Empty droplets filter

In droplet-based data, barcodes can correspond to droplets containing one cell, more cells or no cells at all. In the first dialog of QC for Single Cell, the Empty droplets filter can be enabled and customized to remove the droplets that are detected to not contain any cells. This filter should be skipped for single-cell protocols that are not droplet-based.

Note that each droplet is assigned one barcode and these terms can be used interchangeably for droplet-based protocols.

Non-zero counts in empty droplets are obtained from ambient (i.e., extracellular) RNA, that can be captured and sequenced during the protocol. Sequenced empty droplets contain significantly fewer reads, and this can be seen as a sharp transition in the rank plot, shown in figure 7.9.

Droplets can be classified in three categories (see figure 7.9):

Droplets with a low number of reads are removed as ambient droplets. The threshold for this is usually obtained automatically from the histogram of number of reads, see The cell calling algorithm for details.

Droplets with a high number of reads are automatically retained as cell-containing droplets. The threshold for this is usually obtained from the automatically inferred knee from the rank plot, see figure 7.9 and The cell calling algorithm for details.

To detect cells with low RNA content, first an ambient RNA profile is estimated from the ambient droplets. The remaining droplets with an intermediate number of reads can be tested against this profile and are assigned simulation-based FDR-corrected p-values, from which non-empty droplets are identified.

Image qc-empty-droplets
Figure 7.5: The default settings in the Empty droplets filter dialog.

The following options can be adjusted in the Empty droplets filter dialog (figure 7.5):

The generated rank plot and summary (see figures 7.9 and 7.10) can be used to identify when the automatic thresholds are not suitable and manual thresholds are required.

After applying the Empty droplets filter, only droplets that are identified as non-empty are retained for the remaining filters (see Count-based and extra-chromosomal filters). Note that this filter does not concern the quality of the retained cells. The Empty droplets filter already removes cells with low number of reads, or, by association, low number of expressed features, and enabling the Count-based filters is not strictly necessary. The Extra-chromosomal filters provide the most additional benefit in this situation.

For removing droplets containing more than one cell, the Doublets filter can be used, see Doublets filter.