Droplet Digital PCRAssays for Droplet Digital PCRWORKFLOW

A VERSATILE
AND SCALABLEWORKFLOW

Bio-Rad’s Droplet Digital PCR (ddPCR) technology is based on water-oil emulsion droplet
technology to partition the sample. The ddPCR technology is composed of a droplet
generator, a droplet reader, and associated consumables. The droplet generator partitions
each sample into 20,000 uniform nanoliter-sized droplets containing target and backgroundDNA in random distribution. Each droplet contains a separate reaction. Droplets aretransferred to a 96-well PCR plate and PCR is performed to end point in a thermal cycler.
The droplets are singulated and pass by an optical detection system. Up to 96 samples
can be processed per run.

234Sample

1OilOilDroplets

1

Prepare ddPCR r eaction mix■■

 ombine DNA/RNA sample, primers,
Cand/or probes with one of Bio-Rad’s
ddPCR supermixes■■

Fully validated ddPCR assays can be used

2 | Bio-Rad Laboratories, Inc.

2

3 4 5 6 7
DG8 Cartridge

8Generate droplets

Perform PCR

Read and a nalyze results■■

 oad the ddPCR reaction mix into the wellsLof a droplet generator cartridge■■

 ransfer the droplets to a 96-well PCR plate
Tand seal the plate■■ fter PCR, load the 96-well PCR plateA
into the QX200 Droplet Reader■■

 x 20,000 droplets are generated from each8
run in the QX200 Droplet Generator■■

Run the PCR protocol■■

 ositive and negative droplets in eachPsample are read■■

Target DNA () and background DNA (are randomly distributed in droplets■■Analyze concentrations)ASSAYS FOR DROPLET DIGITAL PCR

EXPERTLY DESIGNED ASSAYSARE A CLICK AWAY THROUGH
OUR ASSAY DESIGN ENGINE
Go to bio-rad.com/Digital-Assays for more information.

Bio-Rad Laboratories, Inc. | 3

A
16,000

MUTATION DETECTION ASSAYS

14,000

THE NEW GOLD STANDARD
IN QUANTIFYING RARECANCER MUTATIONS

FAM amplitude

12,00010,000
8,000
6,000
4,000
2,000
00

1,000

2,000

3,000

4,000
5,000
HEX amplitude

6,000

7,000

8,000

9,000

0

1,000

2,000

3,000

4,000
5,000
HEX amplitude

6,000

7,000

8,000

9,000

B
16,00014,000

FAM amplitude

12,00010,000
8,000
6,000
4,000
2,000
0C100detection, where a biomarker exists within a background of
a highly abundant counterpart that can differ by only a single
nucleotide. Many methods for mutation analysis have poor
selectivity and fail to detect mutant sequences with abundancesof less than one in 100 wild-type sequences. Mutation detection
assays on the ddPCR platform enable detection of minute mutant
fractions without pre-amp because partitioning increases sensitivity
by isolating the target signal from competing background.

4 | Bio-Rad Laboratories, Inc.

Fractional abundance, %One application that harnesses the power of ddPCR is mutation

1021

0.942
0.482

0.1

0.01

0.15
0.0823No template Wild typecontrol		0.1%
mutant0.2%
mutant0.5%
mutant1%
mutant2%mutantHigh sensitivity of ddPCR mutation detection assays provides precise
quantification of JAK2 V617F mutation in the presence of wild-type DNA. A, 2-D
fluorescence amplitude plot shows duplicate wells of a mixed mutant: wild-type sample
across multiple percentage mutant blends. The black cluster on the plot represents thenegative droplets, the green cluster represents the droplets that are positive for wild-type
DNA only, the blue cluster represents the droplets that are positive for mutant DNA only,
and the orange cluster represents the droplets that are positive for both targets.
B, 2-D fluorescence amplitude plot shows duplicate wells of a wild-type–only sample.
C, fractional abundance plot shows the percentage frequency (orange markers) of the mutant
DNA in a wild-type DNA background. All error bars represent the 95% confidence interval.

A
16,000COPY NUMBER ASSAYS

14,000AT LAST — IDENTIFYINGCOPY NUMBER VARIATIONSWITH CONFIDENCE

FAM amplitude

12,00010,000
8,000
6,000
4,000
2,000
00

1,000

2,000

3,000

4,000 5,000 6,000
HEX amplitude

7,000

8,000

9,000

10,000BCopy number

32.04

22.02

2.11

2.01

2.02

10
MergedCThe major technical challenge in copy number assessment ishybridization (aCGH), quantitative PCR (qPCR), and sequencing.
These methods lack the sensitivity and resolution needed for thisfine degree of quantitative discrimination in CNV analysis. The
massive partitioning of a ddPCR reaction into 20,000 droplets
enables the discrimination required to resolve small fold changes.

6Copy numbercopy number variation (CNV) include array comparative genomic

ERBB2

MDM2Well 2

EGFR

FGFR1Well 3

FGFR2Well 4KRASMET7

the ability to discriminate, with statistical confidence, between
consecutive copy number states. Current methods to analyze

MYC8Well 1

6.41
5.84

54
3

3.87
3.04

3.2

3.06

2.93
2.02

2
10

CRL-5807D CRL-5807D CRL-5807D CRL-5807D CRL-5807D CRL-5807D CRL-5807D CRL-5807DSample

Droplet Digital PCR copy number assays provide superior resolution and precision.
A, 2-D fluorescence amplitude plot shows four replicate wells of a copy number sample
duplexed with ERBB2 and RPP30 assays. The black cluster on the plot represents thenegative droplets, the green cluster represents the droplets that are positive for RPP30
reference only, the blue cluster represents the droplets that are positive for ERBB2 only,
and the orange cluster represents the droplets that are positive for both ERBB2 and RPP30
targets. B, copy number plot shows multiple wells at copy number 2 with precise replicate
and merged well values. C, copy number plot shows multiple oncogene copy number
determinations for various genes with the same NCI-H358 lung cancer sample. All error
bars represent the 95% confidence interval.

Bio-Rad Laboratories, Inc. | 5

GAPDHHMBS

ACTB

RPLP0UBC

HSP90AB1

RPL13A

HBB

HPRT1

B2M

TFRC

IPO8GUSB

TBP

G6PD

YWHAZ

GENE EXPRESSION ASSAYS

PGK1

ALAS1A

BETTER GENE EXPRESSIONDATA FOR SIMILAR COST

FAM amplitude

30,00020,00010,0000050,000
100,000		150,000			
Event number

200,000

250,000

B
575

420

Concentration, copies/µl

2,960

1,483

1,000

540

247

219

100

55.3
12.9

1081

68.7

44.4

33

24.7

2,510

746

11.8

8.1

10.1

HBB

RPL13A

HSP90AB1

UBC

RPLP0

ACTB

HMBS

GAPDH

HBB

RPL13A

HSP90AB1

UBC

RPLP0

ACTB

HMBS

GAPDH

HPRT1

B2M

TFRC

IPO8GUSB

TBP

G6PD

YWHAZ

PGK1

ALAS10.01

Reference geneC

With its high precision, ddPCR can be used to

100,000

detect small fold changes in expression of a target■■■■

 inglicate multiplexed ddPCR studies make much better use ofS
rare samples than SYBR® qPCR roplet Digital PCR allows advanced new strategies such asD
SELFIE Digital PCR

6 | Bio-Rad Laboratories, Inc.

HPRT1

B2M

TFRC

IPO8GUSB

1TBP

 ontrary to expectations, multiplexed ddPCR gene expression
Cstudies have similar costs to singleplex SYBR® qPCR studies

G6PD

■■

10

YWHAZ

Supermix and modified thermal cycling conditions.

100

PGK1

which can be used with QX200 ddPCR EvaGreen

®

1,000

ALAS1

existing human and mouse gene expression assays,

Brain RNA, copies/ng

gene between samples. Choose from over 40,000

10,000

Reference gene

Robust gene expression assays used in ddPCR with EvaGreen® provide precise quantification of18 reference genes. A, 1-D fluorescence amplitude plot shows results of a two-step reverse transcription
ddPCR reference assay panel run on human brain RNA. The black clusters on the plot represent the negative
droplets and the blue clusters represent the droplets that are positive for the respective reference assay.
B, concentration plot shows various gene concentrations of cDNA calculated as copies/µl on a log10 scale.
C, calculated RNA concentrations in the original human brain RNA sample for each reference gene. All error
bars represent the 95% confidence interval.

A
6,000

GENOME EDIT DETECTION ASSAYS
FAM amptitude

5,000

FAST. SIMPLE. PRECISE.

4,000
3,000
2,000
1,000
0

0

1,000

2,000

3,000

4,000 5,000
HEX amptitude

6,000

7,000

8,000

0

1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 9,000
HEX amplitude

B
9,000
8,000

FAM amplitude

7,000
6,000
5,000
4,000
3,000
2,000
1,0000Droplet Digital PCR enables a rapid, cost-effective, andultrasensitive method for detecting genome editing events createdusing nucleases that cause double-stranded breaks in DNA, such

HDR and NHEJ assay readouts. A, HDR mutation-positive control (WT + 20% gblocks
gene fragment containing a single base pair substitution) with HDR GED + HDR REF
Assay. The HDR edits are FAM+HEX–, while the HEX+ and FAM+HEX+ positive clusters
represent the total number of copies, including WT, NHEJ, and HDR edits. B, NHEJ
mutation-positive control (WT + 1% gblocks gene fragment containing a one base pairdeletion at the predicted cut site). The WT cluster is positive for both FAM and HEX, while
the NHEJ mutant single-positive droplets are positive for FAM only.

as CRISPR-Cas9 or other gene editing tools.
■■

■■

■■

 etect HDR (homology directed repair) and NHEJ (non-homologous end joining) eventsDpresent at frequencies of ≤0.5%
Provide absolute quantification of genome editing from as little as 5 ng of total gDNA
 esign and order ddPCR Assays to detect HDR and NHEJ edit events for any target on
DBio-Rad’s Digital Assay Site

Bio-Rad Laboratories, Inc. | 7

A
25,000

MULTIPLEXING STRATEGIESHIGHER-ORDER MULTIPLEXING —SAME COLOR CHANNELS,
MORE ANSWERS

Channel 1 amplitude

20,00015,00010,000

5,000

00

2,000

4,000

6,000
8,000
10,000Channel 2 amplitude

12,00014,000B
10,0009,000

Channel 1 amplitude

8,000
7,000
6,000
5,000
4,000
3,000
2,000
1,000
0–1,000
0

The number of targets measured in an assay need not be limited
to the number of color channels available on Bio-Rad’s Droplet■■■■■■ mplitude-Based Multiplexing — targets are detected via probes conjugated withA
a single dye at different final concentrations in an endpoint reaction
 adial/Ratio-Based Multiplexing — targets are detected using probes mixed atR
different probe ratios to create unique endpoint fluorescence.
Nondiscriminating Multiplexing — targets are detected but not uniquely identified

3,000
4,000
Channel 2 amplitude

5,000

6,000

16,00014,000Channel 1 amplitude

be employed to maximize data generated from each channel:

2,000

CDigital PCR Systems. Multiple targets can be measured in a single
reaction, enabling higher-order multiplexing. Various strategies can

1,000

12,00010,000
8,000
6,000
4,000
2,00000

2,000

4,000
Channel 2 amplitude

6,000

8,000

Multiplexing with two color channels: A, detection of four targets by amplitude
multiplexing. B, detection of three targets by radial/ratio-based multiplexing. Assay used
the following ratios of FAM to HEX: T1, 1:0; T2, 0:1; T3, 1:1. C, detection of five targets byradial/ratio-based multiplexing. Assay used the following ratios of FAM to HEX: T1, 1:0; T2,3:1; T3, 1:1; T4, 1:3; T5, 0:1.

8 | Bio-Rad Laboratories, Inc.

A
12,000

MULTIPLEXING STRATEGIESADVANCED MULTIPLEXING —MORE COLOR CHANNELS,EVEN MORE ANSWERS

FAM amplitude

10,000
8,000
6,000
4,000
2,000
00

2,000

4,000

6,000
8,000
HEX amplitude

10,00012,000B
14,00012,000Cy5 amplitude

10,000
8,000
6,000
4,000
2,000
0

Bio-Rad’s QX ONE Droplet Digital PCR System offers four colorchannels — FAM, HEX, Cy5, and Cy5.5 — thereby providing
additional multiplexing flexibility. By using Bio-Rad’s ddPCR
Multiplex Supermix on the QX ONE ddPCR System, as many
as eight targets can be detected and measured in a single
reaction. Such advanced multiplexing is made possible using

05,00010,000Cy5.5 amplitude

15,00020,000C
Dye Name

TargetConcentration, copies/µl

CNV

FAM Lo

PLAU

668

1.96

FAM Hi

CCND1658

1.95
1.96

HEX Lo

VCL662HEX Hi

REN665

1.97

Cy5 LoBRCA1658

1.95

strategies such as amplitude multiplexing in conjunction withCy5 Hi

KLF8691

2.05

four color channels. Extract as much information as possible

Cy5.5 LoSORL1672

1.99

Cy5.5 HiPTEN675—with high sensitivity, using as little sample as possible in a fast,
cost-effective manner.

8-plex amplitude multiplexing on QX ONE System. A, detection of four targets on
FAM/HEX 2-D plot. PLAU (FAM Lo), CCND1 (FAM Hi), VCL (HEX Lo), REN (HEX Hi).
B, detection of four targets on Cy5/Cy5.5 2-D plot. BRCA1 (Cy5 Lo), KLF8 (Cy5 Hi),
SORL1 (Cy5.5 Lo), PTEN (Cy5.5 Hi). C, measured concentration of each target incopies/µl. PTEN (Cy5.5 Hi) is reference.

Bio-Rad Laboratories, Inc. | 9

COMPREHENSIVE ASSAY PORTFOLIOSOLUTIONS OFFERED FOR
NUMEROUS APPLICATIONS

Droplet Digital PCR Assays and Kits are made for numerous applications, including mutation detection, copy number
determination, genome edit detection, gene expression, residual DNA quantification, and library quantification.
CAGTGTCA

Multiplex Mutation Screening

Copy Number

Residual DNA Quantification

Library Quantification

For rapid screening of several key cancer
mutations in a single reaction.

For the detection of SMN1 and SMN2
gene copy number.

For detection of CHO and E. coli
host cell DNA.

For the quantification of NGS
sequencing libraries.

■■

■■

■■

 igh sensitivity (limit of detectionH≤ 0.5%)
 ork with low amounts of input DNAW
without pre-amplification
 ssays available for BRAF V600,AKRAS G12/G13, KRAS Q61, NRAS
G12, NRAS G12/G13, RNAS Q61,
EGFT exon 19 deletions

10 | Bio-Rad Laboratories, Inc.

■■■■ chieve superior performance withAaccurate and reproducible copy
number calls for the gene of interest
 erform high-throughput screening byP
processing several hundred samples
in a single day■■■■

 ighly precise, femtogram-level
H
quantification of residual CHO orE. coli DNA irect quantification without DNA
Dpurification step

■■

■■

■■

 rovide information about library
Pquality, such as adapter dimers,
and indicate library insert size rovide more efficient and consistentPloading of libraries for sequencing runs
 nable balancing of pooled
Elibrary samples

AN ASSAY PRODUCT LINE FOR EVERY NEEDFLEXIBLE ASSAYDESIGN OPTIONS

ATGCCustomAssay Design Service

Expert Design

Know your oligo sequences and justwant to order it from a trusted source?

Have a unique assay need? Contact
your sales representative and workwith a specialist. We’ll design it for you.

See a list of our most sought after specialty
assays, such as the Microsatellite Instability
Assay for detection of MSI markers (BAT25/BAT26, NR21/NR24, and Mono27).

bio-rad.com/ddPCRCustomAssays

bio-rad.com/ddPCRAssayDesignService

bio-rad.com/ddPCRExpertDesign

Visit bio-rad.com/DropletDigitalPCRAssays to learn more.

Bio-Rad Laboratories, Inc. | 11

BIO-RAD, DDPCR, and DROPLET DIGITAL PCR are trademarks of Bio-Rad Laboratories, Inc. in certain jurisdictions.
The QX ONE/QX200 Droplet Digital PCR Systems, and the consumables and reagents designed to work with these systems, and/or their use is covered by claims of U.S. patents and/orpending U.S. and non-U.S. patent applications owned by or under license to Bio-Rad Laboratories, Inc. See bio-rad.com/en-us/trademarks for details. Purchase of the product includes alimited, non-transferable right under such intellectual property for use of the product for internal research purposes in the field of digital PCR only. No rights are granted for diagnostic uses.
No rights are granted for use of the product for commercial applications of any kind, including but not limited to manufacturing, quality control, or commercial services, such as contract
services or fee for services. Information concerning a license for such uses can be obtained from Bio-Rad Laboratories. It is the responsibility of the purchaser/end user to acquire any
additional intellectual property rights that may be required.
EvaGreen is a trademark of Biotium, Inc. Bio-Rad Laboratories, Inc. is licensed by Biotium, Inc. to sell reagents containing EvaGreen Dye for use in real-time PCR, for research purposes only.
SYBR is a trademark of Thermo Fisher Scientific Inc. Bio-Rad Laboratories, Inc. is licensed by Thermo Fisher Scientific Inc to sell reagents containing SYBR Green I for use in real-time PCR,
for research purposes only.
All trademarks used herein are the property of their respective owner.

Bio-RadLaboratories, Inc.
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