Normalizing and denoising protein expression data from droplet-based single cell profiling

Abstract

Recent methods enable simultaneous measurement of protein expression with the transcriptome in single cells by combining protein labeling with DNA barcoded antibodies followed by droplet based single cell capture and sequencing (e.g. CITE-seq). While data normalization and denoising have received considerable attention for single cell RNA-seq data, such methods for protein data have been less explored. Here we showed that a major source of noise in CITE-seq data originated from unbound antibody encapsulated in droplets. We also found that the counts of isotype controls and those of the "negative" population inferred from all protein counts of each cell are significantly correlated, suggesting that their covariation likely reflects cell-to-cell differences due to technical factors such as non-specific antibody binding and droplet-to-droplet differences in capture efficiency of the DNA tags. Motivated by these observations, we developed a normalization method for CITE-seq protein expression data called Denoised and Scaled by Background (DSB). DSB corrects for 1) protein-specific background noise as reflected by empty droplets, 2) the technical cell-to-cell variation as captured by the latent noise component described above. DSB normalization improves separation between positive and negative populations for each protein, centers the negative-staining population around zero, and can improve unbiased protein expression-based clustering. DSB is available through the open source R package "DSB" via a single function call and can be readily integrated with existing single cell analysis workflows, including those in Bioconductor and Seurat.

Improving oligo-conjugated antibody signal in multimodal single-cell analysis

“…Free-floating antibodies in the solution have been shown to be one of the major contributors to background signal for ADTs ( Mulè et al, 2020 ). Similar to cell-free RNA, background ADT signal can be assayed from empty droplets.…”

“…Empty droplets have been shown to be useful for determining the background signal of CITE-seq ( Mulè et al, 2020 ). This suggests that the major source of background signal for ADT libraries can be attributed to free-floating antibodies (or oligos) in the solution rather than nonspecific antibody binding to cell surfaces.…”

“…Such concentrations are much more sensitive to the number of available epitopes (i.e., cell number and cell composition) than an optimized flow cytometry panel. Unlike flow and mass cytometry, where the major source of background is autofluorescence, spillover between neighboring channels, and nonspecific binding of the antibodies ( Hulspas et al, 2009 ; Au-Yeung et al, 2019 ), a major source of background signal for oligo-conjugated antibodies appears to be free-floating antibodies in the cell suspension ( Mulè et al, 2020 ). In droplet-based single-cell sequencing methods, these free-floating antibodies will be distributed between cell-containing and empty droplets.…”

Improving oligo-conjugated antibody signal in multimodal single-cell analysis

“…Reducing staining volume for 10 6 PBMCs from 50 µl to 25 µl only showed minor effect on signal and this minimal impact was primarily observed for antibodies used at very low concentrations (0.0125 to 0.025 µg/mL) targeting highly expressed epitopes (such as CD31, CD44 and CD45). This effect was readily counteracted by concomitantly reducing the number of cells at staining to 0.2x10 6 PBMCs in 25 µl. In flow cytometry, while the binding of antibody is strictly dependent on the concentration, background signal is dependent on the ratio between the total amounts of antibody and epitopes [3].…”

“…cell number and cell composition) than an optimized flow cytometry panel. Unlike flow and mass cytometry where the major source of background is autofluorescence, spillover between neighboring channels and nonspecific binding of the antibodies [5, 10], a major source of background signal for oligo-conjugated antibodies appears to be free-floating antibodies in the cell suspension [11]. In droplet-based single-cell sequencing methods, these free-floating antibodies will be distributed between cell-containing and empty droplets.…”