determining doublets in single-cell RNA-seq
Expected rates of doublets / duplets / multiplets


Fluidigm C1 doublet rate: around 1-5% depending on chip type used.
More information: Fluidigm white paper: Redesign of C1 Medium-Cell 96 IFCs Improves Single-Cell Capture Efficiency (PN 101-3328 B1)
10x Genomics Chromium 3'mRNA-Seq doublet rate: around 1% per 1000 cells captured


Rates in the 'Chromium Single Cell 3’ Reagent Kits v2 User Guide':

Multiplet Rate (%)  # of Cells Loaded   # of Cells Recovered
0.40%   870 500
0.80%   1700    1000
1.60%   3500    2000
2.30%   5300    3000
3.10%   7000    4000
3.90%   8700    5000
4.60%   10500   6000
5.40%   12200   7000
6.10%   14000   8000
6.90%   15700   9000
7.60%   17400   10000




Detection and identification of doublets and multiplets

Experimental


Visual inspection (e.g. Fluidigm C1)
FACS index sorting data can be used to identify doublets.
Mixed species experiment.


Illumina | BioRad White paper Single-Cell RNA Data Analysis Workflow  
Zheng et al., 2017: Massively parallel digital transcriptional profiling of single cells

Cell "hashing" with oligo-tagged antibodies (Stoeckius et al., 2017): cells (barcodes) positive for two or more oligos are multiplets.


Computational


Mixed gene expression (e.g. lymphoid and myeloid, or T and B cell)
Ilicic et al. describe an approach for detecting low quality cells (incl. multiplets). They train a support vector machine (SVM) model on a dataset annotated by microscopy inspection. They find, among others, that


"wells containing multiple cells (multiples) show similar expression and noise patterns to broken cells", suggesting "that multiple cells contain a mixture of broken and high quality cells."
difference in proportion of duplicated reads
higher ERCC spike-in ratios

demuxlet (Kang et al., 2017, Nature Biotechnology)
DoubletDetection