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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/172—Haplotypes

Definitions

• the invention generally relates to droplet based digital PCR and methods for analyzing a target nucleic acid using the same.
• any abnormal nucleic acids containing mutations of interest are often present in small amounts (e.g., less than 1%) relative to a total amount of nucleic acid in the bodily fluid sample. This can result in a failure to detect the small amount of abnormal nucleic acid due to stochastic sampling bias.
• PCR and real-time PCR methodologies have greatly improved the analysis of nucleic acids from both throughput and quantitative perspectives. While traditional PCR techniques typically rely on end-point, and sometimes semi-quantitative, analysis of amplified DNA targets via agarose gel electrophoresis, real-time PCR (or qPCR) methods are geared toward accurately quantifying exponential amplification as the reaction progresses. qPCR reactions are monitored either using a variety of highly sequence specific fluorescent probe technologies, or by using non-specific DNA intercalating fluorogenic dyes.
• Digital PCR is an alternative quantitation method in which dilute samples are divided into many separate reactions. See for example, Brown et al. (U.S. patent numbers 6,143,496 and 6,391,559) and Vogelstein et al. (U.S. patent numbers 6,440,706, 6,753,147, and 7,824,889), the content of each of which is incorporated by reference herein in its entirety.
• the distribution from background of target DNA molecules among the reactions follows Poisson statistics, and at so called “terminal dilution" the vast majority of reactions contain either one or zero target DNA molecules for practical intents and purposes.
• the invention generally relates to the manipulation of nucleic acid in droplets, and in particular, nucleic acid amplification and detection.
• the invention provides a droplet that contains a single nucleic acid template and a plurality of primer pairs specific for multiple target sites on the template.
• the single nucleic acid template can be DNA (e.g., genomic DNA, cDNA, etc.) or RNA.
• the template is amplified in the droplet for detection; and may preferably be amplified using a plurality of primer pairs as described herein.
• the ability to amplify and detect single nucleic acids in droplets enables digital PCR, detection, counting, and differentiation among nucleic acids, especially those present in heterogeneous samples.
• the invention applies to digital amplification techniques and, in specific embodiments enables multiplex PCR in droplets.
• multiplexing primers in droplets enables the simultaneous increase in the number of PCR droplets while keeping the amount of input DNA the same or lower and generate the same or greater amplicon yield. This results in an overall increase in the amount of PCR positive droplets and amplicon yield without the consumption of more DNA. Even though the number of PCR primer pairs per droplet is greater than one, there is only one template molecule per droplet, and thus, in some
• Microfluidic droplets for multiplex analysis according to the invention contain a plurality of probes that hybridize to amplicons produced in the droplets.
• the droplet contains two or more probes, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 60, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 500, or more probes.
• Certain members of the plurality of probes include a detectable label.
• Members of the plurality of probes can each include the same detectable label, or a different detectable label.
• the detectable label is preferably a fluorescent label.
• the plurality of probes can include one or more groups of probes at varying concentrations.
• the one or more groups of probes can include the same detectable label which will vary in intensity upon detection, due to the varying probe concentrations.
• the droplets of the invention can further contain one or more reagents for conducting a polymerase chain reaction, such as a DNA or RNA polymerase, and/or dNTPs.
• the present invention additionally relates to a method for detecting a plurality of targets in a biological sample using digital PCR in microfluidic droplets.
• the sample may be a human tissue or body fluid.
• One or more droplets are formed, each containing a single nucleic acid template and a heterogeneous mixture of primer pairs and probes, each specific for multiple target sites on the template.
• a first fluid either continuous, or discontinuous as in droplets
• a second fluid also either continuous, or discontinuous as in droplets
• the second fluid can also contain reagents for conducting a PCR reaction, such as a polymerase and dNTPs.
• Certain members of the plurality of probes include a detectable label.
• Members of the plurality of probes can each include the same detectable label, or a different detectable label.
• the detectable label is preferably a fluorescent label.
• the plurality of probes can include one or more groups of probes at varying concentrations.
• the one or more groups of probes can include the same detectable label which varies in intensity upon detection, due to the varying probe concentrations.
• the first and second fluids can each be in droplet form. Any technique known in the art for forming droplets may be used with methods of the invention.
• An exemplary method involves flowing a stream of the sample fluid containing the nucleic acid template such that it intersects two opposing streams of flowing carrier fluid.
• the carrier fluid is immiscible with the sample fluid. Intersection of the sample fluid with the two opposing streams of flowing carrier fluid results in partitioning of the sample fluid into individual sample droplets containing the first fluid.
• the carrier fluid may be any fluid that is immiscible with the sample fluid.
• An exemplary carrier fluid is oil.
• the carrier fluid includes a surfactant, such as a fluorosurfactant.
• the same method may be applied to create individual droplets from the second fluid containing the primer pairs (and, in some implementations, the amplification reagents).
• Either the droplets containing the first fluid, the droplets containing the second fluid, or both, may be formed and then stored in a library for later merging, aspects of certain implementations of which are described in U.S. Patent Application Serial No. 12/504,764, hereby incorporated herein in its entirety for all purposes.
• droplets containing the first and second fluids can be merged to form single droplets containing the single nucleic acid template and heterogeneous mixture of primer pairs and probes. Merging can be accomplished, for example, in the presence of an electric field.
• both fluids be in the form of droplets when merging takes places.
• One exemplary method for merging of fluid portions with droplets is taught, for example, in co-pending U.S. Patent Application No. 61/441,985, filed on even date herewith.
• the nucleic acid template in each of the merged/formed droplets is amplified, e.g., by thermocycling the droplets under temperatures/conditions sufficient to conduct a PCR reaction.
• the resulting amplicons in the droplets can then be analyzed. For example, the presence of absence of the plurality of targets in the one or more droplets is detected optically, e.g., by the detectable label on the plurality of probes.
• the invention further relates to methods for analyzing a target nucleic acid. More particularly, methods of the invention are able to detect polymerase errors that occur during a PCR reaction and are able to exclude from analysis amplification products that are a result of a polymerase error. Methods of the invention are particularly useful in digital PCR where a polymerase error may result in a partitioned section of sample being incorrectly identified as containing a mutant allele, i.e., a false positive. Such false positives greatly impact the validity and precision of digital PCR results. Methods of the invention are able to uniquely detect multiple targets with the same optical color. Methods of the invention are particularly useful in digital PCR where it is desirable to identify multiple different target molecules that may be present in the starting test fluid.
• Methods of the invention involve forming sample droplets containing target nucleic acid.
• methods of the invention comprise forming droplets for digital PCR.
• Preferred digital PCR droplets contain one copy of a nucleic acid to be amplified, although they may contain multiple copies of the same nucleic acid sequence.
• Any technique known in the art for forming sample droplets may be used with methods of the invention.
• One exemplary method involves flowing a stream of sample fluid including nucleic acids such that it intersects two opposing streams of flowing carrier fluid. The carrier fluid is immiscible with the sample fluid.
• the carrier fluid may be any fluid that is immiscible with the sample fluid.
• An exemplary carrier fluid is oil.
• the carrier fluid includes a surfactant, such as a fluorosurfactant.
• the targets are then amplified in the droplets.
• Any method known in the art may be used to amplify the target nucleic acids either linearly or exponentially.
• a preferred method is the polymerase chain reaction (PCR).
• PCR polymerase chain reaction
• any amplification technique commonly known in the art may be implemented such as rolling circle amplification, isothermal amplification, or any combination of amplification methods using loci specific primers, nested- primers, or random primers (such primers, and/or primers used for PCR, are included in the term "amplification reagents").
• One method to exclude droplets that contain a heterogeneous population of amplicons from droplets that contain a homogeneous population of amplicons includes hybridizing detectably-labeled probes to the amplicons, flowing the droplets through a microfluidic channel, and excluding those droplets in which both amplicon from the target and amplicon from a variant of the target are detected.
• analyzing the droplets involves determining a number of droplets that contain only wild-type target, and determining a number of droplets that contain only a variant of the target. Generally, the presence of droplets containing only the variant is indicative of a disease, such as cancer.
• the variant may be an allelic variant.
• An exemplary allelic variant is a single nucleotide polymorphism.
• the variant may also be a specific haplotype. Haplotypes refer to the presence of two or more variants on the same nucleic acid strand.
• Haplotypes can be more informative or predictive than genotypes when used to determine such things as the presence or severity of disease, response to drug therapy or drug resistance of bacterial or viral infections. Because each droplet contains only one template strand it is an ideal vessel for the determination of haplotypes.
• the detection of two or more variants in a single droplet that contains a single intact nucleic acid strand identifies the haplotype of the variants on that strand.
• the presence of two or more markers in the same droplet can be identified by such methods as the presence of dyes of multiple colors or the increase in the intensity of a single dye or a combination of both. Any method that allows the identification of multiple variants in a single droplet enables the determination of a samples haplotype.
• a method for analyzing a target nucleic acid that includes compartmentalizing a first fluid into portions, each portion containing a single target nucleic acid; amplifying the target in the portions; excluding portions containing amplicon from the target and amplicon from a variant of the target; and analyzing target amplicons.
• the invention generally provides methods for detecting a recurrence of a cancer in a patient. Those methods may involve forming sample droplets containing a single target nucleic acid derived from a patient sample, flowing the sample droplets through a channel, amplifying the target in the droplets, detecting amplified target in the droplets, excluding droplets including a heterogeneous population of amplicons, and analyzing non-excluded droplets to determine the presence of mutant alleles indicative of recurrence.
• the analyzing step includes capturing amplicon obtained from the droplets using labeled capture probes.
• the sample may be a human tissue or body fluid.
• Exemplary body fluids are pus, sputum, semen, urine, blood, saliva, stool, and cerebrospinal fluid.
• Exemplary body fluids are pus, sputum, semen, urine, blood, saliva, stool, and cerebrospinal fluid.
• Such methods may also be practiced using fluids compartmentalized in containers other than or in addition to droplets.
• Figure 1 depicts a droplet formation device.
• Figure 2 depicts a portion of the droplet formation device of Figure 1.
• Figure 3 depicts an exemplary microfluidic system for droplet generation and readout.
• Figure 3a depicts the droplet generation chip;
• Figure 3b depicts the droplet spacing for readout;
• Figure 3c depicts a cartoon of droplet readout by fluorescence.
• Figure 4 depicts the serial dilution of template DNA quantified by dPCR.
• Figure 4a shows droplet fluorescence during readout for the most concentrated sample. Each discrete burst of fluorescence corresponded to an individual droplet. Two different groups of droplets were evident: PCR(+) droplets peaking at -0.8 V and PCR(-) droplets at -0.1 V;
• Figure 4b shows a histogram of the peak fluorescence intensities of droplets from the complete data trace in (a).
• PCR(+) and PCR(-) droplets appeared as two very distinct populations centered at 0.78 and 0.10 V, respectively;
• Figure 5A is a schematic representation of a droplet having 5 sets of primers for PCR amplification of a template sequence and 5 probes, each labeled with a fluorescent dye, that binds specifically to the amplified sequences;
• Figure 5B is a time trace of fluorescence intensity detected from droplets after PCR amplification;
• Figure 5C is a scatter plot showing clusters representing droplets that contain specific amplified sequences (TERT, RNaseP, El a, SMNl and SMN2).
• Figure 6A is a schematic representation of a droplet having 5 sets of primers for PCR amplification of a template sequence and 5 probes, each labeled with a fluorescent dye, that binds specifically to the amplified sequences;
• Figure 6B is a scatter plot showing clusters representing droplets that contain specific amplified sequences (TERT, 815A, RNaseP, El a, and 815G);
• Figure 6C is a table showing the copy number of specific sequences shown in Figure 6B.
• Figure 7 is a schematic depicting one-color detection of a genetic sequence with a microfluidic device.
• Figure 8 is a schematic depicting two-color detection of two genetic sequences with a microfluidic device.
• Figure 9 is a schematic depicting two-color detection of three genetic sequences with a microfluidic device.
• Figure 10 shows two dot plots depicting clusters of genetic sequences detected through fluorescence intensity.
• Left panel is a dot plot showing four clusters.
• Block for SMNl sequence was present.
• Top left microdroplets containing the reference sequence (SMARCC1); bottom left: microdroplets not containing any sequence; bottom middle: microdroplets containing sequence for SMN1; and bottom right: microdroplets containing sequence for SMN2.
• Right panel is a dot plot showing four clusters. No block for SMN1 sequence was present.
• the shift of the bottom middle cluster in right panel as compared to left panel confirms that fluorescence intensity provides a very sensitive measurement for the presence of a sequence.
• Figure 11 depicts histograms of a duplex gene copy number assay using only one type of fluorophore by digital PCR;
• Figure 11a depicts a histogram of droplet peak fluorescence intensities;
• Figure l ib shows a comparison of gene copy numbers measured by monochromatic dPCR.
• Figure 12 is a schematic for tuning the intensity of a detectable label to a particular target with a microfluidic device.
• Figure 14 depicts a 5-plex dPCR assay for spinal muscular atrophy with only two fluorophores.
• Figure 14a is a 2D histogram of droplet fluorescence intensities, shown as a heat map, for the 5-plex assay against the synthetic model chromosome for validation. The six well resolved droplet populations corresponded to the five individual assays plus the empty droplets;
• Figure 14b shows the results of the SMA pilot study.
• Figure 15 depicts a 9-plex dPCR assay for spinal muscular atrophy with only two fluorophores, showing the process of optimizing droplet intensities.
• Figure 16 depicts an optical schematic for combining optical labels with multiplexing.
• Figure 17 depicts a dPCR assay combining multiplexing with optical labels using co-flow microfluidic s. The contributions from all droplets are shown, that is, from three different triplex assays.
• 2-D histograms shown as heat maps with hotter colors representing higher droplet counts.
• Hist panel histogram of optical labels, i.e. fluorescence intensities of droplets measured at wavelengths for the two fluorophores comprising the optical labels.
• (Right panel) assay histogram i.e. fluorescence intensities of droplets measured at wavelengths suitable for FAM detection (x-axis), and VIC detection (y-axis). Both histograms were compensated for spectral overlap by standard techniques.
• Figure 18 shows single assay selections using optical labels. Selections were taken from all of the droplets from Figure 17. Each of the three different selections in panels A-C were for optical labels encoding the same assay (TERT, SMNl, and SMN2). Histograms are as described in Figure 17. (Left histograms, optical labels) Superimposed lines demark the bounding box for selecting a single optical label. (Right histograms, assay) Only droplets containing the selected optical label are displayed.
• Figure 19 shows single assay selections using optical labels. Selections were taken from all of the droplets from Figure 17. Each of the three different selections in panels A-C was for optical labels encoding the same assay (TERT, c.5C from SMNl, and BCKDHA). Histograms are as described in Figure 17. (Left histograms, optical labels) Superimposed lines demark the bounding box for selecting a single optical label. (Right histograms, assay) Only droplets containing the selected optical label are displayed.
• Figure 20 shows single assay selections using optical labels. Selections were taken from all of the droplets from Figure 17. Each of the three different selections in panels A-C was for optical labels encoding the same assay (TERT, C.88G from SMNl, and RNaseP). Histograms are as described in Figure 17. (Left histograms, optical labels) Superimposed lines demark the bounding box for selecting a single optical label. (Right histograms, assay) Only droplets containing the selected optical label are displayed.
• Figure 21 depicts a dPCR assay combining multiplexing with optical labels using droplet merging.
• Figure 22 is a schematic showing haplotype detection in droplets.
• the invention provides materials and methods for analysis of biomolecules.
• the invention provides for digital analysis in droplets, such as microfluidic droplets.
• the invention allows digital PCR to be conducted and provides for significantly reduced or eliminated errors.
• digital PCR is performed in aqueous droplets separated by oil using a microfluidics system.
• the oil is a fluorinated oil such as the Fluorinert oils (3M).
• the fluorinated oil contains a surfactant, such as PFPE-PEG-PFPE triblock copolymer, to stabilize the droplets against coalescence during the amplification step or at any point where they contact each other.
• Microfluidic approaches allow the rapid generation of large numbers (e.g. 10 6 or greater) of very uniformly sized droplets that function as picoliter volume reaction vessels (see reviews of droplet-based microfluidics).
• the invention is not limited to dPCR performed in water-in-oil emulsions, but rather is general to all methods of reaction compartmentalization for dPCR.
• the invention is described in terms of the use of droplets for compartmentalization, but it is understood that this choice of description is not limiting for the invention, and that all of the methods of the invention are compatible with all other methods of reaction
• Methods of the invention involve novel strategies for performing multiple different amplification reactions on the same sample simultaneously to quantify the abundance of multiple different DNA targets, commonly known to those familiar with the art as "multiplexing".
• Methods of the invention for multiplexing dPCR assays promise greater plexity—the number of simultaneous reactions— than possible with existing qPCR or dPCR techniques. It is based on the singular nature of amplifications at terminal or limiting dilution that arises because most often only a single target allele is ever present in any one droplet even when multiple
• primers/probes targeting different alleles are present. This alleviates the complications that otherwise plague simultaneous competing reactions, such as varying arrival time into the exponential stage and unintended interactions between primers.
• the invention provides materials and methods for improving amplicon yield while maintaining the sensitivity and specificity in droplet based digital PCR. More specifically, the invention provides droplets containing a single nucleic acid template and multiplexed PCR primers and methods for detecting a plurality of targets in a biological sample by forming such droplets and amplifying the nucleic acid templates using droplet based digital PCR.
• adding multiple colors increases the number of possible reactions geometrically, rather than linearly as with qPCR, because individual reactions can be labeled with multiple fluorophores.
• fluorophores VIC and FAM
• VOC and FAM two fluorophores
• Methods of the invention are able to detect polymerase errors that occur during an amplification reaction and are able to exclude from analysis those products that are a result of polymerase errors. In essence, methods of the invention increase the sensitivity of digital PCR by identifying amplification products that are false positives, and excluding those products from analysis.
• Methods of the invention involve forming sample droplets containing a single target nucleic acid, amplifying the target in the droplets, excluding droplets containing amplicon from the target and amplicon from a variant of the target, and analyzing target amplicons.
• Nucleic acid molecules include deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA). Nucleic acid molecules can be synthetic or derived from naturally occurring sources. In one embodiment, nucleic acid molecules are isolated from a biological sample containing a variety of other components, such as proteins, lipids and non-template nucleic acids. Nucleic acid template molecules can be obtained from any cellular material, obtained from an animal, plant, bacterium, fungus, or any other cellular organism. In certain embodiments, the nucleic acid molecules are obtained from a single cell. Biological samples for use in the present invention include viral particles or preparations.
• Nucleic acid molecules can be obtained directly from an organism or from a biological sample obtained from an organism, e.g., from blood, urine, cerebrospinal fluid, seminal fluid, saliva, sputum, stool and tissue. Any tissue or body fluid specimen may be used as a source for nucleic acid for use in the invention. Nucleic acid molecules can also be isolated from cultured cells, such as a primary cell culture or a cell line. The cells or tissues from which template nucleic acids are obtained can be infected with a virus or other intracellular pathogen. A sample can also be total RNA extracted from a biological specimen, a cDNA library, viral, or genomic DNA.
• the nucleic acid molecules are bound as to other target molecules such as proteins, enzymes, substrates, antibodies, binding agents, beads, small molecules, peptides, or any other molecule and serve as a surrogate for quantifying and / or detecting the target molecule.
• target molecules such as proteins, enzymes, substrates, antibodies, binding agents, beads, small molecules, peptides, or any other molecule and serve as a surrogate for quantifying and / or detecting the target molecule.
• nucleic acid can be extracted from a biological sample by a variety of techniques such as those described by Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., pp. 280-281 (1982). Nucleic acid molecules may be single-stranded, double-stranded, or double- stranded with single- stranded regions (for example, stem- and loop- structures).
• Methods of the invention involve forming sample droplets where some droplets contain zero target nucleic acid molecules, some droplets contain one target nucleic acid molecule, and some droplets may or may not contain multiple nucleic acid molecules (corresponding to limiting or terminal dilution, respectively, as defined above).
• the distribution of molecules within droplets obeys the Poisson distribution.
• methods for non-Poisson loading of droplets are known to those familiar with the art, and include but are not limited to active sorting of droplets, such as by laser-induced fluorescence, or by passive one-to- one loading.
• the description that follows assumes Poisson loading of droplets, but such description is not intended to exclude non-Poisson loading, as the invention is compatible with all distributions of DNA loading that conform to limiting or terminal dilution.
• the droplets are aqueous droplets that are surrounded by an immiscible carrier fluid. Methods of forming such droplets are shown for example in Link et al. (U.S. patent application numbers 2008/0014589, 2008/0003142, and 2010/0137163), Stone et al. (U.S. patent number 7,708,949 and U.S. patent application number 2010/0172803), Anderson et al. (U.S. patent number 7,041,481 and which reissued as RE41,780) and European publication number
• Figure 1 shows an exemplary embodiment of a device 100 for droplet formation.
• Device 100 includes an inlet channel 101, and outlet channel 102, and two carrier fluid channels 103 and 104. Channels 101, 102, 103, and 104 meet at a junction 105.
• Inlet channel 101 flows sample fluid to the junction 105.
• Carrier fluid channels 103 and 104 flow a carrier fluid that is immiscible with the sample fluid to the junction 105.
• Inlet channel 101 narrows at its distal portion wherein it connects to junction 105 (See Figure 2).
• Inlet channel 101 is oriented to be perpendicular to carrier fluid channels 103 and 104. Droplets are formed as sample fluid flows from inlet channel 101 to junction 105, where the sample fluid interacts with flowing carrier fluid provided to the junction 105 by carrier fluid channels 103 and 104.
• Outlet channel 102 receives the droplets of sample fluid surrounded by carrier fluid.
• the sample fluid is typically an aqueous buffer solution, such as ultrapure water (e.g., 18 mega-ohm resistivity, obtained, for example by column chromatography), 10 mM Tris HCl and 1 mM EDTA (TE) buffer, phosphate buffer saline (PBS) or acetate buffer. Any liquid or buffer that is physiologically compatible with nucleic acid molecules can be used.
• the carrier fluid is one that is immiscible with the sample fluid.
• the carrier fluid can be a non-polar solvent, decane (e g., tetradecane or hexadecane), fluorocarbon oil, silicone oil or another oil (for example, mineral oil).
• the carrier fluid contains one or more additives, such as agents which increase, reduce, or otherwise create non-Newtonian surface tensions (surfactants) and/or stabilize droplets against spontaneous coalescence on contact.
• Surfactants can include Tween, Span, fluoro surfactants, and other agents that are soluble in oil relative to water.
• performance is improved by adding a second surfactant, or other agent, such as a polymer or other additive, to the sample fluid.
• Surfactants can aid in controlling or optimizing droplet size, flow and uniformity, for example by reducing the shear force needed to extrude or inject droplets into an intersecting channel. This can affect droplet volume and periodicity, or the rate or frequency at which droplets break off into an intersecting channel.
• the surfactant can serve to stabilize aqueous emulsions in fluorinated oils from coalescing.
• the droplets may be coated with a surfactant or a mixture of surfactants.
• Preferred surfactants that may be added to the carrier fluid include, but are not limited to, surfactants such as sorbitan-based carboxylic acid esters (e.g., the "Span” surfactants, Fluka Chemika), including sorbitan monolaurate (Span 20), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span 60) and sorbitan monooleate (Span 80), and perfluorinated polyethers (e.g., DuPont Krytox 157 FSL, FSM, and/or FSH).
• surfactants such as sorbitan-based carboxylic acid esters (e.g., the "Span” surfactants, Fluka Chemika), including sorbitan monolaurate (Span 20), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span 60) and sorbitan monooleate (Span
• non-ionic surfactants which may be used include polyoxyethylenated alkylphenols (for example, nonyl-, p-dodecyl-, and dinonylphenols), polyoxyethylenated straight chain alcohols,
• polyoxyethylenated polyoxypropylene glycols polyoxyethylenated mercaptans, long chain carboxylic acid esters (for example, glyceryl and polyglycerl esters of natural fatty acids, propylene glycol, sorbitol, polyoxyethylenated sorbitol esters, polyoxyethylene glycol esters, etc.) and alkanolamines (e.g., diethanolamine-fatty acid condensates and isopropanolamine-fatty acid condensates).
• carboxylic acid esters for example, glyceryl and polyglycerl esters of natural fatty acids, propylene glycol, sorbitol, polyoxyethylenated sorbitol esters, polyoxyethylene glycol esters, etc.
• alkanolamines e.g., diethanolamine-fatty acid condensates and isopropanolamine-fatty acid condensates.
• the carrier fluid may be caused to flow through the outlet channel so that the surfactant in the carrier fluid coats the channel walls.
• the fluoro surfactant can be prepared by reacting the perflourinated polyether DuPont Krytox 157 FSL, FSM, or FSH with aqueous ammonium hydroxide in a volatile fluorinated solvent. The solvent and residual water and ammonia can be removed with a rotary evaporator. The surfactant can then be dissolved (e.g., 2.5 wt %) in a fluorinated oil (e.g., Flourinert (3M)), which then serves as the carrier fluid.
• a fluorinated oil e.g., Flourinert (3M)
• a device called a lambda injector involves forming a droplet, and contacting the droplet with a fluid stream, in which a portion of the fluid stream integrates with the droplet to form a mixed droplet. In this approach, only one phase needs to reach a merge area in a form of a droplet. Further description of such method is shown in the co-owned and co-pending U.S. patent application to Yurkovetsky, et al. (U.S. patent application serial number 61/441,985), the content of which is incorporated y reference herein in its entirety.
• a droplet is formed as described above. After formation of the sample droplet from the first sample fluid, the droplet is contacted with a flow of a second sample fluid stream. Contact between the droplet and the fluid stream results in a portion of the fluid stream integrating with the droplet to form a mixed droplet.
• the droplets of the first sample fluid flow through a first channel separated from each other by immiscible carrier fluid and suspended in the immiscible carrier fluid.
• the droplets are delivered to the merge area, i.e., junction of the first channel with the second channel, by a pressure-driven flow generated by a positive displacement pump. While droplet arrives at the merge area, a bolus of a second sample fluid is protruding from an opening of the second channel into the first channel.
• the channels are oriented perpendicular to each other. However, any angle that results in an intersection of the channels may be used.
• the bolus of the second sample fluid stream continues to increase in size due to pumping action of a positive displacement pump connected to channel, which outputs a steady stream of the second sample fluid into the merge area.
• the flowing droplet containing the first sample fluid eventually contacts the bolus of the second sample fluid that is protruding into the first channel. Contact between the two sample fluids results in a portion of the second sample fluid being segmented from the second sample fluid stream and joining with the first sample fluid droplet to form a mixed droplet.
• each incoming droplet of first sample fluid is merged with the same amount of second sample fluid.
• an electric charge is applied to the first and second sample fluids.
• Description of applying electric charge to sample fluids is provided in Link et al. (U.S. patent application number 2007/0003442) and European Patent Number EP2004316 to
• Electric charge may be created in the first and second sample fluids within the carrier fluid using any suitable technique, for example, by placing the first and second sample fluids within an electric field (which may be AC, DC, etc.), and/or causing a reaction to occur that causes the first and second sample fluids to have an electric charge, for example, a chemical reaction, an ionic reaction, a photocatalyzed reaction, etc.
• an electric field which may be AC, DC, etc.
• the electric field in some embodiments, is generated from an electric field generator, i.e., a device or system able to create an electric field that can be applied to the fluid.
• the electric field generator may produce an AC field (i.e., one that varies periodically with respect to time, for example, sinusoidally, sawtooth, square, etc.), a DC field (i.e., one that is constant with respect to time), a pulsed field, etc.
• the electric field generator may be constructed and arranged to create an electric field within a fluid contained within a channel or a microfluidic channel.
• the electric field generator may be integral to or separate from the fluidic system containing the channel or microfluidic channel, according to some embodiments.
• an electric field is produced by applying voltage across a pair of electrodes, which may be positioned on or embedded within the fluidic system (for example, within a substrate defining the channel or microfluidic channel), and/or positioned proximate the fluid such that at least a portion of the electric field interacts with the fluid.
• the electrodes can be fashioned from any suitable electrode material or materials known to those of ordinary skill in the art, including, but not limited to, silver, gold, copper, carbon, platinum, copper, tungsten, tin, cadmium, nickel, indium tin oxide (“ITO”), etc., as well as combinations thereof. In some cases, transparent or substantially transparent electrodes can be used.
• the electric field facilitates rupture of the interface separating the second sample fluid and the droplet. Rupturing the interface facilitates merging of bolus of the second sample fluid and the first sample fluid droplet.
• the forming mixed droplet continues to increase in size until it a portion of the second sample fluid breaks free or segments from the second sample fluid stream prior to arrival and merging of the next droplet containing the first sample fluid.
• the segmenting of the portion of the second sample fluid from the second sample fluid stream occurs as soon as the shear force exerted on the forming mixed droplet by the immiscible carrier fluid overcomes the surface tension whose action is to keep the segmenting portion of the second sample fluid connected with the second sample fluid stream.
• the now fully formed mixed droplet continues to flow through the first channel.
• the rupture of the interface can be spontaneous, or the rupture can be facilitated by surface chemistry.
• the invention is not limited in regard to the method of rupture at the interface, as rupture can be brought about by any means.
• the first sample fluid contains nucleic acid templates. Droplets of the first sample fluid are formed as described above. Those droplets will include the nucleic acid templates. In certain embodiments, the droplets will include only a single nucleic acid template, and thus digital PCR can be conducted.
• the second sample fluid contains reagents for the PCR reaction. Such reagents generally include Taq polymerase, deoxynucleotides of type A, C, G and T, magnesium chloride, and forward and reverse primers, all suspended within an aqueous buffer.
• the second fluid also includes detectably labeled probes for detection of the amplified target nucleic acid, the details of which are discussed below.
• a droplet containing the nucleic acid is then caused to merge with the PCR reagents in the second fluid as described above, producing a droplet that includes Taq polymerase, deoxynucleotides of type A, C, G and T, magnesium chloride, forward and reverse primers, detectably labeled probes, and the target nucleic acid.
• the first fluid can contain the template DNA and PCR master mix (defined below), and the second fluid can contain the forward and reverse primers and the probe.
• the invention is not restricted in any way regarding the constituency of the first and second fluidics for PCR or digital PCR.
• the template DNA is contained in the second fluid inside droplets.
• Methods of the invention further involve amplifying the target nucleic acid in each droplet.
• Amplification refers to production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction or other technologies well known in the art (e.g., Dieffenbach and Dveksler, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y. [1995]).
• the amplification reaction may be any amplification reaction known in the art that amplifies nucleic acid molecules, such as polymerase chain reaction, nested polymerase chain reaction, ligase chain reaction (Barany F. (1991) PNAS 88: 189-193; Barany F.
• the amplification reaction is the polymerase chain reaction.
• Polymerase chain reaction refers to methods by K. B. Mullis (U.S. patent numbers 4,683,195 and 4,683,202, hereby incorporated by reference) for increasing concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification.
• the process for amplifying the target sequence includes introducing an excess of oligonucleotide primers to a DNA mixture containing a desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase.
• the primers are complementary to their respective strands of the double stranded target sequence.
• primers are annealed to their complementary sequence within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands.
• the steps of denaturation, primer annealing and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one cycle; there can be numerous cycles) to obtain a high concentration of an amplified segment of a desired target sequence.
• the length of the amplified segment of the desired target sequence is determined by relative positions of the primers with respect to each other and by cycling parameters, and therefore, this length is a controllable parameter.
• the sample droplet may be pre-mixed with a primer or primers, or the primer or primers may be added to the droplet.
• droplets created by segmenting the starting sample are merged with a second set of droplets including one or more primers for the target nucleic acid in order to produce final droplets.
• the merging of droplets can be accomplished using, for example, one or more droplet merging techniques described for example in Link et al. (U.S. patent application numbers 2008/0014589, 2008/0003142, and 2010/0137163) and European publication number EP2047910 to Raindance Technologies Inc.
• a first droplet formation module produces the sample droplets consistent with limiting or terminal dilution of target nucleic acid.
• a second droplet formation or reinjection module inserts droplets that contain reagents for a PCR reaction.
• Such droplets generally include the "PCR master mix” (known to those in the art as a mixture containing at least Taq polymerase, deoxynucleotides of type A, C, G and T, and magnesium chloride) and forward and reverse primers (known to those in the art collectively as "primers”), all suspended within an aqueous buffer.
• PCR master mix known to those in the art as a mixture containing at least Taq polymerase, deoxynucleotides of type A, C, G and T, and magnesium chloride
• primers forward and reverse primers
• the second droplet also includes detectably labeled probes for detection of the amplified target nucleic acid, the details of which are discussed below.
• detectably labeled probes for detection of the amplified target nucleic acid, the details of which are discussed below.
• Different arrangements of reagents between the two droplet types is envisioned.
• the template droplets also contain the PCR master mix, but the primers and probes remain in the second droplets. Any arrangement of reagents and template DNA can be used according to the invention.
• Primers can be prepared by a variety of methods including but not limited to cloning of appropriate sequences and direct chemical synthesis using methods well known in the art (Narang et al., Methods Enzymol., 68:90 (1979); Brown et al., Methods Enzymol., 68: 109 (1979)). Primers can also be obtained from commercial sources such as Operon Technologies, Amersham Pharmacia Biotech, Sigma, and Life Technologies. The primers can have an identical melting temperature. The lengths of the primers can be extended or shortened at the 5' end or the 3' end to produce primers with desired melting temperatures. Also, the annealing position of each primer pair can be designed such that the sequence and, length of the primer pairs yield the desired melting temperature.
• Another method for determining the melting temperature of primers is the nearest neighbor method
• Computer programs can also be used to design primers, including but not limited to Array Designer Software (Arrayit Inc.), Oligonucleotide Probe Sequence Design Software for Genetic Analysis (Olympus Optical Co.), NetPrimer, and DNAsis from Hitachi Software Engineering.
• the TM (melting or annealing temperature) of each primer is calculated using software programs such as Oligo Design, available from Invitrogen Corp.
• the droplet formation modules are arranged and controlled to produce an interdigitation of sample droplets and PCR reagent droplets flowing through a channel.
• Such an arrangement is described for example in Link et al. (U.S. patent application numbers 2008/0014589, 2008/0003142, and 2010/0137163) and European publication number EP2047910 to Raindance Technologies Inc.
• a sample droplet is then caused to merge with a PCR reagent droplet, producing a droplet that includes the PCR master mix, primers, detectably labeled probes, and the target nucleic acid.
• Droplets may be merged for example by: producing dielectrophoretic forces on the droplets using electric field gradients and then controlling the forces to cause the droplets to merge;
• a single droplet formation module, or a plurality of droplet formation modules are arranged to produce droplets from a mixture already containing the template DNA, the PCR master mix, primers, and detectably labeled probes.
• co-flow upstream from a single droplet formation module two channels intersect allowing two flow streams to converge.
• One flow stream contains one set of reagents and the template DNA, and the other contains the remaining reagents.
• the template DNA and the PCR master mix are in one flow stream, and the primers and probes are in the other.
• the invention is not limited in regard to the constituency of either flow stream.
• one flow stream contains just the template DNA, and the other contains the PCR master mix, the primers, and the probes.
• the flow streams may or may not mix before the droplet generation nozzle.
• some amount of fluid from the first stream, and some amount of fluid from the second stream are encapsulated within a single droplet. Following encapsulation, complete mixing occurs.
• the droplets are thermal cycled, resulting in amplification of the target nucleic acid in each droplet.
• the droplets are collected off- chip as an emulsion in a PCR thermal cycling tube and then thermally cycled in a conventional thermal cycler. Temperature profiles for thermal cycling can be adjusted and optimized as with any conventional DNA amplification by PCR.
• the droplets are flowed through a channel in a serpentine path between heating and cooling lines to amplify the nucleic acid in the droplet.
• the width and depth of the channel may be adjusted to set the residence time at each temperature, which can be controlled to anywhere between less than a second and minutes.
• the three temperature zones are used for the amplification reaction.
• the three temperature zones are controlled to result in denaturation of double stranded nucleic acid (high temperature zone), annealing of primers (low temperature zones), and amplification of single stranded nucleic acid to produce double stranded nucleic acids
• intermediate temperature zones The temperatures within these zones fall within ranges well known in the art for conducting PCR reactions. See for example, Sambrook et al. (Molecular
• the three temperature zones are controlled to have temperatures as follows: 95°C (T H ), 55°C (T L ), 72°C (T M ).
• the prepared sample droplets flow through the channel at a controlled rate.
• the sample droplets first pass the initial denaturation zone (T R ) before thermal cycling.
• the initial preheat is an extended zone to ensure that nucleic acids within the sample droplet have denatured successfully before thermal cycling.
• the requirement for a preheat zone and the length of denaturation time required is dependent on the chemistry being used in the reaction.
• the samples pass into the high temperature zone, of approximately 95°C, where the sample is first separated into single stranded DNA in a process called denaturation.
• the sample then flows to the low temperature, of approximately 55°C, where the hybridization process takes place, during which the primers anneal to the complementary sequences of the sample.
• the third medium temperature of approximately 72°C, the polymerase process occurs when the primers are extended along the single strand of DNA with a thermostable enzyme.
• Methods for controlling the temperature in each zone may include but are not limited to electrical resistance, peltier junction, microwave radiation, and illumination with infrared radiation.
• the nucleic acids undergo the same thermal cycling and chemical reaction as the droplets passes through each thermal cycle as they flow through the channel.
• the total number of cycles in the device is easily altered by an extension of thermal zones or by the creation of a continuous loop structure.
• the sample undergoes the same thermal cycling and chemical reaction as it passes through N amplification cycles of the complete thermal device.
• the temperature zones are controlled to achieve two individual temperature zones for a PCR reaction.
• the two temperature zones are controlled to have temperatures as follows: 95°C (T H ) and 60°C (T L ).
• the sample droplet optionally flows through an initial preheat zone before entering thermal cycling.
• the preheat zone may be important for some chemistry for activation and also to ensure that double stranded nucleic acid in the droplets are fully denatured before the thermal cycling reaction begins.
• the preheat dwell length results in approximately 10 minutes preheat of the droplets at the higher temperature.
• the sample droplet continues into the high temperature zone, of approximately 95°C, where the sample is first separated into single stranded DNA in a process called denaturation.
• the sample then flows through the device to the low temperature zone, of approximately 60°C, where the hybridization process takes place, during which the primers anneal to the
• the droplets are created and/or merged on chip followed by their storage either on the same chip or another chip or off chip in some type of storage vessel such as a PCR tube.
• the chip or storage vessel containing the droplets is then cycled in its entirety to achieve the desired PCR heating and cooling cycles.
• the droplets are collected in a chamber where the density difference between the droplets and the surrounding oil allows for the oil to be rapidly exchanged without removing the droplets.
• the temperature of the droplets can then be rapidly changed by exchange of the oil in the vessel for oil of a different temperature. This technique is broadly useful with two and three step temperature cycling or any other sequence of
• the invention is not limited by the method used to thermocycle the droplets. Any method of thermocycling the droplets may be used.
• droplets are flowed to a detection module for detection of amplification products.
• the droplets require re-injection into either a second fluidic circuit for read-out—that may or may not reside on the same chip as the fluidic circuit or circuits for droplet generation— or in certain embodiments the droplets may be reinjected for read-out back into the original fluidic circuit used for droplet generation.
• the droplets may be individually analyzed and detected using any methods known in the art, such as detecting the presence or amount of a reporter.
• the detection module is in communication with one or more detection apparatuses.
• the detection apparatuses can be optical or electrical detectors or combinations thereof.
• detection apparatuses include optical waveguides, microscopes, diodes, light stimulating devices, (e.g., lasers), photo multiplier tubes, and processors (e.g., computers and software), and combinations thereof, which cooperate to detect a signal representative of a characteristic, marker, or reporter, and to determine and direct the measurement or the sorting action at a sorting module.
• light stimulating devices e.g., lasers
• processors e.g., computers and software
• amplified target are detected using detectably labeled probes.
• the detectably labeled probes are optically labeled probes, such as fluorescently labeled probes.
• fluorescent labels include, but are not limited to, Atto dyes, 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine and derivatives:
• acridine, acridine isothiocyanate 5-(2'-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate; N-(4-anilino- 1- naphthyl)maleimide; anthranilamide; BODIPY; Brilliant Yellow; coumarin and derivatives; coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifluoromethylcouluarin (Coumaran 151); cyanine dyes; cyanosine; 4',6-diaminidino-2- phenylindole (DAPI); 5'5"-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red); 7- diethylamino-3-
• Phenol Red Phenol Red
• B-phycoerythrin o-phthaldialdehyde
• pyrene and derivatives pyrene, pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; Reactive Red 4 (Cibacron.TM.
• the droplets of the invention contain a plurality of detectable probes that hybridize to amplicons produced in the droplets.
• Members of the plurality of probes can each include the same detectable label, or a different detectable label.
• the plurality of probes can also include one or more groups of probes at varying concentration.
• the groups of probes at varying concentrations can include the same detectable label which vary in intensity, due to varying probe concentrations.
• the detection can occur by the scanning of droplets confined to a monolayer in a storage device that is transparent to the wavelengths or method or detection. Droplets stored in this fashion can be scanned either by the movement of the storage device by the scanner or the movement of the scanner over the storage device.
• the invention is not limited to the TaqMan assay, as described above, but rather the invention encompasses the use of all fluorogenic DNA hybridization probes, such as molecular beacons, Solaris probes, scorpion probes, and any other probes that function by sequence specific recognition of target DNA by hybridization and result in increased fluorescence on amplification of the target sequence.
• Fluorogenic DNA hybridization probes such as molecular beacons, Solaris probes, scorpion probes, and any other probes that function by sequence specific recognition of target DNA by hybridization and result in increased fluorescence on amplification of the target sequence.
• Digital PCR performance in the emulsion format was validated by measuring a serial dilution of a reference gene, branched chain keto acid dehydrogenase El (BCKDHA). Mixtures of the PCR master mix, lx primers and probe for BCKDHA, and varying concentrations of a mixture of human genomic DNA (1: 1 NA14091 and NA13705) were compartmentalized into over one million 5.3 pL droplets in a water- in-fluorinated oil emulsion using the droplet generation micro fluidic chip. The emulsion was thermally cycled off-chip and afterwards the fluorescence of each droplet was analyzed by fluorescence in the readout chip (see Fig. 3).
• BCKDHA branched chain keto acid dehydrogenase El
• FIG. 3 An exemplary microfluidic system for droplet generation and readout is depicted in Fig. 3.
• the microfluidic system for droplet generation and readout As shown in Fig. 3a (droplet generation chip), a continuous aqueous phase containing the PCR master mix, primers, and probes, and template DNA flowed into the fluidic intersection from the left, and the carrier oil entered from the top and bottom. An emerging bolus of aqueous liquid was imaged inside the intersection just prior to snapping off into a discrete 4 pL droplet as the fluidic strain began to exceed the surface tension of the aqueous liquid. The steady train of droplets leaving the intersection toward the right was collected off chip as a stable emulsion for thermal cycling.
• Figure 3b depicts the droplet spacing for readout. Flows were arranged as in 3a, except instead of a continous phase, the emulsion from (a) was injected from the left into the intersection after thermal cycling. The oil drained from the emulsion during off-chip handling, hence the emulsion appeared tightly packed in the image before the intersection. The oil introduced in the intersection separated the droplets and the fluorescence of each droplet was measured at the location marked by the arrow.
• Figure 3c depicts a cartoon of droplet readout by fluorescence. The relatively infrequent PCR(+) droplets (light gray) flow along with the majority of PCR(-) droplets (dark gray) toward the detector. The droplets were interrogated sequentially by laser induced fluorescence while passing through the detection region.
• the occupancy was calculated from Poisson statistics using the following equation well known to those experienced in the art:
• oecuoancv In I— , (1) where P and N are the numbers of PCR(+) and PCR(-) droplets respectively.
• PCR(+) and PCR(-) droplets were analyzed by fluorescence while flowing through the readout chip to count the numbers of PCR(+) and PCR(-) droplets (see Fig.3c). As each droplet passed the detection zone (marked with an arrow in Fig. 3b), a burst of fluorescence was observed. To account for small run-to-run differences in the fluorescence intensity that can occur due to different chip positioning, etc., each set of data was scaled such that the average fluorescence intensity of the empty droplets was 0.1 V.
• Fig. 4a shows a very short duration of a typical trace of fluorescence bursts from individual droplets for the sample with the highest DNA concentration in the series. PCR(+) and PCR(-) droplets were easily discriminated by fluorescence intensity.
• Droplet based digital PCR technology as described in Link et al. (U.S. patent application numbers 2008/0014589, 2008/0003142, and 2010/0137163), Anderson et al. (U.S. patent number 7,041,481 and which reissued as RE41,780) and European publication number EP2047910 to Raindance Technologies Inc, (the contents of each of which are incorporated by reference herein in their entireties) utilizes a single primer pair per library droplet.
• This library droplet is merged with a template droplet which contains all the PCR reagents including genomic DNA except for the primers. After merging of the template and the primer library droplets the new droplet now contains all the reagents necessary to perform PCR.
• the droplet is then thermal cycled to produce amplicons.
• the template DNA is diluted in the template mix such that on average there is less than one haploid genome per droplet.
• Having only one haploid genome (i.e., one allele) per droplet gives droplet PCR advantages over standard singleplex or multiplex PCR in tubes or micro wells. For example, in traditional PCR, both alleles are present in the reaction mix so if there is a difference in the PCR efficiency between alleles, the allele with the highest efficiency will be over represented.
• a variance in the primer hybridization sequence can cause that primer to have a lower efficiency for hybridization for the allele that has the variance compared to the allele that has the wild type sequence. This can also cause one allele to be amplified
• a certain amount of DNA is required to generate either a specific quantity of DNA and/or a specific number of PCR positive droplets to achieve sufficient sequencing coverage per base. Because only a percentage of the droplets are PCR positive, approximately 1 in 3 in the standard procedure, it takes more DNA to achieve the equivalent PCR yield per template DNA molecule.
• the number of PCR positive droplets and thus the amplicon yield can be increased by adding more genomic DNA. For instance, increasing the amount of genomic DNA twofold while maintaining the number of droplets constant will double the amplicon yield.
• One way to allow the input of more genomic DNA is by generating more droplets to keep the haploid molecules per droplet ratio constant. For instance doubling the amount of DNA and doubling the amount of droplets increases the amplicon yield by 2x while maintaining the same haploid genome per droplet ratio. However, while doubling the number of droplets isn't problematic, increasing the amount of DNA can be challenging to users that have a limited amount of DNA.
• the multiplexing of PCR primers in droplets enables the simultaneous increase in the number of PCR droplets while keeping the amount of input DNA the same or lower to generate an equal or greater amplicon yield. This results in an overall increase in the amount of PCR positive droplets and amplicon yield without the consumption of more DNA.
• the number of droplets containing the 2x multiplexed primers is doubled and the DNA kept constant, then the number of PCR positive droplets drops back to 1 in 4, but the total number of PCR droplets remains the same because the number of droplets have been doubled. If the multiplexing level in each droplet is increased to 4x and the input DNA is the same, the chance of the correct template molecule being present in each droplet doubles. This results in the number of PCR positive droplets being increased to 1 in 2 which doubles the amount of amplicon yield without increasing the amount of input DNA. Thus, by increasing the multiplexing of PCR primers in each droplet and by increasing the number of droplets overall, the amplicon yield can be increased by 4-fold without increasing the amount of input DNA.
• the amplicon yield is already sufficient, by increasing the multiplexing level for the PCR primers in each droplet, the amount of input genomic DNA can be dropped without sacrificing amplicon yield. For example if the multiplexing level of the PCR primers goes from lx to 2x, the amount of input genomic DNA can be decreased by 2x while still maintaining the same overall amplicon yield.
• the fluorescence emission from each droplet was determined and plotted on a scattered plot based on its wavelength and intensity.
• Six clusters, each representing droplets having the corresponding fluorescence wavelength and intensity were shown.
• the TERT, RNaseP and Ela clusters showed the fluorescence of the VIC dye at three distinct intensities and SMNl and SMNl clusters showed the fluorescence of the FAM dye at two distinct intensities ( Figure 5).
• the number of droplets, each having one or more sequences selected from TERT, RNaseP, Ela, SMNl and SMN2 can be determined from the scattered plot.
• the droplets containing both the primers and probes were fused with droplets containing the template.
• PCR reactions were conducted with the fused droplets to amply the sequences for TERT, RNaseP, Ela, 815A and 815G.
• the PCR was conducted with a standard thermal cycling setting.
• the fluorescence emission from each fused droplet was determined and plotted on a scattered plot based on its wavelength and intensity.
• Six clusters, each representing droplets having the corresponding fluorescence wavelength and intensity were shown.
• the TERT and 815A clusters showed the fluorescence of the VIC dye at two distinct intensities;
• the 815G clusters showed the fluorescence of the FAM dye;
• the RNaseP and Ela clusters showed the fluorescence of both the FAM and the VIC dye at distinct intensities ( Figure 6).
• the number of droplets, each having one or more sequences selected from TERT, RNaseP, Ela, 815A and 815G, can be determined from the scattered plot.
• the copy number of RNaseP, Ela, 815A and 815G in the template were determined by the ratio between the number of droplets having the RNaseP, Ela, 815A and/or 815G sequences and the number of droplets having the TERT sequence ( Figure 6).
• droplet library A was generated where each droplet contained only one primer pair; and droplet library B was generated where the primer pairs were multiplexed at 5x level in each droplet.
• HapMap sample NA18858 was processed in duplicate with droplet libraries A or B using standard procedures. Two ⁇ g sample DNA was used for droplet library A and one ⁇ g sample DNA was used for the 5x multiplex droplet library B. After PCR amplification, both droplet libraries were broken and purified over a Qiagen MinElute column and then run on an Agilent Bioanalyzer.
• the results obtained from the 5x multiplexed droplet library B were equivalent or better than what was obtained from droplet library A.
• the multiplexing of primers delivers the same sequencing results for base coverage, specificity and uniformity that the singleplexing does with the added advantage of reduced input DNA.
• Total reads total number of sequencing read found within the provided sample data.
• Mapped reads (%): percentage of total reads that mapped to the human genome.
• the target includes all amplicon sequences with primer sequences excluded.
• Mean base coverage average base coverage within the target.
• the target includes all amplicon sequences with primer sequences excluded.
• CI % of target that has at least lx base coverage. Note: non-unique sequencing reads are mapped randomly.
• C20 % of target that has at least 20x base coverage.
• CIOO % of target that has at least lOOx base coverage.
• Base coverage (0.2x of mean): % of target that has at least 20% of mean base coverage.
• FIG. 7 is a schematic depicting one-color detection of a target sequence using droplet based digital PCR.
• a template DNA is amplified with a forward primer (Fl) and a reverse primer (Rl).
• Probe (PI) labeled with a fluorophore of color 1 binds to the target genetic sequence (target 1).
• Microdroplets are made of diluted solution of template DNA under conditions of limiting or terminal dilution. Droplets containing the target sequence emit fluorescence and are detected by laser (Panels B and C). The number of microcapsules either containing or not containing the target sequence is shown in a histogram (D) and quantified (E).
• Figure 8 is a schematic depicting two-color detection of two genetic sequences with a microfluidic device.
• a template DNA is amplified with two sets of primers: forward primer (Fl) and a reverse primer (Rl), and forward primer (F2) and a reverse primer (R2).
• Probe (PI) labeled with a fluorophore of color 1 binds to the target 1
• probe (P2) labeled with a fluorophore of color 2 binds to the target 2 (Panels B and C).
• Droplets are made of diluted solution of template DNA under conditions of limiting or terminal dilution. Droplets containing the target sequence 1 or 2 emit fluorescence of color 1 or 2 respectively and are optically detected by laser (Panels B and C). The number of microcapsules containing target 1 or 2 is shown by histogram in Panel D.
• FIG. 9 is a schematic depicting two-color detection of three genetic sequences with a microfluidic device.
• a template DNA is amplified with three sets of primers: forward primers (Fl, F2 and F3) and reverse primers (Rl, R2 and R3).
• Probes (PI, P2 and P3) are labeled with fluorophores (color 1, color 2 and color 1) and bind to the target genetic sequences (target 1, target 2 and target 3) (Panels B and C).
• Microdroplets are made of diluted solution of template DNA under conditions of limiting or terminal dilution. Microdroplets containing target sequence
• microdroplets containing target sequence 2 emit fluorescence of color 2.
• dPCR droplet digital PCR
• the results are depicted in Figure 10.
• the left-side dot plot in Figure 10 depicts the effect of having the SMN1 blocker present in the reaction.
• the four clusters depicted in the leftside dot plot are as follows: the top left cluster includes microdroplets containing the reference sequence (SMARCC1); the bottom left cluster includes microdroplets not containing any sequence; the bottom middle cluster includes microdroplets containing sequence for SMN1; and the bottom right cluster includes microdroplets containing sequence for SMN2.
• the dot plot on the right-side of Figure 10 depicts four clusters where no SMN1 blocker was present in the reaction: the top left cluster includes microdroplets containing the reference sequence
• the bottom left cluster includes microdroplets not containing any sequence
• the bottom middle cluster includes microdroplets containing sequence for SMN1
• the bottom right cluster includes microdroplets containing sequence for SMN2.
• the shift of the bottom middle cluster in right panel as compared to left panel confirms that fluorescence intensity provides a very sensitive measurement for the presence of a sequence.
• the cluster arises from weak association of the SMN2 probe to the SMN1 gene despite the presence of a blocker to that gene (a nonfluorescent complementary probe to the SMN1 gene).
• the probe hybridization does not reach equilibrium before exonuclease activity. In this case, the association rates would play a more dominant role.
• the binding rate to the matching site is likely to be faster than to the mismatch site, and the blocker would act to decelerate probe binding to the mismatch site.
• the binding of SMN2 probe to SMNl DNA might be detectable by conventional bulk qPCR, especially in absence of SMN2, but highly quantitative results like those shown here are very unlikely.
• qPCR or any other technique quantifying two different DNA sequence motifs with the same color fluorophore. Sequestration of the individual reactions by single molecule amplification within droplets eliminates any confusion regarding mixed contributions to the signal.
• a multiplexes assay can require a more dilute sample. For instance, at 10% occupancy a duplex reaction would have double occupancy 1% of the time. Hence 1 in 10 PCR+ droplets would be doubles, resulting in a final intensity at least as high and possibly higher than the brighter of the two probes. For a simple duplex system the contribution from each probe could be recovered. In this example the total number of PCR+ droplets for probe 1 would be (Probe 1) + (Probe l+Probe2).
• a single fluorophore was used in a gene copy number assay for both the reference and the target DNA.
• a model system was used with varying concentrations of plasmid DNA to represent a change in the target gene copy number, relative to a reference gene, equivalent to 0-16 copies of the target gene per cell.
• BCKDHA and SMN2 plasmid DNA served as the reference and target with lx and 0.5x primers and probes respectively.
• the sample was diluted serially by 2x into a solution of BCKDHA at the same concentration to vary just the amount of SMN2.
• the resultant samples were emulsified, thermally cycled, and over 10 5 droplets were analyzed for each sample as described in the previous section. The process was repeated in triplicate.
• Methods of the invention also include analytical techniques for identification of fluorescence signatures unique to each probe.
• histograms of the droplet fluorescence intensities are shown in Fig. 11a for three different template DNA samples: a no template control (dotted line), BCKDHA only (solid line), and 1: 1 BCKDHA to SMN2 (dashed line).
• the histograms are shown both overlapped to highlight the similarity for certain peaks, and offset from each other to reveal all of the features.
• 1: 1 BCKDHA to SMN2 three populations were readily apparent: a dominant feature appeared at 0.08 V, and two smaller peaks were evident at 0.27 and 0.71 V.
• TaqMan assays can be designed that are specific for each of the exons in an RNA transcript. After the RNA is turned into cDNA it can be encapsulated into a droplet at 1 copy or less per droplet. The droplet would also contain the multiplexed TaqMan assay for each of the exons. Each of the TaqMan assays would contain a different probe but all the probes would have the same fluorescent dye attached. The droplets would be thermocycled to generate signal for each of the TaqMan assays. If there are multiple splice variants in the sample they each will contain a different number of exons depending on the splicing events. The fluorescent intensity of each droplet would be different depending on the number of exons present. By counting the number of droplets with different intensities it would be possible to identify the presence and abundance of different splice variants in a sample.
• a heterogeneous sample contained components with different copy level numbers. If the copy number variants to be assayed were spaced close enough along the chromosome, the DNA from a sample could be fragmented and encapsulated in droplets at a level of one haploid genomic equivalent or less per droplet.
• the droplet would also contain a TaqMan assay specific for the copy number variant. The intensity of the signal in each droplet would depend on the number of copy number variants are present for the sample. Counting of the number of droplets of different intensities would indicate things like how many cells in a particular sample had what level of copy number variants.
• Identifying probes by fluorescence intensity often requires adjusting the brightness of the probes, particularly for higher-plex assays with dense probe patterns.
• the probes for the gene copy number assay yielded very well resolved peaks (Fig. 1 la).
• a method for adjusting the fluorescence intensity of the new probes is required to avoid interference with the existing assay.
• One method of the invention involves varying the probe and primer concentrations together as a very simple technique to optimize relative intensities in higher-plex reactions.
• Figure 12 is a schematic for tuning the intensity of a detectable label to a particular target with a microfluidic device.
• a template DNA is amplified with two sets of primers: forward primers (Fl and F2) and reverse primers (Rl and R2).
• Probes (PI and P2) are labeled with fluorophore of color 1 and bind to target 1 and target 2 respectively. Fluorescence from target 2 is lower in intensity than that from target 1 due to single base mismatch between P2 and target 2.
• template DNA is amplified with two sets of primers: forward primers (Fl and F2) and reverse primers (Rl and R2) (Panel B).
• Fluorescence from target 2 is lower in intensity than that from target 1 due to the presence of a competing probe 2 that is not labeled with the fluorophore.
• template DNA is amplified with two sets of primers: forward primers (Fl and F2) and reverse primers (Rl and R2).
• Probes (PI and P2) are labeled with fluorophore of color 1 and bind to target 1 and target 2 respectively. Fluorescence from target 2 is lower in intensity than that from target 1 due to the presence of a competing probe 2 that is labeled with a different fluorophore.
• Fig. 13 shows probe fluorescence intensities throughout a serial dilution of the probes and primers for a different reference gene, ribonuclease P (RNaseP), against a constant amount of genomic DNA from the Coriell cell line NA3814 at an occupancy of 0.02 target DNA molecules per droplet.
• probe intensities can be varied by dilution over a small but adequate range for the purpose of tuning multiplexed assays without affecting the amplification itself.
• the invention is not limited to this method alone for varying probe intensity.
• Other methods known to those familiar with the art for varying probe intensities are also considered. Such methods include varying just the probe concentration; varying just the primer concentrations; varying just the forward primer concentration; varying just the reverse primer concentration; varying the probe, forward, and reverse primers concentrations in any way; varying the thermal cycling program; varying the PCR master mix; incorporating into the assay some fraction of probes that lack fluorophores; or incorporating into the assay any hybridization-based competitive inhibitors to probe binding, such as blocking oligomer nucleotides, peptide nucleic acids, and locked nucleic acids.
• the invention incorporates the use of these methods adjusting probe fluorescence intensity, or any other methods for adjusting probe fluorescence intensity, used either by themselves or in any combination.
• probe fluorescent intensities can be adjusted by a variety of means such that each intensity level uniquely identifies a DNA target.
• targets Tl, T2, T3, and T4 might be uniquely identified by intensity levels II, 12, 13, and 14.
• the maximum number of intensity levels possible for unique identification of targets is related to the resolution of the different intensity levels—that is the spread of intensities for each particular probe compared to the separation between the average intensities of the probes— and it is also related to the intensity of the empty droplets that tends to grow with increasing numbers of probes.
• the number of intensity levels can be 0, or 1, or 2, or 3, or 4, or up to 10, or up to 20, or up to 50, or up to 100.
• the number of intensity levels can be higher than 100. In the examples show below, as many as three intensity levels are
• Another method of the invention involves performing higher-plex assays using multiple different probe colors (i.e. fluorophores).
• probe colors i.e. fluorophores
• multiple targets can be identified based on intensity.
• multiple colors that are spectrally separable can be used simultaneously.
• a single droplet might contain four different probes for measuring four different targets. Two probes might be of color A with different intensities (say, Al and A2), and the other two probes of color B with different intensities (say Bl and B2).
• the corresponding targets are Tl, T2, T3, and T4 for Al, A2, Bl, and B2 respectively. If a droplet shows an increase in fluoresce in color A, the droplet therefore contained either targets Tl or T2.
• the target could be identified as Tl or the target could be identified as T2. If, however, a droplet shows an increase in fluorescence in color B, the droplet therefore contained either targets T3 or T4. Then, based on the fluorescence intensity of color B, the target could be identified as T3 or the target could be identified as T4.
• the maximum number of different colors possible is limited by spectral overlap between fluorescence emission of the different fluorophores.
• the maximum number of colors can be 1, or 2, or 3, or 4, or up to 10, or up to 20.
• the maximum number of colors can be higher than 20. In the demonstrations that follow, the largest number of colors is two.
• Another method of the invention involves performing higher-plex assays using multiple different probe colors (i.e. fluorophores), however unlike the strategy above where each target is identified by single type of probe with a unique color and intensity, instead in this method a single target may be identified by multiple probes that constitute a unique signature of both colors and intensities.
• a single droplet might contain four different probes for measuring three different targets (say, Tl, T2, and T3). Two probes might be of color A (say, Al, and A2), and two probes might be of color B (say, Bl and B2). Tl is measured by probe Al, T2 is measured by probe Bl, but T3 is measured by both probes A2 and B2.
• Tl is measured by probe Al
• T2 is measured by probe Bl
• T3 is measured by both probes A2 and B2.
• the above three methods for higher-plex dPCR are simplest to implement under conditions of terminal dilution, that is when the probability of multiple different target molecules co-occupying the same droplet is very low compared to the probability of any single target occupying a droplet.
• multiple occupancy arises the complexity of simultaneous assays competing within the same reaction droplet, and also complexity of assigning the resulting fluorescence intensity that involves a combination of fluorescence from two different reaction products that may or may not be equal to the sum of the two fluorescence intensities of the individual reaction products.
• methods of the invention can accommodate these complications arising from multiple occupancy.
• Methods of the invention for higher-plex reactions also include methods for primer and probe pairing.
• targets are unlikely to reside on the same DNA fragments, such as when targets are from different cells; or when targets are from different chromosomes within a single cell type; or when targets are distant from each other within a single chromosome such that they become physically separated during DNA fragmentation; or when targets are very close to each other within a chromosome, but nevertheless become separated by targeted cleavage of the DNA, such as by restriction enzyme digestion; or for any other reason.
• each probe can be paired with a single set of primers (forward and reverse).
• the target regions might frequently reside on the same DNA fragments, for example when targets reside within the same codon, or for any other reason.
• a single set of primers might serve for multiple probes (for an example, see Pekin et al.).
• Higher multiplex reactions can be performed to distinguish the haplotypes of two SNPs. For example, assume that at position one there can be genotypes A or A' and at position two there can be genotypes of B or B' . In a diploid genome four unique haplotypes are possible (A,B; A,B' ;A' ,B; and ⁇ ' , ⁇ '). If for example A' and B' represent drug resistant mutations for infection, it is often the case that A'B and AB' are less sever and treated differently than A'B' which represents a significant drug resistance that must be treated with extreme care. Digital PCR with intensity discrimination is ideally suited for identifying low prevalence of A'B' in a background of mixtures of the other three haplotypes.
• Haplotyping information is also important for construction of haplotypes in HLA.
• One way that the present example can be constructed is by assay design such that color one is used for A and is of high or low intensity indicative of allele A or A' respectively and color two is used for B and is of high or low intensity
• the multiplex intensity detection of multiple simultaneous targets can be used as surrogate markers for multiple types of binding interactions or labeling of target materials.
• This technique is also not limited to single molecule detection and can be used for haplotype detection in single cells (e.g., bacteria, somatic cells, etc.).
• a sorting step may be applied prior to haplotyping.
• SMA spinal muscular atrophy
• SMN2 copy number is important for clinical prognosis and genetic counseling.
• a number of single point mutations or short deletions / duplications within the same gene also account for -4% of cases of SMA.
• the multiplexed dPCR assay demonstrated here contains both copy number assays (for SMN1 & 2) and an assay for one of the prevalent SNPs (c.815A>G).
• One embodiment of the invention is a 5-plex assay for SMA diagnostics.
• the 5-plex assay quantifies common genetic variants impacting SMA including two copy number assays for the SMN1 and SMN2 genes with BCKDHA as a reference, and a SNP assay for the c.815A>G mutation.
• Two differently colored fluorophores, FAM and VIC were used to uniquely identify each of the assays.
• the probes for SMN1 and SMN2 contained only FAM, and for C.815A only VIC. However, mixtures of VIC and FAM-labeled probes were used for BCKDHA and C.815G.
• VIC and FAM fluorophores in this example does not limit the invention, rather the 5-plex assay can be used with any spectrally separable fluorophores compatible with the TaqMan assay, or any other fluorogenic hybridization-based probe chemistries.
• a model chromosome was synthesized containing a single target region for each of the different primer/probe pairs. EcoRV restriction sites flanked each target, allowing separation of the fragments.
• histogram-based data presentation and analysis is incorporated into the invention for identifying and characterizing statistically similar populations of droplets that arise from one probe signature (color and intensity), and for discriminating one population of droplets from the others.
• Fig. 14a shows a 2-dimensional histogram of droplet fluorescence intensities as a contoured heat map, with hotter colors representing higher occurrences. Standard techniques were used to compensate for spectral overlap of the FAM and VIC signals. Samples were run at 0.006 occupancy per target. Six populations were clearly evident, five for the assay and one for PCR(-) droplets. As one method of the invention, the populations were assigned by selective exclusion of assay components.
• SMN2 primers and probe eliminated the population at the bottom right in the histogram, but otherwise the distribution remained unchanged. Assignments are labeled in Fig. 14a.
• the assay worked immediately with well resolved or at least distinguishable populations for each target.
• the relative positions of the different populations in the histogram were then adjusted into a regularly spaced rectangular array by tuning the probe concentration as described in the previous section. Usually no more than two iterations are required for optimization.
• the different populations were sufficiently well resolved to allow droplets within each population to be counted by integration across rectangular boundaries.
• the boundaries were positioned at mid-sections between neighboring peaks.
• the methods of the invention are not constrained to rectangular boundaries, or to specific boundary locations between peaks. Rather, any closed or unclosed boundary condition can suffice.
• Boundary conditions do not need to be "binary" either, in the sense that weighted integrations can also be performed across the boundaries to arrive at droplet counts.
• the peak position of each cluster varied by no more than 2% from run to run after normalization to the intensity of the empty droplets to account for variations in detection efficiency (data not shown).
• the methods of the invention are not limited to fixed boundary positions. Dynamic population identification and boundary selection in between samples or studies is anticipated. Twenty different patient samples from the Coriell cell repositories were analyzed with this assay: 4 afflicted with SMA, 1 SMA carrier, and 15 negative controls. Assay results are shown in Fig. 14b.
• the measured genotypes of the different patients were consistent with their disease conditions (unafflicted, carrier, or afflicted).
• the patients afflicted with SMA each had zero copies of SMNl (numbers SMA 1-4 in Fig. 14b), the carrier had just one copy, and the negative controls all had two or three copies (numbers 1-15).
• Three unrelated individuals (numbers 6, 8, and 9) had three copies of SMNl, occurring at a rate of 20% which is similar to a previous report for healthy individuals. Variability in SMNl copy number is not surprising since it lies within an unstable region of chromosome 5ql3. A larger variety of SMN2 copy numbers was observed.
• SMA carrier and afflicted patients had elevated copy numbers of SMN2 on average: 5 for the carrier, two afflicted with 3 copies, and the others with 2 copies.
• the afflicted patients were all diagnosed as SMA Type I, the most severe form, based on clinical observations according to the Coriell repository.
• the strong genotype/phenotype correlation between SMN2 copy number and disease severity suggests that the two individuals with three copies of SMN2 might have an improved Type II prognosis, especially for the patient SMA 1 who had survived to three years at the time of sampling, much beyond the typical maximum life expectancy for SMA Type I of 2 years.
• the SNP assay revealed that all patients carried the normal C.815A genotype and no instances of C.815G were observed. The mutation is relatively rare and hence was not expected to appear in a small patient panel. Of interest, however, was the presence of an apparent extra gene fragment in two unrelated individuals that was uncovered with the SNP assay.
• the c.815A>G assay does not discriminate between SMNl and SMN2 due to their high sequence similarity, and hence the total copies of C.815A and G should equal the sum of the copies of SMNl and SMN2. This was true for all patients except for healthy patients number 1 and 2, both of whom had one extra copy of C.815A. c.815 lies on exon 6, and the SNP that
• FIG. 15a shows the various droplet populations in 2-D histograms before optimization of probe concentrations. The identity of the different targets is shown on the figure itself.
• the identification of the different populations was made as before, by selective exclusion and/or addition of one or more assays. Most of the populations were already well resolved, with the exception of the probe for the C.815A genotype that was in close proximity with the cluster corresponding to empty droplets.
• Methods of the invention include any method that can cause an identifiable displacement, appearance, or disappearance of one or more populations within the histograms including changing the probe and primer concentrations together, either by the same factor or by different factors; changing the probe concentration alone; changing the primer concentrations alone; changing the thermal cycling conditions; and changing the master mix composition.
• Another method of the invention takes advantage of prior knowledge of the position of an assay within a histogram to assist assignment.
• the level of multiplexing demonstrated in the preceding SMA example was 9x, significantly exceeding the maximum practicable number with qPCR.
• the two main limitations are the resolution between assays and the increasing fluorescence intensity of empty droplets with higher loading of probes.
• a method of the invention involves optimizing the pattern of colors and intensities of the different probes for maximum multiplexing while still achieving adequate specificity for each individual reaction.
• rectangular arrays of droplet populations were demonstrated for the 5- and 9-plex reactions, another desirable pattern is the tight-packed hexagonal array.
• the invention is not constrained to any particular array strategy.
• primers and probes can be loaded individually into droplets along with an optical label to uniquely identify the assay.
• the optical label is a fluorophore, or a combination of different fluorophores, that are spectrally distinct from the probe fluorophore.
• Various different types of droplets, each containing different assays that are uniquely identified by different optical labels, can be mixed into a "library" of droplets. Then, according to methods of the invention above, library droplets are merged one-to-one with droplets containing template DNA. After thermal cycling, some droplets that contain template DNA will exhibit brighter fluorescence at the emission wavelengths of the probes.
• the specific target DNA molecules giving rise to these PCR(+) signals are subsequently identified by the optical probes.
• the six common mutations in KRAS codon 12 were screened in parallel in a single experiment by one-to-one fusion of droplets containing genomic DNA with any one of seven different types of droplets (a seven-member library), each containing a TaqMan ® probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code.
• optical labeling can be combined with the various methods for multiplexing dPCR already incorporated into this invention.
• a single optical label might code for the entire 5-plex SMA assay, above, instead of just a single assay as in the KRAS example above.
• other optical labels might code for different screening assays for newborn infants.
• a single DNA sample from an infant could then be analyzed with all of the assays simultaneously by merging droplets containing the DNA one-to-one with library droplets containing the optically encoded assays.
• the fluorescence from the FAM and VIC fluorophores were recorded simultaneously with the fluorescence from the optical labels, requiring modifications to the optical layout of the instrumentation described for the SMA assay (the optical schematic for two-laser excitation and 4-color detection is shown in entirety in Figure 16).
• co-flow microfluidics were used in this example (the use of co-flow based microfluidics for this application is one of the methods of the invention described above).
• the template DNA was introduced into the chip in one flow, and the PCR master mix, the primers and probes for one triplex assay, and the unique composition of fluorophores for the optical label were introduced into the chip in another flow simultaneously.
• each droplet formed contained the contents of both flow streams.
• Methods to implement co-flow microfluidics are well known to those in the art.
• the droplets were collected, and then the procedure was repeated with the next triplex assay and optical label. The procedure was repeated a total of nine times, once for each pair of assays and optical labels. All of the droplets were collected into a single PCR tube and thermally cycled off chip. The mixture of thermally cycled droplets was reinjected into the same read-out chip as used for the SMA assay, above, and the fluorescence intensities of the assays from all four fluorophores was recorded.
• Figure 17 shows the cumulative results from all droplets in the 3x3x3 assay using co-flow microfluidics.
• the figure shows two 2-D histograms of droplet fluorescence intensities, the histogram on the left from all of the optical labels, and the histogram on the right from the assays. Standard methods were used to compensate for spectral overlap.
• the histograms are shown as a heat maps, with hotter colors designating larger numbers of droplets.
• Methods of the invention involve selecting individual populations of droplets all containing the same optical labels, or groups of optical labels.
• boundaries of fluorescence intensity were used to specify populations.
• a rectangular boundary was used specifying the minimum and maximum fluorescence intensities for each fluorophore.
• the methods of the invention are not restricted to rectangular boundaries. Any boundary, closed or unclosed, can be employed.
• selections of droplet populations can be made by any method, and is not restricted to threshold-based methods such as boundary selection.
• Figure 18A shows the droplet fluorescence intensities for the assay (right histogram) when only one optical label was selected (left histogram).
• the lines overlaid on the histogram of the optical labels identify the rectangular boundary used to select just the optical label with the lowest fluorescence for both fluorophores. Both histograms showed only the droplets that were selected. After selection, four distinct clusters of droplets appeared in the assay histogram, three for the different assays (in this case, assays for SMN1, SMN2, and TERT, where TERT is another common reference gene) and one for the empty droplets.
• the copy numbers for SMN1 and SMN2 were measured by the same methods of the invention as described above for the 5-plex SMA assay, with values of 1.8 and 0.94 close to the expected values of 2 and 1, respectively.
• the same assay was encoded with two other optical labels, and their selections are shown in Figures 18B and C. Similar results were achieved, with an overall measurement of 1.9+0.1 and 0.9+0.1 copies of SMN1 and SMN2 respectively, showing the measurement to be accurate within experimental uncertainty.
• Figures 19A, B, and C show optical label selections for a different assay (TERT, c.5C in the SMN1 gene, and BCKDHA (labeled Ela in the figure)). In each case four distinct clusters also appeared, and by the same methods of the invention above, accurate measurements of gene copy number were made for c.5C and BCKDHA, referenced to TERT, of 2.9+0.1 and 2.0+0.2 compared to 3 and 2, respectively.
• Figures 20 A, B, and C show optical label selections for a third assay (TERT, C.88G in the SMN1 gene, and RNaseP, where RNaseP is a common reference gene). Accurate gene copy numbers of 2.1+0.1 were measured for both C.88G and RNaseP, referenced to TERT, compared to the expected value of 2.
• the demonstration here shows use of nine different optical labels to enable independent measurement of three triplex assays in a single experiment.
• some of the optical labels encoded for redundant assays in this example (there were only three different assays despite having nine optical labels), the invention is not constrained to any particular formatting of assays and optical labels.
• Embodiments of the invention include formats where all of the assays are the same across all of the optical labels; where none of the assays are the same across all of the optical labels; where some of the assays are the same across all of the optical labels; where some of the assays have greater plexity than others across all of the optical labels; where all of the assays have the same plexity across all of the optical labels; and any other arrangements of assays across all of the optical labels are considered.
• optical labels are not constrained to any particular number of fluorophores comprising the optical labels.
• Embodiments of the invention include optical labels comprised of 1 fluorophore, or 2 fluorophores, or 3 fluorophores, or 4 fluorophores, or up to 10 fluorophores, or up to 20 fluorophores.
• Optical labels can also comprise more than 20 fluorophores.
• Embodiments of the invention include plexities of the following amounts when used with optical labels: single plex, duplex, triplex, 4-plex, up to 10-plex, up to 20-plex, up to 50-plex, and up to 100-plex.
• Embodiments of the invention also include plexities exceeding 100 when used with optical labels.
• Another method of the invention involves the use of droplet merging, instead of co-flow, for combining multiplexing with optical labels.
• a demonstration using droplet merging was performed with the same 3x3x3 assay as in the preceding example with co-flow.
• the assays probes and primers combined with their unique optical labels were first encapsulated into droplets along with the PCR master mix.
• a library containing a mixture of droplets from all nine optically labeled assays was merged one-to-one with droplets containing template DNA from the same patient as in the preceding example.
• the droplet merge was performed using a lambda- injector style merge module, as described in U.S.
• methods of the invention include using either microfluidics with co-flow or droplet merging, the invention is not limited in this regard. Any fluidic method capable of generating optically labeled droplets that also contain fluorogenic DNA hybridization probes are considered.
• other embodiments well known in the art are mixing optical labels and assays in the macrofluidic environment before injection into a droplet generating chip; and mixing optical labels and assays thoroughly upstream from the droplet forming module in dedicated mixing modules, such as with a serpentine mixer.
• One method of the invention involves histogram-based data presentation and analysis for identifying and characterizing populations of statistically similar droplets that arise from unique probe signatures (color and intensity), and for discriminating one population of droplets from the others.
• Another method of the invention involves histogram-based data presentation and analysis for identifying and selecting populations of droplets based on unique signatures from optical labels. Examples of one and two-dimensional histograms have been provided for these methods, but the invention is not limited in this regard. As described above, it is anticipated that greater numbers of colors will be used for both multiplexing and for optical labels.
• embodiments of the invention include histograms of dimensionality greater than two, such as 3, or 4, or up to 10, or up to 20. Histograms of dimensionality greater than 20 are also incorporated into the invention.
• Another method of the invention involves the selection of droplets within histograms, either for counting, or for assay selection as in the use of optical labels, or for any other purpose.
• Methods of the invention include selections by boundaries, either closed or unclosed, of any possible shape and dimension.
• Methods of the invention also include selections of droplets that exhibit fluorescence from single types of fluorophores, or from multiple types of fluorophores, such as arising from multiple probes against a common DNA target.
• each droplet contains only a single target nucleic acid, if any at all. In the preferred embodiment, this is accomplished under the conditions of terminal dilution.
• Droplets that contain amplification products that are a wild- type of the target are detected based on emission from the fluorophore that is released from the probe that hybridizes to the wild-type of the target.
• Droplets that contain the variant of the target are detected based on emission from the fluorophore that is released from the probe that hybridizes to the variant of the target. Since each droplet starts with only a single nucleic acid molecule, the resultant amplification products in each droplet are either homogeneous for the target or homogenous for the variant of the target.
• Polymerase-mediated errors at a frequency of 1 mutation per 10,000 nucleotides per cycle are an important consideration for any PCR application that begins with a small amount of starting material (e.g., less than a total of 10,000 nucleotides of target DNA) or that focuses on individual DNA molecules in the final PCR population.
• the proportion of DNA molecules that contain sequence changes is a function of the error rate per nucleotide per cycle, the number of amplification cycles and the starting population size.
• the population of altered DNA molecules arises during PCR from two sources: (1) new errors at each PCR cycle; and (2) amplification of DNA molecules containing errors from previous cycles.
• a polymerase error that converts a wild-type of the target to a variant of the target during an early round of amplification results in a heterogeneous population of target and target variant in a droplet, and may lead to a droplet being incorrectly identified as containing a variant of the target, i.e., a false positive. Such false positives greatly impact the validity and precision of digital PCR results.
• Methods of the invention are able to detect which droplets contain a heterogeneous population of molecules and are able to exclude those droplets from analysis. As droplets containing amplified product flow in a channel through the detector module, the module is able to detect the fluorescent emission in each droplet. Droplets that produce only a single signal are classified as droplets that contain a homogeneous population of target. Since probes that hybridize to the wild-type of the target have a different fluorophore attached than probes that hybridize to a variant of the wild-type of the target, methods of the invention can classify each droplet as containing either a homogeneous population of amplicons of the target or a homogeneous population of amplicons of the variant of the target.
• Droplets that produce two signals are classified as droplets that contain a heterogeneous population of molecules. Since each droplet started with at most a single target nucleic acid, a droplet that includes amplification products that are both amplicons of the target and amplicons of a variant of the target are droplets in which the variant of the target was produced by a polymerase error during the PCR reaction, most likely a polymerase error during an early cycle of the PCR reaction. Such droplets are detected and excluded from analysis.
• Analyze is then performed on only the droplets that contain a homogeneous population of molecules.
• the analysis may be based on counting, i.e., determining a number of droplets that contain only wild-type target, and determining a number of droplets that contain only a variant of the target.
• counting i.e., determining a number of droplets that contain only wild-type target, and determining a number of droplets that contain only a variant of the target.
• Such methods are well known in the art. See, e.g., Lapidus et al. (U.S. patent numbers 5,670,325 and 5,928,870) and Shuber et al. (U.S. patent number 6,203,993 and
• the presence of droplets containing only variant is indicative of a disease, such as cancer.
• the variant is an allelic variant, such as an insertion, deletion, substitution, translocation, or single nucleotide polymorphism (SNP).
• Biomarkers that are associated with cancer are known in the art. Biomarkers associated with development of breast cancer are shown in Erlander et al. (US 7,504,214), Dai et al. (US 7,514, 209 and 7,171,311), Baker et al. (US 7,056,674 and US 7,081,340), Erlander et al. (US 2009/0092973). The contents of the patent application and each of these patents are incorporated by reference herein in their entirety. Biomarkers associated with development of cervical cancer are shown in Patel (US 7,300,765), Pardee et al. (US 7,153,700), Kim (US 6,905,844), Roberts et al.
• Biomarkers associated with development of brain cancers e.g., glioma, cerebellum, medulloblastoma, astrocytoma, ependymoma, glioblastoma
• D'Andrea US 2009/0081237
• Murphy et al. US 2006/0269558
• Gibson et al. US 2014/0269558
• Biomarkers associated with development of renal cancer are shown in Patel (US 7,300,765), Soyupak et al. (US 7,482,129), Sahin et al. (US 7,527,933), Price et al. (US 7,229,770), Raitano (US 7,507,541), and Becker et al. (US 2007/0292869).
• Biomarkers associated with development of hepatic cancers e.g., hepatocellular carcinoma
• home et al. US 6,974,667
• Biomarkers associated with development of head-and-neck and thyroid cancers are shown in Sidransky et al. (US 7,378,233), Skolnick et al. (US 5,989,815), Budiman et al. (US 2009/0075265), Hasina et al. (Cancer Research, 63:555-559, 2003), Kebebew et al. (US 2008/0280302), and Ralhan (Mol Cell Proteomics, 7(6): 1162-1173, 2008). The contents of each of the articles, patents, and patent applications are incorporated by reference herein in their entirety. Biomarkers associated with development of colorectal cancers are shown in Raitano et al.
• Biomarkers associated with development of prostate cancer are shown in Sidransky (US 7,524,633), Platica (US 7,510,707), Salceda et al. (US 7,432,064 and US
• Biomarkers associated with development of leukemia are shown in Ando et al. (US 7,479,371), Coignet (US 7,479,370 and US 7,449,303), Davi et al. (US 7,416,851), Chiorazzi (US 7,316,906), Seshi (US 7,049,072), Van Baren et al. (US 6,130,052), Taniguchi (US 5,643,729), Hans et al. (US 2009/0131353), and Van Bockstaele et al. (Blood Rev. 23(l):25-47, 2009).
• Biomarkers associated with development of lymphoma are shown in Ando et al. (US 7,479,371), Levy et al. (US 7,332,280), and Arnold (US 5,858,655). Biomarkers associated with development of bladder cancer are shown in Price et al. (US 7,229,770), Orntoft (US 6,936,417), Haak-Frendscho et al. (US 6,008,003), Feinstein et al. (US 6,998,232), Elting et al. (US 2008/0311604), and Wewer et al. (2009/0029372). The content of each of the above references is incorporated by reference herein in its entirety.
• methods of the invention may be used to monitor a patient for recurrence of a cancer. Since the patient has already been treated for the cancer, the genetic profile and particular mutation(s) associated with that patient's cancer are already known.
• Probes may be designed that specifically hybridize to the region of the nucleic acid that contains the mutation(s) that is indicative of the cancer for which the patient was previously treated.
• a patient's sample e.g., pus, sputum, semen, urine, blood, saliva, stool, or cerebrospinal fluid
• Methods of the invention may further include sorting the droplets based upon whether the droplets contain a homogeneous population of molecules or a heterogeneous population of molecules.
• a sorting module may be a junction of a channel where the flow of droplets can change direction to enter one or more other channels, e.g., a branch channel, depending on a signal received in connection with a droplet interrogation in the detection module.
• a sorting module is monitored and/or under the control of the detection module, and therefore a sorting module may correspond to the detection module.
• the sorting region is in communication with and is influenced by one or more sorting apparatuses.
• a sorting apparatus includes techniques or control systems, e.g., dielectric, electric, electro-osmotic, (micro-) valve, etc.
• a control system can employ a variety of sorting techniques to change or direct the flow of molecules, cells, small molecules or particles into a predetermined branch channel.
• a branch channel is a channel that is in communication with a sorting region and a main channel.
• the main channel can communicate with two or more branch channels at the sorting module or branch point, forming, for example, a T-shape or a Y-shape. Other shapes and channel geometries may be used as desired.
• a branch channel receives droplets of interest as detected by the detection module and sorted at the sorting module.
• a branch channel can have an outlet module and/or terminate with a well or reservoir to allow collection or disposal (collection module or waste module, respectively) of the molecules, cells, small molecules or particles.
• a branch channel may be in communication with other channels to permit additional sorting.
• a characteristic of a fluidic droplet may be sensed and/or determined in some fashion, for example, as described herein (e.g., fluorescence of the fluidic droplet may be determined), and, in response, an electric field may be applied or removed from the fluidic droplet to direct the fluidic droplet to a particular region (e.g. a channel).
• a fluidic droplet is sorted or steered by inducing a dipole in the uncharged fluidic droplet (which may be initially charged or uncharged), and sorting or steering the droplet using an applied electric field.
• the electric field may be an AC field, a DC field, etc.
• a channel containing fluidic droplets and carrier fluid divides into first and second channels at a branch point.
• the fluidic droplet is uncharged. After the branch point, a first electrode is positioned near the first channel, and a second electrode is positioned near the second channel. A third electrode is positioned near the branch point of the first and second channels. A dipole is then induced in the fluidic droplet using a combination of the electrodes. The combination of electrodes used determines which channel will receive the flowing droplet. Thus, by applying the proper electric field, the droplets can be directed to either the first or second channel as desired. Further description of droplet sorting is shown for example in Link et al. (U.S. patent application numbers 2008/0014589, 2008/0003142, and 2010/0137163) and European publication number EP2047910 to Raindance Technologies Inc.
• heterogeneous population of molecules are sorted away from droplets that contain a
• Droplets may be further sorted to separate droplets that contain a homogeneous population of amplicons of the target from droplets that contain a homogeneous population of amplicons of the variant of the target.
• Methods of the invention may further involve releasing amplified target molecules from the droplets for further analysis.
• Methods of releasing amplified target molecules from the droplets are shown in for example in Link et al. (U.S. patent application numbers 2008/0014589, 2008/0003142, and 2010/0137163) and European publication number EP2047910 to RainDance Technologies Inc.
• sample droplets are allowed to cream to the top of the carrier fluid.
• the carrier fluid can include a perfluorocarbon oil that can have one or more stabilizing surfactants.
• the droplet rises to the top or separates from the carrier fluid by virtue of the density of the carrier fluid being greater than that of the aqueous phase that makes up the droplet.
• the perfluorocarbon oil used in one embodiment of the methods of the invention is 1.8, compared to the density of the aqueous phase of the droplet, which is 1.0.
• the creamed liquids are then placed onto a second carrier fluid which contains a destabilizing surfactant, such as a perfluorinated alcohol (e.g. lH,lH,2H,2H-Perfluoro-l-octanol).
• a destabilizing surfactant such as a perfluorinated alcohol (e.g. lH,lH,2H,2H-Perfluoro-l-octanol).
• the second carrier fluid can also be a perfluorocarbon oil.
• the released amplified material can also be subjected to further amplification by the use tailed primers and secondary PCR primers.
• the primers in the droplet contain an additional sequence or tail added onto the 5' end of the sequence specific portion of the primer.
• the sequences for the tailed regions are the same for each primer pair and are incorporated onto the 5' portion of the amplicons during PCR cycling.
• another set of PCR primers that can hybridize to the tail regions of the amplicons can be used to amplify the products through additional rounds of PCR.
• the secondary primers can exactly match the tailed region in length and sequence or can themselves contain additional sequence at the 5' ends of the tail portion of the primer.
• additional regions also become incorporated into the amplicons.
• additional sequences can include, but are not limited to adaptor regions utilized by sequencing platforms for library preparation and sequencing, sequences used as a barcoding function for the identification of samples multiplexed into the same reaction, molecules for the separation of amplicons from the rest of the reaction materials such as biotin, digoxin, peptides, or antibodies and molecules such as fluorescent markers that can be used to identify the fragments.
• the amplified target molecules are sequenced.
• the sequencing is single-molecule sequencing-by-synthesis. Single-molecule sequencing is shown for example in Lapidus et al. (U.S. patent number 7,169,560), Quake et al. (U.S. patent number 6,818,395), Harris (U.S. patent number 7,282,337), Quake et al. (U.S. patent application number 2002/0164629), and Braslavsky, et al., PNAS (USA), 100: 3960-3964 (2003), the contents of each of these references is incorporated by reference herein in its entirety.
• a single- stranded nucleic acid e.g., DNA or cDNA
• a single- stranded nucleic acid e.g., DNA or cDNA
• oligonucleotides attached to a surface of a flow cell.
• the single- stranded nucleic acids may be captured by methods known in the art, such as those shown in Lapidus (U.S. patent number 7,666,593).
• the oligonucleotides may be covalently attached to the surface or various attachments other than covalent linking as known to those of ordinary skill in the art may be employed.
• the attachment may be indirect, e.g., via the polymerases of the invention directly or indirectly attached to the surface.
• the surface may be planar or otherwise, and/or may be porous or non-porous, or any other type of surface known to those of ordinary skill to be suitable for attachment.
• the nucleic acid is then sequenced by imaging the polymerase-mediated addition of fluorescently-labeled nucleotides incorporated into the growing strand surface oligonucleotide, at single molecule resolution.
• Table 2 5'-exonuclease genotyping assay design. Assay conditions in column 5 are specific to the multiplexed SMA assay. References: D. Anhuf, T. Eggermann, S. Rudnik-Schoneborn and K. Zerres, Hum Mutat. , 2003, 22, 74-78; D. DiMatteo, S. Callahan and E. B. Kmiec, Exp Cell Res. , 2008, 15, 878- 886.
• Human DNA was obtained in already purified form from cell lines (See Table 3; Coriell, Camden, NJ) and fragmented before use with a K7025-05 nebulizer following manufacturer's instructions (Invitrogen, Carlsbad, CA). DNA concentration was quantified by measuring absorbance at 260 nm on a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE).
• Microfluidic chips were manufactured by conventional soft lithography. Molding masters were fabricated by spin coating SU-8 negative photoresist (MicroChem Corp., Newton, MA) onto 6 inch silicon wafers and transferring the fluidic features from photomasks (CAD/ Art Services, Bandon, OR) by contact lithography with an OAI Hybralign Series 200 aligner (OAI, San Jose, CA). Chips contained channels with two depths: deep channels with low
• hydrodynamic resistance 100 + 10 um
• shallow channels (20 + 1 um) for droplet manipulation and detection.
• SU-8 photoresists 2100 and 2025 were used for deep and shallow channels respectively.
• Polydimethylsiloxane (PDMS) (Sylgard ® 184, Dow Corning, Midland, MI) chips were molded from the negative masters within mold housings of custom design. Glass cover slides were permanently bonded to the fluidic side of the chips by surface activation in an AutoGlowTM oxygen plasma system (Glow Research, Phoenix, AZ) followed by immediate contact bonding.
• microfluidic channels were exposed for ⁇ 2 min to lH,lH,2H,2H-perfluorodecyltrichlorosilane (Alfa Aesar, Ward Hill, MA) dissolved in FC-3283 (3M Specialty Materials, St. Paul, MN) prepared as a mixture of 18 g silane in 100 uL solvent.
• FC-3283 (3M Specialty Materials, St. Paul, MN) prepared as a mixture of 18 g silane in 100 uL solvent.
• the droplet generation chip created an emulsion of uniformly sized aqueous droplets of template DNA and PCR master mix that were suspended in an inert fluorinated oil with an emulsion stabilizing surfactant, called "carrier oil” from this point forward (REB carrier oil; RainDance Technologies, Lexington, MA). Droplets were generated in a cross-shaped microfluidic intersection, or "nozzle".
• aqueous phase flowed into the nozzle from the right (160 uL/hr), joining flows of the carrier oil from the top and bottom (750 uL/hr of total oil), and producing 4 pL droplets at a rate of 11 kHz.
• the channel widths at the intersection measured 15 um for the aqueous inlet, 12.5 for the oil inlets, and 15 um widening to 40 um at the outlet.
• Flow was driven by custom OEM pumps (IDEX Corporation, Northbrook, IL).
• Approximately 25 uL of the PCR reaction mixture was collected as an emulsion from the droplet generation chip and thermally cycled in a DNA Engine (Bio-Rad, Hercules, CA).
• the reaction mixture contained lx TaqMan ® universal PCR master mix (Applied Biosystems, Carlsbad, CA), 0.2 mM dNTP (Takara Bio, Madison, WI), and various amounts of primer pairs and probes as described in the results, lx assay concentration is defined as 0.2 ⁇ probes with 0.9 ⁇ primers. In all cases, when varied from the lx concentration, the primers and probes were varied by the same amount.
• the cycler program included a 10 min hot start at 95 °C, and 45 cycles of 15 s at 95 °C and 60 s at 60 °C.
• the droplets became concentrated during off-chip handling because the carrier oil is more dense than the aqueous phase and drained down from the emulsion. Hence the droplets were reinjected into the readout chip as a tightly packed emulsion that required dilution prior to readout to properly distinguish one droplet from another.
• a "spacer" nozzle similar to the droplet generation nozzle above was used to inject uniform plugs of extra carrier oil between droplets immediately before readout. As shown in Fig. 3b, the droplet entrance into the nozzle tapered down into a constriction about the size of an individual droplet forcing the droplets to enter the nozzle in single file and consequently at a stable rate. Opposed flow of the carrier oil from the top and bottom channels separated the droplets uniformly.
• the channel leaving the spacer nozzle increased in width along the direction of flow, and the droplets were interrogated by laser induced fluorescence at the location along the channel where the width was smaller than or equal to the droplet diameter (marked with an arrow in Fig. 3b).
• the nozzle dimensions were 15 um for the droplet entrance and exit, and 20 um for the oil lines.
• Fluorescence readout was performed by conventional epifluorescence microscopy with a custom microscope.
• a 20 mW, 488 nm laser source (Cyan; Picarro, Sunnyvale, CA) was expanded 2x and focused by the objective lens (20x / 0.45 NA; Nikon, Japan) onto the microfluidic channel.
• Two band pass filters discriminated the fluorescence collected through the objective lens: 512/25 nm and 529/28 nm for FAM and VIC fluorophores respectively

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