Contents

1 Introduction

In this vignette, we provide an overview of the basic functionality and usage of the scds package, which interfaces with SingleCellExperiment objects.

2 Installation

Install the scds package using Bioconductor:

if (!requireNamespace("BiocManager", quietly = TRUE))
    install.packages("BiocManager")
BiocManager::install("scds", version = "3.9")

Or from github:

library(devtools)
devtools::install_github('kostkalab/scds')

3 Quick start

scds takes as input a SingleCellExperiment object (see here SingleCellExperiment), where raw counts are stored in a counts assay, i.e. assay(sce,"counts"). An example dataset created by sub-sampling the cell-hashing cell-lines data set (see https://satijalab.org/seurat/hashing_vignette.html) is included with the package and accessible via data("sce").Note that scds is designed to workd with larger datasets, but for the purposes of this vignette, we work with a smaller example dataset. We apply scds to this data and compare/visualize reasults:

3.1 Example data set

Get example data set provided with the package.

library(scds)
library(scater)
library(rsvd)
library(Rtsne)
library(cowplot)
set.seed(30519)
data("sce_chcl")
sce = sce_chcl #- less typing
dim(sce)
## [1] 2000 2000

We see it contains 2,000 genes and 2,000 cells, 216 of which are identified as doublets:

table(sce$hto_classification_global)
## 
##  Doublet Negative  Singlet 
##      216       83     1701

We can visualize cells/doublets after projecting into two dimensions:

logcounts(sce) = log1p(counts(sce))
vrs            = apply(logcounts(sce),1,var)
pc             = rpca(t(logcounts(sce)[order(vrs,decreasing=TRUE)[1:100],]))
ts             = Rtsne(pc$x[,1:10],verb=FALSE)

reducedDim(sce,"tsne") = ts$Y; rm(ts,vrs,pc)
plotReducedDim(sce,"tsne",col="hto_classification_global")

3.2 Computational doublet annotation

We now run the scds doublet annotation approaches. Briefly, we identify doublets in two complementary ways: cxds is based on co-expression of gene pairs and works with absence/presence calls only, while bcds uses the full count information and a binary classification approach using artificially generated doublets. cxds_bcds_hybrid combines both approaches, for more details please consult (this manuscript). Each of the three methods returns a doublet score, with higher scores indicating more “doublet-like” barcodes.

#- Annotate doublet using co-expression based doublet scoring:
sce = cxds(sce,retRes = TRUE)
sce = bcds(sce,retRes = TRUE,verb=TRUE)
sce = cxds_bcds_hybrid(sce)
par(mfcol=c(1,3))
boxplot(sce$cxds_score   ~ sce$doublet_true_labels, main="cxds")
boxplot(sce$bcds_score   ~ sce$doublet_true_labels, main="bcds")
boxplot(sce$hybrid_score ~ sce$doublet_true_labels, main="hybrid")

3.3 Visualizing gene pairs

For cxds we can identify and visualize gene pairs driving doublet annoataions, with the expectation that the two genes in a pair might mark different types of cells (see manuscript). In the following we look at the top three pairs, each gene pair is a row in the plot below:

scds =
top3 = metadata(sce)$cxds$topPairs[1:3,]
rs   = rownames(sce)
hb   = rowData(sce)$cxds_hvg_bool
ho   = rowData(sce)$cxds_hvg_ordr[hb]
hgs  = rs[ho]

l1 =  ggdraw() + draw_text("Pair 1", x = 0.5, y = 0.5)
p1 = plotReducedDim(sce,"tsne",col=hgs[top3[1,1]])
p2 = plotReducedDim(sce,"tsne",col=hgs[top3[1,2]])

l2 =  ggdraw() + draw_text("Pair 2", x = 0.5, y = 0.5)
p3 = plotReducedDim(sce,"tsne",col=hgs[top3[2,1]])
p4 = plotReducedDim(sce,"tsne",col=hgs[top3[2,2]])

l3 = ggdraw() + draw_text("Pair 3", x = 0.5, y = 0.5)
p5 = plotReducedDim(sce,"tsne",col=hgs[top3[3,1]])
p6 = plotReducedDim(sce,"tsne",col=hgs[top3[3,2]])

plot_grid(l1,p1,p2,l2,p3,p4,l3,p5,p6,ncol=3, rel_widths = c(1,2,2))

4 Session Info

sessionInfo()
## R version 3.6.1 (2019-07-05)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 18.04.3 LTS
## 
## Matrix products: default
## BLAS:   /home/biocbuild/bbs-3.10-bioc/R/lib/libRblas.so
## LAPACK: /home/biocbuild/bbs-3.10-bioc/R/lib/libRlapack.so
## 
## locale:
##  [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
##  [3] LC_TIME=en_US.UTF-8        LC_COLLATE=C              
##  [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
##  [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
##  [9] LC_ADDRESS=C               LC_TELEPHONE=C            
## [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       
## 
## attached base packages:
## [1] parallel  stats4    stats     graphics  grDevices utils     datasets 
## [8] methods   base     
## 
## other attached packages:
##  [1] cowplot_1.0.0               Rtsne_0.15                 
##  [3] rsvd_1.0.2                  scater_1.14.0              
##  [5] ggplot2_3.2.1               SingleCellExperiment_1.8.0 
##  [7] SummarizedExperiment_1.16.0 DelayedArray_0.12.0        
##  [9] BiocParallel_1.20.0         matrixStats_0.55.0         
## [11] Biobase_2.46.0              GenomicRanges_1.38.0       
## [13] GenomeInfoDb_1.22.0         IRanges_2.20.0             
## [15] S4Vectors_0.24.0            BiocGenerics_0.32.0        
## [17] scds_1.2.0                  BiocStyle_2.14.0           
## 
## loaded via a namespace (and not attached):
##  [1] Rcpp_1.0.2               lattice_0.20-38         
##  [3] assertthat_0.2.1         digest_0.6.22           
##  [5] R6_2.4.0                 plyr_1.8.4              
##  [7] evaluate_0.14            pillar_1.4.2            
##  [9] zlibbioc_1.32.0          rlang_0.4.1             
## [11] lazyeval_0.2.2           data.table_1.12.6       
## [13] irlba_2.3.3              Matrix_1.2-17           
## [15] rmarkdown_1.16           labeling_0.3            
## [17] BiocNeighbors_1.4.0      stringr_1.4.0           
## [19] RCurl_1.95-4.12          munsell_0.5.0           
## [21] compiler_3.6.1           vipor_0.4.5             
## [23] BiocSingular_1.2.0       xfun_0.10               
## [25] pkgconfig_2.0.3          ggbeeswarm_0.6.0        
## [27] htmltools_0.4.0          tidyselect_0.2.5        
## [29] gridExtra_2.3            tibble_2.1.3            
## [31] GenomeInfoDbData_1.2.2   bookdown_0.14           
## [33] viridisLite_0.3.0        crayon_1.3.4            
## [35] dplyr_0.8.3              withr_2.1.2             
## [37] bitops_1.0-6             grid_3.6.1              
## [39] gtable_0.3.0             magrittr_1.5            
## [41] pROC_1.15.3              scales_1.0.0            
## [43] stringi_1.4.3            XVector_0.26.0          
## [45] viridis_0.5.1            DelayedMatrixStats_1.8.0
## [47] xgboost_0.90.0.2         tools_3.6.1             
## [49] glue_1.3.1               beeswarm_0.2.3          
## [51] purrr_0.3.3              yaml_2.2.0              
## [53] colorspace_1.4-1         BiocManager_1.30.9      
## [55] knitr_1.25