In the pursuit of understanding human cellular processes, and ultimately to decipher complex disease mechanisms and develop more effective disease treatments, the need for multi-omic techniques is at the forefront. Single-cell analysis has become key to the study of cellular heterogeneity and function, with single-cell RNA-seq (scRNA-seq) revolutionizing single-cell transcriptome analysis. However, true multi-omics analysis in single cells, specifically meaning the simultaneous analysis of whole genome, transcriptome, proteomic and epigenomic information from the same individual cell, has not yet been possible.  Previous attempts have relied on techniques that offered limited multiplexing and processed a limited number of cells. Other techniques have used sequential and separate analysis of transcriptomic and proteomic content in single cells that are then correlated later during analyses.

New products now coming to the market enable high-throughput, high-multiplex, and simultaneous measurement of transcriptome wide mRNA and cell-surface proteins in single cells, offering new insight into the relationship between the genotype and phenotype. These new products also mark the next stage in commercialization of oligo-conjugated antibody products, which are now becoming available as standardized, ready-to-use solutions tailored for single-cell specific applications. Two companies at the forefront of bringing integrated solutions for simultaneous measurement of mRNA and cell-surface protein in single cells to the market are BD (Becton, Dickinson and Company) and 10x Genomics, which is working in partnership with antibody supplier BioLegend.

A new set of solutions aims for a closer integration of pre-conjugated oligo antibodies with instrumentation in order to simplify and standardize the use of new methods for simultaneous proteomic and transcriptomic analyses in single cells.

Oligo-conjugated antibodies associate an oligonucleotide barcode to the epitope the antibody recognizes, thus translating the specificity of that antibody for its target protein into a signal that can be amplified by PCR, and detected via NGS. Oligo-conjugated antibodies have been available for several years for cell analysis applications, including the simultaneous analysis of proteins and mRNA in single cells; however, these tools were offered as unrelated standalone products in the form of kits or reagents for do-it-yourself approaches. A new set of solutions aims for a closer integration of pre-conjugated oligo antibodies with instrumentation in order to simplify and standardize the use of new methods for simultaneous proteomic and transcriptomic analyses in single cells.

Describing BioLegend’s oligo-conjugate offerings, Miguel A. Tam, Senior Product Realization and Marketing manager at BioLegend, told IBO, “We currently offer more than 170 conjugates and keep on expanding our portfolio. Our goal is to facilitate biomedical discovery by making available as many conjugates as possible, based on the needs of the scientific community.”

Utilizing new experimental methods, antibody conjugates have become key to addressing single-cell proteomic analysis.  “Analytical tools such as flow cytometry or Western blot can be used to study and characterize proteins, whereas NGS can efficiently be employed to quantify mRNA at a high-throughput scale, and even at single-cell levels,” explained Dr. Tam. “Protein analysis technologies have been lagging behind in their capacity to visualize multiple targets, as compared to single-cell RNA sequencing, for example, that can retrieve thousands of RNA readings in a single experiment.”

New methods are enabling even higher-throughput, highly multiplex measurement of both mRNA and surface-cell proteins in tens of thousands cells.

But new methods are enabling even higher-throughput, highly multiplex measurements of both mRNA and surface-cell proteins in tens of thousands cells. These techniques include CITE-seq [cellular indexing of transcriptomes and epitopes by sequencing] and REAP-seq [RNA expression and protein sequencing assay], which are essentially the same, according to Dr. Tam. “Last year, in October, two papers were published describing a tremendously innovative approach to apply the high-throughput capacity of NGS to protein analysis,” he explained. “The authors linked oligonucleotides that mimic mRNA to antibodies, and used single-cell platforms and NGS to convert the linked oligo into a readable sequencing signal.”

The methods mark a leap forward in the speed and number of cells that can be analyzed at the same time for mRNA and surface cell proteins as part of a scRNA-seq protocol. “As the oligo-antibody conjugates can be integrated into the single-cell workflow, both transcriptomics and protein data can be obtained in the same experiment, with the high-throughput capacity inherent to this process,” stated Dr. Tam. The methods are also more accessible, as CITE-seq is compatible with existing RNAseq protocols.

Earlier this year, BioLegend announced an exclusive agreement with the New York Genome Center (NYGC) to commercialize static barcodes for its oligo-conjugated antibodies, creating a standardized approach for CITE-seq. “We have been since working in close collaboration with this group, and BioLegend is currently offering antibodies directly conjugated to oligonucleotides compatible with single-cell platforms based on poly-T capture systems. Conjugates can be found under our TotalSeq brand,” noted Dr. Tam. New developments in the design of oligo-conjugated antibodies address the need for faster, lower-cost analysis. “We are also offering what is known as Hashtag conjugates, which allow researchers to process multiple samples in the same experiment, mixed together, to optimize their protocol and minimize the costs.”

In addition to BioLegend’s antibodies, the NYGC is using the 10x Genomics Solution as part of its commercial offerings. In July, 10x Genomics announced an agreement with BioLegend to become a member of the 10x Compatible Partners program. BioLegend’s forthcoming ready-to-use TotalSeq B and TotalSeq C oligo-conjugated antibody products will be compatible with 10x Genomics’ droplet-based instrumentation, consumables for scRNA-seq, and analysis and visualization tools for ready to use solutions. Specifically, the assays will work with 10x’s Chromium Single Cell Gene Expression Solution and Chromium Single Cell Immune Profiling Solutions in combination with its new Feature Barcoding technology, which will be released later this year.

“The Feature Barcoding technology from 10x Genomics enables simultaneous analysis of a specific biological features of interest together with either unbiased gene expression or immune repertoires within that same cell.”

Describing the Feature Barcoding technology, Giovanna Prout, director of Strategic Marketing, 10x Genomics, told IBO, “The Feature Barcoding technology from 10x Genomics enables simultaneous analysis of a specific biological features of interest together with either unbiased gene expression or immune repertoires within that same cell.” Such biological features include surface cell proteins. “Feature Barcoding technology allows the researcher to increase the complexity of their analysis, by simultaneously measuring gene expression profiles and cell surface protein expression in the same cell at high cell throughput scale,” she added. “By enabling the detection of protein isoforms or post-translational modifications, researchers now can use additional data points to identify cells, adding more dimensions to their cell profiling analyses.”

In addition to unbiased mRNA and surface cell protein measurements, Feature Barcoding technology enables simultaneous highly multiplexed single-cell characterization of other cellular contents and functions. “The new technology can support a wide variety of research applications depending upon which biological feature is being targeted, and can include cell surface proteins, MHC [major histocompatibility complex] receptors, CRISPR-mediated perturbations or other targets of interest to the researcher.”

Featuring Barcoding technology is compatible with multiple single-cell analysis solutions offered by 10x Genomics, complimenting and extending the scientific applications of their current offering for single cell analysis. “The Feature Barcoding technology used with the Chromium Single Cell Gene Expression Solution moves beyond methods published in peer-reviewed publications, like CITE-seq, REAP-seq, CROP-seq [CRISPR droplet sequencing] and Perturb-seq [which combines single-cell RNA-seq and CRISPR-based perturbations],” explained Ms. Prout. To make the methods more accessible, 10x has partnered with established reagent companies. “10x Genomics has partnered with key providers to complement these applications for the Single Cell Gene Expression and Immune Profiling Solutions, including products for oligo-conjugated antibodies (BioLegend), guide RNA with complementary feature barcode sequences embedded, and oligo-conjugated Dextramers (Immudex) that are compatible with Feature Barcoding technology.”

For measuring gene expression and protein characterization, Feature Barcoding technology and the Chromium Single Cell Gene Expression Solution can be used with BioLegend’s TotalSeq B oligo-conjugated antibody or researcher’s own custom conjugated antibodies.

For measuring gene expression and protein characterization, Feature Barcoding technology and the Chromium Single Cell Gene Expression Solution can be used with BioLegend’s TotalSeq B oligo-conjugated antibody or researcher’s own custom conjugated antibodies. 10x’s Chromium Single Cell Immune Profiling Solution and Feature Barcoding will be compatible with BioLegend’s TotalSeq C antibody panels.

For characterizing MHC complexes, the Chromium Single Cell Immune Profiling Solution and Feature Barcoding technology can be used with Immudex’s dCODE oligo-tagged dextramers, which enables the detection of antigen-specific T cells using MHC molecules and fluorochromes. Describing this application, Ms. Prout told IBO, “A researcher has the capability to simultaneously profile immune cells at much higher resolution by measuring unbiased gene expression, the complete T- and B-cell repertoire, and the clonotype-specific mapping of MHC complexes from each single cell.” These capabilities differentiate the offering from similar products. “When we refer to the complete T- and B-cell repertoire, it is important to know that this includes the paired and full-length nucleotide sequences of expressed T- and B-cell receptors. This is very unique, and all other commercial methods include only some aspect of this capability,” she stated.

Analyses of T- and B-cells at the molecular and cellular level are one of the single-cell applications addressing current trends in immuno-oncology research. Other applications in immuno-oncology research for 10x’s Single Cell Feature Barcode technology described by Ms. Prout were to: “map the immune response in the tissue microenvironment; determine the identity and heterogeneity of cell types in the tumor microenvironment; screen antigens with high specificity and sensitivity in the context of the immune response; characterize the molecular genetic basis for cancers; and identify potential candidates for targeted and/or immune-therapies and tumor sub-clones and clonal evolution that may evade immuno-therapies.”

“While single-cell RNA-seq and flow cytometry have each made significant strides towards understanding the heterogeneity within complex biological samples, the convergence of these two methods under a single solution can provide even greater insight in the rapidly expanding field of genomic cytometry.”

BD is addressing the simultaneous high-throughput measurement of mRNA and surface cell proteins in single cells using a standardized platform for the analysis of tens of thousands of cells. This fall, the company launched the BD AbSeq assay for use with its BD Rhapsody microfluidic system, which is utilized for targeted single-cell gene expression analysis for RNA-seq. The assay complements traditional techniques for mRNA- and protein-based single cell analysis offerings from BD, according to Brian Lilhanand, Global Platform Lead, Single Cell Multi-Omics at BD. “While single-cell RNA-seq and flow cytometry have each made significant strides towards understanding the heterogeneity within complex biological samples, the convergence of these two methods under a single solution can provide even greater insight in the rapidly expanding field of genomic cytometry,” he said. In this way, the BD AbSeq assay combines many of the same principles as other BD cellular measurement techniques. “In this workflow, cells are incubated with BD AbSeq antibody-oligonucleotide conjugates in the same way that cells are labeled with antibodies before typical cytometry workflows. When run through the BD Rhapsody single-cell analysis system, multi-omic-driven projections revealed cleaner cell separation and more defined cell clustering,” he continued.

Oligo-conjugated antibody technology from BD incorporates antibody-specific sequences. “BD AbSeq assay utilizes monoclonal antibodies conjugated to unique DNA barcodes called Ab-Oligos. This allows for the profiling of many cell surface proteins using high-throughput sequencing where detection of antibody-specific DNA sequences is used to infer the amount of each target protein,” explained Mr. Lilhanand.

“This ability to examine cell surface proteins directly, rather than deducing protein levels based on mRNA information, is advantageous in situations where mRNA is difficult to detect due to low expression or mRNA level does not correlate with protein expression.”

Access to proteomic information in the same cell supplements the insights gained through the use of targeted scRNA-seq, such as for more complete mRNA detection. “This technology, when used with the BD Rhapsody platform, can identify novel biomolecules that cannot be defined by RNA-seq alone. This ability to examine cell surface proteins directly, rather than deducing protein levels based on mRNA information, is advantageous in situations where mRNA is difficult to detect due to low expression or mRNA level does not correlate with protein expression,” said Mr. Lilhanand. “The ability to concurrently examine protein and mRNA expression is important when studying responses to cell stimulation and gene regulatory mechanisms.”

Compared to methods such as CITE-seq and REAP-seq, the BD Abseq assay provides important differentiators, specifically its integration with the BD Rhapsody system, according to Mr. Lilhanand. “Although the BD AbSeq assay is designed to be platform agnostic, combining this assay with the automated, microwell-based BD Rhapsody single-cell analysis system can provide an improved workflow and QC.” The system also offers distinct features for workflow monitoring, “In addition, the microwell configuration within the BD Rhapsody technology includes an optically clear cartridge-based technology that allows for visualization during the workflow to ensure maximal experimental control,” he noted. “This enhances data quality by providing valuable information about cell counts and viability by simple automatic imaging during the multiple QC steps of the process. “

Applications in immuno-oncology for scRNA-seq include the investigation of immune cell subpopulations. As an example, Mr. Lilhanand described the study of the FCD4+ T-helper cell population. “[It] was once assumed to consist of only a few cell subtypes, but with single-cell methods novel intermediate subtypes and subpopulations have been identified.”  Such discoveries have opened new avenues for immuno-oncology research. “By being able to look at individual cells in parallel rather than the response of an entire heterogeneous cell population, researchers have been able to untangle the incredible complexity and uniqueness at the cellular level of the immune system. This has led to new findings about the kinetics of gene expression, differential splicing pattern, the immune response in single T-cells and much more.”

With proteomic data, single-cell analyses can further explore tumor heterogeneity and its role in immune-oncology. “In addition to providing a deeper understanding of the immune system, single-cell analysis is also applicable for protein profiling in heterogeneous biological systems, such as tumors. Through the data that the BD AbSeq assay provides at both the RNA and protein level, immuno-oncologists can now make progress in cancer research by understanding more about tumor heterogeneity, cancer progression, associated immune responses and responses to immuno-therapeutics.”