Sponsored By:

DIGITAL PCR,
REIMAGINED

Page 3 Page 4 Page 5 Page 6

Digital PCR with the Ease A Small Consumable with Absolute Q Workflow Vs. Digital PCR Applications
of Quantitative PCR Large Value Droplet-Based Digital PCR
Digital PCR Reimagined.
Digital
Know PCR
Faster. Reimagined.
Know More.
Digital
Know PCR
Faster. Reimagined.
Know More.
Know Faster. Know More.

The Absolute Q
Our
Thenew digital PCR
Absolute Q platform
combines simple qPCR-like
Our
The new digital PCR
Absolute Q platform
workflow with 4-color multiplexing.
combines simple qPCR-like
Enhance your nucleic
Our new digital acid
PCR platform
workflow with 4-color multiplexing.
quantification
combines without
simple wasting time,
qPCR-like
Enhance your nucleic acid
reagents
workflowor precious
with samples.
4-color multiplexing.
quantification without wasting time,
Enhance your nucleic acid
reagents or precious samples.
quantification without wasting time,
reagents or precious samples.

Enhanced Walk-away Under 90 minute

multiplexing workflow run time
Enhanced Walk-away Under 90 minute
Four color capability. Load the plate & run. Rapid turn-around.
multiplexing workflow run time
Enhanced Walk-away Under 90 minute
Four color capability. Load the plate & run. Rapid turn-around.
multiplexing workflow run time
Four color capability. Load the plate & run. Rapid turn-around.

Learn More
Visit
Learn us at combinati.com or send us
More
an email at info@combinati.com
Visit us at combinati.com or send us combinati.com
Learn More
an email at info@combinati.com
Visit us at combinati.com or send us
combinati.com
an email at info@combinati.com combinati.com
DIGITAL PCR, REIMAGINED
Digital PCR
with the Ease of
Quantitative PCR
Q
uantitative PCR (qPCR) is the gold standard
for measuring cDNA and gDNA levels, but
the resulting data can be highly variable due to
inadequate sample dilution or chemical contamination. The
most susceptible targets are genes with low abundance or
RNA samples extracted from limited sources. Furthermore,
scientists performing qPCR must rely on standard curves
or references to perform quantification. Conventional next-
generation sequencing library quantification methods using
qPCR are also limited by inefficient amplification of certain
nucleotide sequences (for example, AT- and GC- rich
regions). Scientists struggle to detect rare genetic variants
using qPCR, and their results may be prone to bias.
Digital PCR (dPCR) enables scientists to divide the
sample and assay mixture into a very large number of
separate small volume reactions, such that zero or one
target molecule is present in any individual reaction. This
solves the problem of variability, and eliminates the need
for quantification with standard curves. However, many
currently available technologies are time-consuming and
have complicated, multi-step workflows that may contribute
to sample and reagent waste.
A Simpler dPCR Workflow
Researchers typically perform dPCR by generating
droplets in a tube using specialized reagents and a droplet
generator. They transfer those droplets to a 96-well plate,
which they place into a thermocycler for PCR amplification.
A droplet reader directly counts positive “hits.” This hands-
on process involves many steps, each with the potential for
user errors and sample contamination.
Combinati’s dPCR platform, however, consists of just one
instrument, with every step of the reaction taking place in
a single plate. The platform essentially turns a multi-step
dPCR process into one resembling a simple qPCR
3
workflow for preparing the sample, loading the plate, and
analyzing the sample, using only one instrument. The
run time is typically less than 90 minutes, and the system
analyzes more than 90% of the sample, minimizing sample
and reagent waste.
Although the workflow is almost identical to that of qPCR,
Combinati’s dPCR platform enables scientists to directly
count positive results just like any other dPCR method,
rather than calculating them using curves. The simplicity
of the system drives down laboratory costs.
Multiplexing
The majority of dPCR systems are based on detection
in two discrete optical channels, allowing quantification
of one or two targets within a single reaction. Combinati’s
platform allows for multiplexing up to four different
colors, greatly expanding the possibilities for creating
different assays for different applications. Researchers
need multiplexing for various research purposes.
These can include applications such as quantification
of genetically modified organism (GMO) crop events1,
and other broad applications. Digital PCR’s ability to
detect rare targets in a high background, combined with
multiplexing, drives down analysis costs in GMO testing
laboratories2, and many other research areas.
References
1. D. Dobnik et al., “Multiplex quantification of four DNA targets in
one reaction with Bio-Rad droplet digital PCR system for GMO
detection,” Sci Rep, 6:35451, 2016.
2. T. Demeke et al., “Critical assessment of digital PCR for the detection
and quantification of genetically modified organisms,” Anal Bioanal
Chem, 410:4039-50, 2018.
DIGITAL PCR, REIMAGINED

A Small Consumable
with Large Value

R
esearchers using dPCR benefit from a standard-
curve-free method to precisely quantify
sequencing libraries. While many platforms
involve a great deal of sample handling, Combinati’s
dPCR platform consists of one instrument with one
consumable—an injection molded microfluidic siphoning
array with a semi-permeable membrane. The fixed
microchamber array with defined geometry ensures a
consistent number of partitions and partition volume for
all samples across experiments. The Combinati array
has a familiar microplate format for easy handling so
scientists can pipet their sample, cap the plate, load the
plate, and then walk away.
Consistency in dPCR
Quantification by dPCR hinges on random distribution
of molecules in many partitions following a Poisson
distribution. Each partition essentially acts as an
individual PCR microreactor, such that the proportion
of PCR-positive (fluorescent) partitions determines the
concentration of the target sequence. Sample partitioning
efficiently concentrates the target sequences within
the isolated microreactors. This concentration reduces
template competition and thus enables the detection of
rare mutations in a background of wild-type sequences.
Furthermore, it may also allow for a higher tolerance to
inhibitors present in a sample1. However, the efficiency
and consistency of dPCR relies on the method
of partitioning.
There are two main mechanisms for partitioning: using
nanofluidics to load the reaction into prefabricated
chambers (plate-based dPCR), or generating water-in-oil
emulsions (droplet-based dPCR). The partition volume
can vary from 4.4 μL down to 5 pL and the partition
number in a reaction can vary from 496 up to 10 million
partitions depending on the dPCR platform2. Droplet-
based dPCR technologies suffer from a lack of droplet-to-
droplet volume uniformity that can affect quantification
accuracy and hinder conformance to clinical regulatory
requirements3. Common plate-based techniques that rely
on user-applied thin films on the consumable can also
impede uniformity in partition size3. This, in turn, creates
uncertainty in downstream analysis and challenges with
reproducibility.
The Combinati system’s plate is different; it ensures
high consistency because of the fixed partition size.
Fixed partition arrays ensure high reproducibility across
experiments, leading to confidence in downstream
analysis.
Saving Time, Sample, and Effort
Because the partitioning consumable plate is the only
consumable required for dPCR using the Combinati
system, the system minimizes waste, time, and effort
for scientists performing dPCR. The core microfluidic
technology enables partitioning of samples with minimal
sample waste, which is especially relevant for scarce
samples, such as formalin-fixed paraffin-embedded
(FFPE) tissue or circulating tumor DNA. The workflow
has limited steps compared to traditional dPCR, thus
requiring less user interaction.
References
1. P-L. Quan et al., “dPCR: A technology review,” Sensors (Basel),
18(4):1271, 2018.
2. J.F. Huggett et al., “Digital PCR and its potential application to
microbiology,” D.H. Persing, F.C. Tenover, Y. Tang, F.S. Nolte, R.T.
Hayden, A. van Belkum, eds., Molecular Microbiology: Diagnostic
Principles and Practice (3rd ed.), ASM Press Washington DC, 2016,
pp. 49-57.
3. M.E. Dueck et al., “Precision cancer monitoring using a novel, fully

4
 A O N E - S T E P WO R K F LOW :

Absolute Q vs. Digital Droplet-based dPCR

C O M B I N AT I ’ S A B S O L U T E Q d P C R D R O P L E T- B A S E D d P C R

PREP SAMPLE PREP SAMPLE

-" / -" /

P L AT E S A M P L E O N T O
D R O P L E T G E N E R AT O R

LOA D S A M P L E

C R E AT E D R O P L E T S
1 2 3 4
A

B
P L AT E S A M P L E
C
Target
D
LOA D S A M P L E
AND RUN
D I G I TA L P C R

R U N D I G I TA L P C R

A N A LY Z E P L AT E

• 1 instrument, • Multiple
1 consumable, instruments &
1 step plate transfer
• Minimal • >20% wasted
waste reagents
V I E W R E S U LT S • Consistent • Variable V I E W R E S U LT S
number of number of
partitions droplets
• Defined • Variable
volume droplet size

Target 1 Target 2

Multiplexing Multiple target types can be

quantified within the same sample
with Up to PCR by multiplexing with different
colors, such as hydrolysis probes
Four Colors PCR+ PCR- PCR+
with different dyes.
DIGITAL PCR, REIMAGINED
Digital PCR
Applications
D
igital PCR is becoming the standard for tracing
disease-relevant biomarkers over time. It is well
suited to non-invasive prenatal diagnosis, viral
load detection, low frequency mutation validation,
pre-sequencing quality control, environmental applications
such as plant karyotyping, and for PCR assays with
limited sample.
Detecting Rare Sequence Events
Digital PCR is ideal for identifying rare sequences that
are lost in the background when using standard qPCR.
For example, standard PCR techniques have consistently
underestimated low abundance organisms in microbiome
studies1, and dPCR reliably outperforms qPCR in low viral
load detection2. Detecting rare sequence events is important
for diagnosing and treating myriad diseases. One step reverse
transcription (RT)-dPCR, which occurs in a single tube to
minimize experimental variation, may be of particular interest
for researchers detecting rare sequences in where only small
amounts of bacterial mRNA are available..
Infectious Diseases
Drug resistance in Mycobacterium tuberculosis—also known
as heteroresistance—occurs infrequently, but presents an
enormous threat to public health. Detecting small numbers
of drug resistant bacteria is challenging using traditional
methods. Using dPCR to assess heteroresistance ensures
discovery of drug resistance in M. tuberculosis populations3.
Digital PCR also aids in detecting influenza resistance to
the anti-viral treatment, oseltamivir. In a study comparing
dPCR to qPCR for studying viral resistance, researchers
showed a 50-fold improvement in sensitivity for detecting
SNPs, due to reduced off-target amplification, on the qPCR
assay previously used in the clinic4.
First, this study was able to recognize and reduce off-target
amplification in dPCR quantification, thereby enabling
technical sensitivities down to 0.1% SNP abundance at a
6
range of template concentrations, a 50-fold improvement on
the qPCR assay used routinely in the clinic. 
Cancer Applications
Digital PCR shows greater precision and reproducibility
compared to qPCR for microRNA analysis5, which translates
to superior cancer diagnostic performance. Researchers
performing cancer stratification benefit from dPCR’s ability
to detect rare mutations6. In the field of liquid biopsy,
researchers perform dPCR to non-invasively characterize
solid tumors based on tumor-related nucleic acids in blood or
other anatomical fluids7-9.
Prenatal Screening
Because dPCR readily detects aneuploidy, does not depend
on allelic distribution or gender, and distinguishes signals in
the presence of mosaics or contaminating maternal DNA, it is
ideal for non-invasive prenatal disease and karyotype screening.
Scientists use dPCR with maternal blood to identify circulating
cell-free fetal DNA carrying trisomy 21 mutations (Down
syndrome)10 and for diagnosing single-gene diseases such as
sickle cell anemia11 or X-linked disorders such as hemophilia12.
In cases where invasive prenatal testing is traditionally
performed, dPCR offers an attractive alternative12.
Graft Rejection
Organ transplant recipients sometimes carry cell-free DNA
from grafts in their circulation, a potential biomarker of
rejection. Researchers use dPCR to rapidly detect graft-
associated cell-free DNA, which could enable more effective
therapeutic interventions13.
Environmental Applications
Plant karyotyping helps researchers select varieties of plants
that are likely to flower at certain times, which assists them in
growing well-adapted varieties. They can accurately measure
copy number variation using dPCR. For example, researchers
use dPCR to monitor the type and number of flowering-
associated genes or alleles to develop plant species adapted to
different or changing environments14,15.
See references on page 7.
DIGITAL PCR, REIMAGINED

References
1. J.M. Gonzalez et al., “Amplification by PCR artificially reduces that
proportion of the rare biosphere in microbial communities,” PLOS ONE,
7:e29973, 2012.
2. R.H. Sedlak, K.R. Jerome, “Viral diagnostics in the era of digital
polymerase chain reaction,” Diagn Micr Infec Dis, 75(1):1-4, 2014.
3. S. Pholwat et al., “Digital PCR to detect and quantify heteroresistance
in drug resistant Mycobacterium tuberculosis,” PLOS One, 8:e57238,
2013.
4. A.S. Whale et al., “Detection of rare drug resistance mutations by
digital PCR in a human influenza A virus model system and clinical
samples,” J Clin Microbiol, 54:392–400, 2016.
5. C.M. Hindson et al., “Absolute quantification by droplet digital PCR
versus analog real-time PCR,” Nat Methods, 10(10):1003-1005, 2013.
6. V. Taly et al., “Multiplex picodroplet digital PCR to detect KRAS
mutations in circulating DNA from the plasma of colorectal cancer
patients,” Clin Chem, 59(12):1722-31, 2013.
7. G.R. Oxnard et al., “Noninvasive detection of response and resistance
in EGFR-mutant lung cancer using quantitative next-generation
genotyping of cell-free plasma DNA,” Clin Cancer Res, 20:1698-1705,
2014.
8. I. Garcia-Murillas et al., “Mutation tracking in circulating tumor
DNA predicts relapse in early breast cancer,” Sci Transl Med,
7:302ra133, 2015.
9. G. Siravegna, A. Bardelli, “Minimal residual disease in breast cancer:
in blood veritas,” Clin Cancer Res, 20:2505-2507, 2014.
10. H.C. Fan, S.R. Quake, “Detection of aneuploidy with digital
polymerase chain reaction,” Anal Chem, 79(19):7576-9, 2007.
11. A.N. Barrett et al., “Digital PCR analysis of maternal plasma for
noninvasive detection of sickle cell anemia,” Clin Chem, 58(6):1026-32,
2011.
12. N. Lench et al., “The clinical implementation of non-invasive prenatal
diagnosis for single-gene disorders: challenges and progress made,”
Prenat Diagn, 33:555-62, 2013.
13. J. Beck et al., “Digital droplet PCR for rapid quantification of donor
DNA in the circulation of transplant recipients as a potential universal
biomarker of graft injury,” Clin Chem, 59(12):1732-41, 2013.
14. R. Nitcher et al., “Increased copy number at the HvFT1 locus is
associated with accelerated flowering time in barley,” Mol Genet Genom,
288:261-75, 2013.
15. A. Zmienko et al., “Copy number polymorphism in plant genomes,”
Appl Genet, 127:1-18, 2014.

7
 Absolute Quantitation
te Quantitation
Absolute
of SARS-CoV-2Quantitation
Absolute Quantitation
Absolute
S-CoV-2
ofstudy
A usingQuantitation
SARS-CoV-2
of
theSARS-CoV-2
new
of
A
the SARS-CoV-2
singAbsolute
study Q dPCR
newusingAthe Platform
study
new using the new
Q dPCR
Absolute
Platform
A study Q dPCR
using Absolute
Platform
the new Q dPCR Platform
Absolute Q dPCR Platform
Study Highlights:
• 1st demonstration of a true 1-step RT-PCR on
lights:
Study Highlights: Study Highlights:
a digital PCR platform in under 90 minutes
tion of• a1sttrue
demonstration
1-step RT-PCR of•on
a1sttrue
demonstration
1-step RT-PCR of on
a true 1-step RT-PCR on
platform
Study
• Low limitHighlights:
a in
digital
of detection for viral load
underPCR 90 minutes
platformain digital
underPCR 90 minutes
platform in under 90 minutes
• quantification
1st demonstration downof atotrue
5 target
1-step copies
RT-PCR on
tection
• Low
forreaction
per viral
limit load
of detection• Low
for viral
limit load
of detection for viral load
a digital PCR platform in under 90 minutes
down quantification
to 5 target copiesdown to quantification
5 target copiesdown to 5 target copies
•• Potential
Low limit to detection
of enable more for efficient
viral load
per reaction per reaction
quantification
quantification of viraltotargets
down 5 targetwith up to
copies
able more
• Potential
efficient
4-color multiplexingreagents or preciousmore efficient
to enable more
• Potential
efficient
to enable
per reaction
of viralquantification
targets with up
samples. ofto
viralquantification
targets with up ofto
viral targets with up to
• Potential
lexingreagents to enable more
4-color multiplexingreagents efficient
or precious 4-color multiplexingreagents
or precious or precious
quantification
samples. of viral targets
samples. with up to
4-color multiplexingreagents or precious
samples.

Digitalization, PCR,
Set Up Reaction Load Plate Analysis
and Data Collection

Digitalization, PCR, Digitalization, PCR, Digitalization, PCR,

n SetLoad
Up Reaction
Plate SetLoad
Up Reaction
Plate Load
Analysis
Plate Analysis
and Data Collection and Data Collection and Data Collection
Digitalization, PCR,
Set Up Reaction Load Plate Analysis
Read More and Data Collection

Visit us at combinati.com/covid-19
Read More
to learn more.
Read More
binati.com/covid-19
Visit us at combinati.com/covid-19
Visit us at combinati.com/covid-19 combinati.com
Read More
to learn more. to learn more.
Visit us at combinati.com/covid-19 combinati.comcombinati.com com
to learn more.
combinati.com