1 reshape2_1.4.4         
 [70] later_1.1.0.1           munsell_0.5.0           tools_4.0.5            
 [73] dbscan_1.1-5            generics_0.1.0          ggridges_0.5.3         
 [76] evaluate_0.14           stringr_1.4.0           fastmap_1.0.1          
 [79] yaml_2.2.1              knitr_1.28              fs_1.5.0               
 [82] fitdistrplus_1.1-3      rgl_0.100.54            purrr_0.3.4            
 [85] RANN_2.6.1              readbitmap_0.1.5        pbapply_1.4-3          
 [88] future_1.21.0           nlme_3.1-152            whisker_0.4            
 [91] mime_0.10               ggiraph_0.7.7           compiler_4.0.5         
 [94] plotly_4.9.3            png_0.1-7               e1071_1.7-3            
 [97] Morpho_2.8              tibble_3.1.0            stringi_1.5.3          
[100] gdtools_0.2.2           lattice_0.20-41         Matrix_1.3-2           
[103] classInt_0.4-3          shinyjs_1.1             vctrs_0.3.7            
[106] LearnBayes_2.15.1       pillar_1.5.1            lifecycle_1.0.0        
[109] lmtest_0.9-38           RcppAnnoy_0.0.18        data.table_1.14.0      
[112] cowplot_1.1.1           irlba_2.3.3             Rvcg_0.19.1            
[115] raster_3.1-5            httpuv_1.5.2            patchwork_1.1.1        
[118] colorRamps_2.3          R6_2.5.0                imager_0.42.1          
[121] promises_1.2.0.1        KernSmooth_2.23-18      gridExtra_2.3          
[124] bmp_0.3                 parallelly_1.24.0       codetools_0.2-18       
[127] gtools_3.8.2            boot_1.3-27             MASS_7.3-53.1          
[130] assertthat_0.2.1        rprojroot_1.3-2         withr_2.4.1            
[133] sctransform_0.2.1       expm_0.999-4            parallel_4.0.5         
[136] grid_4.0.5              tidyr_1.1.3             coda_0.19-3            
[139] class_7.3-18            rmarkdown_2.1           Rtsne_0.15             
[142] git2r_0.27.1            sf_0.9-7                shiny_1.4.0.2