CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/368,537 filed July 15, 2022, which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

[0002] Detection of nucleic acid sequences are used for a wide variety of purposes. Detection of a particular nucleic acid sequence in a gene of a subject may indicate that the subject may have a particular disorder or be more prone to having a particular disorder. Detection of nucleic acid sequences maybe used to detect an infection by detecting a gene or nucleic acid sequence of a pathogen. PCR may be used to amplify nucleic acids for analysis.

INCORPORATION BY REFERENCE

[0003] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

SUMMARY

[0004] Disclosed herein, in some aspects, are methods, systems and compositions for detection and analytes. The method, systems, and composition may allow for a reduction in the number of channels required to detect targets. A multiplex assay of the systems and methods disclosed herein enable a fast and efficient method of screening for certain common mutations (e.g., oncogene detection) or bacterial or viral subtypes. These mutations and subtype may reveal vulnerabilities or resistances in a disease, which can enable faster treatment with therapeutics that have a higher likelihood of beneficial effects.

[0005] In some aspects of the method and systems disclosed herein, is a method of detecting a first nucleic acid sequence variation and a second nucleic acid sequence variation of a nucleic acid target may be disclosed. In some embodiments, the method may comprise providing a reaction mixture comprising a sample potentially comprising said nucleic acid target comprising said first nucleic acid sequence variation. In some embodiments, the reaction mixture may comprise a first oligonucleotide probe, wherein said first oligonucleotide probe hybridizes to a first region of said nucleic acid target in a presence or absences of said first nucleic acid sequence variation. In some embodiments, the reaction mixture may comprise a second oligonucleotide probe, wherein said second oligonucleotide probe hybridize to a second region of said nucleic acid target in a presence of said first nucleic acid sequence variation, wherein said second region potentially comprises said first nucleic acid sequence variation of the nucleic acid target In some embodiments, the reaction mixture may comprise a third oligonucleotide probe, wherein said third oligonucleotide probe hybridizes to a third region of said nucleic acid target in a presence of said second nucleic acid sequence variation, wherein said third region potentially comprises said second nucleic acid sequence variation of the nucleic acid target. In some embodiments, the method may comprise partitioning said mixture into a plurality of partitions. In some embodiments, the method may comprise generating a plurality of signals in said plurality of partitions from said first oligonucleotide probe, said second oligonucleotide probe, and said third oligonucleotide nucleotide probe. In some embodiments, the method may comprise detecting from multiple partitions of said plurality of partitions said plurality of signals. In some embodiments, the method may comprise determining the presence of said first nucleic acid sequence variation and said second nucleic acid sequence variation of said nucleic acid target. In some embodiments, the plurality of signals maybe detected in a single channel. [0006] In some embodiments, the method may comprise a detection of Y numbers of nucleic acid sequence variation of the nucleic acid target. In some embodiments, the method may comprise an X number of oligonucleotide probe, wherein the X number of oligonucleotide probe hybridizes to an X number regions of a nucleic acid target in a presence of Y numbers of nucleic acid sequence variations. In some embodiments, the method may comprise X numbers regions potentially comprises Y number of nucleic acid sequence variations of the nucleic acid target. In some embodiments, the method may comprise determining the presence of Y numbers of nucleic acid sequence variations of the nucleic acid target. In some embodiments, Y may be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more and X may be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0007] In some embodiments, the method may comprise quantifying the number of nucleic acid sequences comprising a first sequence variation, a second sequence variation, or both. In some embodiments, at least 10 partitions positive for said nucleic acid sequence variation maybe generated. In some embodiments, at least 100 partitions positive for nucleic acid sequence variation may be generated. In some embodiments, the number of partitions positive for a nucleic acid sequence variation may b e greater than 10% of the total number of partitions. In some embodiments, the number of partitions positive for nucleic acid sequence variation may be greater than 20% of the total number of partitions.

[0008] In some embodiments, quantifying may comprise determining a total number of partitions. In some embodiments, quantification may be performed without determining a total number of partitions. In some embodiments, a first oligonucleotide probe or a second oligonucleotide probe may comprise a fluorophore. In some embodiments, a plurality of signals may be generated by excitation of a fluorophore. In some embodiments, a plurality of signals may be generated by degradation of a first oligonucleotide probe or a second oligonucleotide probe. In some embodiments, the method may comprise subjecting the reaction mixture to an amplification reaction, thereby generating said plurality of signals. In some embodiments, the amplification reaction may comprise a polymerase chain reaction. In some embodiments, the reaction amplification may comprise an isothermal amplification reaction.

[0009] In some embodiments, a sample maybe derived from a biological sample. In some embodiments, the biological sample may be blood or plasma. In some embodiments, the biological sample may be a plasma sample of an individual suffering from cancer. In some embodiments, the biological sample may be a plasma sample from an individual suffering from an infection. In some embodiments, the sample may be generated by diluting said biological sample by no more than 10%. In some embodiments, a sample maybe generated by diluting said biological sample by no more than 20%. In some embodiments, a sample may be generated by diluting said biological sample by no more than 50%. In some embodiments, a biological sample may be not diluted prior to addition to a sample.

[0010] In some embodiments, the nucleic acid target may comprise a oncogene nucleic acid sequence. In some embodiments, the oncogene nucleic acid sequence maybe chosen from among the nucleic acid sequences ofEGFR, BRAF, HER2, RET, PIK3CA, FGFR1, DDR2, ROS1, RAS, MYC, SRC, hTERT, BCL2, or ALK. In some embodiments, a nucleic acid target may comprise a nucleic acid encoding a fusion protein. In some embodiments, the fusion protein may be chosen from among the nucleic acid sequences of ALK, ATIC, CARS, CCDC6, CD74, CLIP1, CLTC, DCTN1, EML4, ERC1, ETV6, EZR, FN1, GOLGA5, GOPC, HIP1, HLA A, HOOK3, KIF5B, KLC1, KTN1, LMNA, LRIG3, MET13, MET15, MSN, MY05A, NACC2, NCOA4, NTRK1, 2, 3, PCM1, PPFIBP1, PWWP2A, QKI, RANBP2, RELCH, RET, ROS1, SDC4, SEC31, SHTN1, SLC34A2, SQSTM1, STRN, TFG, TPR, TP53, TPM3, TPM4, TRIOM24, TRIM27, TRIM33, VCL, WDCP, ZCCHC8.

[0011] In some embodiments, a nucleic acid target may be derived from a virus or bacteria. In some embodiments, a nucleic acid target may comprise RNA. In some embodiments, a nucleic acid target may comprise cDNA. In some embodiments, a nucleic acid target may be processed to generate cDNA. In some embodiments, a nucleic acid target may comprise nucleic acids derived from a cancer. In some embodiments, a nucleic acid target may be circulating free DNA (cfDNA).

[0012] In some embodiments, a plurality of signals may be a plurality of fluorescent signals or a plurality of chemiluminescent signals. [0013] In some embodiments, the method may comprise absolute quantification of a first nucleic acid sequence variation of the nucleic acid target or a nucleic acid second sequence variation of the nucleic acid target. In some embodiments, quantifying may comprise a relative quantification of a first nucleic acid sequence variation of the nucleic acid target or a second nucleic acid sequence variation of the nucleic acid target. In some emb odiments, quantifying may comprise generating an average number of nucleic acid molecules per partition. In some embodiments, quantifying may comprise generating a probability that one or more nucleic acid molecules comprising said first nucleic acid sequence variation or said second nucleic acid sequence variation may be present in a given partition. In some embodiments, quantifying may comprise, for a plurality of partitions, generating a probability that one or more nucleic acid molecules comprising a first nucleic acid sequence variation or a second nucleic acid sequence variation may be present in a given partition of said plurality of partitions, thereby generating a plurality of probabilities. In some embodiments, quantifying may further comprise summing two or more probabilities of a plurality of probabilities. In some embodiments, a plurality of partitions may comprise a plurality of droplets.

[0014] In some embodiments, a first nucleic acid sequence variation or a second nucleic acid sequence variation may be indicative of a cancer. In some embodiments, a first sequence variation or a second sequence variation may be indicative of an autoimmune disease. In some embodiments, a first nucleic acid sequence variation or said second nucleic acid sequence variation may be indicative of a vulnerability or resistance to a treatment method. In some embodiments, a first nucleic acid sequence variation or a second nucleic acid sequence variation may be indicative of a genus, species, sub-species or variant.

[0015] In some aspects of the method and systems disclosed herein, a method of treatment for a disease is disclosed. In some embodiments, the method may comprise performing a method as disclosed herein on a sample from a subject. . In some embodiments, the method may comprise determining based on presence or absences of said first nucleic acid sequence variation and/or said second nucleic acid sequence variation of said nucleic acid target whether a treatment will be effective. In some embodiments, the method may comprise, if a treatment is determined to be effective, administering to a subject the treatment.

[0016] In some aspects of the method and systems disclosed herein, a method of detecting a first nucleic acid sequence variation and a second nucleic acid sequence variation of a nucleic acid target is disclosed. In some embodiments, the method may comprise providing a reaction mixture comprising a sample potentially comprising said nucleic acid target comprising said first nucleic acid sequence variation. In some embodiments, the reaction mixture may comprise a first oligonucleotide probe, wherein said first oligonucleotide probe hybridizes to a first region of said nucleic acid target in a presence or absences of said first nucleic acid sequence variation or said second nucleic acid sequence variation In some embodiments, the reaction mixture may comprise a second oligonucleotide probe, wherein the second oligonucleotide probe hybridize to a second region of the nucleic acid target in an absence of said first nucleic acid sequence variation, and the second region potentially comprises said first nucleic acid sequence variation of the nucleic acid target. In some embodiments, the reaction mixture may comprise a third oligonucleotide probe, wherein the third oligonucleotide probe hybridizesto a third region of the nucleic acid target in an absence of the second nucleic acid sequence variation, wherein the third region potentially comprises said second nucleic acid sequence variation of the nucleic acid target. In some embodiments, the method may comprise partitioning said mixture into a plurality of partitions. In some embodiments, the method may comprise generating a plurality of signals in said plurality of partitions from said first oligonucleotide probe and said second oligonucleotide probe. In some embodiments, the method may comprise detecting from multiple partitions of said plurality of partitions said plurality of signals. In some embodiments, the method may comprise determining the presence of said first nucleic acid sequence variation or said second nucleic acid sequence variation of said nucleic acid target. In some embodiments, the method may comprise a detection of an Yth nucleic acid sequence variation of the nucleic acid target. In some embodiments, the method may comprise an Xth oligonucleotide probe, wherein the Xth oligonucleotide probe hybridizes to an Xth region of the nucleic acid target in an absence of an Yth nucleic acid sequence variation, wherein the Xth region potentially comprises an Yth nucleic acid sequence variation of the nucleic acid target. In some embodiments, the method may comprise determining the presence of an Yth nucleic acid sequence variation of the nucleic acid target. In some embodiments, Y may be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more and X may be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0018] FIG. 1 . shows a schematic and representative plots of an assay distinguishing a wild type and a mutant using signals of probes of different colors. [0019] FIG. 2A-2B show schematicsand representative plots of an assay distinguishing a wild type and mutant using signals of probes of one color.

[0020] FIG. 3 show schematics and representative plots of an assay show distinguishing a wild type and two mutants using signals of probes of one color.

[0021] FIG. 4 A illustrates the ability to target multiple loci on a single chromosome with a digital PCR assay as shown in a single channel of droplet intensities. FIG. 4B illustrates the ability to target multiple loci on a single chromosome with a digital PCR assay as shown in a single channel of fluorescent intensity versus partition count.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The following description provides specific details for a comprehensive understanding of, and enabling description for, various embodiments of the technology. It is intended that the terminology used be interpreted in its broadest reasonable manner, even where it is being used in conjunction with a detailed description of certain embodiments.

[0023] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, and as such, may vary. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including,” “includes,” “having,” “has,” “with,” “such as,” or variants thereof, are usedin either the specification and/or the claims, such terms are not limiting and are intended to be inclusive in a manner similar to the term “comprising.” Unless specifically noted, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of’ or “consisting essentially of’ the recited components.

[0024] Where values are described as ranges, it will be understood that such disclosure includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific subrange is expressly stated Drop-Off Assay

[0025] Digital drop-off assay s may be used to detect sequence variations such as insertions, deletions, duplications, and sequence polymorphisms. The general concept involves two or more differentially labeled probe sequences between a common set of primers. A schematic for this version of the assay is displayed in FIG. 1. Standard ddPCR assays use probes specific for a particular sequence variant (e.g., a specific single-base pair exchange) and thus require prior knowledge of the variant to be detected. To circumvent this limitation, drop-off assays may be used. In some embodiments, drop-off ddPCR may use a labeled detection probe designed to hybridize to a target sequence in the vicinity of a known variational regions and a labeled reference probe able to hybridize to a wild-type sequence of a second region of the target nucleic acid. Thus, in some embodiments, a double-positive fluorescence signal from both the reference and detection probes would indicate the presence of a wild-type variation lacking any variation in the region of interest. In some embodiments, a fluorescence signal for only the reference probe may indicates the presence of a sequence variant in the target region that interferes with the ability of the detection probe to hybridize to the target sequence. In some embodiments, the drop-off assay may use two probes that comprise the same fluorophore. In such an embodiment, a difference in the magnitude of the fluorescence may be indicative of whether a sequence variation is present in the nucleic acid target as displayed in the schematic of FIG. 2A. FIG. 2B demonstrates the difference in the output between a drop-off assay using a single channel versus two channels, in which both assay are detecting a 5% rate of sequence variation of the same target nucleic acid.

[0026] In some aspects of the methods and systems disclosed herein, a drop-off assay may be used to detect variations at two distinct regions of interest co-located within a region spanned by a single PCR amplicon. In some embodiments, one probe binds to the wild-type sequence of one region of the sequence of interest. In some embodiments, another probe may bind to the wildtype sequence of the second sequence of interest located within the same PCR amplicon or sequence of interest. Each probe may then independently be used to simultaneously detection variation in separate regions of the same sequence of interest.

[0027] In some aspects of the methods and systems disclosed herein, comprise a drop-off assay that may be used to detect multiple specific nucleic acid sequence variations. These assays allow detection of greater numbers of sequence variation than optical channels available on real-time or digital PCR machine, (e.g., 2, 3 or more targets in one channel; 3, 4, 5, 6 or more targets in two channels; 4, 5, 6, 7, 8, 9, or more targets in three channels; 5, 6, 7, 8, 9, 10, 11, 12 ormore targets in four channels). In some embodiments one probe may bind to a specific variation of a wild type nucleic acid sequence. In some embodiments, another probe may bind to a specific variation of a second sequence of interest located within the same PCR amplicon or sequence of interest. Each probe may then independently be used to simultaneously detection specific variations in separate regions of the same sequence of interest. A potential schematic of using three probes comprising the same fluorophore to detect two possible nucleic acid sequence variations is displayed in FIG. 3.

[0028] In some aspects of the methods and systems disclosed herein, a drop-off assay may be used to simultaneously detectboth a non-specific variations of a first region of a portion of a sequence of interest and a second specific variation of a second region of a portion of the same sequence of interest. In some embodiments, one probe binds to the wild-type sequence of one region of the sequence of interest. In some embodiments, another probe may bind to a specific variation of a second sequence of interest located within the same PCR amplicon or sequence of interest. Each probe may then independently be used to simultaneously detection both a specific variations and a non-specific variation in separate regions of the same sequence of interest. [0029] In some aspects of the methods and systems disclosed herein, a drop-off assay using two or more oligonucleotide probes. The drop-off assay may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more oligonucleotide probes. The drop-off assay may comprise detecting 2 or more nucleic acid variations. The drop-off assay may comprise detecting 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleic acid variations. The nucleic acid variations may be specific nucleic acid variations, non-specific nucleic acid variations, or a combination of both.

Multiplexed PCR

[0030] The present disclosure provides methods for detecting more than one nucleic acid target variation using digital PCR in a single optical channel. In some embodiments, the methods of the present disclosure are used for detecting two, three, four, or more nucleic acid region variations using digital PCR in a single optical channel.

[0031] In some embodiments, detection of multiple variations in a nucleic acid target variations in a single optical channel can be achieved by providing multiple nucleic acid probes, each specific for one region of the target nucleic acid, where the probes are labeled with the same color signal tag but are provided at varying concentrations, thereby identifying each variation of the target with a unique signal readout (e.g., signal intensity value). For example, in order to detect two variations nucleic acid target variations with digital PCR in a single optical channel, a first nucleic acid probe may be provided at a concentration of X, a second nucleic acid probe may be provided at a concentration of at least about 2X, and a third nucleic acid probe may be provided at a concentration of at least about 4X, thereby generating signals in a digital PCR reaction where each signal uniquely corresponds to the presence of each target or combination thereof in a partition. Multiple resolvable levels of intensity maybe defined within a single channel. FIG. 4A and FIG.4B display an exemplary diagram of a single channel assay that has four resolvable intensity levels.

[0032] In some embodiments, detection of more than one variation of a nucleic acid target using a single optical channel can be achieved by providing oligonucleotide probes, each specific for one of region of the target, where each probe is labeled with one or more copies of a luminescent signal tag, such that the probes have varying intensity levels, thereby identifying each target with a unique signal readout (e.g., signal intensity value). For example, in order to detect two nucleic acid target variations with digital PCR in a single optical channel, a first nucleic acid probe having an intensity level of X may be provided, a second nucleic acid probe having an intensity level of 2X may be provided, and a third nucleic acid probe having an intensity level of 4X may be provided, thereby generating signals in a digital PCR reaction where each signal uniquely corresponds to the presence of each target variation and combination thereof in a partition.

[0033] In addition to the use of varying intensities within a single color channel to identify multiple targets, multiple color channels can be combined to further increase the number of detectable targets in a single reaction. This can be achieved by providing probes specific to a given target which comprise different signal tags at varying concentrations and/or with varying intensity levels.

[0034] By combining the detection of more than one target variation within a single color channel with the use of multiple color channels, the amount of targets and variations that can be detected in a single reaction can be increased significantly.

[0035] In some embodiments, each target variation or combination thereof is assigned a specific fluorescence signature (color combination and color intensity) by titrating the fluorogenic probes in specific ratios associated with that target regions. The probe and primer concentrations are chosen to be significantly higher than the target concentration, to mitigate for statistical sample partitioning variation (e.g., Poisson distribution of reagents amongst partitions).

[0036] In embodiments where each partition comprises at most one nucleic acid target, each partition may be measured individually, and its fluorescence signature used to identify the target present in that partition. Table 1 shows reagent concentrations for an example digital assay which can be used to accomplish the following signal output for multiple target regions: [0037] 4x signal level in channel 1 identifies a wild type target sequence with no variations [0038] 3x signal level in channel 1 identifies a target sequence with a variation in the first target region of the target nucleic acid

[0039] 2x signal level in channel 1 identifies a target sequence with a variation in the second target region of the target nucleic acid lx signal level in channel 1 identifies a target sequence with both a variation in the first target region and the second target region of the target nucleic acid

Table 1 Digital Assays

[0040] In some aspects, the present disclosure provides assays for unambiguously detecting the presence or absence of multiple variations of a nucleic acid target in a sample. Nucleic acid target detection may be accomplished by the use of two or more reactions. For example, an assay for measuring a plurality of nucleic acid target variations may comprise a first reaction and a second reaction. Both a first and second reaction may, alone, fail to non -degenerately detect the presence or absence of any combination of nucleic acid target variations. The results of the first and second reactions may together unambiguously detect the presence or absence of each of the nucleic acid target variations.

[0041] Any number of nucleic acid target variations may be detected using assays of the present disclosure. In some embodiments, an assay may unambiguously detect at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50 nucleic acid target variations, or more. In some embodiments, an assay may unambiguously detect at most 50, 40, 30, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 nucleic acid target variations. An assay may comprise any number of reactions, where the results of the reactions together identify a plurality of nucleic acid target variations or variations, in any combination of presence or absence. An assay may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 reactions, or more. Each reaction may be individually incapable of non- degenerately detecting the presence or absence of any combination of nucleic acid target variations. However, the results of each reaction together may unambiguously detect the presence or absence of each of the nucleic acid target variations.

[0042] Reactions may be performed in the same sample solution volume. For example, a first reaction may generate a fluorescent signal in a first color channel, while a second reaction may generate a fluorescent signal in a second color channel, thereby generating two measurements for comparison. Alternatively, reactions maybe performed in different sample solution volumes. For example, a first reaction maybe performed in a first sample solution volume and generate a fluorescent signal in a given color channel, and a second reaction may be performed in a second sample solution volume and generate a fluorescent signal in the same color channel or a different color channel, thereby generating two measurements for comparison

[0043] In some aspects, the present disclosure provides methodsfor performing a digital assay. A method for performing a digital assay may comprise partitioning a plurality of nucleic acid target variations and a plurality of oligonucleotide probes into a plurality of partitions. In some embodiments, two, three, four, five, or more nucleic acid target variations maybe partitioned into a plurality of partitions together with two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more oligonucleotide probes. Following partitioning, the nucleic acid target variations may be amplified in the partitions, for example, by polymerase chain reaction (PCR). Next, N signals may be generated from the oligonucleotide probes. Each signal of the N signals may correspond to the presence of a unique combination of nucleic acid target variations in a partition. Following signal generation, the N signals may be detected in a single optical channel. The signals may be detected using, for example, fluorescence detection in a single-color channel.

[0044] A method for performing a digital assay may comprise amplifying nucleic acid target variations derived from a sample in a plurality of partitions comprising oligonucleotide probes complementary to one or more regions of nucleic acid target. Each oligonucleotide probe may be labeled with a fluorophore. The fluorophores may be capable of being detected in a single optical channel. For example, the fluorophores may each comprise similar emission wavelength spectra, such that they can be detected in a single optical channel. Following partitioning, N signals may be detected from the plurality of partitions if one or more of the nucleic acid target variations is present. Each of the N signals may correspond to a unique combination of one or more of the nucleic acid target variations present in a partition. From the N signals, the presence or absence of each of the nucleic acid target variations in the sample maybe determined.

[0045] A reaction may comprise generating a cumulative signal measurement. Assays of the present disclosure may comprise comparing two or more cumulative signal measurements to unambiguously detect any combination of nucleic acid target variations in a sample. A cumulative signal measurement may comprise one or more signals generated from one or more probes provided to a sample solution. A cumulative signal measurement may be a signal intensity level which corresponds to the sum of signals generated from multiple oligonucleotide probes. For example, two probes may each bind to a nucleic acid molecule, where each probe generates a signal of a given wavelength at lx intensity. Measurement of these signals would generate a cumulative signal measurement corresponding to the sum of both signal intensities, namely a 2x signal intensity.

[0046] A reaction may comprise an ambiguity. An ambiguity may be a signal that fails to unambiguously identify a single combination of nucleic acid target variations in a sample. For example, a reaction may generate a signal at2x intensity level. Based on the encoding of the reaction (e.g., the concentration of hybridization probes present in the reaction), a 2x intensity level may correspond to more than one combination of nucleic acid target variations, thereby comprising an ambiguity. An ambiguity may be resolved by performing one or more additional reactions, thereby resolving the ambiguity. For example, a second reaction may generate a 3x signal intensity level, where the presence of both a 2x signal intensity level from a first reaction and a 3x signal intensity level from a second reaction uniquely identifies a given combination of nucleic acid target variations from a sample. [0047] An assay may comprise selecting two or more reactions from a selection of reactions, depending on the information necessary to resolve an ambiguity. For example, a first reaction may comprise an ambiguity at a first signal level and a second signal level. Results corresponding to the first signal level may identify a first additional reaction as necessary to resolve the ambiguity, while results corresponding to the second signal level may identify a second additional reaction as necessary to resolve the ambiguity.

Amplification

[0048] In some aspects, the disclosed methods comprise nucleic acid amplification. Amplification conditions may comprise thermal cycling conditions, including temperature and length in time of each thermal cycle. The use of particular amplification conditions may serve to modify the signal intensity of a signal, thereby enabling a signal (or plurality of signals) to correspond to a unique combination of nucleic acid target variations. Amplification may comprise using enzymes such to produce additional copies of a nucleic. The amplification reaction may comprise using oligonucleotide primers as described elsewhere herein. The oligonucleotide primers may use specific sequences to amplify a specific sequence. The oligonucleotide primers may amplify a specific sequence by hybridizing to a sequence upstream and downstream of the primers and result in amplifying the sequence inclusively between the upstream and downstream primer. The oligonucleotide may be able to amplify more than one sequence analyte by hybridizing upstream or downstream of multiple different sequences. The amplification reaction may comprise the use of nucleotide tri -phosphate reagents. The nucleotide tri-phosphate reagents may comprise using deoxyribo-nucleotide tri-phosphate (dNTPs). The nucleotide tri-phosphate reagents may be used as precursors to the amplified nucleic acids. The amplification reaction may comprise using oligonucleotide probes as described elsewhere herein. The amplification reaction may comprise using enzymes. Nonlimiting examples of enzymes include thermostable enzymes, DNA polymerases, RNA polymerases, and reverse transcriptases. The amplification reaction may comprise generating nucleic acid molecules of a different nucleotide types. For example, a target nucleic acid may comprise DNA and an RNA molecule may be generated. In another example, an RNA molecule may be subjected to an amplification reaction and a cDNA molecule may be generated.

[0049] The amplification may be performed in an isothermal process. "Isothermal" means conducting a reaction ata substantially constant temperature, i.e., without varying the reaction temperature in which a nucleic acid polymerization reaction occurs. Isothermal temperatures for isothermal amplification reactions are generally below the melting temperature (Tm; the temperature at which half of the potentially double-stranded molecules in a mixture are in a single-stranded, denatured state) of the predominant reaction product, i.e., generally 90°C or below, usually between about 50°C and 75°C, and preferably between about 55°C to 70 °C, or 60 °C to 70°C, or more preferably at about 65°C. Although the polymerization reaction may occur in isothermal conditions, an isothermal process may optionally include a pre -amplification heat denaturation step to generate a single -stranded target nucleic acid to be used in the isothermal amplifying step.

[0050] The isothermal amplification may be linear isothermal amplification or exponential isothermal amplification. Isothermal linear amplification processes amplify template nucleic acid and not amplification products under isothermal conditions, may be conducted using only one amplification primer, and generally amplify a target sequence by about 1,000 fold within one hour. Isothermal exponential amplification processes use a product of an amplification reaction as a substrate in a subsequent step in the amplification reaction that uses isothermal conditions to amplify a target sequence about 10,000-fold to 100,000-fold within one hour. "Amplification conditions" refer to the cumulative biochemical and physical conditions in which an amplification reaction is conducted, which may be designed based on well-known standard methods.

Partitioning

[0051] Methods of the present disclosure may comprise partitioning a sample or mixture into a plurality of partitions. A sample of mixture may comprise nucleic acids, oligonucleotide probes, and/or additional reagents into a plurality of partitions. A partition may be a droplet (e.g., a droplet in an emulsion). A partition may be a microdroplet. A partition may be a well. A partition may be a microwell. Partitioning may be performed using a microfluidic device. In some embodiments, partitioning is performed using a droplet generator. Partitioning may comprise dividing a sample or mixture into water-in-oil droplets. A droplet may comprise one or more nucleic acids. A droplet may comprise a single nucleic acid. A droplet may comprise two or more nucleic acids. A droplet may comprise no nucleic acids. Each droplet of a plurality of droplets may generate a signal. A plurality of signals may comprise the signal(s) generated from each of a plurality of droplets comprising a subset of a sample.

[0052] The plurality of partitions may be atleast 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000 or more partitions. The plurality of partitions may be no more than 100,000, 90,000, 80,000, 70,000, 60,000, 50,000, 40,000, 30,000, 29,000, 28,000, 27,000, 26,000, 25,000, 24,000, 23,000, 22,000, 21,000, 20,000, 19,000, 18,000, 17,000, 16,000, 15,000, 14,000, 13,000, 12,000, 11,000, 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 orfewer. The plurality of partitions may be defined by a range of any of the foregoing values.

[0053] In some embodiments, each partition may receive a single template of the nucleic acid target sequence. In some embodiments, some partitions may receive more than one copy of a nucleic acid target template, while other partitions may not receive any target template. Following partitioning, each partition may be subject to end-point PCR. Partitions emitting a fluorescent signal may be marked “positive” and scored as “ 1 ,” whereas partitions without detectable fluorescence may be deemed “negative” and scored as “0.”

[0054] In some embodiments, the drop-off assay may generate at least 1, 10, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 7,500, 10,000, or more partitions that are positive for a nucleic acid sequence or a nucleic acid sequence variation. In some embodiments, the drop-off assay may generate partitions that are positive for a nucleic acid sequence or a nucleic acid sequence variation in at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the total number of partitions.

Thermal cycling

[0055] Methods of the present disclosure may comprise thermal cycling. Thermal cycling may comprise one or more thermal cycles. Thermally cycling may be performed under reaction conditions appropriate to amplify a template nucleic acid with PCR. Amplification of a template nucleic acid may require binding or annealing of oligonucleotide primer(s) to the template nucleic acid. Appropriate reaction conditions may include appropriate temperature conditions, appropriate buffer conditions, and the presence of appropriate reagents. Appropriate temperature conditions may, in some embodiments, be such that each thermal cycle is performed at a desired annealing temperature. A desired annealing temperature maybe sufficient for annealing of an oligonucleotide probe(s) to a nucleic acid target. Appropriate buffer conditions may, in some embodiments, be such that the appropriate salts are present in a buffer used during thermal cycling. Appropriate salts may include magnesium salts, potassium salts, ammonium salts. Appropriate buffer conditions may be such that the appropriate salts are present in appropriate concentrations. Appropriate reagents for amplification of each member of a plurality of nucleic acid target variations with PCR may include deoxytriphosphates (dNTPs). dNTPs may comprise natural or non-natural dNTPs including, for example, dATP, dCTP, dGTP, dTTP, dUTP, and variants thereof.

[0056] In various aspects, primer extension reactions are utilized to generate amplified product. Primer extension reactions generally comprise a cycle of incubating a reaction mixture at a denaturation temperature for a denaturation duration and incubating a reaction mixture at an elongation temperature for an elongation duration. In any of the various aspects, multiple cycles of a primer extension reaction can be conducted. Any suitable number of cycles may be conducted. For example, the number ofcycles conducted may be less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 cycles. The number of cycles conducted may depend upon, for example, the number of cycles (e.g., cycle threshold value (Ct)) used to obtain a detectable amplified product (e.g., a detectable amount of amplified DNA product that is indicative of the presence of a target DNA in a nucleic acid sample). For example, the number of cycles used to obtain a detectable amplified product (e.g., a detectable amount of DNA product that is indicative of the presence of a target DNA in a nucleic acid sample) may be less than about or about 100 cycles, 75 cycles, 70 cycles, 65 cycles, 60 cycles, 55 cycles, 50 cycles, 40 cycles, 35 cycles, 30 cycles, 25 cycles, 20 cycles, 15 cycles, 10 cycles, or 5 cycles. Moreover, in some embodiments, a detectable amount of an amplifiable product (e.g., a detectable amount of DNA product that is indicative of the presence of a target DNA in a nucleic acid sample) maybe obtained at a cycle threshold value (Ct) of less than 100, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5.

[0057] The time for which an amplification reaction yields a detectable amount of amplified nucleic acid may vary depending upon the nucleic acid sample, the sequence of the target nucleic acid, the sequence of the primers, the particular nucleic acid amplification reactions conducted, and the particular number of cycles of the amplification, the temperature of the reaction, the pH of the reaction. For example, amplification of a target nucleic acid may yield a detectable amount of product indicative to the presence of the target nucleic acid at time period of 120 minutes or less; 90 minutes or less; 60 minutes or less; 50 minutes or less; 45 minutes or less; 40 minutes or less; 35 minutes or less; 30 minutes or less; 25 minutes or less; 20 minutes or less; 15 minutes or less; 10 minutes or less; or 5 minutes or less.

[0058] In some embodiments, amplification of a nucleic acid may yield a detectable amount of amplified nucleic acids at time period of 120 minutes or less; 90 minutes or less; 60 minutes or less; 50 minutes or less; 45 minutes or less; 40 minutes or less; 35 minutes or less; 30 minutes or less; 25 minutes or less; 20 minutes or less; 15 minutes or less; 10 minutes or less; or 5 minutes or less.

Sample and Targets

[0059] A nucleic acid target of the present disclosure may be derived from a biological sample. A biological sample may be a sample derived from a subject. A biological sample may comprise any number of macromolecules, for example, cellular macromolecules. A biological sample may be derived from another sample. A biological sample may be a tissue sample, such as a biopsy, core biopsy, needle aspirate, or fine needle aspirate. A biological sample may be a fluid sample, such as a blood sample, urine sample, or saliva sample. A biological sample may be a skin sample. A biological sample may be a cheek swab. A biological sample may be a plasma or serum sample. A biological sample may comprise one or more cells. A biological sample may be a cell-free sample. A cell-free sample may comprise extracellular polynucleotides. Extracellular polynucleotides may be isolated from a bodily sample that may be selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool, and tears. The nucleic acid targets may be derived from an individual afflicted with or suspected of being afflicted with cancer. The nucleic acid targets may be derived from an individual before or after a cancer treatment has been administered. Such cancer treatments may suitably be radiation therapy, chemotherapy, surgery, or any combination thereof.

[0060] A nucleic acid target may be derived from one or more cells. A cell may be a tumor cell. A cell may be a cell suspected of comprising a viral pathogen. In some embodiments, a nucleic acid target is derived from a cell -free sample (e.g., serum, plasma). A nucleic acid target may be cell-free nucleic acid. Cell -free nucleic acid may be, for example, cell -free tumor DNA, cell-free fetal DNA, cell-free RNA, etc. A nucleic acid target may comprise deoxyribonucleic acid (DNA). DNA may be any kind of DNA, including genomic DNA. A nucleic acid target may be viral DNA. A nucleic acid target may comprise ribonucleic acid (RNA). RNA may be any kind of RNA, including messenger RNA, transfer RNA, ribosomal RNA, andmicroRNA. RNA may be viral RNA. Nucleic acid target variations may comprise one or more members. A member may be any region of a nucleic acid target. A member may be of any length. A member may be, for example, up to 1, 2, 3, 4, 5, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50000, or 100000 nucleotides, or more. In some instances, a member may be a gene. A nucleic acid target may comprise a gene whose detection may be useful in diagnosing one or more diseases. A gene may be a viral gene or bacterial gene whose detection may be useful in identifying the presence or absence of a pathogen in a subject. In some embodiments, the methods of the present disclosure are useful in detectingthe presence or absence or one or more infectious agents (e.g., viruses) in a subject.

[0061] In some embodiments, the presence or absence of sequence variations maybe indicative of the presences of a disease. In some embodiments, the disease may be a bacteria or virus. In some embodiments the disease may be cancer. The cancer may be bladder cancer, breast cancer, colon or rectal cancer, endometrial cancer, kidney cancer, leukemia, liver or bile duct cancer, lung cancer, melanoma, lymphoma, pancreatic cancer, prostate cancer, or thyroid cancer. In some embodiments, the presence or absence of sequence variations may be indicative of a prognosis of a disease. In some embodiments, the presence or absence of sequence variations may be predictive of the expected efficacy of a treatment for a disease. The treatment may be a chemotherapeutic, a antibacterial agent, a steroid, a hormone, radiation, surgery, or other form of treatments.

[0062] In some embodiments, a nucleic acid target is a chromosome. One or more nucleic acid molecules analyzed by methods of the present disclosure may correspond to a chromosome. For example, nucleic acid molecules may be fragments of a chromosome. In another example, nucleic acid molecules maybe amplification products of a chromosome. Nucleic acid molecules corresponding to a chromosome may be obtained from one or more cells. Nucleic acid molecules corresponding to a chromosome may be obtained from a cell-free sample (e.g., serum, plasma, blood, etc.).

[0063] A nucleic acid target may be gene. The gene may be an oncogene. The assays described herein may detect mutations in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more oncogenes in a single assay. The assays described herein may detect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations in a single oncogene in a single assay. The oncogene may be EGFR, BRAF, HER2, RET, PIK3CA, FGFR1, DDR2, ROS1, RAS, MYC, SRC, hTERT, BCL2, ALK, or any combination thereof. The target nucleic acid may be the site of a specific mutation of an oncogene. The target nucleic acid may be a site of mutation in EGFR. The EGFR mutation may be T790M, S768I, L858R, L861Q, G719X, an exon 19 deletion, an exon 20 insertion or a combination thereof. The target nucleic acid maybe a site of V600E mutations in BRAF. The target nucleic acid may be a site of G12C mutations in KRAS. The nucleic acid target may be a fusion protein. The fusion protein may be ALK, ATIC, CARS, CCDC6, CD74, CLIP1, CLTC, DCTN1, EML4, ERC1, ETV6, EZR, FN1, GOLGA5, GOPC, HIP1, HLA A, HOOK3, KIF5B, KLC1, KTN1, LMNA, LRIG3, MET13, MET15, MSN, MY05A, NACC2, NCOA4, NTRK1, 2, 3, PCM1, PPFIBP1, PWWP2A, QKI, RANBP2, RELCH, RET, ROS1, SDC4, SEC31, SHTN1, SLC34A2, SQSTM1, STRN, TFG, TPR, TP53, TPM3, TPM4, TRIOM24, TRIM27, TRIM33, VCL, WDCP, ZCCHC8, or a combination thereof.

Sample processing

[0064] A sample may be processed concurrently with, prior to, or sub sequent to the methods of the present disclosure. A sample may be processed to purify or enrich for nucleic acids (e.g., to purify nucleic acids from a plasma sample). A sample comprising nucleic acids may be processed to purity or enrich for nucleic acid of interest. A sample comprising nucleic acids may be processed to enrich for fetal nucleic acid. A sample may be enriched for nucleic acid of interest (e.g., fetal nucleic acid) by various methods including, for example, sequence -specific enrichment (e.g., via use of capture sequences), epigenetic-specific enrichment (e.g., via use of methylation-specific capture moieties, such as antibodies). Enrichment may comprise isolation of nucleic acid of interest and/or depletion of a nucleic acid that is not of interest. In some embodiments, a sample is not processed to purify or enrich for nucleic acid of interest prior to performing methods of the present disclosure (e.g., amplification of nucleic acids from a sample). For example, a sample may not be processed to enrich for fetal nucleic acid prior to mixing a sample with oligonucleotide primers and oligonucleotide probes, as described elsewhere herein.

Nucleic acid enzymes

[0065] Mixtures and compositions of the present disclosure may comprise one or more nucleic acid enzymes. A nucleic acid enzyme may have exonuclease activity. A nucleic acid enzyme may have endonuclease activity. A nucleic acid enzyme may have RNase activity. A nucleic acid enzyme may be capable of degrading a nucleic acid comprising one or more ribonucleotide bases. A nucleic acid enzyme may be, for example, RNase H or RNase III. An RNase III may be, for example, Dicer. A nucleic acid may be an endonuclease I such as, for example, a T7 endonuclease I. A nucleic acid enzyme may be capable of degrading a nucleic acid comprising a non-natural nucleotide. A nucleic acid enzyme may be an endonuclease V such as, for example, an E. coli endonuclease V. A nucleic acid enzyme maybe a polymerase (e.g., a DNA polymerase). A polymerase may be Taq polymerase or a variant thereof. A nucleic acid enzyme may be capable, under appropriate conditions, of degrading an oligonucleotide probe. A nucleic acid enzyme may be capable, under appropriate conditions, of releasing a quencher from an oligonucleotide probe.

Reactions

[0066] A reaction may comprise contacting nucleic acid target variations with one or more oligonucleotide probes. A reaction may comprise contacting a sample solution volume (e.g., a droplet, well, tube, etc.) with a plurality of oligonucleotide probes, each corresponding to one of a plurality of nucleic acid target variations, to generate a plurality of signals generated from the plurality of oligonucleotide probes. A reaction may comprise polymerase chain reaction (PCR). A reaction may be a digital PCR reaction.

[0067] In some embodiments, one or more signals from a plurality of signals fail to non- degenerately identify the presence or absence of any combination of a plurality of nucleic acid molecules (e.g., a signal corresponds to two or more combinations of nucleic acid molecules in a sample volume). As disclosed herein, two or more signals may be compared, thereby non- degenerately indicating the presence or absence of a plurality of nucleic acid target variations, in any combination.

[0068] In some embodiments, one or more synthetic (or otherwise generated to be different from the target of interest) primer/probe sets may be used to calibrate the location of multiple signals in a reaction (e.g., a digital PCR reaction). A reaction may include a known amount of template. A ratio of a known template to the other targets in a reaction may be used to normalize the locations of target clusters in an assay.

Oligonucleotide primers

[0069] An oligonucleotide primer (or “amplification oligomer”) of the present disclosure may be a deoxyribonucleic acid. An oligonucleotide primer maybe a ribonucleic acid. An oligonucleotide primer may comprise one or more non -natural nucleotides. A non-natural nucleotide may be, for example, deoxyinosine.

[0070] An oligonucleotide primer may be a forward primer. An oligonucleotide primer may be a reverse primer. An oligonucleotide primer may be between about 5 and about 50 nucleotides in length. An oligonucleotide primer may be at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 base pairs in length, or more. An oligonucleotideprimer may be atmost 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or 5 nucleotides in length. An oligonucleotide primer may be about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 base pairs in length.

[0071] A set of oligonucleotide primers may comprise paired oligonucleotide primers. Paired oligonucleotide primers may comprise a forward oligonucleotide primer and a reverse oligonucleotide primer. A forward oligonucleotide primer may be configured to hybridize to a first region (e.g., a 3 ’ end) of a nucleic acid sequence, and a reverse oligonucleotide primer may be configured to hybridize to a second region (e.g., a 5’ end) of the nucleic acid sequence, thereby being configured to amplify the nucleic acid sequence under conditions sufficient for nucleic acid amplification. Different sets of oligonucleotide primers may be configured to amplify different nucleic acid target sequences.

[0072] A mixture may comprise a plurality of forward oligonucleotide primers. A plurality of forward oligonucleotide primers may be a deoxyribonucleic acid. Alternatively, a plurality of forward oligonucleotide primers may be a ribonucleic acid. A plurality of forward oligonucleotide primers may be between about 5 and about 50 nucleotides in length . A plurality of forward oligonucleotide primermaybe atleast 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 base pairs in length, or more. A plurality of forward oligonucleotide primermay be atmost 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or 5 nucleotides in length.

[0073] A mixture may comprise a plurality of reverse oligonucleotide primers. A plurality of reverse oligonucleotide primers may be a deoxyribonucleic acid. Alternatively, a plurality of reverse oligonucleotide primers may be a ribonucleic acid. A plurality of reverse oligonucleotide primers may be between about 5 and about 50 nucleotides in length. A plurality of reverse oligonucleotide primer may be atleast 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 base pairs in length, or more. A plurality of reverse oligonucleotide primer may be at most 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or 5 nucleotides in length.

[0074] In some aspects, a mixture may include one or more synthetic (or otherwise generated to be different from the target of interest) primers for digital PCR reactions. The one or more synthetic primers may be used in combination with a template to calibrate the location of multiple clusters in a reaction.

[0075] In some aspects, a mixture may be subjected to conditions sufficient to anneal an oligonucleotide primer to a nucleic acid molecule. In some aspects, a mixture may be subjected to conditions sufficient to anneal a plurality of oligonucleotide primers to a nucleic acid molecule. In some aspects, a mixture may be subjected to conditions sufficient to anneal a plurality of oligonucleotide primers to a plurality of nucleic acid target variations. The mixture may be subjected to conditions which are sufficient to denature nucleic acid molecules.

Subj ecting a mixture to conditions sufficient to anneal an oligonucleotide primer to a nucleic acid target may comprise thermally cycling the mixture under reaction conditions appropriate to amplify the nucleic acid target(s) with, for example, polymerase chain reaction (PCR).

[0076] Conditions may be such that an oligonucleotide primer pair (e.g., forward oligonucleotide primer and reverse oligonucleotide primer) are degraded by a nucleic acid enzyme. An oligonucleotide primer pair may be degraded by the exonuclease activity of a nucleic acid enzyme. An oligonucleotideprimer pair may be degraded by the RNase activity of a nucleic acid enzyme. Degradation of the oligonucleotide primer pair may result in release of the oligonucleotide primer. Once released, the oligonucleotide primer pair may bind or anneal to a template nucleic acid.

Oligonucleotide probes

[0077] Samples, mixtures, kits, and compositions of the present disclosure may comprise an oligonucleotide probe, also referenced herein as a “detection probe” or “prob e” An oligonucleotide probe may be a nucleic acid (e.g., DNA, RNA, etc.). An oligonucleotide probe may comprise a region complementary to a region of a nucleic acid target. The concentration of an oligonucleotide probe may be such that it is in excess relative to other components in a sample.

[0078] An oligonucleotide probe may comprise a non-target-hybridizing sequence. A nontarget-hybridizing sequence may be a sequence which is not complementary to any region of a nucleic acid target sequence. An oligonucleotide probe comprising a non -target-hybridizing sequence may be a hairpin detection probe. An oligonucleotide probe comprising a non -targethybridizing sequence may be a molecular beacon probe. Examples of molecular beacon probes are provided in, for example, U.S. Patent 7,671, 184, incorporated herein by reference in its entirety. An oligonucleotide probe comprising a non -target-hybridizing sequence may be a molecular torch. Examples of molecular torches are provided in, for example, U.S. Patent 6,534,274, incorporated herein by reference in its entirety.

[0079] A sample may comprise more than one oligonucleotide probe. Multiple oligonucleotide probes may be the same, or may be different. An oligonucleotide probe may be at least 5, at least 10, at least 15, at least 20, or at least 30 nucleotides in length, or more. An oligonucleotide probe may be at most 30, at most 20, at most 15, at most 10 or at most 5 nucleotides in length. In some examples, a mixture comprises a first oligonucleotide probe and one or more additional oligonucleotide probes. An oligonucleotide probe maybe a nucleic acid (e.g., DNA, RNA, etc.). An oligonucleotide probemay be atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 nucleotides in length, or more. An oligonucleotide probe may be at mo st 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotides in length.

[0080] In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more different oligonucleotide probes are partitioned into a plurality of partitions. Each oligonucleotide p robe may correspond to (e.g., capable of binding to) a given region of a nucleic acid target (e.g., a chromosome) in a sample. In one example, a first oligonucleotide probe is specific for a first region of a first nucleic acid target, a second oligonucleotide probe is specific for a second region of the first nucleic acid target, and a third oligonucleotide probe is specific for a third region of the first nucleic acid target. Each oligonucleotide probe may comprise a signal tag with about equal emission wavelengths. In some embodiments, each oligonucleotide probe comprises an identical fluorophore. In some embodiments, each oligonucleotide probe comprises a different fluorop hore, where each fluorophore is capable of being detected in a single optical channel.

[0081] In some aspects, a mixture may include one or more synthetic (or otherwise generated to be different from the target of interest) probes for digital PCR reactions. The one or more synthetic probes may have a known fluorescence ratio to the other targets in the reaction and used to calibrate the location of multiple clusters in a reaction. The one or more synthetic probes may be used to normalize the locations of the target clusters in the assay by ensuring that some, but not most, of the droplets generate a “positive” fluorescence in a distinct cluster.

[0082] A probe may correspond to a region of a nucleic acid target. For example, a probe may have complementarity and/or homology to a region of a nucleic acid target. A probe may comprise a region which is complementary or homologous to a region of a nucleic acid target. A probe corresponding to a region of a nucleic acid target may be capable of binding to the region of the nucleic acid target under appropriate conditions (e.g., temperature conditions, buffer conditions, etc.). For example, a probe may be capable of binding to a region of a nucleic acid target under conditions appropriate for polymerase chain reaction. A probe may correspond to an oligonucleotide which corresponds to a nucleic acid target. For example, an oligonucleotide may be a primer with a region complementary to a nucleic acid target and a region complementary to a probe.

[0083] A probe may be a nucleic acid complementary to a region of a given nucleic acid target. Each probe used in the methods and assays of the presence disclosure may comprise at least one fluorophore. A fluorophore may be selected from any number of fluorophores. A fluorophore may be selected from three, four, five, six, seven, eight, nine, or ten fluorophores, or more. One or more oligonucleotide probes usedin a single reaction may comprise the same fluorophore. In some embodiments, all oligonucleotide probes used in a single reaction comprise the same fluorophore. Each probe may, when excited and contacted with its corresponding nucleic acid target, generate a signal. A signal may be a fluorescent signal. A plurality of signals may be generated from one or more probes.

[0084] An oligonucleotide probemay have less than 50%, 40%, 30%, 20%, 10%, 5%, or 1% complementarity to any member of a plurality of nucleic acid target variations. An oligonucleotide probe may have no complementarity to any member of the plurality of nucleic acid target variations.

[0085] An oligonucleotide probe may comprise a detectable label. A detectable label may be a chemiluminescent label. A detectable label may comprise a chemiluminescent label. A detectable label may comprise a fluorescent label. A detectable label may comprise a fluorophore. A fluorophore maybe, for example, FAM, TET, HEX, JOE, Cy3, or Cy5. A fluorophore may be FAM. A fluorophore may be HEX. An oligonucleotide probe may further comprise one or more quenchers. A quencher may inhibit signal generation from a fluorophore. A quencher may be, for example, TAMRA, BHQ-1 , BHQ-2, or Dabcy. A quencher may be BHQ-1. A quencher may be BHQ-2.

[0086] Each nucleic acid probe of a plurality of nucleic acid probes may be provided at a specific concentration, such that each signal generated from the nucleic acid probes in a partition corresponds to a unique combination of target nucleic acid variations. Where each signal of N signals corresponds to a unique combination of target nucleic acid variations, the signals may be described as “non-degenerate”. In one aspect, a first nucleic acid probe is provided at a concentration of about X. Where a first nucleic acid probe is provided at a concentration of about X, additional nucleic acid probes may be provided at a specific concentration greater than X, thereby enabling the generation of non-degenerate signals. [0087] In some cases, a second nucleic acid probe is provided at a concentration of at least about 2X, about 3X, about 4X, about 5X, about 6X, about 7X, about 8X, or more. In some cases, a second nucleic acid probe is provided at a concentration of at most about 8X, about 7X, about 6X, about 5 X, about 4X, about 3 X, or about 2X. In some cases, a second nucleic acid probe is provided at a concentration of about 2X, about 3X, about 4X, about 5X, about 6X, about 7X, or about 8X.

[0088] In some cases, a third nucleic acid probe is provided at a concentration of at least about 4X, about 5X, about 6X, about 7X, about 8X, about 9X, about 10X, about 1 IX, about 12X or more. In some cases, a third nucleic acid probe is provided at a concentration of at most about 12X, about 1 IX, about 10X, about 9X, about 8X, about 7X, about 6X, about 5X, or about 4X. In some cases, a third nucleic acid probe is provided at a concentration of about 4X, about 5X, about 6X, about 7X, about 8X, about 9X, about 10X, about 1 IX, or about 12X.

[0089] In some cases, a fourth nucleic acid probe is provided at a concentration of at least about 8X, about 9X, about 10X, about 1 IX, about 12X, about 13X, about 14X, about 15X, about 20X, or more. In some cases, a fourth nucleic acid probe is provided at a concentration of at most about20X, about 15X, about 14X, about 13X, about 12X, about 1 IX, about 10X, about9X, or about 8X. In some cases, a fourth nucleic acid probe is provided at a concentration of about 8X, about9X, about 10X, about 1 IX, about 12X, about 13X, about 14X, about 15X, or about20X.

[0090] Each nucleic acid probe may be provided at a concentration that is distinct from all other nucleic acid probes. Any combination of nucleic acid probe concentrations which is capable of producing non -degenerate signals (i.e., signals which each uniquely identify a specific combination of nucleic acid targets in a partition) may be used. In one example, a first nucleic acid probe may be provided at a concentration of about X, a second nucleic acid probe may be provided at a concentration of about 3X, and a third nucleic acid probe may be provided at a concentration of about 8X, thereby generating non -degenerate signals in a plurality of partitions corresponding to the presence of a unique combination of nucleic acid targets.

[0091] X may be a concentration of a nucleic acid probe provided in the disclosed methods. In some cases, X is atleast 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350nM, 400 nM, 450 nM, 500 nM, or greater. In some cases, X is at most 500 nM, 450 nM, 400 nM, 350 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100 nM, or 50 nM. X may be any concentration of a nucleic acid probe which is capable of being partitioned.

[0092] Each nucleic acid probe of a plurality of nucleic acid probes maybe labeled with one or more copies of a luminescent signal tag, such that the probes have varying intensity levels, thereby enabling the generation of signals which uniquely identify each combination of nucleic acid targets in a partition. In one aspect, a first nucleic acid probe has an intensity level of about Y. Where a first nucleic acid probe has an intensity level of about Y, additional nucleic acid probes may have an intensity level greater than Y, thereby enabling the generation of non- degenerate signals. A first nucleic acid may comprise a fluorophore with an intensity level of about Y.

[0093] In some cases, a second nucleic acid probe has an intensity level of at least about 2 Y, about 3 Y, about 4 Y, about 5 Y, about 6 Y, about 7 Y, about 8 Y, or more. In some cases, a second nucleic acid probe has an intensity level of at most about 8 Y, about 7Y, about 6Y, about 5 Y, about 4Y, about 3 Y, or about 2Y. In some cases, a second nucleic acid probe has an intensity level of about 2Y, about 3 Y, about 4Y, about 5 Y, about 6Y, about 7Y, or about 8 Y. A second nucleic acid may comprise a single luminescent signal tag (e.g., fluorophore) with an intensity level of at least about 2 Y. Alternatively or in addition, a second nucleic acid may comprise two or more fluorophores each with an intensity level of about Y.

[0094] In some cases, a third nucleic acid probe has an intensity level of at least about 4 Y, about 5Y, about 6Y, about 7Y, about 8Y, about 9Y, about 10Y, about 11Y, about 12Y ormore. In some cases, a third nucleic acid probe has an intensity level of at most about 12Y, about 11 Y, about 10Y, about 9 Y, about 8 Y, about 7 Y, about 6 Y, about 5 Y, or about 4 Y. In some cases, a third nucleic acid probe has an intensity level of about 4Y, about 5 Y, about 6Y, about 7Y, about 8Y, about 9 Y, about 10Y, about 11 Y, or about 12Y. A third nucleic acid may comprise a single luminescent signal tag (e.g., fluorophore) with an intensity level of at least about 4 Y. Alternatively or in addition, a third nucleic acid may comprise four or more fluorophores each with an intensity level of about Y.

[0095] In some cases, a fourth nucleic acid probe has an intensity level of at least about 8 Y, about 9Y, about 10Y, about 11Y, about 12Y, about 13Y, about 14Y, about 15Y, about20Y, or more. In some cases, a fourth nucleic acid probe has an intensity level of at most about 20Y, about 15Y, about 14Y, about 13Y, about 12Y, about 11Y, about 10Y, about 9Y, or about 8Y. In some cases, a fourth nucleic acid probe has an intensity level of about 8 Y, about 9 Y, about 10Y, about 11 Y, about 12Y, about 13Y, about 14Y, about 15Y, or about 20 Y. A fourth nucleic acid may comprise a single luminescent signal tag (e.g., fluorophore) with brightness of at least about 8 Y. Alternatively or in addition, a fourth nucleic acid may comprise eight or more fluorophores each with an intensity level of about Y.

[0096] Y may be a signal intensity level produced by a nucleic acid probe comprising one or more signal tags (e.g., fluorophores). Y may be any signal intensity capable of being detected in an optical channel.

Signal generation [0097] Thermal cycling may be performed such that one or more oligonucleotide probes are degraded by a nucleic acid enzyme. An oligonucleotide probe may be degraded by the exonuclease activity of a nucleic acid enzyme. An oligonucleotide probe may generate a signal upon degradation. In some embodiments, an oligonucleotide probe may generate a signal only if at least one member of a plurality of nucleic acid target variations is present in a mixture.

[0098] A reaction may generate one or more signals. A reaction may generate a cumulative intensity signal comprising a sum of multiple signals. A signal may be a chemiluminescent signal. A signal may be a fluorescent signal. A signal may be generated by an oligonucleotide probe. For example, excitation of a hybridization probe comprising a luminescent signal tag may generate a signal. A signal may be generated by a fluorophore. A fluorophore may generate a signal upon release from a hybridization probe. A reaction may comprise excitation of a fluorophore. A reaction may comprise signal detection. A reaction may comprise detecting emission from a fluorophore.

[0099] A signal may be a fluorescent signal. A signal may correspond to a fluorescence intensity level. Each signal measured in the methods of the present disclosure may have a distinct fluorescence intensity value, thereby corresponding to the presence of a unique combination of nucleic acid target variations in a partition. A signal may be generated by one or more oligonucleotide probes. The number of signals generated in a digital assay may correspond to the number of oligonucleotide probesthat are partitioned, the number of nucleic acid target variations that are partitioned, and, in some embodiments, the partitioning conditions. For example, where three nucleic acid target variations and three oligonucleotide probes are partitioned such that each partition may comprise one, two, or all three nucleic acid target variations, seven signals may be generated, where each signal corresponds to the presence of a unique combination of the three nucleic acid target variations in the partition. N may be a number of signals detected in a single optical channel in an assay of the present disclosure. N may be atleast2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50 or more. N may be at most 50, 40, 30, 24, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2. N may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, or 50.

[00100] As will be recognized and is described elsewhere herein, sets of signals may be generated in multiple different optical channels, where each set of signals is detected in a single optical channel, thereby significantly increasing the number of nucleic acid target variations that can be measured in a single reaction (e.g., digital PCR reaction). In some embodiments, two sets of signals are detected in a single reaction. Each set of signals detected in a reaction may comprise the same number of signals, or different numbers of signals. [00101] In some embodiments, a signal may be generated simultaneous with hybridization of an oligonucleotide probe to a region of a nucleic acid. For example, an oligonucleotide probe (e.g., a molecular beacon probe or molecular torch) may generate a signal (e.g., a fluorescent signal) following hybridization to a nucleic acid. In some embodiments, a signal may be generated subsequent to hybridization of an oligonucleotide probe to a region of a nucleic acid, following degradation of the oligonucleotide probe by a nucleic acid enzyme.

[00102] In embodiments where an oligonucleotide probe comprises a signal tag, the oligonucleotide probe may be degraded when bound to a region of an oligonucleotide primer, thereby generating a signal. For example, an oligonucleotide probe (e.g., a TaqMan® probe) may generate a signal following hybridization of the oligonucleotide probe to a nucleic acid and subsequent degradation by a polymerase (e.g., during amplification, such as PCR amplification). An oligonucleotide probe may be degraded by the exonuclease activity of a nucleic acid enzyme.

[00103] An oligonucleotide probe may comprise a quencher and a fluorophore, such that the quencher is released upon degradation of an oligonucleotide probe, thereby generating a fluorescent signal. Thermal cycling may be used to generate one or more signals. Thermal cycling may generate at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 signals, or more. Thermal cycling may generate atmost 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 signal. Multiple signals may be of the same type or of different types. Signals of different types may be fluorescent signals with different fluorescent wavelengths. Signals of different types maybe generated by detectable labels comprising different fluorophores. Signals of the same type may be of different intensities (e.g., different intensities of the same fluorescent wavelength). Signals of the same type may be signals detectable in the same color channel. Signals of the same type may be generated by detectable labels comprising the same fluorophore. Detectable labels comprising the same fluorophore may generate different signals by nature of being at different concentrations, thereby generating different intensities of the same signal type.

[00104] The methods presented in this disclosure may be used with any quantifiable signal. In some embodiments, this disclosure provides methods to quantify targets using a single component of a signal (e.g., intensity). For example, an analysis may rely on a multiplicity of signal intensity without consideration of color. Although fluorescent probes have been used to illustrate this principle, the disclosed methods are equally applicable to any other method providing a quantifiable signal, including an electrochemical signal and a chemiluminescent signal.

[00105] In some embodiments, quantification may comprise an absolute quantification of a nucleic acid targets or sequence variations thereof. In some embodiments, quantification may comprise a relative quantification of nucleic acid targets or sequence variations thereof. In some embodiments, quantification may comprise generating an average number of nucleic acid molecules per partition. In some embodiments, quantification may comprise generating a probability that one or more nucleic acid molecules comprising one or more nucleic acid sequence variation is present in a given partition. In some embodiments, quantification may comprise, for a plurality of partitions, generating a probability that one or more nucleic acid molecules comprising one or more nucleic acid sequence variation is present in a given partition of a plurality of partitions, thereby generating a plurality of probabilities. In some embodiments, quantification may comprise summing two or more probabilities of a plurality of probabilities. In some embodiments, quantification may comprise determining a total number of partitions. In some embodiments, quantification may be performed without determining a total number of partitions. In some embodiments, a plurality of partitions may comprise a plurality of droplets. [00106] The methods presented in this disclosure may also utilize the measurement of a signal in at least two dimensions, also referred to as the measurement of at least two components of a signal (e.g., color and intensity). In some embodiments, a quantifiable signal comprises a waveform that has both a frequency (wavelength) and an amplitude (intensity). A signal may be an electromagnetic signal. An electromagnetic signal may be a sound, a radio signal, a microwave signal, an infrared signal, a visible light signal, an ultraviolet light signal, an x-ray signal, or a gamma-ray signal. In some embodiments, an electromagnetic signal may be a fluorescent signal, for example a fluorescence emission spectrum that may be characterized in terms of wavelength and intensity.

[00107] In certain portions of this disclosure, the signal is described and exemplified in terms of a fluorescent signal. This is not meant to be limiting, and one of ordinary skill in the art will readily recognize that the principles applicable to the measurement of a fluorescent signal are also applicable to other signals. For example, like fluorescent signals, any of the electromagnetic signals described above may also be characterized in terms of a wavelength and an intensity. The wavelength of a fluorescent signal may also be described in terms of color. The color may be determined based on measuring intensity at a particular wavelength or range of wavelengths, for example by determining a distribution of fluorescent intensity at different wavelengths and/or by utilizing a band pass filter to determine the fluorescence intensity within a particular range of wavelengths. Intensity may be measured with a photodetector. A range of wavelengths may be referred to as a “channel,” “color channel,” or “optical channel.”

[00108] The presence or absence of one or more signals may be detected. One signal may be detected, or multiple signals may be detected. Multiple signals may be detected simultaneously. Alternatively, multiple signals may be detected sequentially. The presence of a signal may be correlated to the presence of a nucleic acid target. The presence of least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more signals may be correlated with the presence of at least one of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleic acid target variations. The absence of a signal may be correlated with the absence of corresponding nucleic acid target variations. The absence of least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more signals may be correlated with the absence of each of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleic acid target molecules.

Kits

[00109] The present disclosure also provides kits for multiplex analysis. Kits may comprise one or more oligonucleotide probes. Oligonucleotide probes may be lyophilized. Different oligonucleotide probes may be present at different concentrations in a kit. Oligonucleotide probes may comprise a fluorophore and/or one or more quenchers.

[00110] Kits may comprise one or more sets of oligonucleotide primers (or “amplification oligomers”) as described herein. A set of oligonucleotide primers may comprise paired oligonucleotide primers. Paired oligonucleotide primers may comprise a forward oligonucleotide primer and a reverse oligonucleotide primer. A set of oligonucleotide primers may be configured to amplify a nucleic acid sequence corresponding to particular targets. For example, a forward oligonucleotide primer may be configured to hybridize to a first region (e.g., a 3 ’ end) of a nucleic acid sequence, and a reverse oligonucleotide primer may be configured to hybridize to a second region (e.g., a 5’ end) of the nucleic acid sequence, thereby being configured to amplify the nucleic acid sequence. Different sets of oligonucleotide primers may be configured to amplify nucleic acid sequences. In one example, a first set of oligonucleotide primers may be configured to amplify a first nucleic acid sequence, and a second set of oligonucleotide primers maybe configured to amplify a second nucleic acid sequence. Oligonucleotide primers configured to amplify nucleic acid molecules may be used in performing the disclosed methods. In some embodiments, all of the oligonucleotide primers in a kit are lyophilized.

[00111] Kits may comprise one or more nucleic acid enzymes. A nucleic acid enzyme may be a nucleic acid polymerase. A nucleic acid polymerase may be a deoxyribonucleic acid polymerase (DNase). A DNase may be a Taq polymerase or variant thereof. A nucleic acid enzyme may be a ribonucleic acid polymerase (RNase). An RNase may be an RNase III. An RNase III may be Dicer. The nucleic acid enzyme may be an endonuclease. An endonuclease may be an endonuclease I. An endonuclease I may be a T7 endonuclease I. Kits may comprise instructions for using any of the foregoing in the methods described herein.

[00112] Additionally, kits may include one or more synthetic (or otherwise generated to be different from the target of interest) primer/probe sets for digital PCR reactions. The one or more synthetic primer/probe sets may be used to calibrate the location of multiple clusters in a multiplex. The kit may also include a known amount of template.

[00113] Kits provided herein may be useful in, for example, calculating at least first and second sums, each being a sum of multiple target signals corresponding with a first and second chromosome.

Systems

[00114] Methods as disclosed herein may be performed using a variety of systems. The systems may be configured such the steps of the method may be performed. For example, the systems may comprise a detector for the detection of signals as described elsewhere herein. The system may comprise a processor configured to process, receive, plot, or otherwise represent the data obtained from the detector. The processor may be configured to process the data as described elsewhere herein. The processor may be configured to generate a report of the results obtained from the assay. The results of the assay may be uploaded into a remote server, or other computer systems as described elsewhere herein. The results may be uploaded and sent to a subject’s medical provider or an institution monitoring the spread of a disease. The results may also be sent to the subject directly. The subject, medical provider, or other institution may be able to access the remote server such review or analyze the results. For example, the results may then be transmitted to another institution/or medical professional for monitoring or for providing recommendations for the subject. In additional to the data generated for the detection of targets, the data may be used to monitor a geographical location of the assay or subject, for example to allow monitoring of the transmission of a disease. These results can then be uploaded into a cloud database or other remote database for storage and transmission to or access by a variety or individuals and institutions which may use the results of the assay. The results may be obtained on a smart phone or other computer system as disclosed elsewhere herein which may display the results.

[00115] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. The computer system can perform various aspects of the present disclosure. The computer system can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[00116] The computer system may include a central processing unit (CPU, also “processor” and “computer processor” herein), which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system may include memory or memory location (e.g., random-access memory, read-only memory, flash memory), electronic storage unit (e.g., hard disk), communication interface (e.g., network adapter) for communicating with one or more other systems, and peripheral devices, such as cache, other memory, data storage and/or electronic display adapters. The memory, storage unit, interface and peripheral devices are in communication with the CPU through a communication bus (solid lines), such as a motherboard. The storage unit can be a data storage unit (or data repository) for storing data. The computer system can be operatively coupled to a computer network (“network”) with the aid of the communication interface. The network can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network in some embodiments is a telecommunication and/or data network. The network can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network, in some embodiments with the aid of the computer system, can implement a peer-to-peer network, which may enable devices coupled to the computer system to behave as a client or a server.

[00117] The CPU can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory. The instructions can be directed to the CPU, which can subsequently program or otherwise configure the CPU to implement methods of the present disclosure. Examples of operations performed by the CPU can include fetch, decode, execute, and writeback.

[00118] The CPU can be part of a circuit, such as an integrated circuit. One or more other components of the system can be included in the circuit. In some embodiments, the circuit is an application specific integrated circuit (ASIC).

[00119] The storage unit can store files, such as drivers, libraries, and saved programs. The storage unit can store user data, e.g., user preferences and user programs, or raw data or processed results from the assays. The computer system in some embodiments can include one or more additional data storage units that are external to the computer sy stem, such as located on a remote server that is in communication with the computer system through an intranet or the Internet.

[00120] The computer system can communicate with one or more remote computer systems through the network. For instance, the computer system can communicate with a remote computer system of a user (e.g., operator). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smartphones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system via the network.

[00121] The computer system may transmit data associated with or collected during operation of the systems and method described herein to one or more computer systems through a network. The data may be results, conclusions, graphical representations of data, raw outputs, inputs, device or program settings, performance analytics, or other information. The data may be transmitted to another computer system for the purpose of further processing, analyzing, or otherwise transforming the data. The data may then be further transmitted back to the originating computer, to another computer system, or both. The transmission may be physical, such as through a wired network or a physical storage medium, wireless, such as through Bluetooth® or a wireless network connection, or a combination of both, such as a wireless internet transmission. The transmission of the data may be executed manually by a user or through an automated processes, such as programs or algorithms, designed to automatically transmit the data.

[00122] The computer system may be accessed by a remote user in order to retrieve data associated with or collected during the operation of the systems and method described herein. The access may be directly to the computer system or through a series of interconnected computer systems. The access and data retrieval may be accomplished manually or automatically by means of a program or algorithms or through a combination of both.

[00123] Methods as described herein can be implemented byway of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system, such as, for example, on the memory or electronic storage unit. The machine executable or machine - readable code can be provided in the form of software. During use, the code can be executed by the processor. In some embodiments, the code can be retrieved from the storage unit and stored on the memory for ready access by the processor. In some situations, the electronic storage unit can be precluded, and machine-executable instructions are stored on memory.

[00124] The code can be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a precompiled or as-compiled fashion.

[00125] Aspects of the systems and methods provided herein, such as the computer system, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine -executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non -transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical, and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links, or the like, also may be considered as media bearing the software. As used herein, unless restricted to non -transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[00126] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH -EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[00127] The computer system can include or be in communication with an electronic display that comprises a user interface (UI) for providing, for example, plots of data, plots of kinetic signatures, information relating to signal amplitude, Examples ofUIs include, without limitation, a graphical user interface (GUI) and web -based user interface.

[00128] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit. The algorithm can, for example, parameterize datapoints or fit data point to specified mathematical functions, in order to quantify analytes.

[00129] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

EXAMPLES

Example 1

[00130] In some cancers, somatic gene mutations are important prognostic markers and increasingly constitute therapeutic targets. Therefore, robust, sensitive, and fast diagnostic assays are needed.

[00131] A drop-off digital droplet PCR assays, which can detect and quantify gene mutations in two different regions of a gene that is commonly mutated in multiple regions in a common cancer is designed. This assay can be used for mutation screening as well as quantification and sequential monitoring.

[00132] A sample of plasma from a patient is separated by centrifugation and the cfDNA is isolated from the sample. A mixture of primers, nucleic acid probes, and polymerase is added to a sample containing free circulating DNA from a patient. The mixture is partitioned into approximately 20,000 partitions. An amplification reaction is performed on the partitions and the signal generated from the partitions are detected. There are 5 distinct populations generated from the reaction. At the lowest intensity is the population of partition that do not contain any of the gene of interest. At the next higher intensity level are partitions that contain both mutations that the assay is designed to detect. At the next higher intensity level are partitions that contain on of the tested mutation. At the next higher intensity level are partitions that contain the other tested mutation. The identity of which mutation is at which intensity level is determined by the design of the assay and the type of multiplexing strategy used. At the highest level of intensity are partitions that contain no mutations.

[00133] Based upon the number of partitions detected at each level, the presence or absence of each of the mutations is determined. The absolute number of mutation in the sample is also calculated and used to determine the frequency of each mutation.

[00134] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.