mini3D STARmap#

library(Giotto)

Install Python Modules#

To run this vignette you need to install all of the necessary Python modules.

Important

Python module installation can be done either automatically via our installation tool (from within R) (see step 2.2A) or manually (see step 2.2B).

See Part 2.2 Giotto-Specific Python Packages of our Giotto Installation section for step-by-step instructions.

Optional: Set Giotto Instructions#

# to automatically save figures in save_dir set save_plot to TRUE
temp_dir = getwd()
temp_dir = '~/Temp/'
myinstructions = createGiottoInstructions(save_dir = temp_dir,
                      save_plot = TRUE,
                      show_plot = FALSE)

1. Giotto Object#

Minimum Requirements:#

  • Matrix with expression information (or path to)

  • x,y(,z) coordinates for cells or spots (or path to)

# giotto object
expr_path = system.file("extdata", "starmap_expr.txt.gz", package = 'Giotto')
loc_path = system.file("extdata", "starmap_cell_loc.txt", package = 'Giotto')
starmap_mini <- createGiottoObject(raw_exprs = expr_path,
                spatial_locs = loc_path,
                instructions = myinstructions)

How to work with Giotto instructions that are part of your Giotto object:

  • Show the instructions associated with your Giotto object with showGiottoInstructions

  • Change one or more instructions with changeGiottoInstructions

  • Replace all instructions at once with replaceGiottoInstructions

  • Read or get a specific giotto instruction with readGiottoInstructions

Note: The python path can only be set once in an R session. See the **reticulate package* for more information.*

# show instructions associated with giotto object (starmap_mini)
showGiottoInstructions(starmap_mini)

2. Processing Steps#

  • Filter genes and cells based on detection frequencies

  • Normalize expression matrix (log transformation, scaling factor and/or z-scores)

  • Add cell and gene statistics (optional)

  • Adjust expression matrix for technical covariates or batches (optional). These results will be stored in the custom slot.

filterDistributions(starmap_mini, detection = 'genes',
        save_param = list(save_name = '2_a_filtergenes'))
2_a_filtergenes 
filterDistributions(starmap_mini, detection = 'cells',
        save_param = list(save_name = '2_b_filtercells'))
2_b_filtercells.png 
filterCombinations(starmap_mini,
       expression_thresholds = c(1),
       gene_det_in_min_cells = c(50, 100, 200),
       min_det_genes_per_cell = c(20, 28, 28),
       save_param = list(save_name = '2_c_filtercombos'))
2_c_filtercombos.png 
starmap_mini <- filterGiotto(gobject = starmap_mini,
           expression_threshold = 1,
           gene_det_in_min_cells = 50,
           min_det_genes_per_cell = 20,
           expression_values = c('raw'),
           verbose = T)
starmap_mini <- normalizeGiotto(gobject = starmap_mini,
            scalefactor = 6000, verbose = T)
starmap_mini <- addStatistics(gobject = starmap_mini)

3. Dimension Reduction#

  • Identify highly variable genes (HVG)

  • Perform PCA

  • Identify number of significant prinicipal components (PCs)

  • Run UMAP and/or TSNE on PCs (or directly on matrix)

starmap_mini <- runPCA(gobject = starmap_mini, method = 'factominer')
screePlot(starmap_mini, ncp = 30,
  save_param = list(save_name = '3_a_screeplot'))
3_a_screeplot.png 
plotPCA(gobject = starmap_mini,
    save_param = list(save_name = '3_b_PCA'))
3_b_PCA.png 
# 2D umap
starmap_mini <- runUMAP(starmap_mini, dimensions_to_use = 1:8)
plotUMAP(gobject = starmap_mini,
    save_param = list(save_name = '3_c_UMAP'))
2_c_filtercombos.png 
# 3D umap
starmap_mini <- runUMAP(starmap_mini, dimensions_to_use = 1:8, name = '3D_umap', n_components = 3)
plotUMAP_3D(gobject = starmap_mini, dim_reduction_name = '3D_umap',
    save_param = list(save_name = '3_d_UMAP_3D'))
3_d_UMAP_3D.png 
# 2D tsne
starmap_mini <- runtSNE(starmap_mini, dimensions_to_use = 1:8)
plotTSNE(gobject = starmap_mini,
    save_param = list(save_name = '3_e_TSNE'))
3_e_TSNE.png

4. Clustering#

  • Create a shared (default) nearest network in PCA space (or directly on matrix)

  • Cluster on nearest network with Leiden or Louvan (kmeans and hclust are alternatives)

starmap_mini <- createNearestNetwork(gobject = starmap_mini, dimensions_to_use = 1:8, k = 25)
starmap_mini <- doLeidenCluster(gobject = starmap_mini, resolution = 0.5, n_iterations = 1000)

# 2D umap
plotUMAP(gobject = starmap_mini,
    cell_color = 'leiden_clus', show_NN_network = T, point_size = 2.5,
    save_param = list(save_name = '4_a_UMAP'))
4_a_UMAP.png 
# 3D umap
plotUMAP_3D(gobject = starmap_mini, dim_reduction_name = '3D_umap',
    cell_color = 'leiden_clus',
    save_param = list(save_name = '4_b_UMAP_3D'))
4_b_UMAP_3D.png 
# 2D umap + coordinates
spatDimPlot(gobject = starmap_mini, cell_color = 'leiden_clus',
    dim_point_size = 2, spat_point_size = 2.5,
    save_param = list(save_name = '4_c_spatdimplot'))
4_c_spatdimplot.png 
# 3D umap + coordinates
spatDimPlot3D(gobject = starmap_mini,
    cell_color = 'leiden_clus', dim_reduction_name = '3D_umap',
    save_param = list(save_name = '4_d_spatdimplot3D'))


# heatmap and dendrogram
showClusterHeatmap(gobject = starmap_mini, cluster_column = 'leiden_clus',
       save_param = list(save_name = '4_e_clusterheatmap'))
4_e_clusterheatmap.png 
showClusterDendrogram(starmap_mini, h = 0.5, rotate = T,
          cluster_column = 'leiden_clus',
          save_param = list(save_name = '4_f_clusterdendrogram'))
4_f_clusterdendrogram.png

5. Differential Expression#

gini_markers = findMarkers_one_vs_all(gobject = starmap_mini,
                      method = 'gini',
                      expression_values = 'normalized',
                      cluster_column = 'leiden_clus',
                      min_genes = 20,
                      min_expr_gini_score = 0.5,
                      min_det_gini_score = 0.5)

# get top 2 genes per cluster and visualize with violinplot
topgenes_gini = gini_markers[, head(.SD, 2), by = 'cluster']
violinPlot(starmap_mini, genes = topgenes_gini$genes,
    cluster_column = 'leiden_clus',
    save_param = list(save_name = '5_a_violinplot'))
5_a_violinplot.png 
# get top 6 genes per cluster and visualize with heatmap
topgenes_gini2 = gini_markers[, head(.SD, 6), by = 'cluster']
plotMetaDataHeatmap(starmap_mini, selected_genes = topgenes_gini2$genes,
        metadata_cols = c('leiden_clus'),
        save_param = list(save_name = '5_b_metaheatmap'))
5_b_metaheatmap.png

6. Cell Type#

6.1 Cell Type Annotation#

clusters_cell_types = c('cell A', 'cell B', 'cell C', 'cell D',
        'cell E', 'cell F', 'cell G', 'cell H')
names(clusters_cell_types) = 1:8
starmap_mini = annotateGiotto(gobject = starmap_mini,
              annotation_vector = clusters_cell_types,
              cluster_column = 'leiden_clus',
              name = 'cell_types')
# check new cell metadata
pDataDT(starmap_mini)

# visualize annotations
spatDimPlot(gobject = starmap_mini, cell_color = 'cell_types',
    spat_point_size = 2, dim_point_size = 2,
    save_param = list(save_name = '6_a_spatdimplot'))
6_a_spatdimplot.png

6.2 Cell Type Gene Expression#

dimGenePlot3D(starmap_mini,
      dim_reduction_name = '3D_umap',
      expression_values = 'scaled',
      genes = "Pcp4",
      genes_high_color = 'red', genes_mid_color = 'white', genes_low_color = 'darkblue',
      save_param = list(save_name = '6_b_dimgeneplot'))
6_b_dimgeneplot.png 
spatGenePlot3D(starmap_mini,
       expression_values = 'scaled',
       genes = "Pcp4",
       show_other_cells = F,
       genes_high_color = 'red', genes_mid_color = 'white', genes_low_color = 'darkblue',
       save_param = list(save_name = '6_c_spatgeneplot'))
6_c_spatgeneplot.png

7. Spatial Grid#

Create a grid based on defined stepsizes in the x,y(,z) axes.

starmap_mini <- createSpatialGrid(gobject = starmap_mini,
              sdimx_stepsize = 200,
              sdimy_stepsize = 200,
              sdimz_stepsize = 20,
              minimum_padding = 10)
showGrids(starmap_mini)

# visualize grid
spatPlot2D(gobject = starmap_mini, show_grid = T, point_size = 1.5,
    save_param = list(save_name = '7_a_spatplot'))
7_a_spatplot.png

8. Spatial Network#

Only the method = delaunayn_geometry can make 3D Delaunay networks. This requires the package geometry to be installed.

  • Visualize information about the default Delaunay network

  • Create a spatial Delaunay network (default)

  • Create a spatial kNN network

plotStatDelaunayNetwork(gobject = starmap_mini, maximum_distance = 200,
        method = 'delaunayn_geometry',
        save_param = list(save_name = '8_aa_delnetwork'))
8_a_plotStatDelaunayNetwork.png 
starmap_mini = createSpatialNetwork(gobject = starmap_mini, minimum_k = 2,
                maximum_distance_delaunay = 200,
                method = 'Delaunay',
                delaunay_method = 'delaunayn_geometry')
starmap_mini = createSpatialNetwork(gobject = starmap_mini, minimum_k = 2,
                method = 'kNN', k = 10)
showNetworks(starmap_mini)

# visualize the two different spatial networks
spatPlot(gobject = starmap_mini, show_network = T,
    network_color = 'blue', spatial_network_name = 'Delaunay_network',
    point_size = 2.5, cell_color = 'leiden_clus',
    save_param = list(save_name = '8_a_spatplot'))
8_b_spatplot.png 
spatPlot(gobject = starmap_mini, show_network = T,
    network_color = 'blue', spatial_network_name = 'kNN_network',
    point_size = 2.5, cell_color = 'leiden_clus',
    save_param = list(save_name = '8_b_spatplot'))
8_c_spatplot.png

9. Spatial Genes#

Identify spatial genes with 3 different methods:

  • binSpect with kmeans binarization (default)

  • binSpect with rank binarization

  • silhouetteRank

Visualize top 4 genes per method.

km_spatialgenes = binSpect(starmap_mini)
spatGenePlot(starmap_mini, expression_values = 'scaled',
    genes = km_spatialgenes[1:4]$genes,
    point_shape = 'border', point_border_stroke = 0.1,
    show_network = F, network_color = 'lightgrey', point_size = 2.5,
    cow_n_col = 2,
    save_param = list(save_name = '9_a_spatgeneplot'))
9_a_spatgeneplot.png 
rank_spatialgenes = binSpect(starmap_mini, bin_method = 'rank')
spatGenePlot(starmap_mini, expression_values = 'scaled',
    genes = rank_spatialgenes[1:4]$genes,
    point_shape = 'border', point_border_stroke = 0.1,
    show_network = F, network_color = 'lightgrey', point_size = 2.5,
    cow_n_col = 2,
    save_param = list(save_name = '9_b_spatgeneplot'))
9_b_spatgeneplot.png 
silh_spatialgenes = silhouetteRank(gobject = starmap_mini) # TODO: suppress print output
spatGenePlot(starmap_mini, expression_values = 'scaled',
    genes = silh_spatialgenes[1:4]$genes,
    point_shape = 'border', point_border_stroke = 0.1,
    show_network = F, network_color = 'lightgrey', point_size = 2.5,
    cow_n_col = 2,
    save_param = list(save_name = '9_c_spatgeneplot'))

10. Spatial Co-Expression Patterns#

Identify robust spatial co-expression patterns using the spatial network or grid and a subset of individual spatial genes.

10.1 Calculate spatial correlation scores#

# 1. calculate spatial correlation scores
ext_spatial_genes = km_spatialgenes[1:20]$genes
spat_cor_netw_DT = detectSpatialCorGenes(starmap_mini,
                 method = 'network',
                 spatial_network_name = 'Delaunay_network',
                 subset_genes = ext_spatial_genes)

10.2. Cluster correlation scores#

# 2. cluster correlation scores
spat_cor_netw_DT = clusterSpatialCorGenes(spat_cor_netw_DT,
                  name = 'spat_netw_clus', k = 6)
heatmSpatialCorGenes(starmap_mini, spatCorObject = spat_cor_netw_DT,
         use_clus_name = 'spat_netw_clus',
         save_param = list(save_name = '10_a_heatmspatcor', units = 'in'))
10_a_heatmspatcor.png 
netw_ranks = rankSpatialCorGroups(starmap_mini,
              spatCorObject = spat_cor_netw_DT,
              use_clus_name = 'spat_netw_clus',
              save_param = list(save_name = '10_b_rankcorgroup'))
10_b_rankcorgroup.png 
top_netw_spat_cluster = showSpatialCorGenes(spat_cor_netw_DT,
                    use_clus_name = 'spat_netw_clus',
                    selected_clusters = 6,
                    show_top_genes = 1)

cluster_genes_DT = showSpatialCorGenes(spat_cor_netw_DT,
                   use_clus_name = 'spat_netw_clus',
                   show_top_genes = 1)
cluster_genes = cluster_genes_DT$clus; names(cluster_genes) = cluster_genes_DT$gene_ID


starmap_mini = createMetagenes(starmap_mini,
               gene_clusters = cluster_genes,
               name = 'cluster_metagene')
spatCellPlot(starmap_mini,
    spat_enr_names = 'cluster_metagene',
    cell_annotation_values = netw_ranks$clusters,
    point_size = 1.5, cow_n_col = 3,
    save_param = list(save_name = '10_c_spatcellplot'))
10_c_spatcellplot.png

11. Spatial HMRF Domains#

hmrf_folder = paste0(temp_dir,'/','11_HMRF/')
if(!file.exists(hmrf_folder)) dir.create(hmrf_folder, recursive = T)

# perform hmrf
my_spatial_genes = km_spatialgenes[1:20]$genes
HMRF_spatial_genes = doHMRF(gobject = starmap_mini,
            expression_values = 'scaled',
            spatial_genes = my_spatial_genes,
            spatial_network_name = 'Delaunay_network',
            k = 6,
            betas = c(10,2,2),
            output_folder = paste0(hmrf_folder, '/', 'Spatial_genes/SG_top20_k6_scaled'))

# check and select hmrf
for(i in seq(10, 14, by = 2)) {
viewHMRFresults2D(gobject = starmap_mini,
        HMRFoutput = HMRF_spatial_genes,
        k = 6, betas_to_view = i,
        point_size = 2)
}

starmap_mini = addHMRF(gobject = starmap_mini,
        HMRFoutput = HMRF_spatial_genes,
        k = 6, betas_to_add = c(12),
        hmrf_name = 'HMRF')

# visualize selected hmrf result
giotto_colors = Giotto:::getDistinctColors(6)
names(giotto_colors) = 1:6
spatPlot(gobject = starmap_mini, cell_color = 'HMRF_k6_b.12',
    point_size = 3, coord_fix_ratio = 1, cell_color_code = giotto_colors,
    save_param = list(save_name = '11_a_spatplot'))

12. Cell Neighborhood: Cell-Type / Cell-Type Interactions#

set.seed(seed = 2841)
cell_proximities = cellProximityEnrichment(gobject = starmap_mini,
                   cluster_column = 'cell_types',
                   spatial_network_name = 'Delaunay_network',
                   adjust_method = 'fdr',
                   number_of_simulations = 1000)
# barplot
cellProximityBarplot(gobject = starmap_mini,
         CPscore = cell_proximities,
         min_orig_ints = 2, min_sim_ints = 2, p_val = 0.5,
         save_param = list(save_name = '12_a_barplot'))
12_a_barplot.png 
## heatmap
cellProximityHeatmap(gobject = starmap_mini, CPscore = cell_proximities,
         order_cell_types = T, scale = T,
         color_breaks = c(-1.5, 0, 1.5),
         color_names = c('blue', 'white', 'red'),
         save_param = list(save_name = '12_b_heatmap', units = 'in'))
12_b_heatmap.png 
# network
cellProximityNetwork(gobject = starmap_mini, CPscore = cell_proximities,
         remove_self_edges = T, only_show_enrichment_edges = T,
         save_param = list(save_name = '12_c_network'))
12_c_network.png 
# network with self-edges
cellProximityNetwork(gobject = starmap_mini, CPscore = cell_proximities,
        remove_self_edges = F, self_loop_strength = 0.3,
        only_show_enrichment_edges = F,
        rescale_edge_weights = T,
        node_size = 8,
        edge_weight_range_depletion = c(1, 2),
        edge_weight_range_enrichment = c(2,5),
        save_param = list(save_name = '12_d_network'))
12_d_network.png

12.1 Visualization of Specific Cell Types#

Option 1#

pDataDT(starmap_mini)
# Option 1
spec_interaction = "cell D--cell H" # needs to be in alphabetic order! first D, then H
cellProximitySpatPlot2D(gobject = starmap_mini,
            interaction_name = spec_interaction,
            show_network = T,
            cluster_column = 'cell_types',
            cell_color = 'cell_types',
            cell_color_code = c('cell H' = 'lightblue', 'cell D' = 'red'),
            point_size_select = 4, point_size_other = 2,
            save_param = list(save_name = '12_e_cellproximity'))
12_e_spatplot.png

Option 2#

# Option 2: create additional metadata
starmap_mini = addCellIntMetadata(starmap_mini,
             spatial_network = 'Delaunay_network',
             cluster_column = 'cell_types',
             cell_interaction = spec_interaction,
             name = 'D_H_interactions')
spatPlot(starmap_mini, cell_color = 'D_H_interactions', legend_symbol_size = 3,
    select_cell_groups =  c('other_cell D', 'other_cell H', 'select_cell D', 'select_cell H'),
    save_param = list(save_name = '12_e_spatplot'))
12_e_cellproximity.png

13. 2D cross sections from 3D object#

# create cross section
starmap_mini = createCrossSection(starmap_mini,
            method="equation",
            equation=c(0,1,0,600),
            extend_ratio = 0.6)

# show cross section
insertCrossSectionSpatPlot3D(starmap_mini, cell_color = 'leiden_clus',
             axis_scale = 'cube',
             point_size = 2,
             save_param = list(save_name = '13_a_insertcross'))

insertCrossSectionGenePlot3D(starmap_mini, expression_values = 'scaled',
             axis_scale = "cube",
             genes = "Slc17a7",
             save_param = list(save_name = '13_b_insertcrossgene'))
13_a_insert.png 
# for cell annotation
crossSectionPlot(starmap_mini,
        point_size = 2, point_shape = "border",
        cell_color = "leiden_clus",
        save_param = list(save_name = '13_c_crossplot'))
13_b_crossplot.png 
crossSectionPlot3D(starmap_mini,
       point_size = 2, cell_color = "leiden_clus",
       axis_scale = "cube",
       save_param = list(save_name = '13_c_crossplot3D'))
13_c_cross.png 
# for gene expression
crossSectionGenePlot(starmap_mini,
        genes = "Slc17a7",
        point_size = 2,
        point_shape = "border",
        cow_n_col = 1.5,
        expression_values = 'scaled',
        save_param = list(save_name = '13_d_crossgeneplot'))
13_d_crossgeneplot.png 
crossSectionGenePlot3D(starmap_mini,
           point_size = 2,
           genes = c("Slc17a7"),
           expression_values = 'scaled',
           save_param = list(save_name = '13_e_crossgeneplot3D'))
13_e_crossgene.png

14. Export Giotto Analyzer to Viewer#

viewer_folder = paste0(temp_dir, '/', 'Mouse_cortex_viewer')

# select annotations, reductions and expression values to view in Giotto Viewer
exportGiottoViewer(gobject = starmap_mini, output_directory = viewer_folder,
       factor_annotations = c('cell_types',
                  'leiden_clus',
                  'HMRF_k6_b.12'),
       numeric_annotations = 'total_expr',
       dim_reductions = c('umap'),
       dim_reduction_names = c('umap'),
       expression_values = 'scaled',
       expression_rounding = 3,
       overwrite_dir = T)