BD Rhapsody Single-Cell ™

Analysis System
Analyze hundreds of genes across tens of thousands
of single cells in parallel
Analyze hundreds of genes across tens of
thousands of single cells in parallel
Single-cell RNAseq is changing how we understand cells. The The BD Rhapsody Single-Cell Analysis System enables
BD Rhapsody™ Single-Cell Analysis System meets your digital quantitation of hundreds of expressed genes across
experimental need to understand cellular form and function tens of thousands of single cells, provides customized assays
on an individual basis with BD’s 40 years of expertise in that are flexible enough to meet any experimental need,
cell biology. and comprises an efficient system that reduces
This new analysis system overcomes the limitations of experimentation time and sequencing costs.
traditional assays, such as microarrays and bulk RNAseq, System components include:
which rely upon averaging measurements across multiple
cells by enabling observation of highly subtle differences • BD Rhapsody scanner
between individual cells. Users can identify and characterize • BD Rhapsody sample loading station
novel and rare cell types, helping further the understanding • BD Rhapsody cartridge
of biological processes in fields ranging from immunology to
• Reagents for Molecular Indexing and library preparation
oncology and beyond.
• Application-specific targeted panels
How does the BD Rhapsody Single-Cell Analysis System work?
Figure 1 shows how the system enables digital, quantitation of gene expression levels of hundreds of genes. It uses a novel
planar array of microwells for cell capture and a bead-based 3’ RNAseq assay.

Microwell
mRNA
Barcoded bead Barcoded bead
Cell Lysed cell

Pair ONE cell with Lyse cell to hybridize Retrieve beads Synthesize cDNA Sequence and construct
ONE barcoded bead mRNA onto barcoded single cell gene
in microwell capture oligos on bead expression profiles

Figure 1. BD Rhapsody system workflow.

Beginnings—cell capture and

isolation steps Beads with hybridized mRNA retrieved from cartridge
Univ = Universal
The BD Rhapsody cartridge enables 3'
poly A mRNA
5' sequence

single-cell capture and molecular bead Univ CL UMI dT

UMI = Unique
molecular
index
indexing of mRNA transcripts on 5' 3'
CL = Cell label
magnetic oligonucleotide barcoded Reverse transcription dT = oligo(dT)
beads. These beads are then pooled
cDNA archived on bead and tagged with cell label and molecular index
into a single tube for cDNA
amplification and library construction. 3'
poly A mRNA
5'

bead Univ CL UMI dT cDNA

5' 3'

Amplification of cDNA using targeted primer panels

Universal oligo

bead Univ CL UMI dT cDNA

BD Rhapsody PCR 1 primer*

Multiplex PCR 1
Universal oligo

Univ CL UMI dT cDNA

BD Rhapsody PCR 2 primer*

Multiplex PCR 2

Library forward primer

Univ CL UMI dT cDNA

Library reverse primer

Final amplification

Read 1

Sequencing adapter Univ CL UMI dT cDNA Sequencing adapter & library index
Read 2

Figure 2. mRNA capture and library preparation steps.

Bypassing PCR bias – BD Molecular Indexing
PCR amplification bias can result in inaccurate quantitation of genes when sequence reads are counted. BD patented Molecular
Indexing technology assigns unique molecular indices (UMI) for counting individual genes in individual cells, also known as
“molecular barcoding.” This allows the experimenter to overcome amplification bias for more accurate enumeration of
transcript levels.

Customized workflows tailored for your experiments

Single-cell experimental workflows often require varying degrees of sample manipulation, quality, and throughput between
users and experiments. BD instrument options can be configured for specific needs. The BD FACS sorter (like BD FACSMelody™),
is a valuable option for the BD Rhapsody System for upstream sample enrichment. The BD Rhapsody Scanner provides
automated cell counting and viability of cell samples to help users prepare samples at optimal concentrations for single-cell
capture on the cartridge. The scanner provides counts of cells captured with beads on the cartridge. The BD Rhapsody Sample
Loading Station is light, portable, and easily moved within a lab. Bioinformatics pipeline and visualization tools are also
provided. These tools consist of UMI analysis algorithms and visualization tools enabling even inexperienced users to analyze
and understand single-cell data.

Optional –
Cell sorting on
BD flow sorters

Sample Single-cell mRNA capture Sequence on BD Genomics data

and amplification by BD Illumina platforms analysis
Rhapsody

Figure 3. Example BD Rhapsody workflow including cell isolation (FACS optional), cell capture and molecular indexing, sequencing and data analysis.

More efficient sequencing yields higher throughput

at lower costs
Single-cell experiments are very expensive due to sequencing costs associated with high-throughput experimental methods.
The BD Rhapsody Single-Cell Analysis System was designed with multiple features that help reduce experimental costs by
utilizing sequencing reads more efficiently.
• Targeted assays—a specific gene panel approach can help generate similar results as a Whole Transcriptome Analysis (WTA)
assay at a fraction of time and cost, due to improved detection sensitivity. This method also improves UMI counting
efficiency, yielding dramatic experimental time and cost savings.
• Archiving—Storing magnetic beads stably with intact cDNA generated from your sample allows storage of precious sample
for up to 12 weeks. This gives users flexibility to sequence samples when time is available, and run multiple assays on the
same sample by aliquoting or subsampling a pool of stored beads.
• Sub-sampling—cDNA archived on magnetic beads may be split into one or more aliquots and amplified using custom or
pre-designed panels. Sub-sampling allows users to reduce cost by providing the option to sequence a portion of the sample
and the flexibility to test different panels on the sample.
Evaluating BD Rhapsody performance human
mouse
To determine the technical performance of the BD Rhapsody
Single Cell Analysis System, a 1:1 mixture of human 293T and
Mouse UMI

mouse NIH/3T3 cells was loaded onto the BD Rhapsody cartridge

at different concentrations. Archived cDNA molecules were
universally amplified and sequenced at ~ 8,700 reads per cell.
At the loading condition in which 1,109 cells were detected in
sequenced data, an average of 99.4% of molecules from each cell Human UMI

were detected in each cell as only human or only mouse,

1.00
indicating minimal crosstalk between microwells. 0.95
Fraction
Multiplet incidence was close to zero at a cell loading density 0.90 single cells
Average
yielding 1,000 or fewer cells detected by sequencing, and was less
Fraction

0.85
single-cell
0.80 purity
than 5% at 15,000 cells. 0.75

0.70

0.65

Figure 4. Minimal cross talk and low multiplet rate between microwells using BD Rhapsody ~17,000 ~9,000 ~1100 ~100
Cell number
Making single-cell sequencing more efficient
The benefits of a targeted approach to single-cell analysis proteins), thereby driving improved assay efficiency and
are demonstrated by profiling peripheral blood mononuclear sensitivity. In comparison to competitor WTA assay data (C),
cells (PBMCs) using a competitor WTA assay and the BD the greater sensitivity of the BD Rhapsody targeted
Rhapsody Immune Response Panel (Human) at a similar approach enabled detection of the low abundant but
sequencing depth. While t-SNE profiling from both assays important marker CD8A, yielding greater resolution of
showed clustering results of known PMBC cell types, interesting T-cell subpopulations (B).
targeted panels showed higher sensitivity (reads/cell) and To test archiving of pooled beads (see Figure 6), two
UMI counting efficiency than WTA. In a PBMC profiling subsamples of 1,000 cells obtained from a lymph node
experiment (Figure 5), the BD Rhapsody assay achieved containing sparse metastatic breast cancer cells were
similiar clustering performance with nearly 10x fewer reads profiled with the BD Rhapsody Onco-BC Targeted Panel.
than a competitor WTA 3’RNA-seq assay (2k reads per cell Subsamples were profiled at 0, 6 and 12 weeks after bead
vs. 20k reads per cell). Unlike using a WTA approach, storage at 4°C. Strong correlations of gene expression
targeted panels avoid sequencing of many genes with high between subsamples indicated minimal batch effect and
expression or low expression variability (e.g. ribosomal high stability of archived cDNA.

Competitor WTA 3’ RNA-seq

A Major cell type B Proportion of transcriptome C Example marker gene
(~20K reads per cell) from ribosomal proteins (CD8A)

40 40 40
483 cells (10.0%)
0.6 0.7

log10 (number of molecule per cell)

30 30 30 695 mols (0.0%)

20 0.5 20 0.6
20
10 10 0.5
10 0.4
proportion
coord 2

coord 2
coord 2

0 0 0 0.4
0.3
-10 -10 -10 0.3
0.2
-20 -20 -20 0.2

-30 -30 0.1 -30 0.1

-40 -40 -40 0

-40 -30 -20 -10 0 10 20 30 40 -40 -30 -20 -10 0 10 20 30 40 -40 -30 -20 -10 0 10 20 30 40
coord 1 coord 1 coord 1

BD Rhapsody Targeted
A Major cell type B Example marker gene
(~2K reads per cell) (CD8A)

40 40 1.2
773 cell (18.3%)
log10 (number of molecule per cell)

30 30 2.465356e+03 mols (0.4%)

1
20 20
0.8
10 10
coord 2

coord 2

0.6
0 0
0.4
-10 -10

-20 -20 0.2

-30 -30 0
-30 -20 -10 0 10 20 30 40 -30 -20 -10 0 10 20 30 40
coord 1 coord 1

Figure 5. Example results showing assay sensitivity and UMI counting efficiency between competitor WTA and BD Rhapsody targeted assays. By avoiding high
expressing proteins with low variance (e.g. ribosomal proteins), targeted assays yield high quality clustering results with dramatically far fewer reads (2K per cell vs. 20K
per cell), thus reducing sequencing costs. Targeted sequencing also allows more sensitive detection of low abundant but important surface marker genes, for instance,
CD8A. Unlike competitor WTA data, this targeted approach enables the distinction of naïve CD8 T cells from naïve CD4 T cells (plot B magnified cluster).
 A Batch effect tSNE B Week 0 vs week 6, r=0.97 Week 0 vs week 12, r=0.95

100 100
Week 0
log10 (mean(week 12 molecules))

Week 6
log10 (mean(week 6 molecules))

20 Week 12

10-1 10-1

0
tSNE 2

10-2 10-2

−20

10-3 10-3

−25 0 25 50 10-3 10-2 10-1 100 10-3 10-2 10-1 100

tSNE 1 log10 (mean(week 0 molecules)) log10 (mean(week 0 molecules))

Figure 6. Magnetic beads are stably stored with intact cDNA from one preparation and show little batch effect over time (A). Correlation of gene
expression in batches prepared 12 weeks apart shows stability of archived sample (B).
Targeted panels for major applications
Targeted approaches help boost single-cell sequencing efficiency, as well as furthering discoveries in some of the most
challenging scientific areas today. Our validated panels show robust and reproducible arrays of cell heterogeneity, and are
useful for exploring cell types related to numerous applications. The BD Rhapsody System includes these panels:
• BD Rhapsody Onco-BC Targeted Panel – 400 genes associated with breast cancer and immune cells
• BD Rhapsody Immune Response Panel – profiling for immune cell types
• BD Rhapsody T-Cell Targeted Panel
• BD Rhapsody Supplemental Panel
• BD Rhapsody Custom Panel

Advanced software and visualization adds flex to your results

Bioinformatics pipeline and visualization tools are also provided with the BD Rhapsody system. These tools consist of UMI
analysis algorithms and visualization tools enabling even inexperienced users to analyze and understand single cell data.

Demultiplex
reads
Sequencer Count unique molecular Perform UMI Perform cell Single-cell
output identifiers (UMI) per adjustment label filtering expression
.fastq files gene per cell algorithms profile

Align reads

BD Rhapsody system data sets are available for download Total end-to-end system for single-cell research
at www.sbgenomics.com/bdgenomics The BD Rhapsody Single-Cell Analysis system empowers and
Example data sets: streamlines your research with a complete system of tools,
• Single-cell profiling of PMBCs from a healthy donor including reagents and analysis software, that work
together to meet your unique single-cell experimental
• T-cell phenotyping analysis needs. Achieve results while saving time and money with
• Single-cell profiling of breast cancer tumor biopsies assays that dramatically reduce experimental cost and
complexity, improving data and increasing efficiency of
sequencing. For 40 years, BD has been a trusted partner in
single-cell biology. You can rely on us to help open new
frontiers in single-cell analysis.

www.bd.com/genomics
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Trademarks are the property of their respective owners.
© 2017 BD. BD, the BD Logo and all other trademarks are property of Becton, Dickinson and Company.

BDGM1012 Rev. 1