Selective digital multiplexing

Abstract

The present invention provides methods for the detection of molecular targets by digital PCR (dPCR) with blockers against non-target molecules. Each target is provided with a unique mixture of probes. The blocker binds to the non-target molecule and prevents the non-target molecule from generating fluorescence from the partition. The fluorescence intensities of the two or more colors are read, and the fluorescence intensities of the two colors are drawn as a 2D map. In this map, different targets facilitate well-resolved clusters. Each cluster in the graph is present substantially along its own radius range, allowing radial multiplexing. The use of blockers is selected for the detection of non-target molecules and increases the resolution of radial multiplexing, thereby allowing multiple targets to be detected.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the detection of molecular targets. BACKGROUND

[0002] Many clinical and research methods involve the detection of specific nucleic acids. For example, cancers are often associated with certain mutations in tumor DNA, and the ability to detect those mutations can inform about the presence or progression of cancer and the success of treatment. In theory, after a patient is treated to remove a tumor, the success of the treatment can be evaluated by performing an assay to detect tumor DNA. It will be appreciated that tumor cells and free tumor DNA can be found circulating in the blood. Detecting such circulating tumor nucleic acids in a laboratory assay would be a valuable diagnostic tool.

[0003] Unfortunately, circulating tumor DNA is only a minor fraction of drawn blood, making its detection challenging. Such a sample also includes a large amount of DNA from non-tumor cells. In addition, such tumor DNA can have a wide variety of different genetic mutations, each of which can be clinically significant. Similar challenges exist in the area of pathogen detection, agriculture, and other endeavors. For example, there is increasing interest in assaying wastewater to monitor patterns of viral transmission in a community. However, in such a sample, the target of interest can only be present in a minor fraction, and among those viral nucleic acids that are present, the target of interest can also have genetic diversity. For example, a wastewater system serving a metropolitan area can have trace amounts of viral nucleic acids that are differentially derived from different variants of viruses that are circulating in the area.

[0004] Some methods for detecting such nucleic acids of interest involve capture and amplification of the targets by, for example, polymerase chain reaction (PCR). However, for certain samples, particularly where the target of interest is only present in small amounts, and especially where there is a large number of similar but less informative molecules or where there is diversity among the targets of interest, PCR-based assays can not be entirely satisfactory. SUMMARY

[0005] The present invention provides methods for detecting molecular targets, such as nucleic acids, using highly multiplexed digital PCR (dPCR) with blocker molecules that inhibit detection of non-target molecules in the sample. For the multiplexed dPCR of the present invention, a sample is divided into a large number (tens of thousands) of aqueous partitions, and a PCR reaction is performed in each partition with primers and probes that produce amplicons and provide a detectable signal when the target of interest is present in the partition. Each partition can include probes specific to several different targets (e.g., three, four, five, six, or seven or more), and the methods of the present invention allow the presence of each target to be read even when fewer detection channels are used than there are targets present. Certain embodiments use fluorescent hydrolysis probes that are read in two or more optical channels at once. A mixture of unique probes is provided for each target, the mixture of probes will fluoresce in two or more channels. The fluorescence intensity of two or more colors is read (using optical detectors, such as photodiodes), and the fluorescence intensity can be stored as a 2D plot with one axis for each color. Each target present in a partition will appear as a cluster of unique points on the plot. With careful probe design by the methods of the present disclosure, the individual clusters are well resolved, and software can be used to model the Poisson distribution of targets into partitions and report which quantities of targets in the original sample produced the observed clusters. Thus, the readout is multiplexed by virtue of the independent detection of multiple targets from one, two (or more) color detection operations.

[0006] Highly multiplexed detection of rare targets is facilitated by the use and incorporation of blockers. A blocker is a molecule or species of molecules that binds to at least one non-target molecule or suppresses the at least one non-target molecule from producing fluorescence from a partition. For example, a blocker can be an oligonucleotide that is perfectly complementary to a nucleic acid that is not an intended target of the dPCR assay. The blocker will bind to the non-target and copies of its amplicon. The blocker can suppress or displace any fluorescent probe from binding to those molecules, or the blocker can hinder one of the primers from binding to those molecules, thereby suppressing amplification of the non-target and removing the non-target molecule from the dPCR readout. As a result of this suppression, the blocker removes the non-target molecule from the dPCR readout. The multiplexed probe combination and blockers work particularly well together to prevent wild-type sequences from being read in a dPCR assay for rare mutations or variants. With the methods of the present invention, multiple cancer-related mutations can be reliably detected in a multiplexed digital PCR assay while blocking two wild-type alleles from being read.

[0007] When reading for variants to be detected in two or more color channels, the resulting plot has well-resolved clusters. Each cluster in the plot exists along its own radius range extending from the cluster of the double negative partition, and detecting those independent clusters can be referred to as radial multiplexing. The use of a blocker selects for detection of non-target molecules and improves the resolution of radial multiplexing, allowing five, six, seven, or more independent targets (e.g., variants) to be detected in the result. Selective digital multiplexing provides reliable quantification of multiple targets in a sample.

[0008] In certain aspects, the present invention provides methods of target detection. The methods of the invention include partitioning a sample into a plurality of aqueous partitions, the sample including, or potentially including, or suspected of including one or more of at least four different nucleic acid variants or being tested for the presence or absence of one or more of at least four different nucleic acid variants. The partitioning is performed such that the partitions include amplification reagents, variant-specific probes, and a blocker that suppresses fluorescence of amplification from a fourth variant of the variants. Each of the variant-specific probes (e.g., fluorescent hydrolysis probes) is specific to one of three variants of the variants and produces fluorescence of a first color or a second color. Significantly, at least some of the targets are targeted by a mixture of probes with two or more different colors of fluorescent reporters. To illustrate, a first target can be targeted using only probes with a carboxyfluorescein reporter (FAM), a third target can be targeted using only probes with a hexachlorofluorescein reporter (HEX), and a second target can be targeted using a mixture or probes, some with FAM and some with HEX. More complex multiplexing schemes using still other mixtures of probes are described herein.

[0009] The partitions are subjected to conditions that promote amplification (e.g., thermal cycling), and the methods include reporting the presence or absence of the three variants of the variants in the sample based on the amounts of the first color and the second color detected from the partitions. The fourth variant of the variants can be wild type, and the three variants of the variants are mutated versions of the wild type, and the blocker comprises an oligonucleotide without a fluorescent label. The methods provide for a digital PCR (dPCR) assay in a sample using a two-color readout in which two targets can each be independently detected. Significantly, where the target of interest is a variant or mutant of some wild type gene (e.g., estrogen receptor 1), a blocker can suppress fluorescence produced by amplification of the wild type, allowing the dPCR readout to have a much stronger discriminatory power for variants that can each be a very minor fraction of the original sample relative to the wild type.

[0010] In support of readouts such as dPCR, these methods involve partitioning the sample and reagents into partitions. The partitions can be droplets, wells in a plate, or other fluidic partitions. The methods preferably include diluting the sample so that each partition receives a limited number of target molecules, such as zero, one, two, sometimes three, and very rarely four or more target molecules. The dilution can be calculated so that a majority of the partitions receive the target number (e.g., zero or one) of targets. In the presence of a fluorescent probe, each target molecule will serve as a template for generating an amplicon. Preferred probes include an oligonucleotide backbone that anneals to the target of the probe in a sequence-specific manner plus a fluorophore and a quencher. The methods can include thermocycling the droplets within the wells of a reaction tube or plate. During amplification using a polymerase, the exonuclease activity of the fluorophore will digest the oligonucleotide backbone of any bound probe, separating the fluorophore from the quencher, which allows the fluorophore to fluoresce during the readout step. To read the fluorescence, the methods can include flowing the droplets (e.g., one at a time) past a detector (and optionally past a light source for exciting the fluorophore).

[0011] The methods can be used to multiplex detection of multiple targets using dual channel readout at a time. For example, five targets can be read in two color channels. In some embodiments, four or six or more color channels are used, with two used at a time during readout. For example, the methods can include using at least six colors to read at least seven variants of a sample, with the detection step reading two of the six colors in two channels at a time. Alternatively, other combinations of channels and read points can be used (e.g., 2 colors at 1 read point, 2 colors at 2 read points, 2 colors at 3 read points, 3 colors at 1 read point, 3 colors at 2 read points, or 4 colors at 1 read point). The six colors can be provided by any suitable fluorophores or fluorescent dyes. Some embodiments use six fluorescent reporters including carboxyfluorescein (FAM), hexachlorofluorescein (HEX), cyanine 5 (CY5), cyanine 5.5 (CY5.5), 5-carboxy-X-rhodamine (5-ROX), and a fluorescent oligonucleotide dye with an adsorption of 594 nm (ATTO590).

[0012] Methods can be used for radial multiplexing, which can be implemented by reading two or more colors from each of a plurality of partitions, plotting the intensities of the two colors on a 2D plot with an axis for intensity of each color and identifying clusters of points on the plot. It will typically be found that each cluster exists along a unique radius extending from a cluster corresponding to a double negative (no significant fluorescence of either color) partition. In radial multiplexing, clusters corresponding to unique targets in the sample can be distinguished according to their radial direction, which differs from the radial direction of the double negative cluster. Clusters can also be distinguished further based on radial distance. Thus, five or more targets can be distinguished by providing a mixture of probes with different amounts of two fluorescent reporters interrogated in two channels. Other multiplexing methods, such as fluorescence intensity multiplexing, are also within the scope of the present disclosure. For radial multiplexing, the reporting step can comprise plotting the amounts of a first color and a second color detected from a partition as points on a plot, and identifying clusters of points on the plot corresponding to the presence of any of the three variants. The number of the corresponding target in the sample can be determined by a Poisson model of the templates entering into the partition, which will yield the observed cluster pattern.

[0013] The amplification step can comprise performing thermal cycling using reagents including PCR primers and dNTPs. Preferably, the variant-specific probes comprise fluorescent hydrolysis probes, each of which anneals to a sequence of one of the three variants. The blocker can comprise an oligonucleotide that anneals to the wild-type version of the variant and inhibits amplification of the wild-type sequence. In some cases, the blocker can comprise an oligonucleotide that anneals to the wild-type version of the variant and prevents binding of the fluorescent probe to the wild-type version or amplicons thereof. The blocker can comprise other features (e.g., one or more locked nucleic acids in the oligonucleotide) or other molecular strategies. For example, in some embodiments, the blocker is a binding protein (such as an RNA-guided binding protein) that binds to a fourth variant of the variants and prevents amplification.

[0014] The reporting step can comprise detecting colors from the partitions one two or more color channels at a time in three detection operations for six colors. The reporting step can comprise reporting the presence or absence of at least seven mutations of a gene present in the sample. In some embodiments, the method is used to detect mutant versions or variants of the gene. The gene can be estrogen receptor 1 (ESR1), and one or more of the seven mutations can be selected from the group consisting of cl 138G>C, c.1387T>C, cl607T>G, c.1609T>A, cl610A>C, cl610A>G, and c.1613A>G in the coding sequence of the ESR1 gene.

[0015] In certain embodiments, the variants are present in tumor DNA in the sample, and the method (i) comprises isolating circulating tumor DNA (ctDNA) from the sample, or (ii) isolates circulating tumor cells (CTCs) from the sample and purifies DNA from the CTCs.

[0016] Preferably, each of the three of the variants is targeted with a corresponding probe or probe combination comprising a sequence specific for the variant and zero or a characteristic amount of the first color or the second color. In some embodiments, a first probe combination specific for a first variant comprises only probes with a first sequence and the first color, a second probe combination specific for a second variant comprises a subset of probes with a second sequence and the first color and a subset of probes with a second sequence and the second color, and a third probe combination specific for a third variant comprises only probes with a third sequence and the second color. Optionally, the amounts of the first color and the second color detected from the partitions are plotted on a graph against respective first and second axes, wherein the three of the variants each form a respective unique cluster on the graph, wherein the unique clusters can be separated by a radius range extending from one point.

[0017] Other aspects of the invention provide a multiplex digital PCR method comprising dividing a sample into a plurality of aqueous partitions, the sample comprising up to four unique nucleic acid targets, the aqueous partitions further comprising detectably labeled probes for three of the targets and a blocking agent that inhibits the generation of a detectable signal from a fourth of the targets during amplification conditions. The method comprises exposing the partitions to the amplification conditions, and detecting the presence or absence of each of the three of the targets in the sample by reading signals in two optical channels. The detecting step can be done by plotting signal intensity from each partition on a graph against axes for each channel, such that each of the three targets forms a unique cluster on the graph in the case that it is present in the sample. For those targets present in the sample, the graph can comprise clusters positioned along their own radius range extending from one point on the graph (the double negative point).

[0018] In some embodiments, where more than three targets are present, the method can detect the presence or absence of each of the targets in the sample by reading signals in more than two optical channels or by using different channels more than once for each read to read signals from the sample.

[0019] In some embodiments, a first of the three targets is detected using only a probe comprising a first oligonucleotide sequence linked to a label that produces a first color, a second of the three targets is detected using a mixture of probes comprising a second oligonucleotide sequence linked to a label that produces the first color and the second oligonucleotide sequence linked to a label that produces a second color; and a third of the three targets is detected using only a probe comprising a third oligonucleotide sequence linked to a label that produces the second color. The blocker can be an oligonucleotide that binds to the fourth of the targets or a copy thereof and inhibits binding of any detectably labeled probe.

[0020] In highly multiplexed embodiments, the sample can comprise up to eight targets, and the method can comprise detecting presence or absence of a minimum of seven of the eight targets by three two-color read operations on a total of six colors. In some embodiments, the method can comprise detecting presence or absence of a minimum of seven of the eight targets by one or more read operations each reading two or more colors. Four of the unique nucleic acid targets can comprise homologous gene sequences. The targets can be variants of a wild type gene, in which case the variants can be, for example, clinically significant, and the blocker serves to suppress digital PCR readout of the wild type. For example, the blocker can inhibit detection of a wild type sequence of a gene, in which case each target comprises a mutated portion of the gene (i.e., comprises a mutation relative to the wild type sequence). Exemplary genes can include BRAF, EGFR, KRAS, NRAS, PIK3CA, or ESRI. In certain embodiments, the sample comprises tumor DNA from a subject, the blocker suppresses detection of a wild type sequence from non-tumor DNA from the subject, and detection of the three targets (presence or absence of each target) indicates presence or grade of a tumor in the subject.

[0021] The blocker can comprise a protein that specifically binds to the fourth of the targets or an amplicon thereof. The blocker can be, for example, an RNA-guided binding protein.

[0022] The method can comprise estimating a number of each of the three of the targets in the sample by modeling a Poisson distribution of the three of the targets using a computer system, the modeling of the Poisson distribution of the three of the targets providing the readout of the signal in the two optical channels.

[0023] The four unique nucleic acid targets can include (i) a first mutation of a wild type sequence, (ii) a second mutation of the wild type sequence, (iii) a third mutation of the wild type sequence, and (iv) the wild type sequence. The detectably labeled probes can include a first probe comprising a first oligonucleotide that anneals to the first mutation and a fluorophore of a first color linked to the first oligonucleotide, a second probe comprising a second oligonucleotide that anneals to the second mutation and the fluorophore of the first color linked to the second oligonucleotide, a third probe comprising the second oligonucleotide that anneals to the second mutation and a fluorophore of a second color linked to the second oligonucleotide, and a fourth probe comprising a third oligonucleotide that anneals to the third mutation and a fluorophore of a third color linked to the third oligonucleotide.

[0024] In certain embodiments, the method involves detecting the presence or absence of each of five unique nucleic acid targets in two optical channels, wherein the five unique nucleic acid targets include target one, target two, target three, target four, and target five, and wherein the detectably labeled probes include fluorescent hydrolysis probes with five corresponding oligonucleotides, wherein: (i) all probes for target one have a first fluorophore, most probes for target two have the first fluorophore, and the remainder of the probes for target two have a second fluorophore, (iii) probes for target three have about equal amounts of the first fluorophore and the second fluorophore, (iv) most probes for target four have the first fluorophore, and the remainder of the probes for target four have a second fluorophore, and (v) all probes for target five have the second fluorophore. In some embodiments, the five unique nucleic acid targets are variants of a wild type gene sequence, and the blocker inhibits annealing of the fluorescent hydrolysis probes to the wild type gene sequence or an amplicon thereof.

[0025] Embodiments of the present invention provide for multiplexing five or more targets in a dual channel dPCR read, for example, by radial multiplexing. Multiplexed reading of at least five targets in two channels can involve detecting the presence or absence of each of five unique nucleic acid targets in two optical channels. The five unique nucleic acid targets can include target one, target two, target three, target four, and target five. Preferably, the detectably labeled probes include fluorescent hydrolysis probes with five corresponding oligonucleotides, each oligonucleotide linked to one of a first fluorophore and a second fluorophore, such that a matching amount of the first fluorophore and the second fluorophore cannot be detected for two of the five unique nucleic acid targets.

[0026] While described as reading in two optical channels, the scope of the application is not limited to reading that number of channels at one time. For example, in any of the embodiments described herein, one, two, three, four, or more channels or colors can be read at a single point in time. Further, the scope of the application also includes multiple sample readings taken within different color channels. For example, a four-channel read can be performed once, or two two-channel reads can be performed. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Steps of the method are illustrated.

[0028] Figure 2 Multiple detection of four variants is shown.

[0029] Figure 3 Plots obtained using a blocker are shown.

[0030] Figure 4 Reagents including probes and a blocker are shown.

[0031] Figure 5 2D plots of FAM and HEX from a partition are shown.

[0032] Figure 6 2D plots of CY5 and CY5.5 are shown.

[0033] Figure 7 2D plots of ROX and ATTO590 are shown.

[0034] Figure 8 Reads of WT ESR1 gene are shown.

[0035] Figure 9 Second reads of WT ESR1 gene are shown.

[0036] Figure 10 Final plot generated by instrument in ESR1 WT assay.

[0037] Figure 11 Results of fluorescence intensity multiplexed assay are shown. DETAILED DESCRIPTION

[0038] The present invention provides methods for detecting target of interest, and specifically provides for the use of digital PCR based detection for multiplex detection of multiple genetic targets in a single readout operation. Preferred embodiments can be used to detect three or more unique genetic targets in a readout operation that employs only two color channels at a time. Additionally, the methods of the present invention utilize a blocker that suppresses or inhibits the ability of one nucleic acid sequence to generate any signal in a digital PCR assay. The blocker can be particularly useful in situations where several sequences are related and those sequences of interest are primarily wild type sequences. When those variants are of clinical significance, the digital PCR assay of the present disclosure can be performed using fluorescent reporters that generate a unique signal for each variant of interest, while the blocker minimizes any signal from the wild type sequence.

[0039] For example, a digital PCR assay can involve amplification of target nucleic acids from a sample using a primer pair in aqueous partitions. The sample can include some number of variants of interest as well as a large number of copies of the wild type sequence. In this case, the templates are genetic homologs. The templates (wild type plus variants) are amplified by the primer pair. The partitions include detectably labeled probes for the variants of interest. Due to the homology between the templates, some variant specific probes can anneal to the wild type template or amplicons thereof. If such binding were to occur, the wild type template would generate a signal that would be detected in the final readout. The present invention uses a blocker to inhibit the signal from the wild type template. For example, the blocker can be an oligonucleotide that specifically binds to the wild type template. To maximize the effect of the oligonucleotide as a blocker, the blocker can include a wild type specific base at one end, and can include one or more locked nucleic acids, can be designed against a GC rich stretch of the wild type template, or can include other such features or combinations thereof. The blocker can include the full wild type sequence. The blocker can include a blocking moiety at one end to prevent extension by a DNA polymerase. For example, the blocker can include a blocking moiety at the 3' end to prevent extension by a DNA polymerase. Such blocking moieties can include modifications such as a minor groove binder, an inverted DNA nucleotide, a 3 carbon spacer, a modified or unmodified nucleotide that does not pair with the template (e.g., 1, 2, 3, 4 or more nucleotides), or other moieties known in the art. In some embodiments, the blocker anneals to the wild type sequence and inhibits amplification and non-specific binding by the variant specific probes during PCR using, for example, a fluorescent hydrolysis probe. In other embodiments, the blocker can anneal to the template and / or amplicons thereof and can not be displaced by the variant specific probes during PCR using, for example, a fluorescent hydrolysis probe. With the aid of the blocker, each aqueous partition will only generate a large amount of fluorescence if the variant that the probe is targeting is the only one in the partition.

[0040] By virtue of the inhibition of detectable signals from the predominant species by the blocker, the blocker facilitates successful multiplexed detection of various minor species in the sample. That is, multiple (e.g., 3, 4, 5, or more) variants can be detected in a single dual-channel readout, and those variants will form well-bounded and distinguishable clusters, in part because the predominant wild-type has been suppressed. While set forth here in terms of mutant variants and wild-type sequences, the methods of the present invention facilitate successful multiplexed detection in a variety of contexts and biological situations, particularly where multiple minor (in quantity) targets of interest are present in a sample that includes a predominant target. For example, in assays directed to tumor mutations, the multiplexed detection according to the methods of the present invention can use a blocker to suppress healthy, non-tumor sequences. In assays directed to circulating fetal nucleic acids, the methods can use a blocker to suppress signal from maternal DNA. The methods of the present invention can be used for multiplexed detection of pathogenic strains of bacteria within a sample dominated by benign strains. The methods of the present invention can be used to detect multiple variants of a virus (e.g., in wastewater or sewage), while blocking detection of variants for which the public has accepted an effective vaccine.

[0041] Figure 1Steps of a method 101 are illustrated. The method 101 includes partitioning 105 a sample into a plurality of aqueous partitions, the sample including, or potentially including, or suspected of including one or more of at least four different nucleic acid variants or being tested for the presence or absence of one or more of at least four different nucleic acid variants. An amplification mixture and a blocking agent are added 109 to the partitions. Alternatively, in some embodiments, the amplification mixture and the blocking agent can be added to the sample prior to partitioning. For example, in some cases, the sample can be combined with the amplification mixture and the blocking agent prior to the partitioning step 105, e.g., such that in step 105, the sample, the amplification mixture, and the blocking agent are partitioned into the plurality of aqueous partitions. Thus, in some cases, step 109 is omitted. The amplification mixture can include reagents for digital PCR, such as at least one primer pair, a polymerase, dNTPs, any cofactors or ions, and detectably labeled variant-specific probes, such as fluorescent hydrolysis probes. Each variant-specific probe has specificity for one of the three of the variants and produces fluorescence of a first color or fluorescence of a second color. For embodiments including more than three variants and / or more than two channels, those elements would typically still be included. For detecting at least three of however many variants, the dPCR would undergo at least one dual-channel readout step. The blocking agent is typically a molecule or complex that suppresses fluorescence from amplification of a fourth of the variants. The reaction mixture is amplified 113 by subjecting the partitions to conditions that promote amplification. A readout operation is performed to detect 119 fluorescence in at least two channels. In fact, the method 101 can be used with a series of N-channel detections 119 to detect >N variants. The method 101 includes reporting 125 the presence or absence of the three of the variants in the sample based on the amounts of the first color and the second color detected from the partitions.

[0042] Figure 2 is a diagram 201 illustrating multiplexed detection of four variants (labeled Mutant 1, Mutant 2, Mutant 3, and Mutant 4) and two wild-type alleles (labeled WT1 and WT2) from a droplet digital PCR assay by a dual-channel readout operation, here depicted without use of a blocking agent. For the readout shown, the reaction mixture includes a hydrolysis probe for Mutant 1 with fluorophore 1 (e.g., FAM) read in channel 1 and such a probe for Mutant 2. For Mutants 3 and 4, probes labeled with FAM can be included as well as a small amount of probes labeled with fluorophore 2 (e.g., HEX) read in channel 2. Each WT1 and WT2 is read using a hydrolysis probe labeled with fluorophore 2.

[0043] For the depicted embodiment, Mutant 1, Mutant 2, Mutant 3, Mutant 4, WT1, and WT2 are present in a sample that is partitioned into aqueous droplets with amplification mix. Those droplets can be formed by any suitable mechanism, instrument, or technique. For example, those reagents can be loaded into a droplet digital PCR system such as the system sold by Bio-Rad Laboratories under the trademark QX200®. That system can flow an aqueous sample with a preferred dilution through a microchannel to a junction at which an aqueous fluid containing amplification mix is added, downstream of which the aqueous mix encounters a cross-flow of an immiscible carrier fluid such as fluorinated oil. Under co-flow conditions with the oil, the aqueous reaction mix breaks apart into monodisperse water-in-oil droplets at the junction. Because of the preferred dilution, each droplet will contain zero or a small number of template molecules of nucleic acid from the sample. For example, zero, one, two, or three molecules per droplet can be common. The dilution can be calculated by reading the number of nucleic acids in the sample (e.g., optical density) and the average fragment length (which can be a known result from sample processing or can be determined by, e.g., a gel). A surfactant such as a fluorosurfactant can be added to promote droplet stability. Those aqueous droplets flow along the channel, surrounded by oil, and can be collected in suitable containers such as the wells of a 96-well plate. On a droplet digital PCR system, the plate can be subjected to thermal cycling so that the approximately tens of thousands of droplets in the wells are subjected to thermal cycling conditions. The template molecules in the droplets are amplified by way of primers, polymerase, and cofactors. During amplification, when those targets are present and the probes are hydrolyzed by the polymerase, the hydrolyzed probes anneal to their targets (or to amplicons thereof), releasing fluorophores into the droplets.

[0044] Each variant-specific probe (e.g., fluorescent hydrolysis probe) is specific to one variant and produces a first or second color of fluorescence that is read in Channel 1 or Channel 2, respectively. Preferably, at least some of the targets are targeted by a mixture of probes with two or more colors of fluorescent reporter. To illustrate this, the first target (Mutant 2) can be targeted using only probes with a carboxyfluorescein reporter (FAM), the third target (WT2) can be targeted using only probes with a hexachlorofluorescein reporter (HEX), and the second target (Mutant 4) can be targeted using a mixture or probes, some with FAM and some with HEX. More complex multiplexing schemes using more complex mixtures of probes are described herein.

[0045] After amplification, the digital PCR system can load the droplets, separated by oil, into a readout channel and flow the droplets through a detector. Some embodiments of the system read two optical channels (Channel 1 and Channel 2) during one readout operation. For example, Channel 1 and 2 can read FAM and HEX or other suitable dyes. Plotting any fluorescence in either channel from each partition results in a dot on plot 201. Plot 201 shows an interesting phenomenon. In a multiplexed assay, all targets can be distinguished. However, clusters of droplets that include only WT1, WT2, or both WT1 and WT2 occupy most of the space allocated along the Channel 2 axis. To improve discrimination of variants from each other, the present invention introduces a blocker.

[0046] As used herein, a blocker is a molecule or complex that suppresses any signal from some selected species that is expected to be present in a sample. The selected species to be blocked can be the predominant (largest quantity) nucleic acid expected in a sample that also includes minor (lesser quantity) other nucleic acids of interest (e.g., variants). Looking at FIG. 201, one can see that blocking any fluorescence from WT1, WT2, or both WT1 and WT2 would improve resolution between variants. In the case of using a blocker, the region of points labeled “WT + Mutant” in FIG. 201 (the ellipse) would not produce the same signal, so each point in that region would not have a significant contribution from either wild type. That is, any droplet containing one molecule of the mutant and one molecule of the wild type would only produce a large amount of fluorescence from the mutant. However, it is to be remembered that some variants (mutants) are detected using a mixture of probes, some are detected in channel 1 and some are detected in channel 2, so it should be understood that in FIG. 201 those mutants are still displaced in the positive direction along the channel 2 axis. Furthermore, it would be misleading to think that resolution or discrimination is improved by simply rescaling the channel 2 axis in the case where signal from wild type is removed. In fact, the blocker reduces the number of free fluorophores that the wild type contributes in some partitions. The total input excitation energy to the instrument can remain the same (e.g., the stimulus LED can run at a constant energy), but the relative proportion of free fluorophores available to absorb those photons has changed due to the blocker. Thus, a partition with two mutant molecules (or three, four, etc.) will absorb the same amount of excitation energy. In the case where wild type is blocked, a partition that includes a mixture of mutant molecules and wild type molecules will absorb a different amount of energy depending on whether the mutant is targeted by zero, one, or two probes specific to channel 2. It is to be remembered that some variants are only targeted by probes that produce a signal in channel 1, some are targeted by probes that produce a signal in channel 2, and some are targeted by a mixture of channel 1 and channel 2 probes, and it is to be remembered that each partition can include 0, 1, 2, 3, 4, or more molecules, so blocking wild type but keeping the excitation energy constant, the plot 201 of droplets would show greater spread in the channel 2 axis (in the case where wild type is blocked) because the variant-specific probes use a larger proportion of the excitation energy.

[0047] Figure 3Figure 301 is shown, which is obtained when WT1 and WT2 are blocked using a blocker while using a set of fluorescent probes with four target-specific oligonucleotides and two fluorophores to probe Mutant 1, Mutant 2, Mutant 3, and Mutant 4. As shown, Figure 301 is generated using an oligonucleotide specific to Mutant 1 (oligonucleotide 1), an oligonucleotide specific to Mutant 2 (oligonucleotide 2), an oligonucleotide specific to Mutant 3 (oligonucleotide 3), and an oligonucleotide specific to Mutant 4 (oligonucleotide 4). The probe set includes only two fluorophores, Hexachlorofluorescein (HEX), Cyanine 5 (CY5). To generate Figure 301, a probe set including the following six probes can be used:

[0048] Oligonucleotide 1-HEX, Oligonucleotide 2-HEX, Oligonucleotide 2-Cy5, Oligonucleotide 3-HEX, Oligonucleotide 3-Cy5, and Oligonucleotide 4-Cy5.

[0049] To achieve cluster separation in Figure 301, Mutant 2 is probed with the majority of Oligonucleotide 2-HEX probes and a small amount of Oligonucleotide 2-Cy5 probes, while Mutant 3 is probed with a small amount of Oligonucleotide 3-HEX probes and the majority of Oligonucleotide 3-Cy5 probes. The results shown on Figure 301 show radial multiplexing using a blocker in dPCR. Due to the blocker, all variants in the sample are displayed in clusters that can be distinguished from each other. In fact, cluster detection can be performed in software and the variant makeup of the sample can be interpreted by the software, the model, the Poisson distribution of variants into partitions during dilution and partitioning, to identify the concentration of the variant in the sample that is most likely to produce the observed clusters.

[0050] Note that clusters labeled "negative" represent partitions that had zero HEX and Cy5 intensity readouts as associated with the assay. Both FIG. 201 and FIG. 301 show a method for digital multiplexing, which is generally referred to herein as detection of multiple different targets in a single digital assay readout or operation. Digital generally refers to a detection method in which any given target is detected as present or absent. Polymerase chain reaction (PCR) has been used in digital assays that fall under the description of digital PCR or dPCR. Digital PCR (dPCR) provides precise, highly sensitive quantification of nucleic acids. Traditional PCR is an end-point assay that is semi-quantitative because amplified product is detected by agarose gel electrophoresis. Real-time PCR (or qPCR) uses fluorescence-based detection to allow measurement of accumulated amplified product as the reaction proceeds. qPCR requires normalization to a control (to a reference or standard curve), allowing only relative quantification. Furthermore, variations in amplification efficiency can affect qPCR results. Digital PCR builds on traditional PCR amplification and fluorescence probe-based detection methods to allow highly sensitive absolute quantification of nucleic acids without the need for a standard curve. In some droplet digital PCR systems (ddPCR), PCR samples are partitioned into 20,000 droplets. After amplification, droplets containing target sequences are detected by fluorescence and these droplets are scored as positive, and droplets with no fluorescence are scored as negative. Poisson statistical analysis of the number of positive and negative droplets gives absolute quantification of the target sequence.

[0051] Sykes et al. described the concept of digital PCR in 1992, acknowledging that the combination of limiting dilution, end-point PCR, and Poisson statistics could give an absolute measure of nucleic acid concentration. See Sykes, 1992, Quantitation of targets for PCR by use of limiting dilution, Biotechniques 13:444-449, which is incorporated by reference. A method was developed to dilute a sample and partition the sample to such a degree that single template molecules could each amplify individually in separate partitions and the product detected using a fluorescent probe. See Vogelstein, 1999, Digital PCR, PNAS 96:9236-9241, which is incorporated by reference.

[0052] Digital PCR offers improved sensitivity over qPCR and provides detection of rare events in a population of wild-type sequences, such as single nucleotide mutations. In conventional qPCR, the signal from wild-type sequences can dominate and mask the signal from rare sequences. By minimizing the effects of competition between targets, digital PCR overcomes the difficulty inherent in amplifying rare sequences and allows sensitive and accurate absolute quantification of nucleic acids. Preferred embodiments of digital PCR involve sample partitioning - division of the sample into discrete subunits prior to PCR amplification. The sample is prepared in a manner similar to real-time PCR, but then is partitioned into, for example, thousands of partitions, each ideally containing zero or one (or at most a few) template molecules. Each partition behaves as a separate PCR reaction, and as with real-time PCR, a fluorescent probe is used to identify amplified target DNA. Then, each partition can be easily analyzed after amplification to determine whether it contains the target sequence. Samples containing amplified product are considered positive (1, fluorescent), and samples without product and thus with little or no fluorescence are negative (0). The ratio of positive to negative in each sample is the basis for quantification.

[0053] The method preferably includes diluting the sample so that each partition receives a limited number of target molecules, such as zero, one, two, sometimes three, and very rarely four or more target molecules. The dilution can be calculated so that a majority of the partitions receive the target number (e.g., zero or one) of targets. In the presence of a fluorescent probe, each target molecule will serve as a template for generating an amplicon.

[0054] The partitioning can be accomplished by any suitable mechanism, and any suitable type of aqueous partition can be used for digital PCR. Exemplary suitable partitions include droplets, wells in a plate, or other fluidic partitioning structures.

[0055] For example, partitions can be pico, nano, or micro droplets on wells, cavities, pockets, or openings or fluidic harbors on a microtiter plate or substrate (see, e.g., US 2010 / 0041046, which is incorporated by reference). Partitions can be wells in a multi-well plate, such as a 96-well plate, a 384-well plate, a 1536-well plate, a 3456-well plate, or a 9600-well plate. Partitions can be individual chambers (see, e.g., US 20210178395, which is incorporated by reference). Partitions can be unique zones defined within a fluidic device (see, e.g., US 20200269248, which is incorporated by reference). In certain embodiments, partitions are a plurality of droplets formed simultaneously by mixing an aqueous fluid and an oil together and vortexing them. In preferred embodiments, partitions are droplets of an emulsion, such as an oil-in-water (W / O) emulsion or a water-in-oil-in-water (W / O / W) emulsion.

[0056] Preferred embodiments use aqueous partitions in immiscible liquids within a microfluidic device, for example, surrounded or separated by a slug or droplet of immiscible carrier fluid, such as oil. Aqueous droplets can be formed in the immiscible carrier liquid by microfluidic handling. The microfluidic device can use channels to mix sample and reagents and form droplets in the immiscible carrier fluid. Droplets can be formed within a partitioning section or subunit of a digital PCR instrument or system that uses, for example, channels to flow the aqueous mixture into intersecting streams of carrier oil.

[0057] The unique sample partitioning step of digital PCR in conjunction with Poisson statistical analysis enables precision that is higher than that of traditional PCR and qPCR methods. Thus, digital PCR is particularly especially useful for applications that require detection of small amounts of input nucleic acid or finer resolution of target amounts in a sample, such as rare sequence detection, copy number variation (CNV) analysis, and gene expression analysis of rare targets.

[0058] Techniques used in digital PCR include PCR amplification on a microfluidic chip. See Ottesen, 2006, Microfluidic digital PCR enables multigene analysis of individual environmental bacteria, Science 314: 1464-1467, which is incorporated by reference. Other systems involve isolation onto microarrays (Morrison, 2006, Nanoliter high-throughput quantitative PCR, Nucleic Acids Res 34:el23, which is incorporated by reference) or spinning microfluidic disks (Sundberg, 2010, Spinning disk platform for microfluidic digital polymerase chain reaction, Anal Chem 82: 1546-1550, which is incorporated by reference) and droplet technology based on oil-water emulsions (Hindson, 2011, High-throughput droplet digital PCR system for absolute quantitation of DNA copy number, Anal Chem 83:8604-8610, which is incorporated by reference), such as droplet digital PCR (ddPCR) systems, such as the ddPCR system by Bio-Rad Laboratories, Inc. (Hercules, CA) sold under the trademark QX200.

[0059] In such digital assays, the presence or absence of a target will be interrogated for each reaction mixture (e.g., partition). Detection of the target can be performed using optical density, intercalating dyes, ethidium bromide, changes in pH, release of pyrophosphate, or any other suitable method for detecting the target.

[0060] Some embodiments discussed herein use fluorescent hydrolysis probes, such as the probes sold by ThermoFisher Scientific (Waltham, MA) under the trademark TAQMAN. These probes produce a fluorescent signal when their complementary target is amplified by PCR in the presence of the probe. Such probes include an oligonucleotide backbone that anneals to the target of the probe in a sequence-specific manner plus a fluorophore and a quencher. In some embodiments, the method can include thermocycling the droplets within the wells of a reaction tube or plate. During amplification using a polymerase, exonuclease activity will digest the oligonucleotide backbone of any bound probe, separating the fluorophore from the quencher, which allows the fluorophore to fluoresce during the readout step. In some specific embodiments, to read the fluorescence, the method can include flowing the droplets (e.g., one at a time) past a detector (and optionally past a light source for exciting the fluorophore).

[0061] Alternatively, the probes include molecular beacon probes that anneal to the sequence of the amplicon, which can or can not be labeled. The tag can be present on the end of the amplicon or in the middle of the amplicon sequence. The molecular beacon probe can include an oligonucleotide loop backbone that anneals to the substrate of the probe in a sequence-specific manner, a complementary stem sequence plus a fluorophore and a quencher. During the amplification reaction, the complementary sequence of the loop sequence is synthesized, followed by hybridization of the loop sequence, separating the fluorophore from the quencher, which allows the fluorophore to fluoresce during the readout step.

[0062] The fluorescent probes can include one or more quenchers and one or more fluorophores in one or more colors.

[0063] In other embodiments, other fluorescent probe systems known in the art can be used, such as any of the probe systems described in Storts DR. Alternative probe-based detection systems in quantitative PCR. J Mol Diagn. 2014 Nov; 16(6):612-4, which is incorporated by reference herein.

[0064] For a fluorescent hydrolysis probe or a molecular beacon probe, the probe can bind to the target sequence between two primers, or can bind to the tail of one or both primers used in the amplification process. While the description of the present invention refers to fluorescent hydrolysis probes, the present invention equally operates with other probe types that also provide a fluorescent signal (e.g., molecular beacon probes, FRET-based probes, Scorpions® probes, etc.). To read the fluorescence, the method can include flowing the droplets (e.g., one at a time) past one or more detectors (and optionally past a light source for exciting the fluorophore). Alternatively, to read the fluorescence, the method can include using immobilized partitions, including but not limited to droplets, within a 2D or 3D array, and imaging with one or more detectors (and optionally a light source for exciting the fluorophore). Alternatively, to read the fluorescence, the method can include using immobilized partitions, including but not limited to physical partitions such as wells, in a 2D array, and imaging with one or more detectors (and optionally a light source for exciting the fluorophore).

[0065] In another embodiment, an intercalating dye, such as SYBR green, can be used in combination with one or more fluorescent probes during the amplification process.

[0066] Some dPCR systems read two or more channels (or two or more colors of fluorescence) together at a time. For example, some ddPCR systems flow droplets past two color detectors, and read both colors simultaneously for each droplet. Some such platforms have multiple (e.g., six) color channels, but typically generate results by reading two colors from each partition at a time, as in FIG. 201 and FIG. 301. Alternatively, some dPCR systems read two or more channels sequentially.

[0067] Both FIG. 201 and FIG. 301 show digital multiplexing, as many different targets are each independently detected by dual color reading, and this digital multiplexing can be seen in both figures. One difference between FIG. 301 and FIG. 201 is that a selection step has been added to the digital multiplexing assay. In the depicted embodiment, a blocker (e.g., to wild type) was used in generating FIG. 301, but was not used in generating FIG. 201. The blocker is selective for the predominant molecules present in the sample by suppressing signal from wild type molecules, and is selective for molecules present in the sample as a minor fraction of the potential target. Thus, FIG. 301 shows selective digital multiplexing. To generate the selective digital multiplexing results shown in FIG. 301, certain reagent series (probes and blockers) can be used.

[0068] Figure 4Reagents 401 used in certain embodiments of selective digital multiplexing are shown, including probes and blockers. In the illustrated embodiment, the sample includes a fragment of a wild-type gene and at most three variants (e.g., 0, 1, 2, or all 3) VI, V2, and V3 (or possibly including, or suspected of including, or being tested for the presence or absence of). Variant VI is drawn as molecule 451, while wild-type V2 and V3 molecules are not shown. Reagents 401 for selective digital multiplexing include blockers 431, VI-dye 1 probe 403, V2-dye 1 probe 405, V2-dye 2 probe 415, and V3-dye 2 probe 416. As shown, all probes have a quencher 409. Some probes have a first fluorophore 407, and some probes have a second fluorophore 417. Reagents 401 include forward primer 435 and reverse primer 436 and other PCR reagents (e.g., polymerase, cofactors, dNTPs) that need not be drawn as will be readily understood by one of skill in the art.

[0069] When the three variants are amplified in partitions, the probes will release free fluorophores 407, 417. Each partition including any variant will absorb a fluorescent excitation light, which can be provided within the dPCR system by a light source such as an LED. Such systems typically include at least two channels, sometimes referred to as color channels, which include detectors such as photodiodes for detecting and recording the light signal from each partition. When each partition is read (e.g., when the droplet flows past the photodiodes of the two channels), the amplitude of the fluorescence from that partition is recorded. The recorded amplitudes of the two colors of fluorescence (e.g., HEX and Cy5) can then be plotted. Each partition will provide one of the points in the plot 301. Note that because of the use of the blocker 431, the different variants resolve into distinct separate clusters on the plot. In fact, from the two (or more) detection channels of the dPCR system, there are available software packages that create 2D plots showing the clusters and even detect and distinguish the clusters and implement a model based on Poisson statistics to provide the number of variants in the sample based on the observed data. One such software package is the dPCR analysis software sold by Bio-Rad Laboratories, Inc. under the trademark QUANTASOFT. Other methods for “calling” a sample (reporting the presence or absence of a variant introducing a probe) include the use of available software packages such as the software package named “dPCR Cluster Predictor” (dPCP), an R package, and a Shiny app for automated analysis of up to 4plex dPCR data. dPCP can analyze and visualize data generated by multiple dPCR systems that are not affected by the amount and integrity of the input of nucleic acids, performing accurate and fast clustering. See De Falco, 2023, Digital PCR cluster predictor: a universal R-package and shiny app for the automated analysis of multiplex digital PCR data, Bioinformatics 39(5): btad282, which is incorporated by reference.

[0070] As discussed, and as shown in connection with FIG. 301, the methods of the application can be used to selectively digitally multiplex detection of multiple targets at once with dual channel readout. For example, five targets can be read in two color channels. Some systems and platforms can use more than two color channels. In some embodiments, four or six or more color channels are used, two at a time during readout. For example, a method can include using at least six colors to read at least seven variants of a sample, where the detecting step reads two of the six colors in two channels at a time.

[0071] Figures 5 to 7 Detection for eight variants is shown to be performed using six color channels for selective digital multiplexing. A selective digital multiplexing assay is performed to detect variants of estrogen receptor 1 encoding the following amino acid substitutions: E380Q, Y537S, D538G, L536R, Y537N, Y537C, S463P, and an internal control. The gene fragments in the sample are 99% wild type, and 1% of the fragments are one of these variants.

[0072] As shown, the gene is estrogen receptor 1 (ESR1), and the probes each have specificity for at least one of: c.1138G>C, c.1387T>C, c1607T>G, c.1609T>A, c1610A>C, c1610A>G, and c.1613A>G in the coding sequence of the ESR1 gene.

[0073] The sample and reagents are partitioned into an aqueous partition. These reagents include a primer pair for the gene, a blocker for the wild type, a mixture of probes for each variant, and PCR reagents. A dPCR system is used to partition, amplify, read, and analyze the sample. The aqueous sample is partitioned by co-flowing into fluorinated oil to form droplets on the order of 20,000 nanoliters in size, which are then held in one well of a 96-well plate. The ddPCR system thermocycles the plate and transfers the droplets into microfluidic channels that flow the droplets one at a time through two photodiodes. A first read detects fluorescence from carboxyfluorescein (FAM) and hexachlorofluorescein (HEX) in each droplet. A software package makes a 2D plot of the fluorescence readout.

[0074] Figure 5 A 2D plot 501 of the FAM and HEX read in each droplet is shown.

[0075] A second read detects fluorescence from cyanine 5 (CY5) and cyanine 5.5 (CY5.5) in each droplet.

[0076] Figure 6 A 2D plot of the CY5 and CY5.5 read in each droplet is shown.

[0077] Figure 7 A 2D plot is shown resulting from the final read of 5-carboxy-X-rhodamine (5-ROX) and fluorescent oligonucleotide dyes with 594 nm absorbance (ATTO590) from each droplet. In the depicted example, where the target of interest is a variant or mutant of the estrogen receptor 1, the blocker suppresses the fluorescence produced by amplification of the wild type, allowing the dPCR to read out a stronger discrimination for variants, which can each be a very minor fraction of the original sample (here 1%) relative to the wild type. As shown, the selective digital multiplexing approach (selectivity by means of the blocker, digitization by means of separation of templates into partitions) includes the use of at least six colors to read at least seven variants of the sample, with the detection step reading two of the six colors in two channels at a time.

[0078] Preferably, each variant is targeted with a corresponding probe or probe combination that includes a sequence specific for that variant and zero or a characteristic amount of the first or second color. Using Figure 501 for illustration only, two channels (FAM and HEX) are used to read variants encoding E380Q, Y537S, D538G, and L536R. Each of those four variants is detected with a probe or probe combination.

[0079] E380Q is referred to as a first variant, the probes for which all include an oligonucleotide specific for the variant and a FAM fluorophore. In the case of D538G as a second variant, the probe combination for the variant includes an oligonucleotide specific for the variant with about 25% linked to HEX and about 75% linked to FAM. For Y537S, about 25% of the probes have FAM and about 75% of the probes have HEX. For L536R, all probes are linked to HEX. The exact percentages are not as important as the principle to be illustrated: multiple variants can have respective probes each carrying one of two fluorophores, with the percentage of probes having a given fluorophore varying gradually with the target. From this probe combination, the amounts of the first and second colors detected from the partitions are plotted against the respective first and second axes on FIG. 501. The variants each form a respective distinct cluster on FIG. 501. Significantly, the distinct clusters can be separated by a radius range extending from one point (the dark, unlabeled cluster corresponding to double-negative droplets). It should be noted that the blocker converts the wild-type droplets to double-negative. Because the clusters can be represented by distinct radius ranges, this readout can conveniently be referred to as radial multiplexing. The assay is selective for mutations (i.e., variants) due to the blocker. As implemented to generate FIG. 501, the assay is performed using droplet digital PCR. Thus, the result is a selective digital PCR assay with radial multiplexing.

[0080] This selective multiplexing assay can be used for any suitable purpose in the fields of research, medicine, investigation, diagnosis, or any other endeavor. For example, the depicted method can be used for analysis of tumor nucleic acids. In some examples, a patient can have or have had a tumor. The method of the present invention can be used to analyze tumor DNA from a sample of the patient. For example, a liquid biopsy sample, such as drawn blood, can be analyzed for circulating tumor DNA (ctDNA), or the liquid biopsy sample can be analyzed to capture circulating tumor cells (CTCs) and extract nucleic acids from the CTCs. The blocker can suppress readout from wild-type, thereby yielding extremely high sensitivity for tumor DNA. Analysis of such a sample by selective digital PCR using radial multiplexing as shown can provide evidence of the presence of a tumor in the patient. This method can be used, for example, to rapidly and economically detect minimal residual disease after cancer treatment.

[0081] As can be seen, the method of the present application can use radial multiplexing, which can be implemented by reading two colors from each of a plurality of partitions, plotting the intensities of the two colors on a 2D plot with an axis for intensity of each color and identifying clusters of points on the plot. It will generally be found that each cluster exists approximately along a unique radius extending from a cluster corresponding to a double negative (no significant fluorescence of either color) partition. In radial multiplexing, clusters corresponding to unique targets in the sample can be distinguished according to their radial direction, which differs from the radial direction of the double negative cluster. Clusters can also be distinguished further based on radial distance. Thus, five or more targets can be distinguished by providing a mixture of probes with different amounts of two fluorescent reporters interrogated in two channels. Other multiplexing methods, such as fluorescence intensity multiplexing, are also within the scope of the present disclosure. For radial multiplexing, the reporting step can comprise plotting the amounts of a first color and a second color detected from a partition as points on a plot, and identifying clusters of points on the plot corresponding to the presence of any of the three variants. The number of the corresponding target in the sample can be determined by a Poisson model of the templates entering into the partition, which will yield the observed cluster pattern.

[0082] Figures 8 to 10 Reads for the WT ESR1 gene without any one nucleic acid substitution at positions 536, 537, 538, or 380 are shown, which were read in the FAM and HEX channels. Figures 5 to 7 Results of a droplet digital PCR assay for the wild type sequence homologous to the variant interrogated in the ESR1 WT assay are shown ("ESR1 WT Assay"). A PCR system with six color channels was used. The system reads two channels at a time, and with the aid of a dPCR software analysis package in the associated computing system (e.g., QUANTASOFT), three plots from one well of a 96-well plate are generated per instrument run, approximately 20,000 sample droplets are held in the well.

[0083] Figure 8 Reads for the WT ESR1 gene without any one nucleic acid substitution at positions 536, 537, 538, or 380 are shown, which were read in the FAM and HEX channels.

[0084] Figure 9 Reads for the WT ESR gene not encoding a substitution at position 463 are shown, which were read in the Cy5 channel (while the instrument reads Cy5 and Cy5.5).

[0085] Figure 10 Is a blank plot generated by the instrument in the ESR1 WT assay, as the instrument was reading in the ROX and ATTO590 channels, but these channels were not used in this assay. The presence of a large amount of WT nucleic acid shown in the ESR1 WT assay highlights the importance of using a blank plot in the generation of a standard curve. Figures 5 to 7the values obtained using a blocker in a mutant or variant assay. Due to the inclusion of a blocker, true radial multiplexing of all cancer related variants can be reliably performed.

[0086] Embodiments of the present application are described and illustrated using radial multiplexing. However, other multiplexing techniques are within the scope of the present application. For example, all probes can have a unique color, and each variant can be read in its respective color channel. Other embodiments involve the use of fluorescence intensity multiplexing.

[0087] For fluorescence intensity multiplexing, two or three or more different targets can each be given their own detectable label, but the assay can provide the label in a form that provides a different unique fluorescence intensity for each target. For example, a blocker can be used to block the nucleic acids of the wild type or similar primary species. Each variant or target of interest can be given its own fluorescent hydrolysis probe. However, each probe can have a unique number of fluorophores (e.g., probes for target 2 can each include an oligonucleotide linked to two fluorophores, with only one fluorophore on the probe for target 1). Additionally or alternatively, the probes can be provided at different concentrations. For example, target 1 can be provided with a stoichiometrically calculated limited number of probes, such that only about 10% of target 1 amplicons are ultimately probe labeled, while target 2 probes can be provided in excess, such that 100% of target 2 amplicons are probe labeled. Additionally or alternatively, the amplification efficiency between targets can also be controlled, such that, for example, the amplification efficiency of target 1 is greater than that of target 2, and thus provides greater fluorescence intensity. This can result from different lengths of amplicons, different GC content, different annealing temperatures to the probes, non-natural bases, and / or different modifications on the backbone of the DNA.

[0088] Figure 11Results of a fluorescence intensity multiplexing assay are shown. Since only one color channel was read, a 2D plot is not required. Due to the blocker, partitions containing wild type do not fluoresce to a large extent. Partitions containing target 1 fluoresce at an intensity of about 0.23 V due to limited probe numbers. Partitions containing target 2 fluoresce at an intensity of about 0.35 V due to ample probe numbers. The spike in fluorescence above 0.45 V indicates target 3. Partitions with very high fluorescence reflect multiple templates in one partition due to Poisson distribution in the partitions. From the fluorescence intensity, one can "call" (calculate, predict, or report) the templates in the original sample by a software package that implements Poisson modeling. As shown, the present disclosure provides a multiplexed digital PCR method comprising partitioning a sample into a plurality of aqueous partitions, the sample comprising, or possibly comprising, or suspected of comprising one or more of at least four unique nucleic acid targets or being tested for the presence or absence of one or more of at least four different nucleic acid targets, the aqueous partitions further comprising detectably labeled probes for three of the targets and a blocker that inhibits the generation of a detectable signal from a fourth of the targets during amplification conditions. The method comprises exposing the partitions to amplification conditions, and detecting the presence or absence of each of the three of the targets in the sample by reading the signal. For radial multiplexing, the signal is preferably read in at least two optical channels. The detection step can be accomplished by plotting the signal intensity from each partition on a graph with an axis for each channel, such that each of the three targets forms a unique cluster on the graph if present in the sample. For those targets present in the sample, the graph can include clusters positioned along their own radius range extending from one point on the graph (the double negative point). For fluorescence intensity multiplexing, only the fluorescence intensity needs to be read in one channel. Some embodiments of the present invention provide multiplexing of five or more targets, for example by radial multiplexing in a two-channel dPCR read or by fluorescence intensity multiplexing in a single channel read.

[0089] In highly multiplexed embodiments of the methods of the disclosure, a sample can comprise, or can possibly comprise, or is suspected of comprising, one or more of at least eight targets, or is being tested for the presence or absence of one or more of at least eight targets, and the method can comprise detecting the presence or absence of a minimum of seven of the eight targets by performing three two-color read operations on a total of six colors. Four of the unique nucleic acid targets can comprise homologous gene sequences. The targets can be variants of a wild-type gene, in which case the variants can have, for example, clinical significance, and the blockers are used to suppress the digital PCR readout beyond the wild-type. For example, the blockers can suppress detection of the wild-type sequence of the gene, in which case each target comprises a mutated portion of the gene (i.e., comprises a mutation relative to the wild-type sequence). Exemplary genes can include BRAF, EGFR, KRAS, NRAS, PIK3CA, or ESRI. The sample can comprise tumor DNA from a subject, the blockers suppress detection of the wild-type sequence from non-tumor DNA from the subject, and detection of three targets (presence or absence of each target) is indicative of the presence or grade of a tumor in the subject.

[0090] The features described above, and those claimed below, can be combined in various ways. The following examples show some possible, non-limiting combinations: (A1) A target detection method comprising partitioning a sample into a plurality of aqueous partitions, the sample comprising, or possibly comprising, or suspected of comprising, one or more of at least four different nucleic acid variants or being tested for the presence or absence of one or more of at least four different nucleic acid variants. The partitions comprise amplification reagents; variant-specific probes, wherein each probe has specificity for one of three of the variants and produces fluorescence of a first color or fluorescence of a second color; and a blocker that suppresses fluorescence from amplification of a fourth of the variants. The partitions are subjected to conditions that promote amplification, and the presence or absence of the three of the variants in the sample is reported based on the amounts of the first color and the second color detected from the partitions.

[0091] (A2) For the method of (A1), the probes comprise fluorescent hydrolysis probes.

[0092] (A3) For the method of (A1), the probes comprise molecular beacon probes.

[0093] (A4) For the method of any one of (Al) to (A3), the fourth of the variants is wild type, and the three of the variants are mutant versions of the wild type, and optionally the blocker comprises an oligonucleotide that does not have a fluorescent label.

[0094] (A5) For the method of any one of (Al) to (A4), the subjecting step comprises thermocycling the partitions; and optionally the partitions are droplets, and the subjecting step comprises thermocycling the droplets, optionally thermocycling within wells of a reaction tube or plate, and further optionally wherein the reporting step comprises flowing the droplets one at a time past an excitation source and a detector.

[0095] (A6) For the method of any one of (Al) to (A5), the method further comprises using at least six colors in the partitions to detect the presence or absence of at least seven variants, wherein the detecting step reads the at least six colors in two or more channels at a time.

[0096] (A7) For the method of (A6), the six colors are provided by six fluorescent reporters, the fluorescent reporters comprising one or more of carboxyfluorescein (FAM), hexachlorofluorescein (HEX), cyanine 5 (CY5), cyanine 5.5 (CY5.5), 5-carboxy-X-rhodamine (5-ROX), and a fluorescent oligonucleotide dye with an adsorption of 594 nm (ATTO590).

[0097] (A8) For the method of any one of (Al) to (A7), the reporting step comprises plotting on a graph the amounts of the first color and the second color detected from the partitions as points, and identifying clusters of the points on the graph that correspond to the presence of any of the three variants.

[0098] (A9) For the method of any one of (Al) to (A8), the subjecting step comprises thermocycling, wherein the amplification reagents comprise PCR primers and dNTPs, wherein the variant-specific probes comprise fluorescent probes, each fluorescent probe annealing to a sequence of one of the three of the variants, and wherein the blocker comprises an oligonucleotide that anneals to a wild type version corresponding to one of the at least four variants and inhibits amplification and signal from the wild type version or amplicons thereof, optionally wherein the blocker comprises an oligonucleotide that anneals to the fourth of the variants and inhibits amplification and signal from the wild type version or amplicons thereof.

[0099] (A10) For the method of any one of (Al) to (A9), the blocker comprises a binding protein that binds to one of the at least four variants and prevents amplification, and optionally the blocker binds to the fourth of the variants and prevents amplification.

[0100] (A11) For the method of any one of (Al) to (A10), the reporting step comprises detecting color from the partitions in two or more color channels at a time in one or more detection operations.

[0101] (A12) For the method of (Al l), the reporting step comprises reporting the presence or absence of one or more mutations of a gene present in the sample, optionally wherein the reporting step comprises reporting the presence or absence of one or more of at least seven mutations of the gene present in the sample.

[0102] (A13) For the method of (A12), the gene is estrogen receptor 1 (ESRl), and the one or more of the seven mutations is selected from the group consisting of cl 138G>C, c.1387T>C, cl607T>G, cl609T>A, cl610A>C, cl610A>G, and c.1613A>G in the coding sequence of the ESRl gene.

[0103] (A14) For the method of any one of (Al) to (A13), the variants are present in tumor DNA in the sample, and the method (i) comprises isolating circulating tumor DNA (ctDNA) from the sample, or (ii) isolates circulating tumor cells (CTCs) from the sample and purifies DNA from the CTCs.

[0104] (A15) For the method of any one of (Al) to (A14), each of the three of the variants is targeted with one or more variant-specific probes or a combination of variant-specific probes comprising a sequence specific for the variant and zero or a characteristic amount of the first color or the second color.

[0105] (A16) For the method of (A15), the first combination of probes specific for a first variant comprises only probes with a first sequence and the first color, the second combination of probes specific for a second variant comprises a subset of probes with a second sequence and the first color and a subset of probes with a second sequence and the second color, and the third combination of probes specific for a third variant comprises only probes with a third sequence and the second color.

[0106] (A17) For the method of any one of (Al) to (A16), plotting on a graph the amounts of the first color and the second color detected from the partitions for the respective first and second axes, wherein the three of the variants each form a respective unique cluster on the graph, wherein the unique clusters can be separated by a radius range extending from a point.

[0107] (A18) For the method of any one of (Al) to (A17), the sample comprises one or more of at least four different nucleic acid variants.

[0108] (A19) For the method of any one of (Al) to (A18), the sample can comprise one or more of at least four different nucleic acid variants.

[0109] (A20) For the method of any one of (Al) to (A19), the sample is suspected of comprising one or more of at least four different nucleic acid variants.

[0110] (A21) For the method of any one of (Al) to (A20), the sample is being tested for the presence or absence of one or more of at least four different nucleic acid variants.

[0111] (A22) For the method of any one of (Al) to (A21), the sample comprises at least the fourth variant.

[0112] (A23) For the method of (A4), the sample comprises at least the fourth variant.

[0113] (B1) A multiplex digital PCR method comprising partitioning a sample into a plurality of aqueous partitions, the sample can comprise one or more of at least three unique nucleic acid targets and a fourth nucleic acid target, the aqueous partitions further comprising detectably labeled probes for the at least three targets and a blocking agent that inhibits the generation of a detectable signal from the fourth target during amplification. Exposing the partitions to amplification conditions and detecting the presence or absence of each of the at least three targets in the sample by reading signals in two or more optical channels.

[0114] (B2) For the method of (Bl), the detecting step comprises plotting on a graph the signal intensity from each partition by axis for each channel, wherein each of the three of the targets forms a unique cluster on the graph when present in the sample.

[0115] (B3) For the method of (B2), for the targets present in the sample, the graph includes respective clusters and the clusters are located along respective radius ranges extending from a point on the graph.

[0116] (B4) For the method of any one of (Bl) to (B3), detecting a first target of the three targets using only a probe comprising a first oligonucleotide sequence linked to a label producing a first color, detecting a second target of the three targets using a mixture of probes comprising a first probe comprising a second oligonucleotide sequence linked to a label producing the first color and a second probe comprising the second oligonucleotide sequence linked to a label producing a second color; and detecting a third target of the three targets using only a probe comprising a third oligonucleotide sequence linked to a label producing the second color.

[0117] (B5) For the method of any one of (Bl) to (B4), the blocking agent is an oligonucleotide that binds to the fourth target of the targets or a copy thereof, and optionally wherein the blocking agent (a) inhibits binding of any of the detectably labeled probes; or (b) inhibits amplification of the fourth target of the targets, or both (a) and (b).

[0118] (B6) For the method of (B5), the blocking agent comprises at least one locked nucleic acid.

[0119] (B7) For the method of any one of (Bl) to (B6), the at least three unique nucleic acid targets comprise at least seven targets, and the method further comprises detecting the presence or absence of the at least seven targets by performing three dual-color read operations on a total of six colors.

[0120] (B8) For the method of any one of (Bl) to (B6), the fourth nucleic acid target is a wild-type sequence of a gene, and each of the at least three targets comprises a portion of the gene that includes a mutation relative to the wild-type sequence.

[0121] (B9) For the method of (B8), the gene is selected from the group consisting of BRAF, EGFR, KRAS, NRAS, PIK3CA, and ESRl.

[0122] (B10) For the method of any one of (Bl) to (B9), the sample comprises tumor DNA from a subject, the blocking agent suppresses detection of a wild-type sequence from non-tumor DNA from the subject, and wherein detecting the presence or absence of each of the three of the targets is indicative of the presence or grade of a tumor of the subject.

[0123] (B11) For the method of any one of (Bl) to (B10), the blocker comprises a protein that specifically binds to the fourth target among the targets or an amplicon thereof.

[0124] (B12) For the method of (Bl l), the blocker comprises an RNA-guided binding protein.

[0125] (B13) For the method of any one of (Bl) to (B12), the method further comprises estimating the number of each of the three targets among the targets in the sample by modeling Poisson distributions of the three targets among the targets using a computer system, the modeling of the Poisson distributions of the three targets among the targets providing the readout of the signal in the two or more optical channels.

[0126] (B14) For the method of any one of (Bl) to (B13), the four unique nucleic acid targets comprise (i) a first mutation of a wild-type sequence, (ii) a second mutation of the wild-type sequence, (iii) a third mutation of the wild-type sequence, and (iv) the wild-type sequence.

[0127] (B15) For the method of (B14), the detectably labeled probes comprise: a first probe comprising a first oligonucleotide that anneals to the first mutation and a fluorophore of a first color attached to the first oligonucleotide; a second probe comprising a second oligonucleotide that anneals to the second mutation and the fluorophore of the first color attached to the second oligonucleotide; a third probe comprising the second oligonucleotide that anneals to the second mutation and a fluorophore of a second color attached to the second oligonucleotide; and a fourth probe comprising a third oligonucleotide that anneals to the third mutation and a fluorophore of a third color attached to the third oligonucleotide.

[0128] (B16) For the method of any one of (Bl) to (B15), the method further comprises detecting the presence or absence of each of five unique nucleic acid targets in two optical channels, wherein the five unique nucleic acid targets comprise Target One, Target Two, Target Three, Target Four, and Target Five, and wherein the detectably labeled probes comprise fluorescent hydrolysis probes with five corresponding oligonucleotides, wherein: all probes for Target One have a first fluorophore, a majority of probes for Target Two have the first fluorophore, and the remainder of the probes for Target Two have a second fluorophore, probes for Target Three have about equal amounts of the first fluorophore and the second fluorophore, a majority of probes for Target Four have the first fluorophore, and the remainder of the probes for Target Four have a second fluorophore, and all probes for Target Five have the second fluorophore.

[0129] (B17) For the method of (B16), the five unique nucleic acid targets are variants of a wild-type gene sequence, and the blocker inhibits primer annealing to the wild-type gene sequence or an amplicon thereof.

[0130] (B18) For the method of any one of (Bl) to (B17), the method further comprises detecting the presence or absence of each of the five unique nucleic acid targets in the two optical channels, wherein the five unique nucleic acid targets comprise Target One, Target Two, Target Three, Target Four, and Target Five, and wherein the detectably labeled probes comprise fluorescent probes having five corresponding oligonucleotides, each oligonucleotide being attached to one of a first fluorophore and a second fluorophore, such that a matching number of the first fluorophore and the second fluorophore are not detectable for two of the five unique nucleic acid targets.

[0131] (B19) For the method of any one of (Bl) to (B18), the four unique nucleic acid targets comprise homologous gene sequences.

[0132] (B20) For the method of any one of (Bl) to (B19), the sample comprises one or more of at least four different nucleic acid variants.

[0133] (B21) For the method of any one of (Bl) to (B20), the sample can comprise one or more of at least four different nucleic acid variants.

[0134] (B22) For the method of any one of (Bl) to (B21), the sample is suspected of comprising one or more of at least four different nucleic acid variants.

[0135] (B23) For the method of any one of (Bl) to (B22), the sample is being tested for the presence or absence of one or more of at least four different nucleic acid variants.

[0136] (B24) For the method of any one of (Bl) to (B23), the sample comprises at least the fourth variant.

[0137] (C1) A target detection method comprising providing a sample comprising a wild-type sequence and optionally one or more of at least three variant sequences. Partitioning the sample into a plurality of partitions, wherein the partitions further comprise: at least three variant-specific probes, each probe specific for one of the variant sequences and producing fluorescence of a first color or a second color; and a blocker that inhibits fluorescence from amplification of the wild-type sequence. Subjecting the partitions to conditions that promote amplification. Detecting levels of the first color and the second color fluorescence from the partitions.

[0138] (C21) For the method of (C1), the variant-specific probes comprise fluorescent hydrolysis probes.

[0139] (C3) For the method of (C1), the variant-specific probes comprise molecular beacon probes.

[0140] (C4) For the method of any one of (C1) to (C3), the blocker comprises an oligonucleotide that does not have a fluorescent label.

[0141] (C5) For the method of any one of (C1) to (C4), the subjecting step comprises thermocycling the partitions; and optionally the partitions are droplets, and the subjecting step comprises thermocycling the droplets, optionally thermocycling within wells of a reaction tube or plate, and further optionally wherein the detecting step comprises flowing the droplets one at a time past an excitation source and a detector.

[0142] (C6) For the method of any one of (C1) to (C5), the sample optionally comprises one or more of at least seven variant sequences, the partitions comprise at least seven variant-specific probes that fluoresce in one of six colors, wherein the detecting step reads the six colors in two or more channels at a time.

[0143] (C7) For the method of (C6), the six colors are provided by six fluorescent reporters including one or more of carboxyfluorescein (FAM), hexachlorofluorescein (HEX), cyanine 5 (CY5), cyanine 5.5 (CY5.5), 5-carboxy-X-rhodamine (5-ROX), and a fluorescent oligonucleotide dye with an adsorption of 594 nm (ATTO590).

[0144] (C8) For the method of any one of (C1) to (C7), the method further comprises plotting the amounts of the first color and the second color detected from the partitions as points on a graph, and identifying clusters of the points on the graph that correspond to the presence of any of the three variants.

[0145] (C9) For the method of any one of (Cl) to (C8), the subjecting step comprises a thermal cycle, wherein the variant-specific probes comprise fluorescent probes, each fluorescent probe annealing to a sequence of one of the three variants, and wherein the blocker comprises an oligonucleotide that anneals to the wild-type sequence to prevent binding of the variant-specific probes to the wild-type sequence.

[0146] (C10) For the method of any one of (Cl) to (C9), the blocker comprises a binding protein that binds to the wild-type sequence and prevents amplification.

[0147] (C11) For the method of any one of (Cl) to (C10), the detecting step detects two or more color channels at a time in one or more detection operations.

[0148] (C12) For the method of (Cl l), the method comprises reporting the presence or absence of one or more variant sequences of a gene in the sample, optionally wherein the method comprises reporting the presence or absence of at least seven variant gene sequences in the sample.

[0149] (C13) For the method of (C12), the gene is estrogen receptor 1 (ESRl), and one or more of the seven variant sequences is a mutation selected from the group consisting of cl 138G>C, c.1387T>C, cl607T>G, cl609T>A, cl610A>C, cl610A>G, and c.1613A>G in the coding sequence of the ESRl gene.

[0150] (C14) For the method of any one of (Cl) to (C13), the variants are present in tumor DNA in the sample, and the method further comprises: (i) isolating circulating tumor DNA (ctDNA) from the sample; or (ii) isolating circulating tumor cells (CTCs) from the sample and purifying DNA from the CTCs.

[0151] (C15) For the method of any one of (Cl) to (C14), each of the three variant sequences is targeted with one or more variant-specific probes comprising a sequence specific for the variant and a characteristic amount of the first color or the second color.

[0152] (C16) For the method of (C15), the first probe combination specific for the first variant sequence includes only probes with a first sequence and the first color, the second probe combination specific for the second variant includes a subset of probes with a second sequence and the first color and a subset of probes with a second sequence and the second color, and the third probe combination specific for the third variant includes only probes with a third sequence and the second color.

[0153] (C17) For the method of any one of (C1) to (C16), the method further comprises plotting on a graph the amounts of the first color and the second color detected from the partitions against respective first and second axes, wherein each variant sequence forms a respective unique cluster on the graph, and wherein the unique clusters can be separated by a radius range extending from a single point.

[0154] (C18) For the method of any one of (C1) to (C17), the subjecting step comprises a thermal cycle, wherein the variant-specific probes comprise fluorescent probes, each fluorescent probe anneals to a sequence of one of the three of the variants, and wherein the blocker comprises an oligonucleotide that anneals to the wild-type sequence to inhibit amplification of the wild-type sequence; optionally wherein the blocker blocks binding of a primer, thereby inhibiting amplification of the wild-type sequence, further optionally wherein the blocker blocks binding of a forward primer or binding of a reverse primer, thereby inhibiting amplification of the wild-type sequence.

[0155] (C19) For the method of (C18), the blocker blocks binding of a forward primer to inhibit amplification of the wild-type sequence.

[0156] (C20) For the method of any one of (C1) to (C19), the sample comprises one or more of at least four different nucleic acid variants.

[0157] (C21) For the method of any one of (C1) to (C20), the sample can comprise one or more of at least four different nucleic acid variants.

[0158] (C22) For the method of any one of (C1) to (C21), the sample is suspected of comprising one or more of at least four different nucleic acid variants.

[0159] (C23) For the method of any one of (C1) to (C22), the sample is being tested for the presence or absence of one or more of at least four different nucleic acid variants.

[0160] (C24) For the method of any one of (C1) to (C23), the sample comprises at least the fourth variant.

[0161] incorporated by reference Throughout this disclosure, other documents have been referenced and cited, such as patents, patent applications, patent publications, journals, books, papers, web content. All such documents are hereby incorporated by reference in their entirety for all purposes.

[0162] equivalents Various modifications to the implementations described in this document, as well as many additional embodiments of the application, will become apparent to those skilled in the art from the entirety of the foregoing description, including the references cited herein and the accompanying drawings. The subject matter described and demonstrated herein contains important information, exemplifications, and directions that can be adapted to put the application into practice in various embodiments and equivalents thereof.

Claims

1. A target detection method, comprising: The sample is divided into multiple aqueous zones, wherein the sample contains, may contain, or is suspected of containing one or more of at least four different nucleic acid variants, or is being tested for the presence or absence of one or more of at least four different nucleic acid variants. The partitions mentioned above include: Amplification reagents; Variant-specific probes, wherein each probe is specific to one of three variants of the variants and produces fluorescence of a first color or a second color; as well as A blocking agent that inhibits fluorescence amplification from a fourth variant of the variants; The partition is subjected to conditions that promote amplification; as well as The presence or absence of the three variants in the sample is reported based on the amount of the first and second colors detected from the partition.

2. The method of claim 1, wherein the fourth variant of the variants is wild-type, and the three variants of the variants are mutant versions of the wild-type, and optionally wherein the blocking agent comprises an oligonucleotide without a fluorescent label.

3. The method of claim 2, wherein the sample comprises the fourth variant.

4. The method according to any one of claims 1 to 3, wherein the probe comprises a fluorescent hydrolysis probe.

5. The method according to any one of claims 1 to 3, wherein the probe comprises a molecular beacon probe.

6. The method according to any one of claims 1 to 3, wherein the undergoing step comprises thermally cycling the partition; Optionally, the partition is a droplet, and the undergoing step includes thermally cycling the droplet, optionally within an orifice of a reaction tube or plate, and further optionally, the reporting step includes passing the droplet one at a time through the excitation source and detector.

7. The method according to any one of claims 1 to 3, further comprising using at least six colors in the partition to detect the presence or absence of at least seven variants, wherein the detection step reads the at least six colors at a time in two or more channels.

8. The method of claim 7, wherein the six colors are provided by six fluorescent reporters comprising one or more of carboxyfluorescein (FAM), hexachlorofluorescein (HEX), cyanine 5 (CY5), cyanine 5.5 (CY5.5), 5-carboxy-X-rhodamine (5-ROX), and a fluorescent oligonucleotide dye having adsorption at 594 nm (ATTO590).

9. The method according to any one of claims 1 to 3, wherein the reporting step comprises plotting the amounts of the first color and the second color detected in the partition as points on a graph, and identifying clusters of points on the graph corresponding to the presence of any of the three variants.

10. The method of any one of claims 1 to 3, wherein the subjected step comprises thermal cycling, wherein the amplification reagent comprises PCR primers and dNTPs, wherein the variant-specific probe comprises a fluorescent probe, each fluorescent probe being annealed to the sequence of one of three variants of the variants, and wherein the blocking agent comprises an oligonucleotide that anneales to a wild-type version corresponding to one of the at least four variants and inhibits amplification and signaling from the wild-type version or its amplicons, optionally wherein the blocking agent comprises an oligonucleotide that anneales to the fourth variant of the variants and inhibits amplification and signaling from the wild-type version or its amplicons.

11. The method according to any one of claims 1 to 3, wherein the blocking agent comprises a binding protein that binds to one of the at least four variants and prevents amplification, optionally wherein the blocking agent binds to the fourth variant of the variants and prevents amplification.

12. The method according to any one of claims 1 to 3, wherein the reporting step comprises detecting color from the partition at one time in one or more detection operations using two or more color channels.

13. The method of claim 12, wherein the reporting step comprises reporting the presence or absence of one or more mutations in the gene present in the sample, optionally wherein the reporting step comprises reporting the presence or absence of one or more of at least seven mutations in the gene present in the sample.

14. The method of claim 13, wherein the gene is estrogen receptor 1 (ESR1), and one or more of the seven mutations are selected from the group consisting of: c1138G>C, c.1387T>C, c1607T>G, c.1609T>A, c1610A>C, c1610A>G, and c.1613A>G in the coding sequence of the ESR1 gene.

15. The method according to any one of claims 1 to 3, wherein the variant is present in tumor DNA in the sample, and the method (i) comprises isolating circulating tumor DNA (ctDNA) from the sample, or (ii) isolating circulating tumor cells (CTCs) from the sample and purifying DNA from the CTCs.

16. The method of any one of claims 1 to 3, wherein each of the three variants of the variants is targeted with one or more variant-specific probes or a combination of variant-specific probes, the one or more variant-specific probes or the combination of variant-specific probes comprising a sequence specific to the variant and zero or a characteristic amount of the first color or the second color.

17. The method of claim 16, wherein the first probe combination specific to the first variant comprises only probes having the first sequence and the first color, the second probe combination specific to the second variant comprises a subset of probes having the second sequence and the first color and a subset of probes having the second sequence and the second color, and the third probe combination specific to the third variant comprises only probes having the third sequence and the second color.

18. The method according to any one of claims 1 to 3, wherein the amounts of the first color and the second color detected from the partition are plotted on a graph with respect to corresponding first and second axes, wherein the three variants of the variants each form a corresponding unique cluster on the graph, wherein the unique clusters can be separated by a radius range extending from a point.

19. The method according to any one of claims 1 to 3, wherein the sample comprises one or more of at least four different nucleic acid variants.

20. The method according to any one of claims 1 to 3, wherein the sample may contain one or more of at least four different nucleic acid variants.

21. The method according to any one of claims 1 to 3, wherein the sample is suspected to contain one or more of at least four different nucleic acid variants.

22. The method according to any one of claims 1 to 3, wherein the sample is being tested for the presence or absence of one or more of at least four different nucleic acid variants.

23. The method according to any one of claims 1 to 3, wherein the sample comprises at least the fourth variant.

24. A multiplex digital PCR method, comprising: The sample is divided into multiple aqueous partitions, the sample may contain one or more of at least three unique nucleic acid targets and a fourth nucleic acid target, the aqueous partitions also include detectably labeled probes for the at least three targets and blocking agents that inhibit the generation of detectable signals from the fourth target during amplification; Expose the partition to amplification conditions; and The presence or absence of each of the at least three targets in the sample is detected by reading signals from two or more optical channels.

25. The method of claim 24, wherein The first of the three targets is detected using only a probe comprising a first oligonucleotide sequence linked to a marker that produces the first color. A mixture of probes is used to detect the second target among the three targets. The mixture of probes includes a first probe and a second probe, the first probe containing a second oligonucleotide sequence linked to a marker that produces the first color, and the second probe containing a second oligonucleotide sequence linked to a marker that produces the second color; and The third target of the three targets is detected using only a probe that includes a third oligonucleotide sequence linked to the marker that produces the second color.

26. The method of any one of claims 24 to 25, wherein the detection step comprises plotting signal intensity from each partition on a graph along an axis for each channel, wherein the targets form unique clusters on the graph when each of the three targets is present in the sample.

27. The method of claim 26, wherein for a target present in the sample, the map comprises a corresponding cluster, and the cluster is located along a corresponding radius range extending from a point on the map.

28. The method of any one of claims 24 to 25, wherein the blocking agent is an oligonucleotide that binds to the fourth target or a copy thereof in the target, and optionally wherein the blocking agent (a) inhibits the binding of any detectably labeled probe in the detectably labeled probe; or (b) inhibits the amplification of the fourth target in the target, or both (a) and (b).

29. The method of claim 28, wherein the blocking agent comprises at least one locked nucleic acid.

30. The method of claim 28, wherein the blocking agent comprises a protein.

31. The method of claim 30, wherein the blocking agent comprises an RNA-guided binding protein.

32. The method of any one of claims 24 to 25, wherein the at least three unique nucleic acid targets comprise at least seven targets, and the method further comprises detecting the presence or absence of the at least seven targets by performing three two-color readings on a total of six colors.

33. The method of any one of claims 24 to 25, wherein the fourth nucleic acid target is a wild-type sequence of a gene, and each of the at least three targets includes a portion of the gene that comprises a mutation relative to the wild-type sequence.

34. The method of claim 33, wherein the gene is selected from the group consisting of BRAF, EGFR, KRAS, NRAS, PIK3CA and ESR1.

35. The method of any one of claims 24 to 25, wherein the sample comprises tumor DNA from a subject, the blocking agent inhibits the detection of wild-type sequences from non-tumor DNA from the subject, and wherein the presence or absence of each of the three targets in the detection of the targets indicates the presence or grade of a tumor in the subject.

36. The method according to any one of claims 24 to 25, further comprising estimating the number of each of the three targets in the sample by modeling the Poisson distribution of the three targets in the target using a computer system, wherein modeling the Poisson distribution of the three targets in the target provides readings of the signals in the two or more optical channels.

37. The method according to any one of claims 24 to 25, wherein the four unique nucleic acid targets comprise (i) a first mutation of the wild-type sequence, (ii) a second mutation of the wild-type sequence, (iii) a third mutation of the wild-type sequence, and (iv) the wild-type sequence.

38. The method of claim 37, wherein the probe comprises: A first probe, comprising a first oligonucleotide annealed to the first mutation and a first-color fluorophore linked to the first oligonucleotide; The second probe comprises a second oligonucleotide annealed with the second mutation and a fluorophore of the first color linked to the second oligonucleotide; The third probe includes the second oligonucleotide annealed with the second mutation and a second-color fluorophore linked to the second oligonucleotide; as well as The fourth probe comprises a third oligonucleotide annealed with the third mutation and a third-color fluorophore linked to the third oligonucleotide.

39. The method according to any one of claims 24 to 25, wherein the method further comprises detecting the presence or absence of each of five unique nucleic acid targets in two optical channels, wherein the five unique nucleic acid targets include target one, target two, target three, target four, and target five, and wherein the detectably labeled probe comprises a fluorescent hydrolysis probe having five corresponding oligonucleotides, wherein: All probes used for target one have a first fluorophore. Most of the probes used for target two have the first fluorophore, and the remainder of the probes used for target two have the second fluorophore. The probe used for target three has approximately equal amounts of the first fluorophore and the second fluorophore. Most of the probes used for target four have the first fluorophore, and the remainder of the probes used for target four have the second fluorophore. All probes used for target five have the second fluorophore.

40. The method of claim 39, wherein the five unique nucleic acid targets are variants of wild-type gene sequences, and the blocking agent inhibits the annealing of the wild-type gene sequence or its amplicon.

41. The method of any one of claims 24 to 25, wherein the method further comprises detecting the presence or absence of each of five unique nucleic acid targets in the two optical channels, wherein the five unique nucleic acid targets include target one, target two, target three, target four, and target five, and wherein the detectably labeled probe comprises a fluorescent probe having five corresponding oligonucleotides, each oligonucleotide being linked to one of a first fluorophore and a second fluorophore such that two of the five unique nucleic acid targets cannot be detected by matching numbers of the first fluorophore and the second fluorophore.

42. The method according to any one of claims 24 to 25, wherein the four unique nucleic acid targets comprise homologous gene sequences.

43. The method according to any one of claims 24 to 25, wherein the sample comprises one or more of at least four different nucleic acid variants.

44. The method according to any one of claims 24 to 25, wherein the sample may contain one or more of at least four different nucleic acid variants.

45. The method according to any one of claims 24 to 25, wherein the sample is suspected to contain one or more of at least four different nucleic acid variants.

46. ​​The method according to any one of claims 24 to 25, wherein the sample is being tested for the presence or absence of one or more of at least four different nucleic acid variants.

47. The method according to any one of claims 24 to 25, wherein the sample comprises at least the fourth variant.

48. A target detection method, comprising: Provide a sample comprising a wild-type sequence and optionally one or more of at least three variant sequences; The sample is partitioned into multiple partitions, wherein the partitions further comprise: At least three variant-specific probes, each probe being specific to one of the variant sequences and producing fluorescence of a first color or a second color; as well as An inhibitor that suppresses fluorescence amplification from the wild-type sequence; The partition is subjected to conditions that promote amplification; The fluorescence levels of the first color and the second color are detected in the partition.

49. The method of claim 48, further comprising: The amounts of the first and second colors detected from the partitions are plotted as points on the graph; and Identify the clusters of points on the graph corresponding to the existence of any of the three variants.

50. The method of any one of claims 48 to 49, wherein the variant-specific probe comprises a fluorescent hydrolysis probe.

51. The method according to any one of claims 48 to 49, wherein the variant-specific probe comprises a molecular beacon probe.

52. The method according to any one of claims 48 to 49, wherein the blocking agent comprises an oligonucleotide without a fluorescent label.

53. The method according to any one of claims 48 to 49, wherein the undergoing step comprises thermally cycling the partition. Optionally, the partition is a droplet, and the undergoing step includes thermally cycling the droplet, optionally within a hole in a reaction tube or plate, and further optionally, the detection step includes passing the droplet one at a time through an excitation source and a detector.

54. The method of any one of claims 48 to 49, wherein the sample optionally comprises one or more of at least seven variant sequences, the partition comprises at least seven variant-specific probes that fluoresce in one of six colors, wherein the detection step reads the six colors in two or more channels at a time.

55. The method of claim 54, wherein the six colors are provided by six fluorescent reporters, the fluorescent reporters comprising one or more of carboxyfluorescein (FAM), hexachlorofluorescein (HEX), cyanine 5 (CY5), cyanine 5.5 (CY5.5), 5-carboxy-X-rhodamine (5-ROX), and a fluorescent oligonucleotide dye having adsorption at 594 nm (ATTO590).

56. The method of any one of claims 48 to 49, wherein the subjected step comprises thermal cycling, wherein the variant-specific probe comprises a fluorescent probe, each fluorescent probe being annealed to a sequence of one of the three variants of the variants, and wherein the blocking agent comprises an oligonucleotide annealed to a wild-type sequence to prevent the variant-specific probe from binding to the wild-type sequence.

57. The method of any one of claims 48 to 49, wherein the blocking agent comprises a binding protein that binds to the wild-type sequence and prevents amplification.

58. The method according to any one of claims 48 to 49, wherein the detection step detects two or more color channels at a time in one or more detection operations.

59. The method of claim 58, wherein the method comprises reporting the presence or absence of one or more variant sequences of a gene in the sample, optionally wherein the method comprises reporting the presence or absence of at least seven variant gene sequences in the sample.

60. The method of claim 59, wherein the gene is estrogen receptor 1 (ESR1), and one or more of the seven variant sequences are mutations selected from the group consisting of: c1138G>C, c.1387T>C, c1607T>G, c.1609T>A, c1610A>C, c1610A>G, and c.1613A>G in the coding sequence of the ESR1 gene.

61. The method according to any one of claims 48 to 49, wherein the variant is present in tumor DNA in the sample, and the method further comprises: (i) Isolate circulating tumor DNA (ctDNA) from the sample, or (ii) Isolate circulating tumor cells (CTCs) from the sample and purify the DNA from the CTCs.

62. The method of any one of claims 48 to 49, wherein each of the three variant sequences is targeted with one or more variant-specific probes, the one or more variant-specific probes comprising a sequence specific to the variant and a first color or a second color of a characteristic amount.

63. The method of claim 62, wherein the first probe combination specific to the first variant sequence comprises only probes having the first sequence and the first color, the second probe combination specific to the second variant comprises a subset of probes having the second sequence and the first color and a subset of probes having the second sequence and the second color, and the third probe combination specific to the third variant comprises only probes having the third sequence and the second color.

64. The method according to any one of claims 48 to 49, further comprising plotting the amounts of the first color and the second color detected from the partition on a graph for corresponding first and second axes, wherein each variant sequence forms a corresponding unique cluster on the graph, and wherein the unique clusters are separable by a radius range extending from a single point.

65. The method of any one of claims 48 to 49, wherein the subjected step comprises thermal cycling, wherein the variant-specific probe comprises a fluorescent probe, each fluorescent probe being annealed to a sequence of one of the three variants of the variants, and wherein the blocking agent comprises an oligonucleotide annealed to a wild-type sequence to inhibit the amplification of the wild-type sequence; optionally wherein the blocking agent blocks primer binding, thereby inhibiting the amplification of the wild-type sequence, and further optionally wherein the blocking agent blocks the binding of a forward primer or a reverse primer, thereby inhibiting the amplification of the wild-type sequence.

66. The method of claim 65, wherein the blocking agent blocks the binding of the forward primer to inhibit the amplification of the wild-type sequence.

67. The method according to any one of claims 48 to 49, wherein the sample comprises one or more of at least four different nucleic acid variants.

68. The method according to any one of claims 48 to 49, wherein the sample may contain one or more of at least four different nucleic acid variants.

69. The method according to any one of claims 48 to 49, wherein the sample is suspected to contain one or more of at least four different nucleic acid variants.

70. The method according to any one of claims 48 to 49, wherein the sample is being tested for the presence or absence of one or more of at least four different nucleic acid variants.

71. The method according to any one of claims 48 to 49, wherein the sample comprises at least the fourth variant.

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