Group 4: Digital PCR (Molbio)

Members:
● Macapallag, Maria Amor
● Maninding, Jerez Xean
● Mendoza, Andrea
● Musico, Van
● Ng, Anjannette
● Pagsanghan, Allaine
● Peralta, Ivan
● Ramones, Haywee Angelie Ericka

Title: “High-Throughput Droplet Digital PCR System for Absolute Quantitation of

DNA Copy Number”
Authors: Benjamin J. Hindson, Kevin D. Ness, Donald A. Masquelier, Phillip Belgrader,
Nicholas J. Heredia, Anthony J. Makarewicz, Isaac J. Bright, Michael Y. Lucero, Amy L.
Hiddessen, Tina C. Legler, Tyler K. Kitano, Michael R. Hodel, Jonathan F. Petersen,
Paul W. Wyatt, Erin R. Steenblock, Pallavi H. Shah, Luc J. Bousse, Camille B. Troup,
Jeffrey C. Mellen, Dean K. Wittmann, Nicholas G. Erndt, Thomas H. Cauley, Ryan T.
Koehler, Austin P. So, Simant Dube, Klint A. Rose, Luz Montesclaros, Shenglong Wang,
David P. Stumbo, Shawn P. Hodges, Steven Romine,
Fred P. Milanovich, Helen E. White, John F. Regan, George A. Karlin-Neumann,
Christopher M. Hindson, Serge Saxonov, and Bill W. Colston

I. INTRODUCTION
(1. What was the paper all about? - RAMONES)

Digital PCR is extremely important in the detection and quantitation of nucleic

acid sequences. There is a lack of available necessary equipment, know-how, and
facilities that delay the mass production and global use and distribution of digital PCR
technology; thus, the said technology is limited in stock as it is both high demand in high
value, and complicated in workflows. This urged the researchers of the paper to provide
in-depth knowledge and understanding of the high-throughput droplet digital PCR or
ddPCR system that enables the processing of 2 million PCR reactions using
conventional TaqMan assays with a 96-well plate workflow. This study aims to
demonstrate the three applications that the ddPCR system provides orders of
magnitude more precision and sensitivity than real-time PCR; to show the accurate
measurement of germline copy number, to show sensitive detection of mutant DNA in a
100, 000-fold excess of wild types background for rare alleles, and lastly, to
demonstrate absolute quantification of circulating fetal and maternal DNA from cell-free
plasma. This paper sets out to allow researchers to explore and acquire knowledge
complex genetic landscapes, discover and corroborate the findings of the study, and to
cause a paradigm shift in molecular.
The authors described a high-throughput droplet digital PCR (ddPCR) device that
can execute 2 million PCR reactions in a 96-well plate workflow using traditional
TaqMan assays. Three applications showed that the ddPCR system's vast partitioning
delivers orders of magnitude better precision and sensitivity than real-time PCR. They
first demonstrated how to accurately assess germline copy number variation. Second,
they demonstrated sensitive detection of mutant DNA in a 100 000-fold excess of
wildtype background for rare alleles. Finally, they showed that absolute quantification of
circulating fetal and maternal DNA from cell-free plasma is possible. Using the ddPCR
technology, they believed that researchers would be able to investigate complicated
genomic landscapes, identify and validate new disease connections, and usher in a new
era of molecular diagnostics.

Objectives Of The Paper

(2. What were the objectives of the paper? - MENDOZA)
The objectives of the experiment are as follows:
● To describe a high-throughput droplet digital PCR system that enables the
processing of ~2 million PCR reactions.
● To accurately acquire the measurement of the germline copy number variation.
● To show sensitive detection of mutant DNA in a 100,000-fold excess of wildtype
background for rare alleles.
● To demonstrate absolute quantification of circulating fetal and maternal DNA from
cell-free plasma.

Significance Of The Paper

(3. How is the paper significant in the contribution of knowledge and to society as a
whole? - PAGSANGHAN)

First, Digital PCR enables us to produce more accurate, precise, and highly
sensitive quantification results of the test of the nucleic acids in a sample. Second, this
makes the process of the test also quicker than it usually is. Moreover, as the name
suggests – “digital”, it will allow researchers to examine complicated genomic
landscapes, detect and verify disease connections, and bring about a new epoch of
clinical techniques. As stated in the paper, digital PCR can withstand wide differences in
amplification efficiencies without compromising DNA copy number estimation because it
uses a binary end-point threshold to classify each replicate reaction as either positive or
negative (one or zero, respectively). Digital PCR by limiting dilution in microwell plates is
still utilized today, despite its low throughput and limited dynamic range. A practical and
 low-cost embodiment will unlock the promise of digital PCR and establish the third
generation of PCR users and applications.

II. METHODOLOGY
(5. What were the methodologies employed by the authors to achieve the objectives?
NG)

The researchers provided 6 steps to know and describe the workflow of the
high-throughput droplet digital PCR system which is also known as ddPCR.

Figure 1. Droplet Digital PCR Workflow

1. Load samples and oil into disposable droplet generator cartilage

Eight assembled PCR reactions are loaded into individual wells of a single-use
injection molded cartridge.
2. Generate Droplets
Droplet generation oil containing stabilizing surfactants is loaded and the
cartridge is placed into the droplet generator. By application of vacuum to the outlet
wells, sample and oil are drawn through a flow-focusing junction where monodisperse
droplets are generated at a rate of ∼1 000 per second
3. Transfer droplets to 96 wells PCR plate
The surfactant-stabilized droplets are pipet transferred to a 96-well PCR plate.
4. Thermal cycle to end-point
After the densely packed droplets, surfactant-stabilized droplets, are pipet
transferred to a conventional 96-well PCR plate, droplet PCR amplification to end-point
(3545 cycles) is performed in a conventional thermal cycle.
5. Read droplet fluorescence
After thermal cycling, the plate is transferred to a droplet reader which sips
droplets from each well and streams them single-file past a two-color detector at the
rate of ∼1 000 per second.
6. Apply amplitude thresholds and calculate concentrations.
Droplets are assigned as positive or negative based on their fluorescence
amplitude. The number of positive and negative droplets in each channel is used to
calculate the concentration of the target and reference DNA sequences and their
Poisson-based 95% confidence intervals.

II. A. EXPERIMENTAL

1. Droplet Digital PCR System (as described in Figure 1.)

● In a final volume of 20 L, a TaqMan PCR reaction mixture was assembled from a

2 ddPCR Mastermix (Bio-Rad), 20 primers and probes (final concentrations of
900 and 250 nM, respectively), and template (variable volume).
● After that, each assembled ddPCR reaction mixture was loaded into a sample
well of an eight-channel disposable droplet generator cartridge (Bio-Rad).
● For each channel, a volume of 60 L of droplet generation oil (Bio-Rad) was
loaded into the oil well. The droplet generator was loaded with the cartridge
(Bio-Rad).
● The cartridge was removed from the droplet generator, and the droplets that
accumulated in the droplet well were manually transferred to a 96-well PCR plate
using a multichannel pipet.
● The plate was heat-sealed with a foil seal and then amplified to the end-point on
a conventional thermal cycler (4055 cycles).
● After PCR, the 96-well PCR plate was loaded onto a droplet reader (Bio-Rad),
which automatically reads the droplets from each well of the plate (32 wells/h).
● Lastly, the ddPCR data was analyzed using the QuantaSoft analysis software
(Bio-Rad) that came with the droplet reader.

2. Determination of Copy number variation in HapMap Samples

 ● 4.4 g of each purified human genomic DNA sample (Coriell) was digested with 10
units of RsaI (NEB) in 50 uL for 1 hour at 37oC for MRGPRX1, ChromosomeX,
and CYP2D6.
● The digest was diluted 8-fold to 400 uL with TE buffer (pH 8.0) before being
assayed at 33 ng (3 L) per 20 L ddPCR reaction. In 10 L with 7.5 units of MseI
(NEB), 815 ng of purified human genomic DNA (Coriell) for CCL3L1, was
digested for 1 hour at 37oC. The digest was diluted 3.5 times with TE buffer to 35
L, and 69 ng (3L) was assayed per 20 L ddPCR reaction.
● The CYPD2D6 (Hs00010001 cn) was purchased as a 20-primer and
FAM-MGBNFQ probe premix (Applied Biosystems).
● All CNV assays were duplexed with an RPP30 reference assay
● Thermal cycling conditions were 95oC for 10 minutes (1 cycle), 94oC for 30
seconds, and 60oC

3. Determination of GRB7 & ERBB2

The restricted DNA was then directly added to the ddPCR Mastermix
● Purified DNA (20 ng) from normal and tumorous breast tissue samples such as
D8235086-1, Biochain was digested for 1 hour at 37 o C in a 10 L using 0.2 units
of Nlalll.
● At a rate of 8.8 ng (4.4 L) per 20 L of ddPCR reaction, the ERBB2 (Hs02803918
cn) and GRB7 (Hs02139994 cn) assays (Applied Biosystems) were purchased
as 20 premixes of primers and FAM-MGBNFQ probe and duplexed with the
RPP30 reference assay described above.
● The thermal cycling conditions were 95oC for 10 minutes which is equivalent for 1
cycle, 94oC for 30 seconds and 60oC for 60 seconds which is equivalent for 40
cycles, 98oC for 10 minutes which is equivalent for 1 cycle, and 12oC hold.

4. Rare Allele Detection (Pagsanghan)

● A high, constant background (5 000 copies/μL) of wildtype DNA (NA19205,

Coriell was used in the preparation of a dilution series of BRAF V600E mutant
DNA (HTB-38D) from a HT-29 cell line (ATCC).
● When the concentration of intact human genomic DNA is less than 66 ng/20 μL
reaction, there is an increase in viscosity which can cause the average droplet
volume to change. This could affect the accuracy of DNA quantitation.
● For samples of this nature, it is recommended to utilize restriction enzyme
digestion that would fragment the DNA and lessen the viscosity of the solution.
 ● According to the researcher’s experience, once fragmented, the human genomic
DNA concentration can exceed 1 μg/20 μL reaction without affecting the average
droplet volume.
● In preparation for ddPCR, each sample of the dilution series was digested with
40 U of HaeIII (NEB) in 100 μL containing 1× NEB buffer 4 and BSA. Common
primers were used in the BRAF V600E/wildtype duplex TaqMan assay.
● For each sample of the dilution series, eight ddPCR wells were utilized.
● Thermal cycling conditions for this portion of the experiment were 95 °C × 10 min
(1 cycle), 94 °C × 30 s and 62.7 °C × 60 s (55 cycles), and 12 °C hold.

5. Quantitation of Cell-Free Faral and Total DNA (Pagsanghan)

● Collecting of whole blood (3x10mL) from healthy pregnant donors was done
between 10-20weeks of gestational age via venipuncture into cell-free DNA BCT
tubes (Streck) basing it w/ the instructions of the manufacturer.
● Within 6 weeks of collecting the sample, the gender of the fetus is identified
through the use of an ultrasound.
● Sample tubes were stored for 48 hrs at room temperature and transferred
overnight at 4°C to Bio-Rad, where processing began once received.
● The whole blood was centrifuged for 10 minutes at 1 600g, the supernatant was
removed and transferred to a new tube, and the cell-free plasma was kept at 80 o
C.
● Cell-free plasma (5 mL) was thawed and cell-free DNA was isolated using the
QIAmp Circulating Nucleic Acid Kit (Qiagen) according to the manufactuerʼs
protocol and before being eluted in AVE buffer (150 μL).
● Parts of the eluate (99 μL) were subjected to a single-tube digest which has HhaI
(30 U), HpaII (60 U), and BstUI (30 U) in 1× NEB buffer 4 in 120 μL total volume.
● A second portion of the eluate (33 μL) was utilized in a no-digest control mixture
where restriction enzymes were substituted for water.
● The mixtures were then incubated for 37 °C for 2 h, 60 °C for 2 h, then 65 °C for
20 min.
● The restriction enzyme digested mixture was split and subjected to three ddPCR
duplexed assays namely SRY/TERT, RASSF1/RNaseP, and RASSF1/β-actin.
● Unmethylated RASSF1 and β-actin TaqMan templates but not SRY, RNaseP, or
TERT, were then cut by the restriction enzyme mixture.
● The no-digest control mixture was split and subjected to two ddPCR duplexed
assays of RASSF1/RNaseP and RASSF1/β-actin.
● β-Actin is hypomethylated in both fetal and maternal DNA, and the enzyme
cocktail completely digests it. RASSF1 and SRY assays have previously been
reported.
 ● Commercially obtainable RNaseP and TERT copy number reference assays
were used (Applied Biosystems).
● 1 GC-Rich Solution (Roche) was used as a component of the assembled ddPCR
reaction mixtures for RASSF1/RNaseP and RASSF1/-actin duplexes. The
thermal cycling conditions were 95 °C for 10 minutes (1 cycle), 95 °C for 30
seconds and 60 °C for 60 seconds (45 cycles), and 4 °C hold.
● One TERT, one-actin, two RASSF1, and two RNaseP assays were used to mix
independent total DNA concentration (G.E/mL) in determination for each sample.
Seven replicate ddPCR wells were used in each assay measurement.
● The droplet counts were combined (positive and negative) from all seven
replicate wells to yield a single “metawell”.
● Each of the 6 measurement metawells’s concentration and confidence intervals
were computed.
● The confidence interval was scaled appropriately after using the necessary
dilution factors to get total cell-free DNA concentration (G.E./mL).
● The six total measurements’s weighted mean was calculated, with the weights
equal to inverses of confidence interval variances of these measurements.
● There are two independent assay measurements for digested RASSF1, which
are also combined in the same way.
● For SRY, one measurement was used directly, with haploidy being accounted for
by scaling by a factor of two.
● The fetal load is then calculated as a ratio with Poisson 95% confidence
intervals.

III. RESULTS & DISCUSSION (MENDOZA, MUSICO, PERALTA)

Immediate utility of ddPCR system was demonstrated through presenting data

on three application areas of increasing interest to researchers: determination of copy
number variation (CNV), detection of rare alleles, and the absolute quantitation of
circulating DNA in cell-free plasma.
 Figure 2. Determination of copy number variation states by droplet digital PCR.

A copy number variation (CNV) is when the number of copies of a particular gene
varies from one individual to the next that have been linked to a variety of human
diseases. Three target genes were examined for CNVs in seven HapMap samples.
Duplex TaqMan test reagents for the target and reference genes were included in each
ddPCR run. From the results of a single well for each sample, the copy number states
from 1 to 6 were entirely resolved for MRGPRX1 (Figure 2a). As indicated, lower CNV
states for CYP2D6 and Chromosome X were easily resolved.

Second, the researchers calculated the copy number of CCL3L1, a gene linked
to HIV/AIDS risk, for 13 HapMap samples (Figure 2b). For next-generation sequencing,
it predicted the CCL3L1 copy number to be 5.725 for DNA sample NA18507, but the
ddPCR technique estimated 6.05. The next-generation sequencing estimate of 5.7 is
likely due to under-sampling because it only has an average read-depth of only 30x but
for ddPCR, it easily obtained an increased accuracy since the number of reads can be
adjusted practically. From a single well, the current ddPCR method can obtain read
depths of up to 20 000 for two genes.

The last application of ddPRC in CNV is the Correlation of measured copy

number alterations of GRB7 and ERBB2 in DNA extracted from normal and tumorous
breast tissues. For a set of normal and tumor breast tissue samples, the measured copy
numbers of ERBB2 and GRB7 correlated with the exception of two samples that
showed lower GRB7 amplification (Figure 2c). These results were expected as the
GRB7 gene is part of the HER2 amplicon and is coamplified in almost all breast tumors.

The results show that the ddPCR technology is particularly suited for CNV
population research, as it allows large numbers of samples to be evaluated against
smaller sets of gene with high accuracy, sensitivity, and precision. Although microarray
technologies are useful for CNV identification, their dynamic range is limited, and
scaling to large numbers of samples for population investigations is costly. Additionally,
real-time PCR techniques have reported technical difficulties in achieving reliable copy
number estimations. Copy number estimations can fluctuate between cases and
controls because of the imprecision of real-time PCR measurements, thereby further
increasing the need for a high-throughput, low cost approach ddPCR to making precise
CNV measurements with increased dynamic range for validation for CNV population
studies. The results also shows that ddPCR method provides the ability to scale the
number of partitions by combining replicate wells to resolve fine copy number
differences in heterogeneous mixtures and could be a better alternative diagnosing
amplifications and deletions since other methods like the fluorescent in situ hybridization
(FISH) method is costly and low-throughput only.

Figure 3. Detection of the BRAF V600E rare mutant allele in the presence of
homologous wildtype DNA by droplet digital PCR.

The successive dilutions of the mutant cell line DNA were generated in a
continuous background of wildtype human genomic DNA, as shown in the results.
Droplet partitioning eliminates some competitive amplification effects, which allows the
determination of mutant fractions as low as 0.001%, which is 1,000 times lower than
real-time PCR. BRAF V600E is present in 35% of the mutant cell line, as determined by
ddPCR.

For the second application, it exhibits how competitive PCR processes can be
greatly reduced in the face of a relatively homogeneous wild-type DNA background,
resulting in enhanced detection of uncommon mutant alleles. Real-time PCR methods
can detect down to the 1% mutant fraction with careful tuning. While, ddPCR divides the
competitive background away from the mutant using the same assays, substantially
boosting the mutant-to-wild-type ratio by 20,000 times. The number of sample partitions
employed is proportional to the effective enrichment of mutant molecules per PCR
experiment. Droplet partitioning finds 0.001% mutant fraction, 1,000 times lower than
real-time PCR, in a duplex TaqMan assay targeting the BRAF V600E mutation29. Due
to the reliance on the amount of DNA retrieved from clinical samples, more DNA can be
put into the ddPCR system, allowing detection limits to be lowered even further. This
method allows researchers to detect extremely low amounts of mutation, potentially
leading to better diagnosis of minor residual disease and less invasive diagnostics.
 Figure 4. Absolute quantitation of circulating fetal and maternal DNA from cell-free
plasma for male and female fetuses.

The absolute quantification of circulating fetal and maternal DNA from cell-free
plasma for male and female fetuses can be separated into three graphs. (a)
Quantification of the fetal concentration using SRY and hypermethylated RASSF1. This
resulted in leaving the hypermethylated fetal DNA being quantified whereas the
hypomethylated fraction, maternal DNA, was selectively digested away through the use
of methylation-sensitive restriction enzymes. (b) Quantification of the total amount of
DNA concentration acquired represented as the weighted average from six independent
assay measurements which includes undigested RASSF1, β-actin, RnaseP, and TERT
(c) The ratio of SRY to the total of male fetuses and RASSF1 to the total of both male
and female fetuses was used to determine fetal loads. The correlation of fetal load
between SRY and RASSF1 was 97.3% for male fetuses. This indicates that the fetal
DNA is not completely hypermethylated. Hence, the RASSF1 fetal load measure for
some samples is lower compared to those determined using SRY.

This portion of the experiment entails evaluating the ability of the ddPCR to
quantify DNA in clinical samples. The cell-free DNA located in the plasma is known to
be highly fragmented, therefore, presents at low levels which can be a challenge for
quantification. In the experiment, the fetal and total DNA in maternal cell-free plasma
were enumerated. As shown in Figure 4a, 19 maternal plasma samples were taken
between 10 and 20 weeks of gestational age. Figure 4b depicts the total DNA that was
measured for both male and female fetuses. As aforementioned, a selective
methylation-sensitive digest enabled the low levels of hypermethylated RASSF1 fetal
DNA to be accurately quantified. Through the absolute measure of SRY, RASSF1, and
total DNA concentrations, the fetal load (Figure 4c) was calculated. As seen in the
results, for male fetuses, there was a correlation of 93.7% between the hypermethylated
RASSF1 fetal DNA and SRY fetal loads which provided confidence in the estimation for
female fetuses. Moreover, the use of this technique or system would increase the
capability of absolute quantification of low-level highly fragmented cell-free DNA found
in clinical samples.

IV. Conclusion

(6. What were the conclusions? - MANINGDING)

In this study, the authors presented data on three application areas to

demonstrate the immediate utility of this ddPCR system: determination of copy number
variation (CNV), detection of rare alleles, and the absolute quantitation of circulating
DNA in cell-free plasma. Each application was chosen to highlight a distinct benefit of
massive droplet partitioning for digital PCR. A large number of replicates for CNV
provides enough precision to accurately measure copy number states. Partitioning the
target mutant DNA away from highly homologous wildtype DNA improves sensitivity for
the detection of rare alleles. Finally, droplet partitioning allows for precise quantification
of nucleic acids in clinical samples over a wide dynamic range without the use of
external calibrators or endogenous controls.

● Determination of copy number variation (CNV)

- The data presented by the authors for CNV determination show that
their ddPCR system is well suited for CNV population studies because it
allows large numbers of samples to be tested against smaller sets of genes.

● Detection of rare alleles

- The authors conclude that, depending on the amount of DNA

recovered from clinical samples, more DNA can be loaded into the ddPCR
system to lower detection limits even further. This method allows researchers
to detect extremely low levels of mutant, which could lead to better detection
of minimal residual disease and less invasive diagnostics.

● The absolute quantitation of circulating DNA in cell-free plasma

- The authors evaluated the ddPCR system's ability to quantify DNA in

clinical samples. Circulating DNA in cell-free plasma has received increasing
levels of attention as a sample type for developing noninvasive prenatal and
oncology diagnostics. The cell-free DNA in plasma is highly fragmented and
present at low levels, making quantification difficult. Hence, after analyzing
the necessary data, the authors concluded that this application demonstrates
the capability of absolute quantitation of highly fragmented cell-free DNA in
clinical samples.

Overall, the data that the authors presented show that ddPCR is a practical solution for
achieving precise estimates of DNA copy number with high throughput. The researchers
hope that this system will enable more researchers to harness the inherent power of
digital PCR for a variety of applications.

(4. Were the objectives met? PERALTA)

Yes, the researchers were able to prove that ddPCR is a practical solution & it provides
precise estimates of DNA copy number with high-throughput. Digital PCR/technology
allows the evaluation of huge amounts of samples in smaller sets of genes, making it
suitable and appropriate for common number variation population studies. In addition,
Droplet division eliminates some competitive amplification effects, which allows the
determination of mutant fractions as low as 0.001%, which is 1000 times lower than
real-time PCR. Lastly, through the absolute measure of SRY, RASSF1, and total DNA
concentrations, the fetal load was calculated. There was a correlation of 93.7% between
the hypermethylated RASSF1 fetal DNA and SRY fetal loads in male fetuses, which
provided confidence in the estimation for female fetuses

Recommendations
(7. Are there any research gaps that may be addressed by the future researchers? -
Macapallag)

In scientific research papers, there would always be research gaps as it is

extremely important to be addressed by the current and future researchers for a more
effective outcome. It takes longer time to promulgate project outcomes as there is a
need for it to be further assessed before being published to the public. That being said, I
think the research gap that can be found in the paper is that the digital PCRs must be
studied further and experimented hands-on by the future researchers in order to
achieve greater precision and produce an in-depth analysis of the functions and benefits
of the digital PCRs in detecting and identifying of bacteria and viruses. This can provide
us a deeper knowledge and understanding of how we can practice and put these into
use in the medical field as they are introducing more sufficient clinical techniques. Also,
researchers who will study the same or similar research topic must take into
consideration the concept or idea of how to improve these digital PCRS with accurate
and well-founded measurements instead of relying on estimates that may or may not in
order to produce even better practical solutions. With this, the future researchers can
also study how they can make these digital PCRs accessible and affordable to the
general public.

Opinion on the Paper

(8. What is your personal opinion about the paper? MUSICO)

Based on the paper, ddPCR is more advantageous and more practical for quantification
results for DNA copies over real-time PCR since the ddPCR showed more accurate,
precise, and high sensitivity results. As students of Medical Laboratory Science, this
paper is also significant in the field of Medical Laboratory because with the help of
ddPCR, it allows researchers to explore complex genetic landscapes, discover and
validate new disease associations that will alow us to further study and improve
molecular diagnostics without doubting if the results are valid or not since ddPCR
showed accurate, precise, and high sensitivity results. This paper also shows how
significant ddPCR to the community is and not just to medical field workers only
because trough ddPCR, as the paper showed promising results, maybe in the future the
ddPCR can help us to prevent or much better, find cure to certain diseases that is
incurable for now. Additionally with the existence of ddPCR, hospital bills can be greatly
reduced since ddPCR can be an alternative for high-cost methods such as FISH
method.