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High-Throughput Droplet Digital PCR System For Absolute PDF
High-Throughput Droplet Digital PCR System For Absolute PDF
High-Throughput Droplet Digital PCR System For Absolute PDF
pubs.acs.org/ac
bS Supporting Information
ABSTRACT: Digital PCR enables the absolute quantitation of nucleic
acids in a sample. The lack of scalable and practical technologies for
digital PCR implementation has hampered the widespread adoption of
this inherently powerful technique. Here we describe a high-throughput
droplet digital PCR (ddPCR) system that enables processing of ∼2
million PCR reactions using conventional TaqMan assays with a
96-well plate workflow. Three applications demonstrate that the massive
partitioning afforded by our ddPCR system provides orders of magni-
tude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number
variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background.
Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR
system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new
era of molecular diagnostics.
r 2011 American Chemical Society 8604 dx.doi.org/10.1021/ac202028g | Anal. Chem. 2011, 83, 8604–8610
Analytical Chemistry ARTICLE
Figure 2. Determination of copy number variation states by droplet digital PCR. (a) Measured copy number variation states in HapMap samples for
MRGPRX1, Chromosome X, CYP2D6, and (b) CCL3L1. (c) Correlation of measured copy number alterations of GRB7 and ERBB2 in DNA extracted
from normal and tumorous breast tissues. Each marker represents a CNV measurement from a single ddPCR well of ∼20 000 droplets. Error bars
indicate the Poisson 95% confidence intervals for each copy number determination.
template, specific cleavage of TaqMan probes generates a strong or extensive upfront assay optimization for new target panels.
fluorescence signal. On the basis of fluorescence amplitude, a CNV investigators using methods based on real-time PCR have
simple threshold assigns each droplet as positive or negative. As reported technical difficulty obtaining accurate copy number
the droplet volume is known, the fraction of positive droplets is measurements.22 Real-time PCR measurements are inherently
then used to calculate the absolute concentration of the target imprecise, and copy number estimates can drift between cases
sequence. For 20 000 droplets, the dynamic range for absolute and controls.
quantitation spans from a single copy up to ∼100 000 copies. For We measured the germline copy number variation of HapMap
human genomic DNA, this equates to an input DNA mass samples by ddPCR. Because increases in gene copy number are
ranging from 3.3 fg to 330 ng per 20 μL reaction. As templates often the result of tandem gene duplications, we used restriction
are randomly distributed across the droplet partitions, a Poisson enzymes to predictably and efficiently separate linked copies of
correction extends the dynamic range into the realm where on the target gene such that each sequence is encapsulated into its
average there are multiple copies per droplet. Statistical models own droplet and counted separately. Restriction enzymes were
are applied to calculate confidence limits of the concentration selected to cut either side of the amplicon sequences avoiding
estimates and their ratios.4,17 known mutation sites23 and methylation sensitivities. Physically
To demonstrate the immediate utility of this ddPCR system, shearing DNA using ultrasound or microfluidic devices is less
we present data on three application areas of increasing interest attractive as it reduces the amount of target that can be amplified
to researchers: determination of copy number variation (CNV), by PCR and can be challenging to implement in high-throughput
detection of rare alleles and the absolute quantitation of circulat- without specialized equipment. Preamplification, an alternative
ing DNA in cell-free plasma. Each application was selected to strategy for separation of linked copies24 has the potential to
highlight a distinct advantage that massive droplet partitioning introduce bias between the target and reference genes.
affords to digital PCR. For CNV, the large number of replicates Seven HapMap samples were screened for CNVs for three
provides sufficient precision to accurately measure copy number target genes. Each ddPCR reaction contained duplex TaqMan
states. For the detection of rare alleles, partitioning the target assay reagents for the target and reference genes. For MRGPRX1,
mutant DNA away from highly homologous wildtype DNA the copy number states from 1 up to 6 were completely resolved
increases sensitivity. Finally, droplet partitioning enables accurate from the results of a single well for each sample (Figure 2a).
quantitation of nucleic acids from clinical samples over a wide Lower CNV states for CYP2D6 and Chromosome X were also
dynamic range without external calibrators or endogenous controls. easily resolved, as shown. For 13 HapMap samples, our system
CNVs are deletions and amplifications of genome segments estimated the copy number of CCL3L1, a gene associated with
ranging from hundreds to millions of base pairs in length that HIV-1/AIDS susceptibility18 (Figure 2b). For DNA sample
have been implicated in a broad spectrum of human disease.18 NA18507, next-generation sequencing estimated the CCL3L1
Microarrays and the next-generation sequencing technologies copy number to be 5.725 whereas our ddPCR system estimated
have enabled and accelerated the discovery of new CNVs,19 6.05. The estimate of 5.7 is likely due to under-sampling since the
thereby further increasing the need for a high-throughput, low- billions of reads of a next-generation sequencing run are dis-
cost approach to making precise CNV measurements with tributed across the entire genome giving an average read-depth of
increased dynamic range for validation and follow-up studies. only 30. Thus, once target genes have been identified, greater
Although microarray technologies are valuable tools for CNV precision can readily be achieved with ddPCR since the number
discovery,20 they have limited dynamic range and are expensive of reads can be scaled almost arbitrarily. The current ddPCR
to scale to large numbers of samples for population studies. system can achieve read depths of up to 20 000 for two genes
Multiplex ligation-dependent probe amplification (MLPA)21 is from a single well. These data show that our ddPCR system is
an assay that allows resolution of deletions or duplications for up well suited for CNV population studies as it enables large
to 40 targets but requires selection from a predefined test menu numbers of samples to be tested against smaller sets of genes.
8606 dx.doi.org/10.1021/ac202028g |Anal. Chem. 2011, 83, 8604–8610
Analytical Chemistry ARTICLE
The second application demonstrates improved detection of droplets that collected in the droplet well were then manually
rare mutant alleles by drastically reducing competitive PCR pro- transferred with a multichannel pipet to a 96-well PCR plate. The
cesses that occur in the presence of a highly homologous wild- plate was heat-sealed with a foil seal and then placed on a
type DNA background. With careful optimization, real-time PCR conventional thermal cycler and amplified to the end-point
assays can detect down to the 1% mutant fraction. With the same (4055 cycles). After PCR, the 96-well PCR plate was loaded
assays, ddPCR partitions the competing background away from on the droplet reader (Bio-Rad), which automatically reads
the mutant, effectively increasing the average mutant-to-wild- the droplets from each well of the plate (32 wells/h). Analysis
type ratio by 20 000 times. On average, the effective enrichment of the ddPCR data was performed with QuantaSoft analysis
of the mutant molecules per PCR reaction is proportional to the software (Bio-Rad) that accompanied the droplet reader.
number of sample partitions used. For a duplex TaqMan assay Determination of Copy Number Variation in HapMap
targeting the BRAF V600E mutation,29 we show droplet parti- Samples. For MRGPRX1, ChromosomeX, and CYP2D6, 4.4 μg
tioning detects 0.001% mutant fraction, 1 000 times lower than of each purified human genomic DNA sample (Coriell) was
real-time PCR (Figure 3 and Supplementary Table 1 and Supple- digested with 10 units of RsaI (NEB) in 50 μL for 1 h at 37 °C.
mentary Figure 1 in the Supporting Information). With depen- The digest was diluted 8-fold to 400 μL with TE buffer (pH 8.0)
dence on the amount recovered from clinical samples, more then 33 ng (3 μL) was assayed per 20 μL ddPCR reaction. For
DNA can be loaded into the ddPCR system to push the detection CCL3L1, 815 ng of each purified human genomic DNA sample
limits down to even lower levels. This approach enables re- (Coriell) was digested with 7.5 units of MseI (NEB) in 10 μL for
searchers to measure extremely low levels of mutant that could in 1 h at 37 °C. The digest was diluted 3.5-fold to 35 μL with TE
turn lead to the improved detection of minimal residual disease buffer and then 69 ng (3 μL) was assayed per 20 μL ddPCR
and less invasive diagnostics. reaction. MRGPRX1 assay sequences36 were (forward primer)
We next evaluated the ability of this ddPCR system to quan- 50 -TTAAGCTTCATCAGTATCCCCCA-30 , (reverse primer)
titate DNA in clinical samples. Circulating DNA in cell-free 50 -CAAAGTAGGAAAACATCATCACAGGA-30 , and (probe)
plasma30 has received increasing levels of attention as a sample 6FAM-ACCATCTCTAAAATCCT-MGBNFQ. Chromosome X
type for developing noninvasive prenatal31 and oncology32 assay sequences37 were (forward primer) 50 -GATGAGGAAGG-
diagnostics. The cell-free DNA in plasma is highly fragmented33 CAATGATCC-30 , (reverse primer) 50 -TTGGCTTTTACCA-
and present at low levels, which present challenges for quantita- AATAGGG-30 , and (probe) 50 -FAM-TGTTTCTCTCTGCC-
tion. We enumerated fetal and total DNA in maternal cell-free TGCACTGG-BHQ1-30 (Integrated DNA Technologies). The
plasma. For 19 maternal plasma samples taken between 10 and CYPD2D6 (Hs00010001_cn) was purchased as a 20 premix of
20 weeks gestational age, the level of fetal (Figure 4a) and total primers and FAM-MGBNFQ probe (Applied Biosystems). Mod-
DNA (Figure 4b) were measured for both male and female ified CCL3L1 assay sequences19 were (forward primer) 50 -GGG-
fetuses. A selective methylation-sensitive digest enabled the low- TCCAGAAATACGTCAGT-30 , (reverse primer) 50 -CATGTT-
levels of hypermethylated RASSF1 fetal DNA34 to be accurately CCCAAGGCTCAG-30 , and (probe) 6FAM-TTCGAGGCC-
quantified using our ddPCR system. With an absolute measure of CAGCGACCTCA-MGBNFQ. All CNV assays were duplexed
SRY, RASSF1, and total DNA concentrations, the fetal load for with an RPP30 reference assay (forward primer) 50 -GATTTG-
each sample was calculated (Figure 4c). For male fetuses, a GACCTGCGAGCG-30 , (reverse primer) 50 -GCGGCTGTCT-
correlation of 93.7% between the hypermethylated RASSF1 fetal CCACAAGT-30 , and (probe) VIC-CTGACCTGAAGGCTCT-
DNA and SRY fetal loads provided confidence in the estimates MGBNFQ. Thermal cycling conditions were 95 °C 10 min
for female fetuses. On the basis of RASSF1 alone, fetal loads (1 cycle), 94 °C 30 s and 60 °C 60 s (40 cycles), 98 °C
ranged from 2.1 to 11.9% and were in general agreement with 10 min (1 cycle), and 12 °C hold.
those data collected by next-generation sequencing35 that is Determination of GRB7 and ERBB2 Copy Number Altera-
currently limited to estimating fetal loads from male fetuses. tions. Purified DNA (20 ng) from each normal and tumorous
This application demonstrates the capability of absolute quanti- breast tissue sample (D8235086-1, Biochain) was digested with
tation of highly fragmented cell-free DNA in clinical samples. 0.2 units of NlaIII in 10 μL for 1 h at 37 °C. The restricted DNA
Overall, these data show that ddPCR offers a practical was added directly to ddPCR Mastermix at 8.8 ng (4.4 μL) per
solution to realize precise estimates of DNA copy number 20 μL of ddPCR reaction. ERBB2 (Hs02803918_cn) and GRB7
with high-throughput. We anticipate this system will unlock (Hs02139994_cn) assays were purchased as 20 premixes of
the inherent power of digital PCR to more researchers for primers and FAM-MGBNFQ probe (Applied Biosystems) and
many applications. duplexed with the RPP30 reference assay described above.
Thermal cycling conditions were 95 °C 10 min (1 cycle),
’ EXPERIMENTAL SECTION 94 °C 30 s and 60 °C 60 s (40 cycles), 98 °C 10 min
(1 cycle), and 12 °C hold.
Droplet Digital PCR Workflow. The ddPCR workflow was Rare Allele Detection. A dilution series of BRAF V600E
described in Figure 1. The TaqMan PCR reaction mixture was mutant DNA (HTB-38D) from a HT-29 cell line (ATCC) was
assembled from a 2 ddPCR Mastermix (Bio-Rad), 20 primer, prepared in a high, constant background (5 000 copies/μL) of
and probes (final concentrations of 900 and 250 nM, respectively) wildtype DNA (NA19205, Coriell). For ddPCR, when the
and template (variable volume) in a final volume of 20 μL. Each concentration of intact human genomic DNA is >66 ng/20 μL
assembled ddPCR reaction mixture was then loaded into the reaction, the accompanying increase in viscosity can cause the
sample well of an eight-channel disposable droplet generator average droplet volume to change, which in turn could affect the
cartridge (Bio-Rad). A volume of 60 μL of droplet generation oil accuracy of DNA quantitation. Therefore, for samples of this
(Bio-Rad) was loaded into the oil well for each channel. The nature, restriction enzyme digestion is recommended to frag-
cartridge was placed into the droplet generator (Bio-Rad). The ment the DNA and reduce solution viscosity. In our experience,
cartridge was removed from the droplet generator, where the once fragmented, the human genomic DNA concentration can
8608 dx.doi.org/10.1021/ac202028g |Anal. Chem. 2011, 83, 8604–8610
Analytical Chemistry ARTICLE
exceed 1 μg/20 μL reaction without affecting the average droplet metawells were computed.4 The appropriate dilution factors
volume. Therefore, prior to ddPCR, each sample of the dilution were applied to yield total cell-free DNA concentration (G.E./mL)
series was digested with 40 U of HaeIII (NEB) in 100 μL con- and the confidence interval is scaled accordingly. The weighted
taining 1 NEB buffer 4 and BSA. The BRAF V600E/wildtype mean of the six total measurements was calculated, where
duplex TaqMan assay used common primers (forward) 50 -CTA- weights are inverses of confidence interval variances of these
CTGTTTTCCTTTACTTACTACACCTCAGA-30 , (reverse) measurements. For digested RASSF1, there are two independent
50 -ATCCAGACAACTGTTCAAACTGATG-30 , and specific assay measurements, which are also combined in the same
probes (BRAF V600E) 6FAM-TAGCTACAGAGAAATC-MG- manner. For SRY, there is one measurement that was used
BNFQ and (wildtype) VIC-CTAGCTACAGTGAAATC-MGB- directly, with scaling by a factor of 2 to account for haploidy.
NFQ. Eight ddPCR wells were used for each sample of the Fetal load is then computed as a ratio with the associated Poisson
dilution series. Thermal cycling conditions were 95 °C 10 min 95% confidence intervals.
(1 cycle), 94 °C 30 s and 62.7 °C 60 s (55 cycles), and
12 °C hold. ’ ASSOCIATED CONTENT
Quantitation of Cell-Free Fetal and Total DNA in Maternal
Plasma. Whole blood (3 10 mL) was collected (ProMedDx)
from healthy pregnant donors, between 10 and 20 weeks of
bS Supporting Information. Additional information as
noted in text. This material is available free of charge via the
gestational age, by venipuncture into cell-free DNA BCT tubes Internet at http://pubs.acs.org.
(Streck) according to the manufacturer’s instructions. Fetus
gender was determined by ultrasound within 6 weeks of sample ’ AUTHOR INFORMATION
collection. The tubes were stored for up to 48 h at room
temperature then shipped overnight at 4 °C to Bio-Rad where Corresponding Author
they were processed upon receipt. The whole blood was cen- *E-mail: ben_hindson@bio-rad.com.
trifuged for 10 min at 1 600g, the supernatant removed and
transferred to a new tube, centrifuged for 10 min at 16 000g, the
supernatant removed, and transferred to a new tube, then the ’ ACKNOWLEDGMENT
cell-free plasma was stored at 80 °C. Cell-free plasma (5 mL) The project described was supported by Grant Number
was thawed and cell-free DNA isolated using the QIAmp R01EB010106 from the National Institute of Biomedical Ima-
Circulating Nucleic Acid Kit (Qiagen) according to the manu- ging and Bioengineering. The content is solely the responsibility
factuer's protocol and eluted in AVE buffer (150 μL). A portion of the authors and does not necessarily represent the official
of the eluate (99 μL) was subjected to a single-tube digest views of the National Institute of Biomedical Imaging and
containing HhaI (30 U), HpaII (60 U), and BstUI (30 U) in 1 Bioengineering or the National Institutes of Health.
NEB buffer 4 in a total volume of 120 μL. A second portion of the
eluate (33 μL) was used in a no-digest control mixture where
restriction enzymes were substituted for water. The mixtures
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