CROSS-REFERENCE

[0001] This application claims the benefit of priority to U.S. Provisional App. No. 63/368,427 filed July 14, 2022, which is incorporated by reference herein in its entirety.

BACKGROUND

[0002] Real-Time PCR (qPCR) is a process of monitoring a PCR reaction by recording the fluorescence generated at the end of amplification each cycle. In fluorescently multiplexed PCR, detection of multiple target nucleic acid sequences in a single reaction is accomplished by associating each nucleic acid target with a distinct fluorescent tag. The present disclosure provides a precise method measuring multiple reporter signals during multiplex qPCR reactions .

SUMMARY

[0003] Disclosed herein, in some aspects, are methods, systems and compositions for fluorometric signal engineering for multiplexed qPCR. In an aspect, the present disclosure provides A method of accessing an amplification signal generated on a polymerase chain reaction instrument holding a biological sample and an assay solution, said method comprising the steps of : (a) providing a sample, wherein the sample further comprises: (i) a plurality of target nucleic acids; (ii) at least a first set of paired amplification oligomers configured to amplify at least a first target nucleic acid sequence; (iii) at least a second set of paired amplification oligomers configured to amplify at least a second target nucleic acid sequence; (iv) at least a first detectable probe configured to anneal to at least the first target nucleic acid sequence; (v) at least a second detectable probe configured to anneal to at least the second target nucleic acid sequence; and (vi) wherein at least the first set of paired amplification oligomers and atleastthe first detectable probe is at a different concentration than at least the second set of paired amplification oligomers and at least the second detectableprobe; (b) amplifyingthe firsttargetmolecule with a polymerase chain reaction using a polymerase having 5 ’ to 3 ’ exonuclease activity in the presence of atleastthe first target nucleic acid sequence to generate at least a first detectable signal, and in the presence of at least the second target nucleic acid sequence to generate at least a second detectable signal, and wherein the amplifying step causes at least the first detectable probe bound to at least the first target nucleic acid and at least the second detectable probe bound to at least the second target nucleic acid to be degraded by the polymerase and permitting generation of at least the first and at least the second detectable signals; (c) measuring the intensity of at least the first detectable signal and at least the second detectable signal upon initiating of the amplification reaction; (d) measuring the intensity of at least the first detectable signal and at least the second detectable signal upon completion of said amplification reaction; (e) determining a reaction cycle at which the amplification signal first satisfies an amplification criterion; (f) detecting signal peak amounts in the signal; (g) identifying a magnitude of the amplification signal; (h) comparing the reaction cycle and the magnitude of the amplification signal to empirically determined posterior prob ability distributions; and (i) determining the presence or absence of at least the first target nucleic acid sequence, and the presence or absence of at least the second target nucleic acid sequence in the sample.

[0004] In some embodiments, a posterior probability distribution A is conditioned on presence of at least the first target nucleic acid sequence and ab sence of at least the second target nucleic acid sequence. In some embodiments, a posterior probability distribution B is conditioned on presence of at least the second target nucleic acid sequence and absence of at least the first target nucleic acid sequence. In some embodiments, the posterior probability distribution B assigns a higher probability to the magnitude of the amplification signal than is assigned by the distribution A. In some embodiments, the higher probability assigned to the distribution B indicates at least the second target nucleic acid sequence is present in the sample, and at least the first target nucleic acid sequence is absent from the sample. In some embodiments, a distribution A is selected from multiple posterior probability distributions, and wherein each of the multiple posterior probability distributions is conditioned upon a distinct reaction cycle or range of reaction cycles .

[0005] In some embodiments, at least the first set of paired amplification oligomers comprises at least a first forward amplification oligomer and at least a first reverse amplification oligomer. In some embodiments, the atleastthe second set of paired amplification oligomers comprises atleast a second forward amplification oligomers and at least a second reverse amplification oligomer. In some embodiments, the at least the first detectable probe is configured to anneal to at least the first target nucleic acid sequence. In some embodiments, the atleastthe second detectable probe is configured to anneal to at least the second target nucleic acid sequence. In some embodiments, the amplification is a polymerase chain reaction. In some embodiments, the polymerase chain reaction is a quantitative polymerase chain reaction.

[0006] In some embodiments, the quantitative polymerase chain reaction comprises a mixture comprising: a) a buffer; b) a salt; c) a set of dNTPs; and d) an enzyme. In some embodiments, the salt is selected from the group consisting of : (a) magnesium chloride; (b) sodium chloride; (c) potassium chloride; or (d) sodium citrate. In some embodiments, the set of dNTPs is selected from the group consisting of: Set A comprising deoxyadenosine triphosphate (dATP), deoxy cytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP); and Set B comprising adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP) and uridine triphosphate (UTP). In some embodiments, said enzyme is selected from the group consisting of : (a) a thermostable DNA polymerase; (b) a reverse transcriptase; and (c) a RNA polymerase.

[0007] In some embodiments, the first detectable probe and at least the second detectable probe are each independently selected from the group consisting of: (a) a dye; (b) a fluorescent molecules; (c) a chemiluminescent label; and (d) a combination thereof. In some embodiments the at least the first detectable probe and at least the second detectable probe is a dye. In some embodiments, the method further comprises during said amplification reaction, releasing said dye from said detection probe, thereby generating said at least a first signal and said at least a second signal. In some embodiments, the mixture comprises primers and probes for at least five nucleic acid targets.

[0008] In some embodiments, the sample comprises: a) a first dye selected from the group consisting of CY3 and ATTO532; b) a second dye selected from the group consisting of ROX and ATTO/RHO 101 ; c) a third dye selected from the group consisting of CY5, ATTO647, and Mustang Purple; d) 6-FAM; and e) ATTO565. In some embodiments, the sample comprises a) a first dye selected from the group consisting of CY3 and ATTO532; b) a second dye selected from the group; c) LC Blue; d) 6-FAM; and e) ATTO565. In some embodiments, the sample containsprimersandprobesforatleastsix nucleic acid targets. In some embodiments, the sample comprises: a) a first dye selected from the group consisting of CY3 and ATTO532; b) a second dye selected from the group consisting of ROX and ATOO/FHO 101 ; c) a third dye selected from the dye consisting of CY5, ATTO647, and Mustang Purple; d) a fourth dye selected from the group consisting of Quasar 705 and CY5.5; e) 6-FAM; and f) ATTO565.

[0009] In some embodiments the at least the first detectable probe and at least the second detectable probe is a fluorescent molecule. In some embodiments, the at least the first detectable probe and atleastthe second detectableprobeis a chromophore. In some embodiments, a plurality of at least two chromophores fluoresces in the in the visible spectrum. In some embodiments, the plurality of at least two chromophores fluorescing in the visible spectrum are attached to a single nucleic acid target. In some embodiments, the plurality of chromophoresfluorescingin the visible spectrum generates a characteristic signal that indicates the presence the single nucleic acid target. In some embodiments, the plurality of characteristic fluorescent signals are measured in distinct optical channels. In some embodiments, the at least the first detectable probe and at least the second detectable probe is a fluorophore. In some embodiments, the sample comprises a plurality of fluorophores. In some embodiments, the at least one fluorophore generates signal at a distinct PCR annealing temperature, and wherein at least one fluorophore generates a second signal at a second distinct PCR annealing temperature in the same reaction. In some embodiments, at least one fluorophore generates a first signal at a first wavelength, and wherein at least one fluorophore generates a second signal at a second wavelength, wherein both the first signal and the second signal are detected in a first optical channel. In some embodiments, the first signal and/or the second signal exhibit bleed through into a second optical channel.

[0010] In one aspect described herein is a method of accessing an amplification signal generated on a polymerase chain reaction instrument holding a biological sample and an assay solution, said method comprising the steps of: (a) determining a reaction cycle at which an amplification signal first satisfies an amplification criterion; (b) detecting signal peak amounts in the signal; (c) identifying a magnitude of the amplification signal; (d) comparing the reaction cycle and the magnitude of the amplification signal to empirically determined posterior probability distributions; and (e) determining the presence or absence of at least a first target nucleic acid sequence, and the presence or absence of at least a second target nucleic acid sequence in the sample. In certain embodiments, a posterior probability distribution A is conditioned on presence of at least the first target nucleic acid sequence and ab sence of at least the second target nucleic acid sequence. In certain embodiments, a posterior probability distribution B is conditioned on presence of at least the second target nucleic acid sequence and absence of at least the first target nucleic acid sequence. In certain embodiments, the posterior probability distribution B assigns a higher probability to the magnitude of the amplification signal than is assigned by the distribution A. In certain embodiments, the higher probability assigned to the distribution B indicates at least the second target nucleic acid sequence is present in the sample, and at least the first target nucleic acid sequence is absentfrom the sample . In certain embodiments, a distribution A is selected from multiple posterior probability distributions, and wherein each of the multiple posterior probability distributions is conditioned upon a distinct reaction cycle or range of reaction cycles. In certain embodiments, the polymerase chain reaction instrument is a real-time polymerase chain reaction instrument.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIGs. 1A-1C illustrate how specific combinations of fluorophores and/or chromophores may be used to reduce cross-channel interference, thereby enabling highly multiplexed assays. [0012] FIGs. 2A-2D illustrate how a single TaqMan® probe can be designed to have multiple reporting fluorophores, thereby reducing cross-channel interference.

[0013] FIGs. 3A-3C illustrate how a single TaqMan® probe can be designed to have multiple reporting fluorophores, thereby reducing interference within a single channel.

[0014] FIGs. 4A-4C illustrate a strategy for engineering signal levels by designing fluorescent tags with multiple fluorophores that are released at different stages of the PCR. DETAILED DESCRIPTION

[0015] 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.

[0016] Described herein is a method of accessing an amplification signal generated on a polymerase chain reaction instrument holding a biological sample and an assay solution, said method comprising the steps of: (a) providing a sample, wherein the sample further comprises:® a plurality of target nucleic acids; (ii) at least a first set of paired amplification oligomers configured to amplify at least a first target nucleic acid sequence; (iii) at least a second set of paired amplification oligomers configured to amplify at least a second target nucleic acid sequence; (iv) atleast a first detectable probe configuredto anneal to atleastthe first target nucleic acid sequence; (v) at least a second detectable probe configuredto anneal to at least the second target nucleic acid sequence; and (vi) wherein at least the first set of paired amplification oligomers and at least the first detectable probe is at a different concentration than at least the second set of paired amplification oligomers and at least the second detectable probe; (b) amplifying the first target molecule with a polymerase chain reaction using a polymerase having 5 ’ to 3 ’ exonuclease activity in the presence of at least the first target nucleic acid sequence to generate at least a first detectable signal, and in the presence of at least the second target nucleic acid sequence to generate at least a second detectable signal, and wherein the amplifying step causes at least the first detectable probe bound to at least the first target nucleic acid and at least the second detectable probe bound to atleastthe second target nucleic acid to be degraded by the polymerase and permittinggeneration of atleastthe firstand at leastthe second detectable signals; (c) measuring the intensity of at least the first detectable signal and at least the second detectable signal upon initiating of the amplification reaction; (d) measuring the intensity of at least the first detectable signal and at least the second detectable signal upon completion of said amplification reaction; (e) determining a reaction cycle at which the amplification signal first satisfies an amplification criterion; (f) detecting signal peak amounts in the signal; (g) identifying a magnitude of the amplification signal; (h) comparing the reaction cycle and the magnitude of the amplification signal to empirically determined posterior probability distributions; and (i) determining the presence or absence of at least the first target nucleic acid sequence, and the presence or absence of at leastthe second target nucleic acid sequence in the sample.

[0017] In another aspect described herein is a method of accessing an amplification signal generated on a polymerase chain reaction instrument holding a biological sample and an assay solution, said method comprising the steps of: (a) determining a reaction cycle at which an amplification signal first satisfies an amplification criterion; (b) detecting signal peak amounts in the signal; (c) identifying a magnitude of the amplification signal; (d) comparingthe reaction cycle and the magnitude of the amplification signal to empirically determined posterior probability distributions; and (e) determining the presence or absence of at least a first target nucleic acid sequence, and the presence or absence of at least a second target nucleic acid sequence in the sample.

[0018] 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 used in 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.

[0019] Polymerase Chain Reaction (PCR) is a method of exponential amplification of specific nucleic acid target in a reaction mix with a nucleic acid polymerase and primers. Primers are short single stranded oligonucleotides which are complementary to the 3 ’ sequences of the positive and negative strand of the target sequence. The reaction mix is cycled in repeated heating and cooling steps. The heating cycle denatures or splits a double stranded nucleic acid target into single stranded templates. In the cooling cycle, the primers bind to complementary sequence on the template. After the template is primed the nucleic acid polymerase creates a copy of the original template. Repeated cycling exponentially amplifies the target 2 -fold with each cycle leading to approximately a billion-fold increase of the target sequence in 30 cycles (Saiki et al 1988).

[0020] Real-Time PCR (qPCR) is a process of monitoring a PCR reaction by recording the fluorescence generated either by an intercalating dye such as SYBR Green or a target -specific reporter probe at each cycle. This is generally performed on a Real-Time PCR instrument that executes thermal cycling of the sample to complete the PCR cycles and ata specified point in each cycle measures the fluorescence of the sample in each channel through a series of excitation/emission filter sets.

[0021] As used herein “bleed through” refers to signal detected in an optical channel of an amplification instrument that is not the primary optical channel for detection of the signal peak produced from the selected fluorophore or chromophore. For example, Fluorescein (FITC) emits light strongly at around 517 nm, but has detectable emission up to about 650 nm. Bleed through would be the emission that is detected by other channels or filters than the preferred filter (e.g, 530 nm with a 30 nm bandwidth).

[0022] Primers, or “amplification oligomers,” used herein interchangeably, refer to an oligonucleotide or nucleic acid configured to bind to another nucleic acid and facilitate one or more reactions, for example, transcription, nucleic acid synthesis, and nucleic acid amplification. A primer can be double-stranded. A primer can be single-stranded. A primer can be a forward primer or a reverse primer. A forward primer and a reverse primer can be those which bind to opposite strands of a double-stranded nucleic acid. For example, a f orward primer can bind to a region of a first strand (e.g. , Watson strand) derived from a nucleic acid, and a reverse primer can bind to a region of a second strand (e.g., Crick strand) derived from the nucleic acid. A forward primer may bind to a region closer to the start site of a gene relative to a reverse primer or may bind closer to the end site of a gene relative to a reverse primer. A forward primer may bind to the coding strand of a nucleic acid or may bind to the non-coding strand of a nucleic acid. A reverse primer may bind to the coding strand of a nucleic acid or may bind to the non-coding strand of a nucleic acid.

[0023] Digital PCR (dPCR) is a process of partitioning a sample containing one or more targets into a plurality of partitions (e.g., wells, droplets, etc.), performing a PCR reaction in each partition, and recordingthe luminescence (e.g., fluorescence) generatedby, for example, a target-specific reporter probe. The use of labeled oligonucleotide probes enables specific detection. dPCR may be used in a variety of nucleic acid detection methods. Digital PCR is generally performed on a digital PCR instrument that measures the fluorescence from each partition in an optical channel through one or more ex citation/emission filter sets.

[0024] Frequently, the target-specific oligonucleotide probe is a short oligonucleotide complementary to one strand ofthe amplified target. The probe lacks a 3' hydroxyl andtherefore is not extendable by the DNA polymerase. TaqMan® (ThermoFisher Scientific) chemistry is a common reporter probe method used for multiplex Real-Time PCR (Holland et al. 1991). The TaqMan oligonucleotide probe is covalently modified with a fluorophore and a quenching tag (i.e., quencher). In this configuration the fluorescence generated by the fluorophore is quenched and is not detected by the real time PCR instrument. When the target of interest is present, the probe oligonucleotide base pairs with the amplified target. While bound, it is digested by the 5' to 3 ' exonuclease activity of the Taq polymerase thereby physically separating the fluorophore from the quencher and liberating signal for detection by the real time PCR instrument.

Overview

[0025] Real-Time PCR (qPCR) is a process of monitoring a PCR reaction by recording the fluorescence generated either by an intercalating dye or a target-specific reporter probe at each cycle. In fluorescently multiplexed PCR, detection of multiple target nucleic acid sequences in a single reaction is accomplished by associating each nucleic acid target with a distinct fluorescent tag such as a TaqMan® probe, FRET probe, or a molecular beacon. However, one limitation of current qPCR machines is the number of channels available for multiple target detection. Contemporary multiplexing instrumentation generally have between two and six channels, enabling detection of two to six different fluorescent reporters, and therefore, two to six targets in a single reaction. However, accurately detecting multiple targets is hindered by overlapping fluorescence signals of the different reporters.

[0026] When imaging multiple fluorescent labels concurrently, emission profiles of each fluorescent lab el often share similar spectral regions, thereby requiring emission filters to limit (or eliminate) unwanted detection of emission from other fluorophores. Specifically, because the visible spectrum region is limited from approximately 400 to 700 nanometers, simultaneous imaging of two or more fluorescent tags, each having emission spectral profiles spanning 150 nanometers, will often result in emission overlap.

[0027] For example, Fig. 1 illustrates how the selection of chromophore combinations for TaqMan® probes affects signal-to-noise ratios in multiplex PCR. Fig. 1 A demonstrates a situation where a TaqMan® probe with a combination of the Fl and Q chromophores generates a signal of 60 in Channel 1 (CHI) and a bleed-through of 30 in Channel 2 (CH2). Fig. IB demonstrates a situation where a probe with a combination of F2 and Q generates a signal of 80 in CHI and a bleed-through of 30 in CH2 and 10 in CH3. Finally, Fig. 1C demonstrates a situation where a probe a combination of F3 and Q generates a signal of 100 in CHI and a bleed -through of 10 in CH2. Not surprisingly, accurately detectingmultiple targets is unduly complicatedby overlapping fluorescence signals of the different reporters across multiple channels. Additionally, because fluorophore signal levels in living cells is generally muted, added limitations may further obstruct accurate detection.

[0028] As the number of fluorescent tags increases, so too does the complexity. Generally, due to limitations in fluorescent protein color choices, imaging three or more fluorophores requires the use of highly restricted excitation and emission filter strategies to reduce spectral overlap. Relatedly, fluorometric multiplexing may be used to simultaneously monitoring multiple PCR reactions in a single chamber. By associating each fluorescence signal with the presence of a single nucleic acid target, the detection of multiple sequences within a single reaction can be accomplished. In a fluorometric multiplexing reaction, each of the fluorophores on tags can be selected to minimize signal interference (/. e. , cross talk) between fluorophores. For example, with a single fluorescent label for a single target, the selection of reporting and quenching fluorophore has a large effect on the signal-to-noise ratios (see Fig. 1). [0029] Disclosed herein is a method of using specific combinations of fluorophores during multiplex qPCRreactionsto reduce cross-channel interference. Specifically, the disclosed method claims a manner of using specific combinations of fluorophores and/or chromophores to reduce cross-channel interference, thereby enabling highly multiplexed assays. In reducing signal interference, the present disclosure enables highly multiplexed assays that reduce optical “bleed- through” (z.e., “dye emission cross-talk”).

[0030] Disclosed herein is a method of engineering signal intensity generated by a fluorescent tag by attaching multiple reporter signals to a single nucleic acid during multiplex qPCR reactions to reduce cross-channel interference. The methodmay include selectingvariousfluorophores on tags to minimize signal interference between fluorophores. The method may include using specific combinations of fluorophores on a single TaqMan probe can be used to generate unique signals. The method may include using a TaqMan® probe attached to a re porting fluorop hore to generate a single signal channel 1 (CHI). The method may include using a TaqMan® probe attached to two reporting fluorophores to generate multiple signals in channel 1 (CHI) and channel 2 (CH2). The method may include using a TaqMan® probe attached to three reporting fluorophores to generate multiple signals in channel 1 (CHI), channel 2 (CH2), and channel 3 (CH3). The method may include using a TaqMan® probe attached to four reporting fluorophores to generate multiple signals in channel 1 (CHI), channel 2 (CH2), channel 3 (CH3), and channel 4 (CH4).

[0031] Disclosed herein is a method of engineering signal intensity generated by a fluorescent tag by attaching multiple reporter signals to a single nucleic acid during multiplex qPCR reactions to reduce interference within a single channel. The method may include using a TaqMan® probe with a combination of chromophores fluorescing in different wavelengths to generates a signal over the course of the PCR. The method may include using a TaqMan® probe with a unique combination of chromophores, wherein some chromophores emit or fluoresce in the same wavelength band, over the course of the PCR.

Disclosed herein is a method of engineering signal intensity generated by a fluorescent tag by designing fluorescent tags with multiple fluorophores that are released at different stages of the PCR. The method may include designing fluorescent tags with multiple fluorophores that are released at different stages of the PCR. The method may include a situation where a TaqMan® probe with a reporting fluorophore, when subject to a PCR protocol with a sufficiently low annealing/extension temperature, will be liberated, generating a fluorescent signal. The method may include a situation where a TaqMan® probe with two reporting fluorophores, when subject to a PCR protocol with a sufficiently low annealing/extension temperature, will be liberated at different times, generating two distinctive fluorescent signals. [0032] Disclosed herein is a method of detecting multiple reporter signals during multiplex qPCR reactions to reduce cross-channel interference.

[0033] Disclosed herein is a method of measuring multiple reporter signals during multiplex qPCR reactions to reduce cross-channel interference.

[0034] Disclosed herein is a method of using specific combinations of fluorophores to reduce cross-channel interference, thereby enabling highly multiplexed assays. First, a mixture may be provided comprising a plurality of nucleic acid molecules and a plurality of oligonucleotide probes. The plurality of nucleic acid molecules may be derived from, and/or may correspond with, the nucleic acid target in the sample. The plurality of oligonucleotide probes may each correspond to a different region of the nucleic acid target. The mixture may further comprise other reagents (e.g., amplification reagents) including, for example, oligonucleotide primers, dNTPs, a nucleic acid enzyme (e.g., a polymerase), and salts (e.g., Ca2+, Mg2+, etc.). Next, the mixture be used in a quantitative Polymerase Chain Reaction, whereby a plurality of signals may be generated. The plurality of signals may be detectable in one color channel. The plurality of signals may be detectable in multiple color channels. Atleast one signal of the plurality of signals may correspond with the presence of a unique combination of two or more of the plurality of nucleic acid molecules. For example, one signal may correspond to the presence of two nucleic acid molecules (e.g. , two copies of a nucleic acid sequence). Based on the detecting, the nucleic acid target in the sample may be quantified.

[0035] The plurality of signals may be generated by one or more of the plurality of probes from the mixture. The plurality of signals may be generated by nucleic acid amplification (e.g., PCR) of the plurality of nucleic acid molecules. Nucleic acid amplification may degrade the plurality of oligonucleotide probes (e.g. , by activity of a nucleic acid enzyme), thereby generatingthe plurality of signals. A plurality of signals may be a plurality of fluorescent signals, a plurality of chemiluminescent signals, or a combination thereof.

[0036] In some cases, the sample further comprises an additional plurality of nucleic acid molecules and an additional plurality of oligonucleotide probes. The additional plurality of nucleic acid molecules may be derived from and/or correspond with an additional nucleic acid target. The additional plurality of oligonucleotide probes may each correspond to a different region of the additional nucleic acid target.

[0037] A sample may be a biological sample. A sample may be derived from a biological sample. A biological sample may be, for example, blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool or tears. A biological sample may be a fluid sample. A fluid sample may be blood or plasma. A biological sample may comprise cell-free nucleic acid (e.g. , cell-free RNA, cell-free DNA, etc.). A nucleic acid target may be a nucleic acid from a pathogen (e.g., virus, bacteria, etc.). A nucleic acid target may be a nucleic acid suspected of comprising one or more mutations. Assays

[0038] In some cases, assays may be run using the reagents in the chemical composition. Assay may use a reagent to perform a reaction. The reaction may comprise a hybridization reaction. For example, the reagent may comprise a nucleic acid and hybridize with another nucleic acid. The nucleic acid and the another nucleic acid may be complementary to one another. The reaction may comprise an extension reaction. For example, the reaction may comprise extending a nucleic molecule by the addition of a nucleotide. The reaction may comprise a polymerase chain reaction. [0039] Any number of nucleic acid targets may be detected using assays of the present disclosure. In some cases, 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 targets, or more. In some cases, 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 targets. An assay may comprise any number of reactions, where the results of the reactions together identify a plurality of nucleic acid targets, 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 targets. However, the results of each reaction together may unambiguously detect the presence or absence of each of the nucleic acid targets.

[0040] 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 may be performed in different sample solution volumes. For example, a first reaction may be 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.

[0041] Assay reactions as described hereinmay be conducted in parallel. In general, parallel assay reactions are reactions that occur in the same reaction vessel and at the same time. For example, parallel nucleic acid amplification reactions may be conductedby includingreagents used for each nucleic acid amplification reaction in a reaction vessel to obtain a reaction mixture and subjecting the reaction mixture to conditions used for each nucleic acid amplification reaction. Any suitable number of nucleic acid amplification reactions may be conducted in parallel. In some cases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleic acid amplification reactions are conducted in parallel. [0042] Each oligonucleotide probemay be labeled with a fluorophore. Fluorescentmoleculesmay be excited at a wavelength at emit light at another wavelength. The fluorescent molecules may be visible to the naked human eye. The fluorescent molecules may visible or identified via spectroscopic methods such to analyze the wavelength of light that are transmitted or absorbed by a solution comprising a fluorescent molecule. 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.

[0043] The fluorescent molecules may have a distinct or known signature of excitation or emission wavelength of electromagnetic radiation. The detection of a fluorescent molecule signature may comprise identifying an amplitude or amplitudes of signal at diff erent wavelengths. The fluorescent molecule signature may comprise a signal at wavelengths that do not overlap with wavelengths that may be generated by reagents in the chemical composition. In some cases, the excitation wavelength of the molecule may comprise a signal that does not overlap with wavelengths that may be generated by reagents in the chemical composition. In some cases, the signals of the reaction and the fluorescent molecule may be simultaneously detected. Non -limiting examples of fluorescent molecules that may be used include Alexa Fluor 350, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, AlexaFluor 680, Alexa Fluor 750, Cy3, Cy5, Texas Red, Fluorescein(FITC), 6-FAM, 5-FAM, HEX, JOE, TAMRA, ROX, BODIPYFL, Pacific Blue, Pacific Green, Coumarin, Oregon Green, Pacific Orange, Trimethylrhodamine (TRITC), DAPI, APC, CyanFluorescentProtein (CFP), Green Fluorescent Protein (GFP), RedFluorescentProtein (RFP), Phycoerythin (PE), quantum dots (for example, Qdot 525, Qdot 565, Qdot 605, Qdot 705, Qdot 800), Mustang Purple, Quasar705, or derivatives thereof.

[0044] In certain embodiments, amplification reactions and assays described herein maybe performed using digital methods. A method for performing a digital assay may comprise amplifying nucleic acid targets derived from a sample in a plurality of partitions comprising oligonucleotide probes complementary to one or more regions of nucleic acid targets. 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 targets is present. Each of the N signals may correspond to a unique combination of one or more of the nucleic acid targets present in a partition. From the N signals, the presence or absence of each of the nucleic acid targets in the sample may be determined. [0045] 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 cases, 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.

[0046] The plurality of partitions may be at least 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 or fewer. The plurality of partitions may be defined by a range of any of the foregoing values.

Amplification

[0047] 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 each signal, thereby enabling each signal to correspond to a unique combination of nucleic acid targets. Amplification may compriseusing 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 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. Non-limiting 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.

Thermal cycling

[0048] 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 cases, be such that each thermal cycle is performed at a desired annealing temperature. A desired annealing temperature may be sufficient for annealing of an oligonucleotide probe(s) to a nucleic acid target. Appropriate buffer conditions may, in some cases, 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 targets with PCR may include deoxyribonucleotide triphosphates (dNTPs). dNTPs may comprise natural or non-natural dNTPs including, for example, dATP, dCTP, dGTP, dTTP, dUTP, and variants thereof.

[0049] 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 extensionreactioncanbeconducted. Any suitable number of cycles may be conducted. For example, the number of cycles 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) may be 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.

[0050] 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.

[0051] In some embodiments, amplification of a nucleic acid may yield a detectable amount of amplified DNA 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. Nucleic acid targets

[0052] 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. Abiological sample may be a skin sample. A biological sample may b e 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, for example, blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool or tears.

[0053] A nucleic acid target may be derived from one or more cells. 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, and microRNA. RNA may be viral RNA. [0054] Nucleic acid targets 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 cases, the methods of the present disclosure are useful in detecting the presence or absence or one or more infectious agents (e.g., viruses) in a subject.

[0055] Nucleic acid targets may be of various concentrations in the reaction. The nucleic acid sample may be diluted or concentrated to achieve different concentrations of nucleic acids. The concentration of the nucleic acids in the nucleic acid sample may at least 0.1 nanograms per microliter (ng/pL), 0.2 ng/pL, 0.5 ng/pL, 1 ng/pL, 2 ng/pL, 3 ng/pL, 5 ng/pL, lO ng/pL, 20 ng/pL, 30 ng/pL, 40, ng/pL, 50 ng/pL, 100 ng/pL, lOOO ng/pL, 10000 ng/pL or more. In some cases, the concentration of the nucleic acids in the nucleic acid sample may be at most ng/pL, 0.2 ng/pL, 0.5 ng/pL, 1 ng/pL, 2 ng/pL, 3 ng/pL, 5 ng/pL, 10 ng/pL, 20 ng/pL, 30 ng/pL, 40, ng/pL, 50 ng/pL, 100 ng/pL, 1000 ng/pL, 10000 ng/pL or less.

Sample Processing

[0056] A sample may be processed concurrently with, prior to, or subsequent 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.

Nucleic acid enzymes

[0057] 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.

[0058] A nucleic acid enzyme may be a polymerase (e.g., a DNA polymerase). A DNA polymerase may be used. Any suitable DNA polymerase may be used, including commercially available DNA polymerases. A DNA polymerase generally refers to an enzyme that is capable of incorporating nucleotides to a strand of DNA in a template bound fashion. A polymerase maybe Taq polymerase or a variant thereof. Non -limiting examples of DNA polymerases include Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerases, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfi polymerase, Platinum Taq polymerases, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment, and variants, modified products and derivatives thereof. For certain Hot Start Polymerase, a denaturation step at 94°C -95°C for 2 minutes to 10 minutes may be required, which may change the thermal profile based on different polymerases. A nucleic acid enzyme may be capable, under appropriate conditions, of degrading an oligonucleotide probe. For example, a nucleic acid enzyme may be a polymerase and comprise exo activity and degrade a probe resulting in a detectable signal. A nucleic acid enzyme may be capable, under appropriate conditions, of releasing a quencher from an oligonucleotide probe.

Reactions

[0059] In various aspects disclosed elsewhere herein, reactions are performed. A reaction may comprise contacting nucleic acid targets 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, eachcorrespondingto one of a plurality of nucleic acid targets, to generate a plurality of signals generated from the plurality of oligonucleotide probes. A reaction may comprise polymerase chain reaction (PCR).

Oligonucleotide primers

[0060] In various aspects disclosed elsewhere herein, oligonucleotide primers are used. An oligonucleotide primer (or “amplification oligomer”) of the present disclosure may be a deoxyribonucleic acid. An oligonucleotide primer may be a ribonucleic acid. An oligonucleotide primer may comprise one or more non -natural nucleotides. A non-natural nucleotide may be, for example, deoxyinosine.

[0061] 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 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. 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. [0062] 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. For example, a first set of oligonucleotide primers may be configured to amplify a first nucleic acid sequence of a given length, and a second set of oligonucleotide primers may be configured to amplify a second nucleic acid sequence of shorter length than the first nucleic acid sequence. In another example, a first set of oligonucleotide primers may be configured to amplify a first nucleic acid sequence of a given length, and a second set of oligonucleotide primers may be configured to amplify a second nucleic acid sequence of longer length than the first nucleic acid sequence.

[0063] 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 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. A plurality of forward 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. [0064] 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 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. 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.

[0065] A set of oligonucleotide primers (e.g, a forward primer and a reverse primer) may be configured to amplify a nucleic acid sequence of a given length (e.g. , may hybridize to regions of a nucleic acid sequence a given distance apart). A pair of oligonucleotide primers may be configured to amplify a nucleic acid sequence of a length of at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, or at least 300 base pairs (bp), or more. A pair of oligonucleotide primers may be configured to amplify a nucleic acid sequence of a length of atmost 300, atmost 275, atmost250, at most225, at most 200, atmost 175, atmost 150, at most 125, atmost 100, at most 75, or at most 50 bp, or less.

[0066] In some aspects, a mixture may include one or more synthetic (or otherwise gene rated to be different from the target of interest) primers for PCR reactions.

[0067] 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. [0068] In some aspects, a mixture may be subjected to conditions sufficient to anneal a plurality of oligonucleotide primers to a plurality of nucleic acid targets. The mixture may be subjected to conditions which are sufficient to denature nucleic acid molecules. Subjecting 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).

[0069] 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 degradedby the exonuclease activity of a nucleic acid enzyme. An oligonucleotide primer pair may be degradedby the RNase activity of a nucleic acid enzyme. Degradation of the oligonucleotide primer pair may resultin release of the oligonucleotideprimer. Once released, the oligonucleotide primer pair may bind or anneal to a template nucleic acid. Oligonucleotide probes

[0070] In various aspects disclosed elsewhere herein, oligonucleotide probes are used. Samples, mixtures, kits, and compositions of the present disclosuremay comprise an oligonucleotide probe, also referenced herein as a “detection probe” or “probe”. 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.

[0071] An oligonucleotide probe may comprise a non-target-hybridizing sequence. A non-targethybridizing 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 -target-hybridizing 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. [0072] 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 may be a nucleic acid (e.g., DNA, RNA, etc.). An oligonucleotide probe may 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 most 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotides in length.

[0073] In some cases, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more different oligonucleotide probes may be used. Each oligonucleotide probe 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 cases, each oligonucleotide probe comprises an identical fluorophore. In some cases, each oligonucleotide probe comprises a different fluorophore, where each fluorophore is capable of being detected in a single optical channel.

[0074] 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.

[0075] A probe may be provided at a specific concentration. 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 5X, about 4X, about 3X, 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. X may be a concentration of a nucleic acid probe provided in the disclosed methods. In some cases, X is at least 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 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.

[0076] 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 fluorop hore may be selectedfrom anynumberof fluorophores. Afluorophoremay be selected from three, four, five, six, seven, eight, nine, or ten fluorophores, or more. One or more oligonucleotide probes used in a single reaction may comprise the same fluorophore. In some cases, 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.

[0077] An oligonucleotide probe may have less than 50%, 40%, 30%, 20%, 10%, 5%, or 1% complementarity to any member of a plurality of nucleic acid targets. An oligonucleotide probe may have no complementarity to any member of the plurality of nucleic acid targets.

[0078] An oligonucleotide probe may comprise a detectable label. A detectable label may be a chemiluminescent label. A detectable label may comprise a fluorescent label. A detectable label may comprise a fluorophore. A fluorophore may be, for example, FAM, TET, HEX, JOE, Cy3, or Cy5. A fluorophore maybe 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, orDabcy. A quencher may be BHQ-1 . A quencher may be BHQ-2.

Signal generation

[0079] Thermal cycling may be performed such that one or more oligonucleotide probes are degraded by a nucleic acid enzyme. An oligonucleotide probe maybe degradedby the exonuclease activity of a nucleic acid enzyme. An oligonucleotide probe may generate a signal upon degradation. In some cases, an oligonucleotide probe may generate a signal only if at least one member of a plurality of nucleic acid targets is present in a mixture.

[0080] 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.

[0081] 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 targets. A signal may be generated by one or more oligonucleotide probes. The number of signals generated in an assay may correspond to the number of oligonucleotide probes and nucleic acid targets present.

[0082] N may be a number of signals detected in a single optical channel in an assay of the present disclosure. N may be at least 2, 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.

[0083] 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 targets that can be measured in a single reaction. In some cases, 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.

[0084] In some cases, 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 cases, 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.

[0085] In cases 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.

[0086] 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 atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 signals, or more. Thermal cycling may generate at most 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 differenttypesmay be fluorescent signals with different flu orescent wavelengths. Signals of different types may be generated by detectable labels comprising different fluorophores. Signals of the same type maybe of different intensities (e.g., different intensities of the same fluorescent wavelength). Signals of the same type maybe 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.

[0087] 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, chemiluminescent signals, magnetic particles, and electrets structures exhibiting a permanent dipole.

[0088] In certain portions of this disclosure, the signal may be a fluorescent signal. For example, like fluorescent signals, any of the electromagnetic signals described above may also be characterized in terms of a wavelength, whereby 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. A range of wavelengths may be referred to as a “channel,” “color channel,” or “optical channel.”

[0089] 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 maybe detected sequentially. A signal may be detected throughout the process of thermal cycling, for example, at the end of each thermal cycle.

[0090] In some cases, the signal intensity increases with each thermal cycle. The signal intensity may increase in a sigmoidal fashion. The presence of a signal may be correlated to the presence of at least one member of a plurality of target nucleic acids. Correlating the presence of a signal to the presence of at least one member of a plurality of target nucleic acids may comprise estab lishinga signal intensity threshold. A signal intensity threshold may be different for different signals. Correlating the presence of a signal to the presence of at least one member of a plurality of target nucleic acids may comprise determining whether the intensity of a signal increases beyond a signal intensity threshold. In some examples, the presence of a signal may be correlated with the presence of at least one of all members of a plurality of target nucleic acids. In other examples, the presence of a first signal may be correlated with the presence of at least one of a first subset of members of a plurality of target nucleic acids, and the presence of a second signal may be correlated with the presence of at least one of a second sub set of members of a plurality of target nucleic acids.

[0091] 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 targets. The absence of a signal may be correlated with the absence of corresponding nucleic acid targets. 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

[0092] The present disclosure also provides kits for 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.

[0093] 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 m ay 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 may be 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 cases, all of the oligonucleotide primers in a kit are lyophilized.

[0094] 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. 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 may be a polymerase (e.g. , a DNA polymerase). Apolymerase 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. Kits may comprise instructions for using any of the foregoing in the methods described herein.

[0095] 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 target nucleic acid.

EXAMPLES

Example 1 - Engineering Signal Levels with Multiple Fluorophores

[0096] In a fluorometric multiplexing reaction, each of the fluorophores on tags can be selected to minimize signal interference between fluorophores. For example, with a single fluorescent label for a single target, the selection of reporting and quenching fluorophore has a large effect on the signal-to-noise ratios (See Fig. 1).

[0097] Fig. 1 illustrates how the selection of chromophore combinations for TaqMan® probes affects signal-to-noise ratios in multiplex PCR. Fig. 1A demonstrates a situation where a TaqMan® probe with a combination of the Fl and Q chromophores generates a signal of 60 in Channel 1 (CHI) and a bleed-through of 30 in Channel 2 (CH2). Fig. IB demonstrates a situation where a probe with a combination ofF2 and Q generates a signal of 80 in CHI and a bleed -through of 30 in CH2 and 10 in CH3. Fig. 1C demonstrates a situation where a probe a combination of F3 and Q generates a signal of 100 in CHI and a bleed -through of 10 in CH2.

[0098] Importantly, the signal intensity generated by a fluorescent tag can also be engineered by attaching multiple reporting fluorophores to a single nucleic acid probe See Fig. 2). Fig. 2 illustrates how a single tagging molecular can be designed to have multiple reportingfluorophores to generate multiple distinct wavelength emissions. During the course of a qPCR reaction, these fluorophores are cleaved during target replication generating signals. Notably, specific combinations of fluorophores on a single TaqMan probe can be used to generate unique signals. Fig. 2D demonstrates a situation where a TaqMan® probe attached to a reporting fluorophores Fl generates a signal of 20 intensity units in optical measurement channel 1 (CHI). Fig. 2C demonstrates a situation where a modified version of the TaqMan® probe wherein the probe has two fluorophores, Fl and F2. When the TaqMan® probe attached to reporting fluorophores Fl and F2 is digested, it generates a characteristic signal of 20 intensity units in CHI (due to the cleaving of Fl) and 30 intensity units in optical measurement in CH2 due to the cleaving of F2. Fig. 2B demonstrates a situation where a TaqMan® probe is attached to reporting fluorophores F1, F2 and F3. When the TaqMan® probe attached to reporting fluorophores Fl, F2, and F3 is digested, it generates a signal of 20 intensity units in optical measurement channel 1 (CHI), a signal of 40 intensity units in optical measurement channel 2 (CH2), and a signal of 20 intensity units in optical measurement channel 3 (CH3) respectively. Fig. 2A demonstrates a situation where a TaqMan® probe attached to reporting fluorophoresFl, F2, F3 and F4. Similarly, when the TaqMan® probe attached to reporting fluorophores Fl, F2, F3, and F4 is digested it generates a signal of 20 intensity units in optical measurement channel 1 (CHI), a signal of 40 intensity units in optical measurement channel 2 (CH2), a signal of 20 intensity units in optical measurement channel 3 (CH3), and a signal of 60 intensity units in optical measurement channel 4 (CH4). This same concept can be extended to multiple fluorophores that report in the same optical channel (See Fig. 3).

[0099] Fig. 3. illustrates how a single TaqMan® probe can be designed to have multiple reporting fluorophores that fluoresce in the same wavelength band, thereby allowing for multiple fluorophores to be used to report within the same optical channel. Fig.3A demonstrates a situation where a TaqMan® probe with a combination of chromophores Fl and Q, generates a signal of 30 over the course of the PCR. Fig. 3B demonstrates a situation where a TaqMan® probe with a combination of a pair of Fl , chromophores and a Q chromophore produces a signal of 50 over the course of the PCR. Fig. 3C demonstrates a situation where a TaqMan® probe with a combination of a Fl and a F2 chromophore, both fluorescing in the same wavelength band, with a Q chromophore, produces a signal of 80 over the course of the PCR.

Example 2 - Multiple Digestion Events

[0100] Another strategy for engineering signal levels is to design fluorescent tags with multiple fluorophores that are released at different stages of the PCR See Fig. 4). Fig. 4A illustrates a situation where a TaqMan® probe with a reporting fluorophore Fl , a length of N bases, and a melt temperature of T1 . When subject to a PCR protocol with a sufficiently low annealing/extension temperature (e.g., < Tl), the fluorophore Fl will be liberated, generating a fluorescent signal. Fig. 4C illustrates a situation where a TaqMan® probe has two reporting fluorophore Fl and F2, a length of N bases, and a melt temperature of Tl . Here, the second fluorophore F2 attached internal, such that the spacing between F2 and Q is M-bases. With a careful selection of the PCR annealing temperature, Fl can be selectively cleaved off before F2 is cleaved. Therefore, when subject to a PCR protocol with a sufficiently low annealing/extension temperature (e.g., Tameai < Tl), the fluorophore Fl will be liberated, generating a fluorescent signal, while F2 remains quenched. Then, in a sub sequent PCR cycle, F2 can be cleaved, generating a second fluorescent signal.