digital PCRtech note 6277

Probing Copy Number Variations Using Bio-Rad’s QX100™
Droplet Digital™ PCR System
George Karlin-Neumann, Luz Montesclaros, Nick Heredia, Shawn Hodges,
Jack Regan, Camille Troup, Dave Mukhopadhyay, Claudia Litterst, and Svilen
Tzonev, Bio-Rad Digital Biology Center, 7068 Knoll Center Parkway, Suite 401,
Pleasanton, CA 94566

Introduction
The Role of Copy Number Variation in Phenotype
and Disease

Cytogenetic studies over the past 50 years have hinted at
the impact that copy number variations (CNVs) can have on
phenotypic traits and disease susceptibility (Beckmann et al.
2008). The advent of holistic discovery methods afforded by
DNA microarrays (Redon et al. 2006) and more recently by
next-generation DNA sequencing (Sudmant et al. 2010)
have allowed us to better understand the extent of genetic
variation in genomes. Using single nucleotide polymorphism(SNP) DNA microarrays, Redon et al. (2006) estimatedthat upwards of 12% of the human genome consists ofcopy number variable regions greater than 1 kb, harboring
approximately 1,000 genes. Next-generation sequencing
analyses from the 1000 Genomes Project indicated that the
majority of CNVs exist as five or fewer copies per cell, but
the 56 most variable gene families have median CNV values
between 5 and 368. Moreover, some gene families show
significant copy number differences among the three HapMap
populations analyzed (Sudmant et al. 2010).
The most recent comparative genome-wide characterizations
estimate that over 29% of the reference human genome
harbors more than 38,000 CNVs greater than 100 bp(Vissers and Stankiewicz 2012). These variants have thepotential to influence phenotype through both gene dosage
and structure and underlie hundreds of inherited microdeletionand microduplication syndromes, including early-onset
Alzheimer’s disease (duplication of the APP gene), familial
Parkinson’s disease (duplication and triplication of the SNCAgene), retinoblastoma (deletion of the RB1 gene), X-linked
mental retardation (deletion of IL1RAPL1 or CASK, or deletionand duplication of SOX3), spinal muscular atrophy (deletionand duplication of the SMN1 and SMN2 genes), and drug
metabolism and transporter abnormalities such as deletionand duplication of CYP2D6 (Vissers and Stankiewicz 2012,
Beckmann et al. 2008). Besides these Mendelian traits,
deletions and amplifications have been found to be associatedwith complex traits and somatic copy number alterations,or CNAs (Beckmann et al. 2008, Speleman et al. 2008).

Need for Improved Methods to Validate, Diagnose,and Monitor Copy Number Variations

Given the high incidence and clinical impact of CNVs,
a precise, rapid, and cost-effective method is needed forhigh-throughput validation of candidate CNV associations
and for subsequent routine deployment in diagnostic
settings. The predominant method used to validate CNVs inlarger populations is real-time or quantitative PCR (qPCR),
which measures the relative rates of fluorescence increases
during the exponential amplification of target and singlecopy reference genes. The accuracy and precision of thesemeasurements can be impacted by multiple factors including
differences in amplification rates between the target andreference genes, variations in their amplification rates during
qPCR, sampling error due to DNA concentration, and analysis
errors (Karlen et al. 2007). Weaver et al. (2010) rigorously
characterized these factors and found that systemicerrors can be addressed by increasing the number of
replicates to achieve the desired precision. However, therequired number of replicates increases rapidly as finer
discrimination is desired, with four replicates required to
distinguish a twofold difference (for example, a CNV of 1 vs. 2)
and up to 18 replicates to distinguish a 1.25-fold difference(for example, a CNV of 4 vs. 5).
Droplet Digital PCR (ddPCR™) is a DNA molecule counting
technique that directly counts the absolute number of target
and reference DNA molecules in a sample, enabling small
fold differences to be determined for higher-order CNV
states (Hindson et al. 2011). In ddPCR, targets of interest
are partitioned into droplets and amplified to endpoint with
TaqMan probes as in qPCR, then their concentrations are
determined based on the number of fluorescently positive andnegative droplets in a sample well. Thus, the fluorescencesignal in qPCR is converted from an analog signal into a
digital one, thereby removing the requirement for standard
curves and allowing the determination of target copies onan absolute scale with high precision (Hindson et al. 2011).
Our studies using ddPCR to analyze CNV status focused on
several clinically important loci including the CCL3L1 locus,
which is thought to influence susceptibility to HIV-1 infection
and has been the subject of conflicting results among
numerous studies using qPCR (Gonzalez et al. 2005, Liu et al.
2010). Our results demonstrate that Bio-Rad’s QX100 Droplet
Digital PCR system enables excellent discrimination of copy
number states.

Materials and Methods
Assays and DNA samples

All TaqMan hydrolysis probe assays were either predesigned
assays obtained from Life Technologies Corporation
or were designed at Bio-Rad Laboratories, Inc. andsynthesized by Integrated DNA Technologies (BHQ) or
Life Technologies (NFQ-MGB). The MRGPRX1 assay
sequences were from Hosono et al. (2008): forward primer,
5'-TTAAGCTTCATCAGTATCCCCCA-3'; reverse primer,
5'-CAAAGTAGGAAAACATCATCACAGGA-3'; probe,5'-FAM-ACCATCTCTAAAATCCT-MGB-3'. CYP2D6_FAMMGB was obtained as a predesigned assay, Hs00010001_cn(Life Technologies). QL_Eichler_CCL3_LG_FAM-BHQ
sequences were adapted from Sudmant et al. (2010):
fwd primer, 5'-GGGTCCAGAAATACGTCAGT-3';
rev primer, 5'-CATGTTCCCAAGGCTCAG-3'; probe,5'-FAM-TTCGAGGCCCAGCGACCTCA-BHQ-3'.
QL_RPP30_1_VIC-MGB reference assay: fwd primer,
5'-GATTTGGACCTGCGAGCG-3'; rev primer,
5'-GCGGCTGTCTCCACAAGT-3'; probe, 5'-VICCTGACCTGAAGGCTCT-MGB-3'. QL_uc473_chX_VICMGB ultra-conserved reference assay: fwd primer,
5'-GGGAACAGAACAAGCTGGTCTT-3'; rev primer,
5'-GCAAGACCCTGTGTAAGAACTTTGA-3'; probe,
5'-VIC-CTATCACATGTGGATCGTA-MGB-3'. DNA HapMap
samples were all obtained as purified DNA from the Coriell
Institute for Medical Research cell repositories and diluted
to 100 ng/µl in 1x TE, pH 8, before use.
DNA Sample Digestion

DNA samples were digested with restriction endonucleases
at a concentration of 1 µg DNA/40 µl reaction using thevendor’s (New England BioLabs Inc.) recommended
conditions except that 5 U of enzyme was added per 1 µg
DNA unless otherwise indicated. Restriction digests werediluted a minimum of 7.5-fold in the ddPCR reaction mix toobtain the desired final DNA concentration. When higher DNA
concentrations (>1 copy per droplet, CPD) were used in the
ddPCR reaction (for example, for the screening of HapMap
DNA populations), DNA was digested at the concentrationof 1 µg/20 µl per reaction. After restriction digestion, thereactions were incubated at 65˚C for 20 min to deactivate
the enzyme but not treated at a higher temperature to
avoid denaturing the DNA target fragments. For restriction
endonuclease titration experiments, the enzyme was first
diluted into the supplied dilution buffer, and the appropriate
enzyme dilution was added to each DNA being digested.
For these titration experiments, 33 ng of digested DNA(0.5 CPD of the RPP30 reference gene) was added per 20 µl
ddPCR reaction.
For experiments with CCL3L1, sample DNAs were doubledigested with 5 U each of RsaI and MseI per µg of DNA andthen added to a concentration of 16 ng/20 µl ddPCR reaction(0.25 CPD of the RPP30 reference gene or 33 ng/20 ul© 2012 Bio-Rad Laboratories, Inc.

(0.5 CPD of the reference genes). For HapMap screening
experiments, 100 ng (1.5 CPD) of digested genomic DNA
was run in each ddPCR reaction.
ddPCR Experiments

Genomic DNA samples, either digested or undigested asindicated, were added to the Bio-Rad 2x ddPCR supermix
at concentrations ranging from 16.5 ng (0.25 CPD) to 100 ng(1.5 CPD) DNA per 20 µl ddPCR reaction. TaqMan assay
primers and probes were present at final concentrationsof 900 nM and 250 nM, respectively. Reaction mixes werebriefly mixed by vortexing while avoiding the formation of
bubbles, microcentrifuged for 20 sec, then kept on ice
until droplet generation.
Samples were converted into droplets, eight samples at a time,by adding 20 µl of sample to each sample well of the DG8™
cartridge followed by addition of 70 µl of droplet generation oil
to each of the corresponding oil wells. The cartridge was thenplaced into the QX100 droplet generator within 2 min after
oil addition for droplet generation. Droplets were transferredfrom the droplet wells in the cartridge with an 8-channel P50
pipetor to a 96-well PCR plate (twin.tec real-time PCR plates,
Eppendorf). As many as 12 sets of samples were successively
prepared in cartridges and transferred to the PCR plate
before it was heat sealed with a pierceable foil seal. The plate
was transferred to a thermal cycler and, unless otherwise
specified, reactions were run under the following standard
cycling conditions: 95˚C for 10 min followed by 40 cycles of
94˚C for 30 sec; 60˚C for 60 sec, 98˚C for 10 min, and 12˚C
infinitely; ramp rate = 50% (3˚C/sec). For gradient experiments,
the annealing/extension temperature was varied from 60˚C,
as specified in Figure 3. After PCR, the plate was loaded
onto the QX100 Droplet Digital reader, which automaticallyreads the droplets from each well of the plate (~36 wells/hr).
Data analysis was performed using Quantasoft™ software(Bio-Rad). Negative droplets, lacking target and/or reference
gene DNA, and positive droplets, containing either or bothDNAs, were counted to give the fraction of positive droplets.
Using Poisson statistics, the concentrations of both DNA
species were determined, and confidence intervals (CI) werecomputed, which were in turn used to compute the CNV valueof the sample.
DNA Loading Considerations

When potentially high copy number samples are tested,
it is important to reduce the input DNA concentration to
avoid exceeding ~5 or 6 CPD for the unknown target
gene. The amount of genomic DNA to introduce into asingle reaction is governed by the number of droplets per
reaction (~20,000), the concentration of the two genes being
measured (the unknown target and the known reference
genes), and the Poisson statistics for the partitioning of thesegene copies into the droplets. We previously calculated(Hindson et al. 2011) that the DNA concentration yielding
the most precise concentration measurements for 20,000

Bulletin 6277

Msel Titration (N = 4)

© 2012 Bio-Rad Laboratories, Inc.

UND
UND

0.01953125

0.004882813

0.31250.004882813

CYP2D60.078125

5

1.25

20

UND

0.01953125

0.004882813

0.31250.078125

5

1.25

20

UND

0.01953125

MRGPRX1

CCL3L1

Average target concentration
Average RPP30 concentration

Average CNV (N = 4)

MRGPRX1

0.01953125

0.31250.078125

1.25

5

20

UND

0.01953125

CYP2D60.004882813

0.31250.078125

1.25

5

20

UND

0.01953125

6
5
4
3
2
1
0
20

CNV

The most robust and reliable approach for separating linked
gene copies is the use of restriction enzymes. We haveextensively investigated the use of endonucleases forfragmentation and found that a wide range of enzymes with
4-base and 6-base recognition sites perform satisfactorily
for this purpose. The benefit of predigestion for CNV
measurement is typically achieved by a wide range of enzyme
concentrations. Three considerations should be taken into
account in the choice of enzyme for a particular locus:(1) the enzyme should not cut within the PCR amplicon itself;(2) it is best to use an enzyme that is insensitive to methylation
to avoid incomplete fragmentation due to methylation of
the target DNA; and (3) in some instances, it is optimal todigest the target copy to the smallest size fragment that fully
contains the amplicon footprint — preferably under a few
hundred base pairs.

0.004882813

1,400
1,200
1,000
800
600
400
200
0
20

Fig. 1. Tandem gene copies require separation before partitioning.
If tandem gene copies (red boxes) remain linked along a DNA fragment(blue bar), they will be partitioned into the same droplet and will result inan underestimated copy number. Their separation onto smaller DNA
fragments prior to partitioning into droplets (right panel) allows them tobe properly counted.

0.004882813

5 positive droplets:
accurate CNV estimate

0.3125

3 positive droplets:
inaccurate CNV estimate

0.078125

3 positive droplets

Our results show that the single-copy RPP30 reference geneis unaffected by the DNA digestion status. Likewise, CYP2D6,
which is also a single-copy gene, shows no difference inconcentration and CNV value (= 2) with or without digestion
by MseI (Figure 2) or XhoI and CviQI (data not shown) when
duplexed with the RPP30 assay. A third locus, CCL3L1, wasexamined and shows five copies per cell, whether digested
by MseI or not, indicating that the copies are unlinked.
Similar results were obtained with XhoI (data not shown).

0.3125

Two unlinked copies

0.078125

Two tandem copies

5

Single copy variant

1.25

Alterations in the copy number of a locus are commonly due
to tandem duplications. It is necessary to physically separate
linked copies to achieve an accurate count of all copies in
Droplet Digital PCR, as illustrated in Figure 1.

1.25

Separating Linked Gene Copies

5

Results and Discussion

The importance of proper target fragmentation for the tandem
copies of MRGPRX1 (Mas-related G-protein coupled receptor
member X1), is illustrated in Figure 2 for the Yoruban HapMap
sample NA19205. In the mock digestion, where the MseI
enzyme is absent, the CNV estimate is ~3.5 copies; however,when the genomic DNA is incubated with as little as 0.078 Uof enzyme at 37˚C for 60 min, the proper CNV value of 5
(Weaver et al. 2010) is obtained when compared with thesingle-copy reference gene RPP30 (ribonuclease P protein
subunit p30). The measured copy number (and concentration)of the MRGPRX1 gene increases with the amount of
restriction enzyme used as the separation of the linked
copies becomes more complete. After complete digestionis achieved, the CNV value is unaffected by the quantity ofenzyme used. Similar results are found with the MRGPRX1/
RPP30 duplexed assays when the DNA is digested by XhoI
or CviQI (data not shown).

Concentration, copies/µl

partitions is 1.6 CPD. This concentration corresponds to~100 ng of human genomic DNA in a 20 µl ddPCR reaction
for a single-copy gene. If the target gene is present at morethan two copies per diploid genome, for example, four copies
per diploid genome, the same 100 ng of DNA will contain
3 CPD of the target gene. If the target gene copy number ismore than approximately eight copies per diploid genome,
it is necessary to reduce the total input DNA per well in orderto maintain a sufficient number of negative droplets so thata reliable concentration and CNV estimate can be obtained.
If higher precision is desired, multiple ddPCR wells can be run.

CCL3L1

Fig. 2. Effect of restriction endonuclease digestion of genomic DNA on
CNV assessment. HapMap sample NA19205 was digested with the indicatedconcentrations of MseI enzyme per 1 µg DNA. Thirty-three ng of digested
DNA (0.5 CPD of the RPP30 reference gene) was added per 20 µl ddPCR
reaction. ddPCR reactions contained one of three target assays indicated —MRGPRX1_FAM, CYP2D6_FAM, or CCL3L1_FAM — each duplexed with
the RPP30_VIC reference assay. Each concentration and CNV value
shown represents the average of four replicates per condition with 95% CI.
CNV, copy number variation; UND, undigested (buffer only).

Bulletin 6277

Optimization of Annealing Temperature of the Duplexed Assays

We recommend establishing the optimal annealing/extension
temperature range for newly designed duplex assays
by running a thermal gradient PCR experiment. Figure 3
shows the behavior of the CCL3L1 assay (QL_Eich_CCL3_
LG_FAM-BHQ) duplexed with the RPP30 reference assay(QL_RPP30_1_VIC-MGB) over the annealing temperature
range of 57–64.8˚C. At lower temperatures, the two assays
exhibit the maximal separation of fluorescence amplitudes
between positive and negative droplets (Figure 3). At higher
temperatures across the tested range, the fluorescence
amplitude of negative droplets is only minimally affected;
however, the amplitude for positive droplets begins todecrease as the temperature is increased above 59.2˚C
for RPP30 and 60.2˚C for CCL3L1. For RPP30, there is stillsatisfactory separation of fluorescence signals at 64.8˚C,
and the measured concentration is constant across theentire temperature range (Figure 3B, green square markers).
However, the separation for CCL3L1 is satisfactory only at or
below 62.3˚C (Figure 3A), as reflected by a measured CNV
value of 5 below 62.3˚C (Figure 3B). These results indicatethat the optimal annealing temperature is between 59 and61˚C in these single-well ddPCR measurements.

57

14,40058

60
63

10,800

14
65

7,200

13
12

3,600

11

0

10

0

27,400 54,800 82,200 109,600 137,000

9CNV

Event number

5

3,240

4

2,160

3

1,080

2
1
27,400 54,800 82,200 109,600 137,000

426

425

5.19

5.17

400

427
5.36

437
416

5.02

5.08

6

431
406

5.31

406

5.16
4.75

4.88

341

335
315

4.18

3.98

3.85

4
3

200
164

164

159

164

166

172

167

171

167

163

168

163

Copy number

300

5

1

Temperature, ˚C:		

A01 A02 A03 A04 A05 A06 A07 A08 A09 A10 A11 A12
57

57.2

57.6

58.3

59.2

60.2

61.2

62.3

63.2

64

64.5

0

64.8

Fig. 3. Annealing temperature optimization of the CCL3L1/RPP30
duplexed assay for CCL3L1 copy number determination in HapMap
sample NA19108. A ddPCR reaction mix containing the QL_Eich_CCL3_
LG_FAM-BHQ and QL_RPP30_1_VIC-MGB assays and 16.5 ng of digested
DNA was converted into droplets, and PCR was performed at the designatedannealing/extension temperatures before droplet reading. A, temporalplots for CCL3L1 (FAM) and RPP30 (VIC) fluorescent droplet amplitudes
at various annealing temperatures; B, concentration values for CCL3L1 (n)and RPP30 (n); CNV values (l).
© 2012 Bio-Rad Laboratories, Inc.

NA18853

NA18854

NA19239

Fig. 4. Robustness of CNV estimates for CCL3L1 among 11 cell lines.
Each DNA was double-digested with RsaI and MseI and assessed for copy
number via ddPCR as described in Figure 3 using the same CCL3L1/RPP30
duplexed assays. Duplicate reactions of each of the samples were tested at
an annealing/extension temperature of 59˚C. CNV values shown are from themerged wells with 95% CIs. CNV, copy number variation.

2

100

0

NA185027437

NA18516

NA18573500

NA191080

Event number

NA191070

Concentration, copies/μl

7
6

4,320

0

B

8

NA18916

VIC amplitude

5,400

NA18501

FAM amplitude

18,000For another HapMap sample, NA18507 (not shown here),
the CNV value for CCL3L1 was previously determined by
ddPCR to be 6 and was independently estimated to be 5.7
by next-generation sequencing (Hindson et al. 2011). The
minor difference in these values likely reflects the many fewer
counts generated by sequencing compared with ddPCR,
which can achieve read depths of 20,000x for each of two
genes per single well. Higher precision can be obtained by
combining measurements from additional ddPCR wells.

NA11994A

To test the robustness of the optimized CCL3L1/RPP30
duplexed assay, 11 samples from the Coriell Institute wereassessed by ddPCR for the number of CCL3L1 copies
present. The CNV values presented in Figure 4 represent
duplicate wells run at 59˚C and include the 95% CIs,
showing robust determination across a diverse sample set
where as few as 1 and as many as 13 copies are present.
Similar results were obtained for the same assay run atannealing temperatures of 60˚C and 61˚C, demonstrating
the robustness of the CNV measurement across multiple
temperatures (data not shown). The assay appears toprovide discrimination between 10 and 11 copies with highconfidence, although the actual CNV values for these samples
have not been independently confirmed.

Robustness of CNV Value to Reference Assay Used

In a separate experiment testing different reference assays,
CCL3L1 was duplexed with either the RPP30 (QL_RPP30_1_
VIC-MGB) reference gene (Chr 10) or an ultraconserved Chr X
marker reference (QL_uc473_chX_VIC-MGB). Figure 5 showsthat both duplexes gave nearly identical CNV values for thecell lines NA18507, NA11994, and NA19221. The slightly higher
value seen for NA18502 with the Chr X reference (8.36) thanwith RPP30 (8.03) may be due to aneuploidy in this EBVimmortalized line, to which the X chromosome is susceptible

Bulletin 6277

Chr X

RPP30

5
4
3
2

10

1

8

0
Sample6

Mexican

4

5

2

4

NA19221

NA18502

NA18507

NA11994

NA19221

NA18502

NA18507

NA119940

Copy number

Copy number

12

BritishCopy number(Redon et al. 2006). Note that the red markers for CCL3L1/Chr
X–derived CNV values denote male samples which have onlya single copy of the X chromosome. Their CNV values weresuitably adjusted in the Quantasoft software to reflect thecorrect copies per diploid genome.

SampleLarge populations of individuals can be profiled with ddPCR
for copy number status of a gene of interest. Figure 6 presents
the results for the CYP2D6 gene measured in nearly 300
HapMap samples from the 1000 Genomes Project, including
96 British, 120 Kenyan, and 72 Mexican samples (1000
Genomes Project Consortium 2010). Examples of clearly
distinguishable CNV values of 1, 2, 3, and 4 are found among
them, although the most frequent copy number state in eachof the populations is a CNV of 2 (British, 96%; Kenyan, 87.5%;
Mexican, 86%). Distinctive features of the tested populations,such as allele distributions, are readily discernible fromthese data. For example, the Mexican population shows asubstantially higher frequency of individuals with a CNV of 3(~12.5% vs. 1% for British and 5% for Kenyans, including
one Kenyan with CNV of 4), a genotype that confers ahigher rate of metabolism for a broad range of drugs andother xenobiotics, often with significant pharmacologicalconsequences for these ultra-metabolizing individuals(Johansson and Ingelman-Sundberg 2008). Additionally,
one British and one Kenyan individual each had no copiesof the CYP2D6 gene, characteristic of poor metabolizers(Table 1). The ability to obtain high-precision CNV values
from single-well measurements — combined with the
96-well format of the QX100 Droplet Digital PCR systemand the reader’s throughput of >32 samples/hr — enables
rapid characterization of 3 x 96 samples in a single day withapproximately 1 hr of hands-on time per plate.

© 2012 Bio-Rad Laboratories, Inc.

0
SampleKenyan 1
6
5
Copy number

High-Throughput Profiling of CNV Status in HapMap Populations

2
1

4
3
2
1
0
SampleKenyan 2
5
4
Copy number

Fig. 5. CCL3L1 CNV estimates using duplex assays with either RPP30
or Chr X_u.c. as VIC-labeled reference genes. Each DNA was doubledigested and processed in ddPCR as described in Figure 3 using anannealing/extension temperature of 59˚C. Samples were assayed with either
the CCL3L1/RPP30 (RPP30) duplex or the CCL3L1/QL_uc473_chX_VIC-MGB(Chr X) duplex. Samples with two copies of reference gene per cell (l);samples with one copy of reference gene per cell (l). The CNV value and
95% CI for each sample are derived from two merged wells.

3

3
2
1
0
SampleFig. 6. High-throughput profiling of the CYP2D6 CNV state in three
populations using single-well ddPCR measurements. 100 ng of MseIdigested DNA (1.5 CPD of the single-copy RPP30 reference gene) of three
HapMap populations was analyzed with the CYP2D6/RPP30 duplexed
TaqMan assays.

Bulletin 6277

Table 1. 1000 Genomes Project samples with CNV not equal to 2.

References

Population

Sample Name

CNV Value

95% CI

BritishBritishBritishBritishMexicanMexicanMexicanMexicanMexicanMexicanMexicanMexicanMexicanMexican
Kenyan 1
Kenyan 1
Kenyan 1
Kenyan 1
Kenyan 1
Kenyan 1
Kenyan 1
Kenyan 1
Kenyan 1
Kenyan 2
Kenyan 2
Kenyan 2
Kenyan 2
Kenyan 2
Kenyan 2

HG00156*A1
HG00099*A1
HG00260*A1
HG00149*A1
NA19783*2
NA19719*1
NA19660*1
NA19761*1
NA19723*1
NA19685*1
NA19752*1
NA19788*1
NA19649*1
NA19785*1
NA19317*A1
NA19035*A1
NA19392*1
NA19338*A1
NA19036*1
NA19430*1
NA19359*1
NA19308*1
NA19378*B1
NA19471*1
NA19461*1
NA19332*1
NA19469*1
NA19452*1
NA19393*1

0
1.06
1.07
3.23
1.03
3.1
3.12
3.12
3.13
3.16
3.21
3.22
3.23
3.32
0
1.02
1.05
1.05
1.05
1.06
1.08
3.27
3.98
1.07
1.10
3.08
3.1
3.23
3.27

0–0
1.01–1.12
1.02–1.11
3.10–3.37
1.00–1.07
3.01–3.19
3.01–3.23
3.03–3.21
3.04–3.23
3.07–3.26
3.12–3.31
3.13–3.31
3.12–3.35
3.22–3.42
0–0
0.98–1.05
1.02–1.09
1.01–1.08
1.02–1.09
1.03–1.1
1.04–1.11
3.18–3.37
3.86–4.1
1.03–1.10
1.07–1.14
2.99–3.17
3.01–3.19
3.14–3.33
3.17–3.37

1000 Genomes Project Consortium (2010). A map of human genome
variation from population-scale sequencing. Nature 467, 1061–1073.

Summary
Droplet Digital PCR is a highly precise counting technology
for simultaneously measuring the concentrations of target andreference genes in DNA samples and for providing precise
and reproducible CNV values. It is easy to perform using
conventional TaqMan hydrolysis probe assays, and because
it directly counts the gene copies in a sample, it does not
require the use of a standard curve and is not plagued by
the PCR amplification irregularities that can contribute tomeasurement error in real-time PCR. Precise measurements
can be made from only a single ddPCR well if sufficient
target DNA is available. Running experiments in a convenient
96-well format makes ddPCR well-suited for high-throughput
studies profiling CNV frequencies in populations of interest(for example, for marker associations).

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duplications on HIV-1/AIDS susceptibility. Science 307, 1434–1440.
Hindson BJ et al. (2011). High-throughput droplet digital PCR system forabsolute quantitation of DNA copy number. Anal Chem 83, 8604–8610.
Hosono N et al. (2008). Multiplex PCR-based real-time invader assay(mPCR-RETINA): A novel SNP-based method for detecting allelic asymmetries
within copy number variation regions. Hum Mutat 29, 182–189.
Johansson I and Ingelman-Sundberg M (2008). CNVs of human genes andtheir implication in pharmacogenetics. Cytogenet Genome Res 123, 195–204.
Karlen Y et al. (2007). Statistical significance of quantitative PCR. BMC
Bioinformatics 8, 131.
Liu S et al. (2010). CCL3L1 copy number variation and susceptibility to HIV-1
infection: A meta-analysis. PLoS One 5, e15778.
Redon R et al. (2006). Global variation in copy number in the human genome.
Nature 444, 444–454.
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FAM and VIC are trademarks of Applera Corporation. TaqMan is a trademarkof Roche Molecular Systems, Inc. twin.tec is a trademark of Eppendorf AG.
Practice of the polymerase chain reaction (PCR) may require a license.

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