Cell type classification with SignacX: CITE-seq PBMCs from 10X Genomics

Compiled 2021-03-02

Source: vignettes/signac-Seurat_CITE-seq.Rmd

In Figure 2-3 of the pre-print, we validated Signac with CITE-seq PBMCs. Here, we reproduced that analysis with Seurat, and provide interactive access to the data here. There are three parts to this vignette: Seurat, SignacX and then visualization. We use an example PBMCs CITE-seq data set from 10X Genomics.

Seurat

Start with the standard pre-processing steps for a Seurat object.

Download data from 10X Genomics.

dir.create("fls")
download.file("https://cf.10xgenomics.com/samples/cell-exp/3.0.0/pbmc_10k_protein_v3/pbmc_10k_protein_v3_filtered_feature_bc_matrix.h5", 
    destfile = "fls/pbmc_10k_protein_v3_filtered_feature_bc_matrix.h5")

Create a Seurat object, and then perform SCTransform normalization. Note:

  • You can use the legacy functions here (i.e., NormalizeData, ScaleData, etc.), use SCTransform or any other normalization method (including no normalization). We did not notice a significant difference in cell type annotations with different normalization methods.
  • We think that it is best practice to use SCTransform, but it is not a necessary step. SignacX will work fine without it.
# load dataset
E = Read10X_h5(filename = "fls/pbmc_10k_protein_v3_filtered_feature_bc_matrix.h5")
pbmc <- CreateSeuratObject(counts = E$`Gene Expression`, project = "pbmc")

# run sctransform
pbmc <- SCTransform(pbmc)

Perform dimensionality reduction by PCA and UMAP embedding. Note:

  • SignacX actually needs these functions since it uses the nearest neighbor graph generated by Seurat.
# These are now standard steps in the Seurat workflow for visualization and clustering
pbmc <- RunPCA(pbmc, verbose = FALSE)
pbmc <- RunUMAP(pbmc, dims = 1:30, verbose = FALSE)
pbmc <- FindNeighbors(pbmc, dims = 1:30, verbose = FALSE)

SignacX

Load the package

Generate SignacX labels for the Seurat object. Note:

  • Optionally, you can do parallel computing by setting num.cores > 1 in the Signac function.
  • Run time is ~10-20 minutes for <100,000 cells.
# Run Signac
labels <- Signac(pbmc, num.cores = 4)
celltypes = GenerateLabels(labels, E = pbmc)
Can we make Signac faster?

Sometimes, training the neural networks takes a lot of time. To make Signac faster, we implemented SignacFast which uses an ensemble of pre-trained neural network models. Note:

  • SignacFast uses an ensemble of 1,800 pre-calculated neural networks using the GenerateModels function together with the training_HPCA reference data.
  • Features that are absent from the single cell data and present in the neural network are set to zero.
# Run Signac
labels_fast <- SignacFast(pbmc)
celltypes_fast = GenerateLabels(labels_fast, E = pbmc)
SignacFast took only ~30 seconds. Relative to Signac, the main difference is that SignacFast tends to leave a few more cells “Unclassified.”
How does SignacFast compare to Signac?
B MPh TNK Unclassified
B 550 0 0 0
MPh 0 2178 0 0
TNK 0 0 4914 0
Unclassified 0 4 2 217

Visualizations

Now we can visualize the cell type classifications at many different levels: Immune and nonimmune

pbmc <- AddMetaData(pbmc, metadata = celltypes_fast$Immune, col.name = "immmune")
pbmc <- SetIdent(pbmc, value = "immmune")
png(filename = "fls/plot1_citeseq.png")
DimPlot(pbmc)
dev.off()

Immune, Nonimmune (if any) and unclassified cells

pbmc <- AddMetaData(pbmc, metadata = celltypes$L2, col.name = "celltypes")
pbmc <- SetIdent(pbmc, value = "celltypes")
png(filename = "fls/plot2_citeseq.png")
DimPlot(pbmc)
dev.off()

Myeloid and lymphocytes

pbmc <- AddMetaData(pbmc, metadata = celltypes$CellTypes, col.name = "celltypes")
pbmc <- SetIdent(pbmc, value = "celltypes")
png(filename = "./fls/plot3_citeseq.png")
DimPlot(pbmc)
dev.off()

Cell types

pbmc <- AddMetaData(pbmc, metadata = celltypes$CellTypes_novel, col.name = "celltypes_novel")
pbmc <- SetIdent(pbmc, value = "celltypes_novel")
png(filename = "./fls/plot4_citeseq.png")
DimPlot(pbmc)
dev.off()

Cell types with novel populations

pbmc <- AddMetaData(pbmc, metadata = celltypes$CellStates, col.name = "cellstates")
pbmc <- SetIdent(pbmc, value = "cellstates")
png(filename = "./fls/plot5_citeseq.png")
DimPlot(pbmc)
dev.off()

Cell states

Identify differentially expressed genes between cell types.

pbmc <- SetIdent(pbmc, value = "celltypes")

# Find markers for all clusters, and draw a heatmap
markers <- FindAllMarkers(pbmc, only.pos = TRUE, verbose = F, logfc.threshold = 1)
library(dplyr)
top5 <- markers %>% group_by(cluster) %>% top_n(n = 5, wt = avg_logFC)

png(filename = "./fls/plot9_citeseq.png", width = 640, height = 720)
DoHeatmap(pbmc, features = unique(top5$gene), angle = 90)
dev.off()

Immune marker genes - cell types

pbmc <- SetIdent(pbmc, value = "cellstates")

# Find markers for all clusters, and draw a heatmap
markers <- FindAllMarkers(pbmc, only.pos = TRUE, verbose = F, logfc.threshold = 1)
top5 <- markers %>% group_by(cluster) %>% top_n(n = 5, wt = avg_logFC)

png(filename = "./fls/plot6_citeseq.png", width = 640, height = 720)
DoHeatmap(pbmc, features = unique(top5$gene), angle = 90)
dev.off()

Immune marker genes - Cell states Add protein expression information

pbmc[["ADT"]] <- CreateAssayObject(counts = E$`Antibody Capture`[, colnames(E$`Antibody Capture`) %in% 
    colnames(pbmc)])
pbmc <- NormalizeData(pbmc, assay = "ADT", normalization.method = "CLR")
pbmc <- ScaleData(pbmc, assay = "ADT")

Identify differentially expressed proteins between clusters

DefaultAssay(pbmc) <- "ADT"
# Find protein markers for all clusters, and draw a heatmap
adt.markers <- FindAllMarkers(pbmc, assay = "ADT", only.pos = TRUE, verbose = F)
png(filename = "./fls/plot7_citeseq.png", width = 640, height = 720)
DoHeatmap(pbmc, features = unique(adt.markers$gene), angle = 90)
dev.off()

Immune marker genes

Save results

saveRDS(pbmc, file = "fls/pbmcs_signac_citeseq.rds")
saveRDS(celltypes, file = "fls/celltypes_citeseq.rds")
saveRDS(celltypes_fast, file = "fls/celltypes_fast_citeseq.rds")
Session Info 
## R version 4.0.0 (2020-04-24)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: CentOS Linux 7 (Core)
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## 
## attached base packages:
## [1] stats     graphics  grDevices utils     datasets  methods   base     
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## loaded via a namespace (and not attached):
##  [1] knitr_1.31        magrittr_2.0.1    R6_2.5.0          ragg_1.1.1       
##  [5] rlang_0.4.10      fastmap_1.1.0     highr_0.8         stringr_1.4.0    
##  [9] tools_4.0.0       xfun_0.21         jquerylib_0.1.3   htmltools_0.5.1.1
## [13] systemfonts_1.0.1 yaml_2.2.1        assertthat_0.2.1  digest_0.6.27    
## [17] rprojroot_2.0.2   pkgdown_1.6.1     crayon_1.4.1      textshaping_0.3.1
## [21] formatR_1.7       sass_0.3.1        fs_1.5.0          memoise_2.0.0    
## [25] cachem_1.0.3      evaluate_0.14     rmarkdown_2.7     stringi_1.5.3    
## [29] compiler_4.0.0    bslib_0.2.4       desc_1.2.0        jsonlite_1.7.2