BD Rhapsody Targeted Immune Multiplex Sequencing Service
CD Genomics offers a BD Rhapsody Targeted Immune Multiplex Sequencing service that combines targeted gene expression panels, surface protein detection via AbSeq, and sample multiplexing in a single streamlined workflow. Designed for researchers with well-defined immune profiling questions, this service delivers focused, high-sensitivity readouts of immune cell populations, activation states, and functional signatures — at lower cost per sample than whole transcriptome approaches.
- Pre-designed immune panels (Human/Mouse Immune Response, T-cell, Onco-BC) or fully custom panel design
- Simultaneous transcriptome + surface proteome profiling with AbSeq (up to 30 markers)
- Sample multiplexing for up to 12 samples per cartridge, reducing batch effects and per-sample cost
- End-to-end service from sample QC to publication-ready immune profiling reports
Services are provided for research use only.
Targeted Immune Multiplex — Technology Overview
BD Rhapsody targeted immune multiplex sequencing combines three layers of single-cell information in one experiment: targeted mRNA profiling of immune-relevant genes, surface protein quantification via AbSeq oligonucleotide-conjugated antibodies, and sample-level barcoding for multiplexed runs.
Targeted gene expression panels. Unlike whole transcriptome analysis (WTA), targeted panels focus sequencing reads on a curated set of immune-relevant genes — from 259 to 399 genes depending on the panel — covering cytokines, transcription factors, checkpoint molecules, activation markers, and lineage-defining transcripts. This focused approach achieves higher molecular tag counts per transcript, improving sensitivity for low-abundance immune genes while reducing sequencing cost. Pre-designed panels are available for human and mouse immune response profiling, T-cell biology, and oncology applications (Onco-BC). Custom panels can be designed to match specific gene sets of interest.
AbSeq surface proteome integration. BD AbSeq uses DNA-barcoded antibodies to convert surface protein expression into a sequencing-readable signal, measured alongside mRNA from the same single cell. The pre-formulated AbSeq Immune Discovery Panel (IDP) covers 30 surface markers spanning major immune lineages (CD3, CD4, CD8, CD19, CD14, CD56), activation states (CD25, CD69, HLA-DR), checkpoint molecules (PD-1), and differentiation markers (CD45RA, CD45RO, CCR7). Additional custom AbSeq markers can be added for project-specific targets.
Sample multiplexing. BD Sample Tags allow up to 12 individually barcoded samples to be pooled and processed within a single BD Rhapsody cartridge. This reduces per-sample library preparation costs, minimizes batch effects, and is particularly advantageous for cohort studies comparing immune profiles across multiple conditions, time points, or patient groups.
Available Panels & Panel Design
CD Genomics offers both pre-designed immune panels and fully custom panel design services for the BD Rhapsody platform.
Pre-designed targeted panels
Immune Response Panel (Human) — 399 genes: cytokines, chemokines, transcription factors, checkpoint molecules, immune lineage markers
Immune Response Panel (Mouse) — 397 genes: murine T/B/NK/myeloid markers, cytokines, signaling
T-Cell Panel (Human) — 259 genes: TCR signaling, exhaustion, activation, differentiation, effector function
Onco-BC Panel (Human) — 389 genes: tumor-associated genes, B-cell markers, proliferation
AbSeq Immune Discovery Panel (IDP)
30 surface markers in a single-tube format: CD3, CD4, CD8, CD19, CD14, CD16, CD56, CD11c, CD123, CD45RA, CD45RO, CCR7, CD25, CD69, CD27, CD38, CD24, CD20, CD40, IgD, IgG, IgM, HLA-DR, PD-1, CD127, CD28, CD62L, CXCR5, CD95, and Cetuximab (isotype control). Custom markers available upon request.
Custom panel design. For projects requiring gene sets beyond pre-designed panels, our team works with you to curate a targeted gene list aligned with your specific biological questions, then validates the panel for specificity and sensitivity on the BD Rhapsody platform.
Targeted Immune Multiplex Workflow
CD Genomics manages the full targeted immune multiplex workflow from sample receipt to data delivery.
- Study design and panel selection
We align on your research goals, select appropriate targeted panels and AbSeq markers, and determine sample multiplexing strategy. For custom panels, gene selection and validation are completed during this phase.
- Sample preparation and QC
Upon receipt, cell viability, concentration, and purity are assessed. Tissue samples undergo optimized enzymatic dissociation. A viability threshold of ≥70% (≥80% for cryopreserved cells) is typically required.
- Single-cell capture, barcoding, and AbSeq staining
Cells are stained with AbSeq antibodies, labeled with sample tags, and loaded onto the BD Rhapsody cartridge. After gravity sedimentation into microwells, barcoded beads are co-loaded. The BD Rhapsody Scanner captures imaging-based QC data before proceeding.
- Targeted library preparation and sequencing
Barcoded cDNA (mRNA targets + AbSeq antibody-oligos + sample tags) is amplified, purified, and converted into Illumina-compatible libraries. Sequencing is performed on Illumina platforms at project-specific depth.
- Bioinformatics analysis and immune profiling report
Data is processed through the BD Rhapsody Analysis Pipeline followed by our customized immune profiling workflow. The final report includes cell-type annotation with surface marker validation, differential expression, and group comparisons.
Immune Multiplex Sample Requirements
Proper sample preparation is critical for high-quality single-cell immune profiling data. Please follow the guidelines below.
| Sample type | Requirement |
|---|---|
| Cells (cryopreserved) | ≥ 5 × 105 viable cells; ≥80% viability after thawing; cryopreservation medium, controlled-rate freezing; −80°C storage, dry ice shipping |
| Fresh tissue | 200–400 mg; cut into ~0.5 × 0.5 × 0.5 cm pieces (2–3 pieces); immediately immerse in 4°C pre-chilled tissue preservation solution (volume ≥ 5× sample volume); 4°C cold chain shipping (do NOT freeze); deliver within 48 hours |
| Blood / PBMCs | 3–5 mL peripheral blood in EDTA anticoagulant tube; keep at 4°C, process within 4–6 hours; alternatively, isolate PBMCs prior to cryopreservation and ship on dry ice; avoid hemolysis and clotting |
Targeted Immune Profiling Bioinformatics
Our bioinformatics team delivers a tiered analysis package that integrates mRNA, AbSeq protein, and sample tag data from your targeted immune multiplex experiment. All outputs include publication-quality figures and fully documented analysis code.
Standard analysis
- Raw data QC: read quality, mapping statistics, molecular tag saturation, per-cell metrics
- Demultiplexing: separation of mRNA, AbSeq, and sample tag libraries
- Normalization and integration: library-size normalization, batch correction (Seurat/Harmony, Scanpy), WNN integration of mRNA + AbSeq modalities
- Dimensionality reduction and clustering: PCA, UMAP (separate and integrated views), graph-based clustering (Louvain/Leiden)
- Cell type annotation: validated against AbSeq surface marker expression for higher-confidence calls
- Differential expression: per-cluster and per-condition comparisons for both mRNA and protein
- Immune cell composition: cell-type proportions per sample with statistical group comparisons
Advanced analysis
- Pseudotime trajectory: Monocle3, Slingshot, or scVelo for differentiation trajectories
- Cell–cell communication: ligand–receptor inference using CellChat, CellPhoneDB, or NicheNet
- Gene set enrichment: GSEA, GSVA, pathway over-representation (GO, KEGG, Reactome)
- Immune repertoire analysis: when combined with BD Rhapsody VDJ (separate assay)
- Custom cohort comparisons: tailored analyses for multi-condition, multi-timepoint studies
See also: Single-Cell Immune Repertoire Sequencing for TCR/BCR profiling and Single-cell Transcriptome + Surface Proteome for broader multiomic strategies.
Data Deliverables
Every targeted immune multiplex project includes a comprehensive set of deliverables ready for direct use in analysis and reporting.
- Raw sequencing data
- FASTQ files, demultiplexed per sample and per library (mRNA, AbSeq, sample tags)
- Processed data
- Gene–cell and protein–cell expression matrices in standard formats (.h5, .h5ad, .rds)
- QC report
- Per-sample QC metrics, library validation traces, imaging QC snapshots
- Bioinformatics report
- Publication-ready figures (clustering, annotation with surface marker overlay, differential expression, group comparisons), methods documentation, reproducible analysis code
- Custom analysis outputs
- As defined in the project-specific analysis plan
Targeted Immune Profiling Applications
Targeted immune multiplex sequencing is ideal for studies where the biological question focuses on specific immune cell populations and pathways, rather than unbiased transcriptome-wide discovery.
Immune cell phenotyping
Define and quantify T cell (CD4+, CD8+), B cell, NK cell, monocyte, and dendritic cell populations with simultaneous transcriptomic and surface proteomic validation. AbSeq surface markers provide orthogonal confirmation of cell identities, increasing annotation confidence over transcriptome-only approaches.
Immuno-oncology and checkpoint studies
Profile checkpoint molecule expression (PD-1, PD-L1, CTLA-4, LAG-3), T cell exhaustion and activation states, and tumor-associated immune signatures. Targeted panels focus on the genes most relevant to immunotherapy response and resistance. See also: Tumor Microenvironment Solutions.
Drug response and pharmacodynamic studies
Compare immune cell composition and activation states across treatment arms, dose levels, or time points. Sample multiplexing enables efficient processing of multi-condition cohort studies, while AbSeq provides cell-surface-level validation of drug-induced phenotypic changes.
Infectious disease and sepsis immunology
Characterize the immune response to infection at single-cell resolution, identifying dysfunctional or hyperactivated cell populations. The targeted approach is well suited to studies where specific immune pathways are hypothesized to be perturbed.
Autoimmune and inflammatory disease
Map immune cell dysregulation, cytokine signatures, and autoreactive populations in diseases such as rheumatoid arthritis, lupus, and inflammatory bowel disease at single-cell resolution.
Case Study: Targeted Multi-Omic Immune Profiling of Sepsis PBMCs
Source: Multi-omic analysis of PBMCs in sepsis reveals widespread cytotoxic dysfunction and an increased population of CD69 expressing naïve CD4+ T cells (Flynn J, Baird AM, et al., Frontiers in Immunology, 2025)
Background
Sepsis is responsible for 1 in 5 deaths globally and is characterized by a dysregulated inflammatory response to infection. Despite its clinical significance, the specific immune cell populations and functional states driving sepsis-associated immune dysfunction remain incompletely characterized at the single-cell level.
Methods
The study used the BD Rhapsody platform with a targeted approach combining the BD Rhapsody Immune Response Panel plus a custom supplementary panel (424 mRNA targets total) with a 46-plex AbSeq protein panel for simultaneous transcriptomic and surface proteomic profiling. Peripheral blood mononuclear cells from an Irish cohort of sepsis patients and healthy controls were labeled with sample tags for multiplexed processing. Three libraries — mRNA, AbSeq, and Sample Tag — were generated per sample.
Results
The multi-omic analysis revealed widespread cytotoxic dysfunction in sepsis patients, with altered expression of granzyme and perforin pathways across multiple immune cell types. A distinct population of CD69-expressing naïve CD4+ T cells was identified as significantly expanded in sepsis, suggesting early activation of T cells that have not yet undergone antigen-driven differentiation. The integrated mRNA + protein data provided orthogonal validation of cell identities and functional states unachievable with either modality alone.
Conclusion
The application of BD Rhapsody targeted multi-omics — combining focused immune gene panels with 46-plex surface proteome detection and sample multiplexing — provided high-resolution characterization of sepsis-associated immune dysfunction. The study demonstrates how targeted immune multiplex sequencing enables efficient, cost-effective profiling of specific immune populations and pathways in human disease cohorts.
Targeted Immune Multiplex vs scWTA — Which Approach?
BD Rhapsody offers two complementary single-cell profiling strategies. The choice depends on whether your research question is hypothesis-driven or exploratory. For context, see our BD Rhapsody scWTA Service.
| Dimension | Targeted Immune Multiplex | scWTA (Whole Transcriptome) |
|---|---|---|
| Gene coverage | 259–399 curated immune targets (panel-dependent) | >10,000 genes, unbiased |
| Best for | Hypothesis-driven immune questions: cell phenotyping, cytokine/checkpoint profiling, drug response, cohort comparisons | Gene discovery, novel cell type identification, unbiased transcriptome exploration |
| Sensitivity per gene | Higher — deeper read depth within targeted gene set | Broader but shallower per-gene coverage |
| Sequencing cost per cell | Lower | Higher |
| AbSeq integration | Standard in targeted workflow | Compatible |
| Sample multiplexing | Up to 12 samples per cartridge | Up to 12 samples per cartridge |
How to decide
Choose Targeted Immune Multiplex when your research focuses on specific immune pathways and cell populations that are well defined in the literature, and when cohort size or budget favors cost-efficient per-sample profiling. Choose scWTA when you need unbiased transcriptome-wide discovery or are studying cell types whose transcriptional programs are not yet fully characterized. CD Genomics offers both workflows and can advise on the best approach for your project.
Frequently asked questions (FAQ)
References
- Flynn J, Baird AM, Breen E, Carty M, McNevin CS, McDermott L, Kenny EM, Coakley JD, Ryan T, Doherty DG, Sheils O. "Multi-omic analysis of PBMCs in sepsis reveals widespread cytotoxic dysfunction and an increased population of CD69 expressing naïve CD4+ T cells." Frontiers in Immunology, vol. 16, 2025, 1667186.
- Gao C, Zhang M, Chen L. "Analytical Workflows for Single-Cell Multiomic Data Using the BD Rhapsody Platform." Current Protocols, vol. 4, no. 2, 2024, e963.
