Developments in Multimodal Single-Cell Analysis
Developments in multimodal single-cell analysis
Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) is a form of multimodal single-cell analysis that uses deoxyribonucleic acid (DNA)-tagged antibodies to measure protein levels while analyzing the transcriptome. 1,2
CITE-seq has provided the data needed to answer important cellular biology questions. However, an issue with CITE-seq is background signaling, which can constitute a sizeable fraction of the sequenced reads complicating analysis and masking biological variations. 1,3
It has recently been postulated that staining with the recommended antibody concentrations causes high background. Therefore, it is thought that concentrations of most antibodies in an optimized CITE-seq panel aren’t supposed to reach a saturation plateau and should instead be within their linear concentration range. 3
This would involve titrating antibodies to an optimal concentration, which is a lengthy process, and it is yet to be seen whether this would resolve the low signal-to-noise issue entirely. In the experiment detailed below, the BD Rhapsody™ Single-Cell Analysis System demonstrated less noise than the 10x Genomics Chromium among other benefits.4
Comparing two CITE-seq platforms
This experiment sought to analyze two common workflows for CITE-seq—the BD Rhapsody™ Whole Transcriptome Analysis (WTA) Amplification Kit and the BD® AbSeq Assay and the 10x Genomics Chromium with BioLegend TotalSeq™ Reagents. The antibodies selected for this experiment had overlapping clones for both systems.
The peripheral blood mononuclear cells were first prepared and stained with their respective reagents prior to cell capture. The two protocols were followed to create whole transcriptome and surface protein libraries following cell capture and these data were subsequently analyzed on their respective platforms.5
Results of this CITE-seq experiment4
The BD Rhapsody™ System:
- Demonstrated higher specificity with more negative cells near-zero molecule detection outside the target population
- Exclusively was able to resolve CD28 expressing population of cells
- Had a higher resolution for all clones tested
- Demonstrated fewer molecules from the negative population detected, contributing to less noise and allowing positive populations to be resolved
Overall, the BD Rhapsody™ System demonstrated fewer reads from non-target cells and high-resolution CITE-seq surface feature data compared to the 10x Genomics Chromium with BioLegend TotalSeq™ Reagents.
Read the full technical note entitled “Comparative analysis of CITE-seq on the BD Rhapsody™ and 10X Genomics Chromium Single-Cell Analysis Systems.
