Doublets filter

In droplet-based data, barcodes can correspond to droplets containing two or more cells. In this dialog of QC for Single Cell, the Doublets filter can be enabled and customized to remove the droplets that are detected as containing two cells. This filter should be skipped for single-cell protocols that are not droplet-based.

Note: The Doublets filter can only be used together with the Empty droplets filter.

Note that QC for Single Cell cannot remove droplets containing more than two cells. However, these are expected to be present at negligible rates.

There are two types of doublets:

Doublet-removal software, which relies on gene expression to detect doublets, cannot identify homotypic doublets, as their expression profiles are indistinguishable from those of other cells. Alternative approaches are required to detect homotypic doublets, such as cell hashing [Stoeckius et al., 2018] and SNPs in multiplexed samples [Kang et al., 2018].

The Doublets filter simulates heterotypic doublets by averaging the expression of two random barcodes that are sufficiently different from each other. These artifical doublets are then used for predicting which of the input barcodes are doublets.

Image qc-doublets
Figure 5.4: The default settings in the Doublets filter dialog.

The following options can be adjusted in the Doublets filter dialog (figure 5.4):

Note: Expected doublets (%) is relative to the number of captured cells and not to the number of high quality cells. This is estimated as the number of barcodes passing the Empty droplets filter. The Doublets filter receives as input only the high quality cells that also pass the Count-based filters and Extra-chromosomal filters.

For more details on how doublets are detected, see The doublet calling algorithm.