The droplet digital PCR (ddPCR) workflow requires the following steps (Figure ): Eight assembled PCR reactions, each comprising template, ddPCR Mastermix and TaqMan reagents, are loaded into individual wells of a single-use injection molded cartridge. Next, droplet generation oil containing stabilizing surfactants is loaded and the cartridge 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. The surfactant-stabilized droplets flow to a collection well where they quickly concentrate due to density differences between the oil and aqueous phases, forming a packed bed above the excess oil. The densely packed droplets are pipet transferred to a conventional 96-well PCR plate and thermal cycled to end-point. After thermal cycling, the plate is transferred to a droplet reader. Here, droplets from each well are aspirated and streamed toward the detector where, en route, the injection of a spacer fluid separates and aligns them for single-file simultaneous two-color detection. TaqMan assays provide specific duplexed detection of target and reference genes. All droplets are gated based on detector peak width to exclude rare outliers (e.g., doublets, triplets). Each droplet has an intrinsic fluorescence signal resulting from the imperfect quenching of the fluorogenic probes enabling detection of negative droplets. For droplets that contain template, specific cleavage of TaqMan probes generates a strong fluorescence signal. On the basis of fluorescence amplitude, a simple threshold assigns each droplet as positive or negative. As the droplet volume is known, the fraction of positive droplets is then used to calculate the absolute concentration of the target sequence. For 20
000 droplets, the dynamic range for absolute quantitation spans from a single copy up to ~100
000 copies. For human genomic DNA, this equates to an input DNA mass ranging from 3.3 fg to 330 ng per 20 μL reaction. As templates are randomly distributed across the droplet partitions, a Poisson correction extends the dynamic range into the realm where on average there are multiple copies per droplet. Statistical models are applied to calculate confidence limits of the concentration estimates and their ratios.4,17
Figure 1 Droplet digital PCR workflow: (a) Samples and droplet generation oil are loaded into an eight-channel droplet generator cartridge. (b) A vacuum is applied to the droplet well, which draws sample and oil through a flow-focusing nozzle where monodisperse (more ...)
To demonstrate the immediate utility of this ddPCR system, we present 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. Each application was selected to highlight a distinct advantage that massive droplet partitioning affords to digital PCR. For CNV, the large number of replicates provides sufficient precision to accurately measure copy number states. For the detection of rare alleles, partitioning the target mutant DNA away from highly homologous wildtype DNA increases sensitivity. Finally, droplet partitioning enables accurate quantitation of nucleic acids from clinical samples over a wide dynamic range without external calibrators or endogenous controls.
CNVs are deletions and amplifications of genome segments ranging from hundreds to millions of base pairs in length that have been implicated in a broad spectrum of human disease.(18
) Microarrays and the next-generation sequencing technologies have enabled and accelerated the discovery of new CNVs,(19
) thereby further increasing the need for a high-throughput, low-cost approach to making precise CNV measurements with increased dynamic range for validation and follow-up studies. Although microarray technologies are valuable tools for CNV discovery,(20
) they have limited dynamic range and are expensive to scale to large numbers of samples for population studies. Multiplex ligation-dependent probe amplification (MLPA)(21
) is an assay that allows resolution of deletions or duplications for up to 40 targets but requires selection from a predefined test menu or extensive upfront assay optimization for new target panels. CNV investigators using methods based on real-time PCR have reported technical difficulty obtaining accurate copy number measurements.(22
) Real-time PCR measurements are inherently imprecise, and copy number estimates can drift between cases and controls.
We measured the germline copy number variation of HapMap samples by ddPCR. Because increases in gene copy number are often the result of tandem gene duplications, we used restriction enzymes to predictably and efficiently separate linked copies of the target gene such that each sequence is encapsulated into its own droplet and counted separately. Restriction enzymes were selected to cut either side of the amplicon sequences avoiding known mutation sites(23
) and methylation sensitivities. Physically shearing DNA using ultrasound or microfluidic devices is less attractive as it reduces the amount of target that can be amplified by PCR and can be challenging to implement in high-throughput without specialized equipment. Preamplification, an alternative strategy for separation of linked copies(24
) has the potential to introduce bias between the target and reference genes.
Seven HapMap samples were screened for CNVs for three target genes. Each ddPCR reaction contained duplex TaqMan assay reagents for the target and reference genes. For MRGPRX1
, the copy number states from 1 up to 6 were completely resolved from the results of a single well for each sample (Figure a). Lower CNV states for CYP2D6
and Chromosome X
were also easily resolved, as shown. For 13 HapMap samples, our system estimated the copy number of CCL3L1
, a gene associated with HIV-1/AIDS susceptibility(18
) (Figure b). For DNA sample NA18507, next-generation sequencing estimated the CCL3L1
copy number to be 5.7(25
) whereas our ddPCR system estimated 6.05. The estimate of 5.7 is likely due to under-sampling since the billions of reads of a next-generation sequencing run are distributed across the entire genome giving an average read-depth of only 30×. Thus, once target genes have been identified, greater precision can readily be achieved with ddPCR since the number of reads can be scaled almost arbitrarily. The current ddPCR system can achieve read depths of up to 20
000× for two genes from a single well. These data show that our ddPCR system is well suited for CNV population studies as it enables large numbers of samples to be tested against smaller sets of genes.
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 (more ...)
Sample heterogeneity can attenuate the measurement of copy number amplifications, which requires more precise measurements to discriminate smaller differences from normal. Somatic copy number alteration is the hallmark of many cancers. Without high-throughput technology for precise copy number quantitation, pathologists use fluorescent in situ hybridization (FISH) for diagnosing amplifications and deletions as this technique affords single-cell resolution. FISH and related techniques are expensive, laborious, and subject to large losses in performance due to other analytical factors.(26
) Specific amplifications define tumor subtypes and guide therapy. For example, Her2
positive breast tumors respond to Trastuzumab (Herceptin). For a set of normal and tumor breast tissue samples, the measured copy numbers of ERBB2
correlated with the exception of two samples that showed lower GRB7
amplification (Figure c). These results were expected as the GRB7
gene is part of the HER2
amplicon and is coamplified in almost all breast tumors with 17q11-21 amplification.27,28
This 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 foreseeably form the basis of more efficient diagnostic tests.
The second application demonstrates improved detection of rare mutant alleles by drastically reducing competitive PCR processes that occur in the presence of a highly homologous wild-type DNA background. With careful optimization, real-time PCR assays can detect down to the 1% mutant fraction. With the same assays, ddPCR partitions the competing background away from the mutant, effectively increasing the average mutant-to-wild-type ratio by 20
000 times. On average, the effective enrichment of the mutant molecules per PCR reaction is proportional to the number of sample partitions used. For a duplex TaqMan assay targeting the BRAF
) we show droplet partitioning detects 0.001% mutant fraction, 1
000 times lower than real-time PCR (Figure and Supplementary Table 1 and Supplementary Figure 1 in the Supporting Information
). With dependence on the amount recovered from clinical samples, more DNA can be loaded into the ddPCR system to push the detection limits down to even lower levels. This approach enables researchers to measure extremely low levels of mutant that could in turn lead to the improved detection of minimal residual disease and less invasive diagnostics.
Figure 3 Detection of the BRAF V600E rare mutant allele in the presence of homologous wildtype DNA by droplet digital PCR. Serial dilutions of the mutant cell line DNA were prepared in a constant background of wildtype human genomic DNA. Droplet partitioning reduces (more ...)
We next evaluated the ability of this ddPCR system to quantitate DNA in clinical samples. Circulating DNA in cell-free plasma(30
) has received increasing levels of attention as a sample type for developing noninvasive prenatal(31
) and oncology(32
) diagnostics. The cell-free DNA in plasma is highly fragmented(33
) and present at low levels, which present challenges for quantitation. We enumerated fetal and total DNA in maternal cell-free plasma. For 19 maternal plasma samples taken between 10 and 20 weeks gestational age, the level of fetal (Figure a) and total DNA (Figure b) were measured for both male and female fetuses. A selective methylation-sensitive digest enabled the low-levels of hypermethylated RASSF1
) to be accurately quantified using our ddPCR system. With an absolute measure of SRY
, and total DNA concentrations, the fetal load for each sample was calculated (Figure c). For male fetuses, a correlation of 93.7% between the hypermethylated RASSF1
fetal DNA and SRY
fetal loads provided confidence in the estimates for female fetuses. On the basis of RASSF1
alone, fetal loads ranged from 2.1 to 11.9% and were in general agreement with those data collected by next-generation sequencing(35
) that is currently limited to estimating fetal loads from male fetuses. This application demonstrates the capability of absolute quantitation of highly fragmented cell-free DNA in clinical samples.
Figure 4 Absolute quantitation of circulating fetal and maternal DNA from cell-free plasma for male and female fetuses. (a) Quantitation of fetal DNA concentration using SRY (red bar) and hypermethylated RASSF1 (blue bar). The RASSF1 gene of circulating fetal (more ...)
Overall, these data show that ddPCR offers a practical solution to realize precise estimates of DNA copy number with high-throughput. We anticipate this system will unlock the inherent power of digital PCR to more researchers for many applications.