Scds Tutorial¶

scds is a transcription-based doublet detection software that uses two different methods to detect doublets - cxds and bcds. The cxds method uses marker genes that are not co-expressed to identify droplets that are likely doublets. bcds simulates doublet by adding droplet transcriptomes together and then uses variable genes to identify the probability a droplet is a doublet with a binary classification algorithm. We typically use the combined score of these two methods but they can be use separately as well. We have provided a wrapper script that takes common arguments for scds and we alsp provide an example script that you can run manually in R if you prefer.

Data¶

This is the data that you will need to have preparede to run scds:

Required

A counts matrix ($MATRIX)
- Scds expects counts to be in the cellranger output format (directory containing barcodes.tsv, genes.tsv and matrix.mtx or barcodes.tsv.gz, features.tsv.gz and matrix.mtx.gz)
  - If you don’t have your data in this format, you can run scds manually in R and load the data in using a method of your choosing.
Output directory ($SCDS_OUTDIR)
- If you don’t provide an $SCDS_OUTDIR, the results will be written to the present working directory.

Run scds¶

You can either run scds with the wrapper script we have provided or you can run it manually if you would prefer to alter more parameters.

singularity exec Demuxafy.sif scds.R -o $SCDS_OUTDIR -t $MATRIX

First, you will have to start R. We have built R and all the required software to run scds into the singularity image so you can run it directly from the image.

singularity exec Demuxafy.sif R

That will open R in your terminal. Next, you can load all the libraries and run scds.

.libPaths("/usr/local/lib/R/site-library") ### This is required so that R uses the libraries loaded in the image and not any local libraries
library(dplyr)
library(tidyr)
library(tidyverse)
library(scds)
library(Seurat)
library(SingleCellExperiment)

## Set up variables and parameters ##
out <- "/path/to/scds/outdir/"
tenX_matrix <- "/path/to/counts/matrix/dir/"

## Read in data
counts <- Read10X(as.character(tenX_matrix), gene.column = 1)

## Account for possibility that not just single cell data
if (is.list(counts)){
  sce <- SingleCellExperiment(list(counts=counts[[grep("Gene", names(counts))]]))
} else {
  sce <- SingleCellExperiment(list(counts=counts))
}

## Annotate doublet using binary classification based doublet scoring:
sce = bcds(sce, retRes = TRUE, estNdbl=TRUE)

## Annotate doublet using co-expression based doublet scoring:
try({
    sce = cxds(sce, retRes = TRUE, estNdbl=TRUE)
})

### If cxds worked, run hybrid, otherwise use bcds annotations
if ("cxds_score" %in% colnames(colData(sce))) {
    ## Combine both annotations into a hybrid annotation
    sce = cxds_bcds_hybrid(sce, estNdbl=TRUE)
    Doublets <- as.data.frame(cbind(rownames(colData(sce)), colData(sce)$hybrid_score, colData(sce)$hybrid_call))
} else {
    print("this pool failed cxds so results are just the bcds calls")
    Doublets <- as.data.frame(cbind(rownames(colData(sce)), colData(sce)$bcds_score, colData(sce)$bcds_call))
}

## Doublet scores are now available via colData:
colnames(Doublets) <- c("Barcode","scds_score","scds_DropletType")
Doublets$scds_DropletType <- gsub("FALSE","singlet",Doublets$scds_DropletType)
Doublets$scds_DropletType <- gsub("TRUE","doublet",Doublets$scds_DropletType)

message("writing output")
write_delim(Doublets, paste0(out,"/scds_doublets_singlets.tsv"), "\t")


summary <- as.data.frame(table(Doublets$scds_DropletType))
colnames(summary) <- c("Classification", "Droplet N")
write_delim(summary, paste0(out,"/scds_doublet_summary.tsv"), "\t")

scds Results and Interpretation¶

After running the scds with the wrapper script or manually you should have two files in the $SCDS_OUTDIR:

.
├── scds_doublets_singlets.tsv
└── scds_doublet_summary.tsv

scds_doublet_summary.tsv
- A sumamry of the number of singlets and doublets predicted by scds.
  
  Classification
  
  Droplet N
  
  doublet
  
  2771
  
  singlet
  
  18211
  - To check whether the numbe of doublets identified by scds is consistent with the expected doublet rate expected based on the number of droplets that you captured, you can use our Expected Doublet Estimation Calculator.

scds_doublets_singlets.tsv

The per-barcode singlet and doublet classification from scds.

Barcode	scds_score	scds_DropletType
AAACCTGAGATAGCAT-1	0.116344358493288	singlet
AAACCTGAGCAGCGTA-1	0.539856378453988	singlet
AAACCTGAGCGATGAC-1	0.0237184380134577	singlet
AAACCTGAGCGTAGTG-1	0.163695865366576	singlet
AAACCTGAGGAGTTTA-1	0.11591462421927	singlet
AAACCTGAGGCTCATT-1	0.0479944175570073	singlet
AAACCTGAGGGCACTA-1	0.374426050641161	singlet
AAACCTGAGTAATCCC-1	0.247842972104563	singlet
…	…	…

Merging Results with Other Software Restults¶

We have provided a script that will help merge and summarize the results from multiple softwares together. See Combine Results.

Citation¶

If you used this workflow for analysis, please reference our paper (REFERENCE) as well as scds.

Classification	Droplet N
doublet	2771
singlet	18211