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Weighted Nearest Neighbor Analysis

BMNC - RNA & ADT

This vignette introduces the weighted nearest neighbor (WNN) workflow for the analysis of multimodal single-cell datasets. The workflow consists of three steps

  • Independent preprocessing and dimensional reduction of each modality individually
  • Learning cell-specific modality 'weights', and constructing a WNN graph that integrates the modalities
  • Downstream analysis (i.e. visualization, clustering, etc.) of the WNN graph

We use the CITE-seq dataset from (Stuart*, Butler* et al, Cell 2019), which consists of 30,672 scRNA-seq profiles measured alongside a panel of 25 antibodies. The object contains two assays, RNA and antibody-derived tags (ADT).

To run this vignette please install Seurat v4, available as a beta release on our github page.

remotes::install_github("satijalab/seurat", ref = "release/4.0.0")
library(Seurat)
library(SeuratData)
library(cowplot)
library(dplyr)
InstallData("bmcite")
bm <- LoadData(ds = "bmcite")

We first perform pre-processing and dimensional reduction on both assays independently. We use standard normalization, but you can also use SCTransform or any alternative method.

DefaultAssay(bm) <- 'RNA'
bm <- NormalizeData(bm) %>% FindVariableFeatures() %>% ScaleData() %>% RunPCA()

DefaultAssay(bm) <- 'ADT'
# we will use all ADT features for dimensional reduction
# we set a dimensional reduction name to avoid overwriting the 
VariableFeatures(bm) <- rownames(bm[["ADT"]])
bm <- NormalizeData(bm, normalization.method = 'CLR', margin = 2) %>% 
  ScaleData() %>% RunPCA(reduction.name = 'apca')

For each cell, we calculate its closest neighbors in the dataset based on a weighted combination of RNA and protein similarities. The cell-specific modality weights and multimodal neighbors are calculated in a single function, which takes ~2 minutes to run on this dataset. We specify the dimensionality of each modality (similar to specifying the number of PCs to include in scRNA-seq clustering), but you can vary these settings to see that small changes have minimal effect on the overall results.

# Identify multimodal neighbors. These will be stored in the neighbors slot, 
# and can be accessed using bm[['weighted.nn']]
# The WNN graph can be accessed at bm[["wknn"]], 
# and the SNN graph used for clustering at bm[["wsnn"]]
# Cell-specific modality weights can be accessed at bm$RNA.weight
bm <- FindMultiModalNeighbors(
  bm, reduction.list = list("pca", "apca"), 
  dims.list = list(1:30, 1:18), modality.weight.name = "RNA.weight"
)

We can now use these results for downstream analysis, such as visualization and clustering. For example, we can create a UMAP visualization of the data based on a weighted combination of RNA and protein data We can also perform graph-based clustering and visualize these results on the UMAP, alongside a set of cell annotations.

bm <- RunUMAP(bm, nn.name = "weighted.nn", reduction.name = "wnn.umap", reduction.key = "wnnUMAP_")
bm <- FindClusters(bm, graph.name = "wsnn", algorithm = 3, resolution = 2, verbose = FALSE)
p1 <- DimPlot(bm, reduction = 'wnn.umap', label = TRUE, repel = TRUE, label.size = 2.5) + NoLegend()
p2 <- DimPlot(bm, reduction = 'wnn.umap', group.by = 'celltype.l2', label = TRUE, repel = TRUE, label.size = 2.5) + NoLegend()
p1 + p2