Quantitative partitioned amplification

Abstract

Provided herein systems, compositions, and methods for nucleic acid quantification that combines the sensitivity and accuracy of digital PCR with the capability for high-throughput multiplexing. The methods integrate digital amplification in compartments with capillary electrophoresis or other methodologies as a readout mechanism.

Description

[0001] QUANTITATIVE PARTITIONED AMPLIFICATION

[0002] FIELD

[0003] Provided herein systems, compositions, and methods for nucleic acid analysis and quantification that combines the sensitivity and accuracy of digital PCR with the capability for high-throughput multiplexing. The technique integrates digital amplification in compartments with one or more of a variety of detection and separation techniques, e.g., capillary electrophoresis or other methodologies, as a readout mechanism.

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims the benefit of U.S. Provisional Application No. 63 / 589,133, filed October 10, 2023, the content of which is herein incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] Molecular detection technologies are featured in many important research and clinical endeavors, including for the detection of cancer and infectious diseases. However, limitations in existing technologies, particularly related to complexity, cost, and efficiency have prevented more widespread use, particularly preventing use for quantitative determination of analytes of interest (e.g., disease biomarkers).

[0008] Today, nearly all infectious disease tests are qualitative, reporting mere presence / absence of a potential pathogen (1-3). Qualitative information is simple to interpret and provides minimal actionable information in the clinical setting. However, it can generate ambiguity, since molecular assays may detect inactive pathogens, ones at levels too low to support disease, or contaminants (4-9). This is common when screening a large panel to detect a causative pathogen, as needed for infections in sepsis and of the urinary, gastrointestinal, and respiratory tracts. In such cases, follow-up tests and quantitative information reporting the load of each pathogen are needed to determine which organism is the likely source of infection (7-9). Obtaining this information today requires re-testing the patient with alternative methods targeting the narrowed list of candidate pathogens, which adds cost and delays efficacious treatment. Quantitative tests are also valuable for tracking infection course or confirming when pathogen load has dropped to a level indicating treatment can be stopped or the patient is no longer infectious (16). Thus, tests that could screen for tens of organisms and report on quantitative load would shorten the time-to-answer when speed can have a major impact on the outcome of the patient and others they may infect (10-13). qPCR dominates infectious disease diagnostics. qPCR is a quantitative, sensitive, and accurate method for analyzing nucleic acids (43). It is relatively inexpensive and easy to automate with robotics, allowing efficient processing of high volumes of samples (44-47). While new sequencing-based techniques are growing in popularity they are expensive and relatively slow (48), and qPCR tests still account for most assays in infectious disease today (22,33). Indeed, the dominant instrument for virologic infections is BioMerieux’s BioFire, a multiplexed qPCR system (44,45). In the laboratory setting, qPCR is quantitative over 4-8 decades of dynamic range (43,49), and used routinely for myriad applications, from gene expression profiling to sequencing library preparation and validation of cellular mutations (50-52). Why then does the BioFire instrument, and nearly every qPCR test used in the clinical setting, report qualitative presence / absence results? The reason has to do with the nature of qPCR, which estimates target concentration based on the rate of amplification (43). Due to exponential growth, small variations in reaction initiation or efficiency compound with each cycle, leading to significant error in the rate estimate (17,18,49). To correct for this, qPCR requires that each reaction be run in parallel with a standard (18,53). While this is effective in a controlled setting, it is difficult to perform in clinical labs, which regularly yield disparate concentration values for “identical” samples (17,19). Consequently, nearly all diagnostic qPCR tests report qualitative presence / absence, including the BioFire which, indeed, is a quantitative test under the hood, but must report binary qualitative results to meet FDA approval rules due to the high Ct value variability (53,54). In all of virologic infection, there are only a few cases in which quantitative reporting was made possible, including: HepC, HIV, and CMV (3,21). This required millions in research over years, the development of strict protocols, training, and licensing requirements, and the shipment of standards from a central facility (19,22). Repeating this costly process for the myriad infections encountered in the clinic daily is currently impractical. If a quantitative modality could be developed that was practical for the clinical setting, it would arm clinicians with powerful information when diagnosing and treating disease.

[0009] Digital PCR, theoretically, affords a practical approach to quantitative testing in clinical labs, but only if limitations can be addressed. In contrast to qPCR, digital PCR (dPCR), quantitates nucleic acids by counting single molecules (23,24). Thus, it is insensitive to reaction efficiency and provides an absolute count that does not require a standard (55). As a result, dPCR is more reproducible than qPCR when compared across labs and testing environments (25,26). Additionally, dPCR is more sensitive (24,56), which is important when assessing the contagiousness of a patient or confirming that the infection has been cured. Consequently, dPCR is already a powerful tool in oncology for detecting minimal residual disease and in cell therapy for verifying therapeutic persistence (57,58). However, it is less important in infectious disease for two reasons: First, current dPCR methods are complex and expensive, requiring custom instrumentation and trained personnel. Unlike qPCR approaches that are often automated from sample-in to answer-out (BioFire), dPCR technologies require many steps to be performed manually, including nucleic acid extraction, cDNA synthesis, and transfer of droplets between instruments for generation, thermocycling, and reading (25,59). These steps add labor costs and can introduce error, and impede use of the technology for common infectious disease testing. Second, current dPCR offers limited multiplexing; while a qPCR assay can be split and processed in well plates and leverage melt-curve analysis to test for tens of pathogens (18,30), with dPCR each must be associated with a fluorescent dye, limiting panels to 5-6 targets (28,29). While dPCR thus has potential for quantitative diagnostics in the clinical setting, its cost, complexity, and limited multiplexing are critical drawbacks.

[0010] SUMMARY

[0011] The present disclosure provides methods for analyzing nucleic acids. Methods of the invention combine nucleic acid amplification and size selection to allow the identification and evaluation of nucleic acid target molecules. According to methods of the invention, target nucleotides are introduced to compartments under limiting dilution with multiplex primers. The Primers are used for target-specific amplification of nucleic acids in the compartment to generate amplicons in proportion to the concentration of the target nucleic acid that was introduced into the compartment. The amplicons are then separated by size selection and matched to a list of targets, known or suspected to be in the sample that was introduced into the compartments.

[0012] In one aspect, detectable labels are introduced during amplification. Labels of different types (e.g., different colored fluorophores) are used to identify target sequence and to distinguish two targets of the same or similar length. In an alternative, the labels are introduced postamplification.

[0013] A preferred method for size selection is capillary electrophoresis. However, any suitable method is useful in the invention. For example, any gel electrophoresis technique, including polyacrylamide gel electrophoresis, agarose gel electrophoresis, gradient gel electrophoresis, isoelectric focusing and others known in the art are useful for implementation of size selection according to the invention. In one preferred embodiment, size selection is performed on a miroCE reader that may be integrated with the compartment housing the amplicons.

[0014] Methods of the invention are applicable to single cell analysis. In preferred methods, single cells are loaded into a compartment and an emulsion is created. The compartment can be a well, a tube, a droplet (e.g., an oil-in-water droplet), or any other means for sequestering the cells. Target nucleic acid in the emulsion is then amplified and the resulting amplicons are size selected by, for example, electrophoresis. Size-selected amplicons are then compared to known targets and quantified. The resulting size-selected amplicons are useful for diagnostics, prognostics, therapeutic selection, therapeutic efficacy, detection of minimal residual disease, and / or recurrence monitoring.

[0015] Thus, in certain embodiments, provided herein are methods for analyzing target nucleic acids in a sample. In some embodiments, the methods comprise: encapsulating target nucleic acids in compartments under limiting dilution conditions with probes from a multiplexed probeset; amplifying nucleic acids targeted by the multiplexed probeset such that certain probe subsets amplify in different compartments, depending on which nucleic acid targets are present in the compartments; and analyzing the amplification products to detect their identifiable signal and / or quantifying the amplification products. In some embodiments, the amplifying generates amplification products in a concentration proportional to the original target nucleic acid. In some embodiments, individual compartments contain at most a single target nucleic acid.

[0016] In some embodiments, the methods further comprise re-encapsulating the amplification products in at least one successive compartment and amplifying the amplification products to generate a successive round of amplification products with an identifiable signal prior to the analyzing.

[0017] In some embodiments, the amplification product comprises an identifiable signal. In some embodiments, the identifiable signal includes a fluorophore, a mass tag, a molecular tag, a peptide, a detectable nucleic acid sequence, a modified nucleotide, a small molecule, a lipid, a nanoparticle, or a combination thereof. In some embodiments, the identifiable signal is included on a probe within the multiplexed probeset.

[0018] In some embodiments, the analyzing comprises detection of one or more of fluorescence, mass, chemical, and / or base composition properties. In some embodiments, the analyzing comprises detection of relative length of the amplification products.

[0019] In some embodiments, the multiplexed probeset generates amplification products of different length. In some embodiments, the multiplexed probeset generates amplification products of same length.

[0020] In some embodiments, one or more or all probes from the multiplexed probeset further comprise a stuffer sequence as the identifiable signal. In some embodiments, the stuffer sequence is a non-hybridizing sequence of nucleotides which aids in controlling the length of the amplification products.

[0021] In some embodiments, one or more or all probes from the multiplexed probeset further comprise a nuclease recognition sequence.

[0022] In some embodiments, the methods further comprise incubating the amplification products with a nuclease.

[0023] In some embodiments, the methods further comprise pooling the amplification products from two or more or all compartments prior to the analyzing.

[0024] In some embodiments, the sample comprises at least 5 (e.g., at least 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, etc.) different target nucleic acids and the method analyzes them simultaneously.

[0025] In some embodiments, the methods further comprise estimating concentrations of the target nucleic acids originally present in the sample based on the quantifying of the amplification products and known or estimated amplification factors.

[0026] In some embodiments, the methods further comprise adding control targets to the compartments; analyzing the control targets by one or more of length, fluorescence, mass, chemical, base composition properties; and determining concentration of the nucleic acid targets based on data transformation and amplification factors of control targets.

[0027] In some embodiments, the compartments (e.g., droplets) are generated by emulsification. In some embodiments, the emulsification comprises one or more of vortexing, pipetting, or homogenization. In some embodiments, the compartments are generated by particle templating, membrane emulsification, or microfluidics to generate an emulsion with reduced size poly dispersity, increased droplet number, and desired droplet volume. In some embodiments, the desired droplet volume confers limiting dilution conditions. In some embodiments, the compartments comprise double and higher order emulsions. In some embodiments, the compartments comprise a carrier phase to introduce reaction components to the compartments. In some embodiments, the reaction components comprise one or more of lysis reagents and amplification reagents.

[0028] In some embodiments, the sample comprises target nucleic acids within cells, viruses, or other biological entities that are encapsulated in the compartments.

[0029] In some embodiments, the amplifying comprises one or more of PCR, MDA, LAMP, RPA, and ligase chain reaction.

[0030] In some embodiments, the multiplexed probeset comprises at least 10 different probes. In some embodiments, the multiplexed probeset comprises at least 100 different probes. In some embodiments, the multiplexed probeset comprises at least 1000 different probes. In some embodiments, the multiplexed probeset comprises at least 10,000 different probes. In some embodiments, individual probes are configured to minimize probe-probe associations across the multiplexed probeset.

[0031] In some embodiments, multiple probesets within single compartments amplify in the same compartment, generating products that indicate their co-amplification. In some embodiments, the amplifying generates fusion amplification products indicating in a single amplicon that the multiple targets were in the same compartment.

[0032] In some embodiments, the amplifying generates amplification products that do not include initial target sequences to which probes bound.

[0033] In some embodiments, the multiplexed probeset comprises molecular inversion probes that generate amplicons of a looping region with a length that is controlled independently of a target region to which the probes are bound. In some embodiments, the multiplexed probeset comprises probes configured for a ligation-based assay and the method further comprises adding a ligase to the compartments prior to the amplifying to generate ligated amplification products.

[0034] In some embodiments, the analyzing comprises quantitative sequencing methods, nanodrop measurements, capillary electrophoresis, mass spectrometry, two-dimensional separation methods, or a combination thereof. In some embodiments, the analyzing comprises analysis of amplification products by capillary electrophoresis.

[0035] In some embodiments, the analyzing comprises separation by length and detection of fluorescence properties. In some embodiments, the fluorescence properties include emission and excitation properties. In some embodiments, the separation by length is at sub -nucleotide resolution due to the identifiable signal.

[0036] In some embodiments, amplification product peak length value is used to determine target identify and peak height to determine target concentration in the sample (number of droplets that amplified).

[0037] In some embodiments, the analyzing further comprises detection of mass tags. In some embodiments, mass tags are analyzed by a mass spectrometer after capillary electrophoresis amplicon separation.

[0038] In some embodiments, the methods further comprise analyzing mass of the amplification products after capillary electrophoresis to obtain information about the amplification product, such as their base composition and inclusion of mass-labeled nucleotides during amplification.

[0039] In some embodiments, amplification products are run at different total dilutions on the capillary electrophoresis range to increase dynamical range of quantitation.

[0040] In some embodiments, capillary electrophoresis peak height data is normalized to the amplifications of the control targets to estimate total reaction efficiency and thereby correct for variances in reaction efficiency.

[0041] In some embodiments, amplification efficiency of each probe within the multiplexed probeset is estimated or determined computationally or empirically and resultant estimates or determinations are used to normalize multiplexed probeset data during the analyzing step.

[0042] In some embodiments, the analyzing comprises reacting amplification products with a microarray printed with associated probes. In some embodiments, binding of amplification products to the associated probes yields a quantitative fluorescence signal for each probe that indicates concentration of associated amplification product. In some embodiments, the nucleic acid targets are derived from pathogens. In some embodiments, the pathogens are one or more of: bacteria, virus, and fungi. In some embodiments, the multiplexed probeset comprises probes sufficient to analyze at least 10 different pathogens simultaneously. In some embodiments, the multiplexed probeset comprises probes sufficient to analyze at least 100 different pathogen targets simultaneously. In some embodiments, the multiplexed probeset comprises probes sufficient to analyze at least 1000 different pathogen targets simultaneously. In some embodiments, the multiplexed probeset comprises probes sufficient to analyze at least 10,000 different pathogen targets simultaneously. In some embodiments, the analyzing comprises determining specific genetic features present in viruses or microbes in a sample by including probes specific for the features. In some embodiments, the features comprise one or more of 16S RNA sequences, sequences associated with virulence factors, and sequences associated with antibiotic resistance. In some embodiments, the multiplexed probeset comprises probes specific for host targets as controls or to provide normalization.

[0043] In some embodiments, the sample is from a patient screened for cancer or suspected of having cancer and wherein the multiplexed probeset comprises a plurality of probes that bind to cancer-specific nucleic acid sequences. In some embodiments, the cancer-specific nucleic acid sequences are one or more of a point mutation, a deletion, an insertion, a translocation, and a methylated base or sequences derived therefrom (e.g., bisulfite converted sequence). In some embodiments, the cancer-specific nucleic acid sequences are specific for a particular type of cancer. In some embodiments, the cancer-specific nucleic acid sequences are specific to a plurality of different types of cancer. In some embodiments, the multiplexed probeset comprises probes sufficient to analyze at least 10 different cancer-specific nucleic acid molecules. In some embodiments, the multiplexed probeset comprises probes sufficient to analyze at least 100 different cancer-specific nucleic acid molecules. In some embodiments, the multiplexed probeset comprises probes sufficient to analyze at least 1000 different cancer-specific nucleic acid molecules. In some embodiments, the multiplexed probeset comprises probes sufficient to analyze at least 10,000 different cancer-specific nucleic acid molecules. In some embodiments, the cancer-specific nucleic acid sequences reveal minimizing residual disease (MRD). In some embodiments, probes in the multiplexed probeset are tailored to a specific patient based on cancer genomic sequencing data from the patient. In some embodiments, the analyzing comprises characterizing gene expression of the sample by estimated gene concentration of a plurality of genes of interest.

[0044] In some embodiments, the analyzing comprises analysis of gene editing or gene editing materials to determine or quantify one or more of editing efficiency, editing yield, editing quality, or off-target editing. In some embodiments, the editing materials comprise cells, vector, nuclease, guides, or accessory proteins.

[0045] In some embodiments, analyzing comprises analysis of one or more genetic features of a fetus, wherein the sample comprises one or more of maternal blood, vaginal swabs, or other maternal samples. In some embodiments, one or more genetic features comprises aneuploidy, point mutations, insertions, deletions, translocations, or methylation status.

[0046] In some embodiments, the target nucleic acids are suspected of having one or more mutations, insertions, deletions, or other sequence variations as compared to a wild-type sequence. In some embodiments, the multiplexed probeset comprises a blocking oligonucleotide or peptide which associates with the wild-type sequence and prevents amplification.

[0047] In some embodiments, at least a portion of the target nucleic acids are recovered from protein labeling techniques. In some embodiments, the method further comprises labeling the sample with one or more affinity probes each directed to a protein of interest, wherein the one or more affinity probes each comprise a unique target nucleic acid or one or more affinity probes are reacted and bound with a coordinating secondary affinity probe which comprises a unique target nucleic acid. In some embodiments, the method further comprises recovering the target nucleic acid from each affinity probe or secondary affinity probe. In some embodiments, the affinity probe is an antibody.

[0048] Also provided herein are methods for identifying an unknown nucleic acid in a sample. In some embodiments, the methods comprise: encapsulating nucleic acids from the sample in compartments under limiting dilution conditions with probes from a multiplexed probeset, wherein the multiplexed probeset comprises specific, degenerate, and / or random probes; amplifying the nucleic acids with the multiplexed probeset to generate amplification products in which the quantity of different amplification products exhibits sequence-specific bias, thereby generating an amplification length fingerprint at least partly unique to target nucleic acids within the sample due to unique sequence biases of the target nucleic acids; pooling the amplification products; analyzing the amplification products to detect the amplification length fingerprint; and comparing the amplification length fingerprint to the fingerprints of known targets, thereby allowing one to identify an unknown nucleic acid from the sample.

[0049] In some embodiments, the comparing comprises projecting high dimensional fingerprint data onto a lower dimensional space (e.g., 2D space) to generate plots that are used for target identification by clustering and association. In some embodiments, the clustering and association comprises use of one or more of Principal Component Analysis (PCA), t-distributed Stochastic Neighbor Embedding (tsNE), or Uniform Manifold Approximation and Projection (UMAP). In some embodiments, characteristic peaks in the fingerprint data are used to identify targets based on look-up tables of known peaks. In some embodiments, nanopore sequencing of the fingerprints is used to identify targets.

[0050] In some embodiments, the comparing comprises determination of identity of pathogens in the sample. In some embodiments, the comparing comprises determination of subsequences associated with specific genetic sequences or virulence factors associated with one or more pathogens.

[0051] In some embodiments, the comparing comprises determination of one or more specific mutations present in nucleic acid in the sample.

[0052] Further provided herein are methods for quantitative targeted proteomics. In some embodiments, the methods comprise: incubating a sample comprising proteins to be detected with reagents comprising affinity probes that bind to protein targets of interest; washing to recover proteins and their bound affinity probes; reacting proteins and their bound affinity probes with secondary affinity probes labeled with oligonucleotides specific to the secondary probes; washing to enrich target proteins and their dual bound affinity probes; and quantitating dual bound target proteins (which may comprise from one to thousands of unique species) by using a method described herein to quantitate oligonucleotides specific to the secondary affinity probes.

[0053] Additionally provided are methods for quantitative targeted protein detection. In some embodiments, the methods comprise labeling proteins present in a sample with unique aptamer sequences; and using a method disclosed herein to detect the aptamer sequences. In some embodiments, the methods comprise generating DNA concatenations that indicate presence of dual bound affinity probes to target proteins and using a method disclosed herein to detect the DNA concatenations. Systems comprising instruments and reagents to conduct the disclosed methods on a sample suspected of containing one or more targets are also provided. In some embodiments, the systems comprise a well plate processing component and is configured for simultaneously analyzing multiple targets in a plurality of different samples.

[0054] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description.

[0055] DESCRIPTION OF FIGURES

[0056] FIGS. 1A-1G show a schematic of an exemplary workflow according to an embodiment of the methods disclosed herein, referred to as Partitioned Amplification Capillary Electrophoresis (PACE). FIG. 1A) Monodispersed polyacrylamide beads that serve as the selfassembled partitioning (SAP) reagent are added to the PCR reaction mix and FIG. IB) soaked with enzymes and molecules FIG. 1C) After removing excess aqueous phase, FIG. ID) oil with stabilizing surfactant is introduced. FIG. IE) The mixture is uniformly emulsified by vortexing. FIG. IF) For digital (droplet) PCR, target sequences are encapsulated and amplified in the droplets, generating a detectable fluorescence signal. FIG. 1G) Droplets are broken and the sample processed onto the capillary electrophoresis (CE) machine to separate out all the amplicons based on size and fluorophore. Amplicon sizes and amplitudes are measured to provide a multiplexed and quantitative read-out. Because droplets provide a quantized amount of DNA, peak heights correspond to the original targets’ abundance in the sample.

[0057] FIGS. 2A-2C show that self-assembled partitioning (SAP) generates monodispersed droplets. FIG. 2A) In contrast to microfluidic emulsification, in which droplets are made serially and thus the number scales with time, with SAP they are made in parallel by bulk shearing, and thus the number scales with container volume. FIG. 2B) SAP works in all multiwell plate formats tested. FIG. 2C) Droplets for all formats are monodispersed (top plots). In contrast to microfluidics (pF) that has a time dependence on volume generation, SAP generates emulsions within 30 s for all volumes (bottom table).

[0058] FIG. 3 shows a comparison of qPCR to dPCR approach. Compartmentalization sequesters inhibitors in droplet subsets in dPCR, allowing efficient amplification and quantitation in other droplets without purification. FIGS. 4A-4C shows data showing qPCR v dPCR. FIG. 4 A) Ct values for qRT-PCR from purified RNA (black) versus RNA prepared from crude lysate (red) across 32 patient samples. PCR efficiency decreased without upfront nucleic acid purification, as evidenced by increase Ct. B) Relative quantification of viral load in crude lysate from FIG. 4B) qRT-PCR and FIG. 4C) digital RT-PCR (dRT-PCR) versus purified RNA. Results show that qPCR, but not dPCR, is inhibited when applied to crude.

[0059] FIG. 5 shows a comparison of qPCR and PACE vs dPCR for quantitating SARS-CoV-2 RNA over a dilution series. The x-axis is the sample concentration estimate based on dPCR. The y-axis shows the corresponding estimates based on qPCR and PACE (gPCR), respectively. While PACE matches dPCR estimates down to a lower limit of detection of -1 / 10,000 molecules, qPCR loses its quantitation below -20 / 10,000 molecules.

[0060] DETAILED DESCRIPTION

[0061] Provided herein are systems, compositions, and methods for nucleic acid quantification that combines the sensitivity and accuracy of digital PCR with the capability for high-throughput multiplexing. The technique integrates digital amplification in compartments with one or more of a variety of detection and separation techniques, e.g., capillary electrophoresis or other methodologies, as a readout mechanism.

[0062] As noted above, in traditional digital PCR methods, an optical reader is necessary to count the number of positive droplets. This introduces additional complexity and costs related to the instrumentation. The disclosed methods sidestep the need for optical reading by using other methods to estimate the number of positive droplets. The key feature is that the number of amplicons generated in a digital PCR reaction is directly proportional to the number of positive droplets, provided that the targets in the droplets are at limiting dilution. This holds true even for endpoint-cycled reactions, which is not the case in bulk PCR. Therefore, by quantifying the amplicon concentration at the end of the digital PCR process, one can infer the number of positive droplets in the initial reaction and thus estimate the target concentration.

[0063] The absence of optical reading equipment simplifies the workflow considerably. However, an even more significant advantage of the disclosed methods lies in its ability to overcome one of the key limitations of digital PCR: low multiplexing capability. Traditional digital PCR is confined to the use of a small number of fluorescent dyes for labeling targets, usually limiting the process to identifying no more than five or so targets simultaneously. In contrast, the methods disclosed herein not only measure amplicon concentration but also identify amplicon length, thus providing a vastly expanded labeling spectrum.

[0064] As one example, capillary electrophoresis systems possess single base-pair resolution for length measurement. This enables the generation of a large number of unique length labels for amplicons. For example, if one uses amplicons ranging from 100 to 200 base pairs with single base-pair length differences, this yields 100 unique length labels for uniquely labeling targets. Primer design and different primer structures are used to produce amplicons of a specific length for each target of interest. Consequently, methods disclosed herein using capillary electrophoresis offer a substantial improvement in multiplexing capabilities without sacrificing the high accuracy, sensitivity, and quantitative nature intrinsic to digital PCR. While capillary electrophoresis is highlighted here due to the wide availability of instruments and the efficiency, the technology described herein can be practiced in any system employing separation media that allows for size separation and visualization of different sized / shaped nucleic acid molecules. This includes any of a wide variety of gel electrophoresis technologies.

[0065] The disclosed methods are not only noteworthy for their capability in terms of lengthbased multiplexing but also for their compatibility with other forms of labeling, significantly expanding its multiplexing prowess. For instance, the use of fluorescently labeled primers can add a color dimension to the amplicons. This means that each amplicon could be identified by both its length and its fluorescent emission and excitation. Combining, for example, four distinct fluorescent colors with 100 different lengths results in a combinatorial space of 400 unique labels for amplicons.

[0066] Further adding to this combinatorial diversity, mass tags can be incorporated into the amplicons (amplification products). These tags, which could be included in the primers used for initiating the reaction, add a mass signal to each amplicon. If one were to include four different mass tags in conjunction with four fluorescent signals and 100 unique lengths, the potential arises for 1,600 unique label combinations.

[0067] Some separation techniques, such as capillary electrophoresis, already commonly use multi-color detection and thus can measure both the lengths and colors of the amplicons. Following this, the pool of amplicons can be sequentially analyzed through mass spectrometry to acquire the mass tag information. This comprehensive approach allows for the gathering of length, color, and mass information, massively extending the target space that can be simultaneously analyzed.

[0068] A valuable advantage of the disclosed methods is their ability to perform targeting, detection, and quantitation simultaneously within a single assay. Unlike conventional multiplex PCR, which often necessitates splitting the sample into multiple reactions to avoid primer competition and skewing of quantitation, the disclosed methods avoid this pitfail by encapsulating the sample in multiple droplet compartments, each likely containing single targets. While each droplet contains the full set of primers, only the primers corresponding to the target in that specific droplet will be selected for amplification. This produces pure amplicons for that target within the droplet.

[0069] This process occurs in parallel across all droplet compartments, generating a spectrum of amplicons with unique lengths and other tag features. As a result, these can be pooled into a single reaction for analysis, using capillary electrophoresis and mass spectrometry as already described, to accurately estimate the target concentration for multiple targets simultaneously.

[0070] In summary, the disclosed methods offer an advanced, integrated approach for nucleic acid quantification that leverages multiple labeling schemes, providing a level of accuracy comparable to traditional digital PCR methods while greatly extending the target space that can be multiplexed, all without necessitating the division of samples into separate reactions.

[0071] Definitions

[0072] To facilitate an understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description. Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise.

[0073] The phrase “in one embodiment” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

[0074] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. However, two or more copies are also contemplated. The singular forms “a,” “and,” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. In addition, as used herein, the term “or” is an inclusive “or” operator and is equivalent to the term “and / or” unless the context clearly dictates otherwise. The term “based on” is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. The meaning of “in” includes “in” and “on.”

[0075] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0076] As used herein, a “nucleic acid” or “nucleic acid molecule” generally refers to any ribonucleic acid or deoxyribonucleic acid, which may be unmodified or modified DNA or RNA. “Nucleic acids” include, without limitation, single- and double-stranded nucleic acids. As used herein, the term “nucleic acid” also includes DNA as described above that contains one or more modified bases. Thus, DNA with a backbone modified for stability or for other reasons is a “nucleic acid”. The term “nucleic acid” as it is used herein embraces such chemically, enzymatically, or metabolically modified forms of nucleic acids, as well as the chemical forms of DNA characteristic of viruses and cells, including for example, simple and complex cells.

[0077] The terms “oligonucleotide” or “polynucleotide” or “nucleotide” or “nucleic acid” refer to a molecule having two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. Typical deoxyribonucleotides for DNA are thymine, adenine, cytosine, and guanine. Typical ribonucleotides for RNA are uracil, adenine, cytosine, and guanine.

[0078] As used herein, the terms “locus” or “region” of a nucleic acid refer to a sub-region of a nucleic acid, e.g., a gene on a chromosome, a single nucleotide, a CpG island, etc. The terms “complementary” and “complementarity” refer to nucleotides (e.g., 1 nucleotide) or polynucleotides (e.g., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence 5'-A-G-T-3' is complementary to the sequence 3'-T-C-A-5'. Complementarity may be “partial,” in which only some of the nucleic acids’ bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands effects the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions and in detection methods that depend upon binding between nucleic acids.

[0079] “Amplification” is a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (e.g., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (e.g., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of “target” specificity. Target sequences are “targets” in the sense that they are sought to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out.

[0080] Amplification of nucleic acids generally refers to the production of multiple copies of a polynucleotide, or a portion of the polynucleotide, typically starting from a small amount of the polynucleotide (e.g., a single polynucleotide molecule, 10 to 100 copies of a polynucleotide molecule, which may or may not be exactly the same), where the amplification products or amplicons are generally detectable. Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes. For example, amplification methods may comprise PCR, LAMP, NASBA, HAD, SDA, NEAR, SPIA, RCA and RPA, isothermal and / or thermal amplification and related methods. The generation of multiple DNA copies from one or a few copies of a target or template DNA molecule during a polymerase chain reaction (PCR) or a ligase chain reaction (LCR; see, e.g., U.S. Patent No. 5,494,810) are forms of amplification. Additional types of amplification include, but are not limited to, allele-specific PCR (see, e.g., U.S. Patent No. 5,639,611), assembly PCR (see, e.g., U.S. Patent No. 5,965,408), helicasedependent amplification (see, e g., U.S. Patent No. 7,662,594), Hot-start PCR (see, e.g., U.S. Patent Nos. 5,773,258 and 5,338,671), intersequence-specific PCR, inverse PCR (see, e.g., Triglia, et al. (1988) Nucleic Acids Res., 16:8186), ligation-mediated PCR (see, e.g., Guilfoyle, R. et al., Nucleic Acids Research, 25: 1854-1858 (1997); U.S. Patent No. 5,508,169), methylation-specific PCR (see, e.g., Herman, et al., (1996) PNAS 93(13) 9821-9826), miniprimer PCR, multiplex ligation-dependent probe amplification (see, e.g., Schouten, et al., (2002) Nucleic Acids Research 30(12): e57), multiplex PCR (see, e g., Chamberlain, et al., (1988) Nucleic Acids Research 16(23) 11141-11156; Ballabio, et al., (1990) Human Genetics 84(6) 571-573; Hayden, et al., (2008) BMC Genetics 9:80), nested PCR, overlap-extension PCR (see, e.g., Higuchi, et al., (1988) Nucleic Acids Research 16(15) 7351-7367), real time PCR (see, e.g., Higuchi, et al., (1992) Biotechnology 10:413-417; Higuchi, et al., (1993) Biotechnology 11 : 1026-1030), reverse transcription PCR (see, e.g., Bustin, S.A. (2000) J. Molecular Endocrinology 25: 169-193), solid phase PCR, thermal asymmetric interlaced PCR, and Touchdown PCR (see, e.g., Don, et al., Nucleic Acids Research (1991) 19(14) 4008; Roux, K. (1994) Biotechniques 16(5) 812-814; Hecker, et al., (1996) Biotechniques 20(3) 478-485). Polynucleotide amplification also can be accomplished using digital PCR (see, e.g., Kalinina, et al., Nucleic Acids Research. 25; 1999-2004, (1997); Vogelstein and Kinzler, Proc Natl Acad Sci USA. 96; 9236-41, (1999); International Patent Publication No. W005023091A2; US Patent Application Publication No. 20070202525).

[0081] The term “amplifiable nucleic acid” refers to a nucleic acid that may be amplified by any amplification method. It is contemplated that “amplifiable nucleic acid” will usually comprise “sample template.”

[0082] The term “primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product that is complementary to a nucleic acid strand is induced, (e.g., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer, and the use of the method. In some embodiments, a primer sequence(s) is incorporated into a probe of the probeset, optionally with other sequences (e.g., non-hybridizable, filler sequences, sequence tags) or moi eties (e.g., tags, labels - based on small molecule or other macromolecules or fragments thereof). However, not all probes within a probeset may contain primer sequences. Probes within the probeset may comprise sequences for use in ligation and other methods described herein in which the primer sequence is being provided by a coordinating probe within the probeset.

[0083] The term “isolated” when used in relation to a nucleic acid, as in “an isolated oligonucleotide” refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids, such as DNA and RNA, are found in the state they exist in nature. Examples of non-isolated nucleic acids include: a given DNA sequence (e.g., a gene) found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, found in the cell as a mixture with numerous other mRNAs which encode a multitude of proteins. However, isolated nucleic acid encoding a particular protein includes, by way of example, such nucleic acid in cells ordinarily expressing the protein, where the nucleic acid is in a chromosomal location different from that of natural cells or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid or oligonucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid or oligonucleotide is to be utilized to express a protein, the oligonucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide may be single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide may be double-stranded). An isolated nucleic acid may, after isolation from its natural or typical environment, be combined with other nucleic acids or molecules. For example, an isolated nucleic acid may be present in a host cell in which into which it has been placed, e.g., for heterologous expression.

[0084] The term “purified” refers to molecules, either nucleic acid or amino acid sequences that are removed from their natural environment, isolated, or separated. An “isolated nucleic acid sequence” may therefore be a purified nucleic acid sequence. “Substantially purified” molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated. As used herein, the terms “purified” or “to purify” also refer to the removal of contaminants from a sample. The removal of contaminating proteins results in an increase in the percent of polypeptide or nucleic acid of interest in the sample. In another example, recombinant polypeptides are expressed in plant, bacterial, yeast, or mammalian host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample.

[0085] The term “sample” is used in its broadest sense. In one sense it can refer to an animal cell or tissue. In another sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from plants or animals (including humans) and encompass fluids, solids, tissues, and gases. The definition encompasses blood and other liquid samples of biological origin, as well as solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as polynucleotides. Environmental samples include environmental material such as surface matter, soil, water, and industrial samples. These examples are not to be construed as limiting the sample types applicable to the present invention.

[0086] As used herein, the term “kit” refers to any delivery system for delivering materials. In the context of reaction assays, such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. in the appropriate containers) and / or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and / or supporting materials. As used herein, the term “fragmented kit” refers to delivery systems comprising two or more separate containers that each contain a sub-portion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides. The term “fragmented kit” is intended to encompass kits containing Analyte Specific Reagents (ASR's) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act, but are not limited thereto. Indeed, any delivery system comprising two or more separate containers that each contains a sub-portion of the total kit components are included in the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system containing all of the components of a reaction assay in a single container (e.g., in a single box housing each of the desired components). The term “kit” includes both fragmented and combined kits.

[0087] A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes as described herein. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non-human). Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non- human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of nonmammals include, but are not limited to, birds, fish, and the like. In one embodiment, the mammal is a human.

[0088] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0089] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0090] FIG. 1 provides a description of exemplary embodiments of the methods disclosed herein, specifically those using capillary electrophoresis and referred to herein as Partitioned Amplification Capillary Electrophoresis (PACE). Step 1 (FIG. 1A-E) mixes the sample with the hydrogel partitioning particles and reagents via pipette, then vortexing (or pipetting) for 30 s. This encapsulates the target molecules in droplet compartments; to enable absolute quantitation. The targets are encapsulated at limiting dilution, which is accomplished by producing ~10-fold more droplets than targets. Because one particle generates one droplet, drop number is controlled by loading the desired number of particles into the reaction (34,35,60). Step 2 (FIG. IF) thermocycles the emulsion, accomplished using a thermocycler. Perfluorinated oils and triblock copolymer surfactants stabilize the droplets at high temperature (35,66). Step 3 (FIG. 1G) demulsifies with perfluorooctanol and 1 min vortexing (or pipetting), followed by CE analysis. CE utilizes electrophoresis to aspirate samples with picoliter-precision (39,42,67). To obtain a quantitative range spanning 7-8 decades, the inherent precision of the CE machine is used to analyze a range of sample volumes processed sequentially (39-41). These automated systems can process 96 to 384 samples simultaneously, with a single run taking 1 to 2 min (63).

[0091] In dPCR, important features are sensitivity, precision, and dynamic range. Sensitivity is proportional to 1 / V_tot, where V tot is the total volume of the sample analyzed: More sample provides more targets for detection (62). With PACE, this is enabled by exploiting the scalability of SAP for generating droplets, allowing the full sample to be analyzed. By contrast, with microfluidic dPCR, sample input is limited due to the inability to partition more volume in a reasonable time and the fixed operating conditions of the microfluidic system (68-70). For example, with PACE, a full 50 pL sample can be partitioned in 30 s and analyzed with CE in 5 min, enhancing Limit of Detection (LOD) by tenfold relative to current dPCR approaches. Precision is proportional to 1 / A / N, where N is the number of drops in the reaction (26). While most commercial dPCR instruments use up to 100,000 drops, with PACE one can use 10 million (~20 pm), increasing precision by ~25X, important for rare target quantitation. Dynamic range of a dPCR is proportional to log A when using a Poisson correction to account for multiple encapsulation (71); thus, while existing instruments achieve 5 decades, PACE can achieve 7-8, comparable to qPCR (61).

[0092] In some embodiments, provided herein are methods for analyzing nucleic acid targets in a sample, comprising: a) encapsulating nucleic acids in compartments under limiting dilution conditions with probes from a multiplexed probeset, wherein the probeset generates amplification products, also referred to herein as amplicons, of different length allowing them to be distinguished by length measurement; b) amplifying nucleic acids targeted by the multiplexed probeset such that certain probe subsets amplify in different compartments, depending on which targets are present in the compartments; c) pooling the amplification products from compartments; and d) analyzing the amplification products to detect their relative length and one or more of fluorescence, mass, chemical, and / or base composition properties. In some embodiments, the methods encoding distinct features, e.g., identifiable signals such as a fluorophore, a mass tag, a molecular tag, a peptide, a detectable nucleic acid sequence, a modified nucleotide, a small molecule, a lipid, a nanoparticle, or a combination thereof, into the amplification products (e.g., through inclusion with a probe within the probe set), making different amplification product for various nucleic acid targets distinguishable. These distinguishing features or identifiable signals can vary and may include the length of the amplicons, the inclusion of fluorescent or mass tags, peptides, or sequence tags, among others. These methods combine two concepts: linear endpoint production, which facilitates the determination of target concentration through a simple endpoint measurement of the concentration of the amplification products; and multiplexing, which involves labeling the amplification products in such a way that they are independently distinguishable in a bulk measurement.

[0093] In some embodiments, the methods comprise a) encapsulating target nucleic acids in compartments under limiting dilution conditions with probes from a multiplexed probeset, b) amplifying nucleic acids targeted by the multiplexed probeset such that certain probe subsets amplify in different compartments, depending on which nucleic acid targets are present in the compartments, wherein the amplifying generates amplification products in a concentration proportional to the original target nucleic acid and wherein the amplification product comprises an identifiable signal; and c) analyzing the amplification products to detect their identifiable signal and / or quantifying the amplification products. In some embodiments, the amplification products are of different length. In some embodiments, the amplification products are of the same length.

[0094] In some embodiments, the analyzing comprises quantifying one or more targets originally present in the sample.

[0095] In some embodiments, the droplet compartments are generated by random emulsification. In some embodiments, the random emulsification comprises one or more of vortexing, pipetting, or homogenization to generate an emulsion with size polydispersity. In some embodiments, compartments are generated by particle templating, membrane emulsification, or microfluidics to generate an emulsion with reduced size polydispersity, increased droplet number, and desired droplet volume. In some embodiments, the desired droplet volume confers limiting dilution conditions. In some embodiments, the compartments comprise double and higher order multiple emulsions. In some embodiments, compartments comprise a carrier phase to introduce reaction components to the compartments.

[0096] In some embodiments, reaction components comprise one or more of lysis reagents and amplification reagents. In some embodiments, the sample comprises target nucleic acids within cells, viruses, or other biological entities that are encapsulated in the compartments. In some embodiments, amplifying comprises one or more of PCR, MDA, LAMP, RPA, and ligase chain reaction.

[0097] In some embodiments, the probeset comprises at least 10 different probes (e.g., at least 100, at least 1000, at least 10,000). In some embodiments, the multiple probesets within single compartments amplify in the same compartment, generating products that indicate their coamplification. In some embodiments, amplifying generates fusion amplification products indicating in a single amplicon that the multiple targets were in the same compartment.

[0098] In some embodiments the methods disclosed herein utilize techniques based on ligation, such as Multiplexed Ligation-dependent Amplification (MLP A), Long Adapter Single Strand Oligonucleotide Probes (LASSO probes), and / or Padlock Probes. These ligation-based techniques generate products only when probes perfectly match and ligate to the target, enhancing the specificity of the assay, particularly beneficial in single nucleotide polymorphism detection. The probes can be designed with loops connecting two A&B probes or have lengths, stuffer sequences, facilitating the generation of products of desired lengths. Taking MLPA as an example, a set of A&B probes would be used for each target in the sample. These probes are annealed to the targets in bulk, followed by a ligation reaction. Ligation occurs only when the probes perfectly bind to the targets, resulting in a linear product. Subsequent PCR using universal primers amplifies only the ligated products, conducted in droplet compartments to maintain linear amplification. Notably, in such reactions, the original target sequences are not required; only the ligated products need to be encapsulated. If desired, the methods can include a purification step to remove unligated products. The resulting amplicons vary in length due to their design in the universal priming step or through inclusion of nuclease digestion sites to produce fragments of the desired lengths. Crucially, this ligation-based approach not only allows for length independence of amplicons relative to the targets but also leverages the specificity of ligation for effective SNP detection. These ligation-based techniques are fully compatible with other techniques described herein, such as multiplexing based on fluorescence or nuclease digestion, producing lengths suitable for analysis. The integration of ligation assays achieves diverse amplicon lengths while maintaining high specificity.

[0099] In some embodiments, all primers for all the targets are present in all the droplet compartments and the targets are present at limiting dilution such that, when present, the associated probesets amplify. Accordingly, the primers and probeset are designed such that primer-primer interactions are minimized.

[0100] In some embodiments, the sample comprises more than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) different target nucleic acids. In select embodiments, the sample comprises more than 5 different target nucleic acids. Accordingly, the sample may comprise 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more target nucleic acids. In some embodiments, the methods disclosed herein analyze each of the different target nucleic acids at the same time.

[0101] The capacity to detect multiple sequences within a single droplet compartment has various applications, particularly in scenarios where co-occurrence information is valuable. This could be essential for studying co-occurring viruses, cells, or specific subsequences within a larger target sequence, such as sets of genes in a viral genome. In conventional digital PCR, this co-occurrence is often detected by multiplexing with different colors and observing the resultant droplets. If one product is labeled green and another red, a droplet containing both targets would emit a yellow fluorescence, indicating the co-occurrence of both sequences within the same droplet. In exemplary methods disclosed herein, similar information is derived, for example, using capillary electrophoresis as the readout. One approach is to employ a system that forms fusion products, which link amplified amplicons present within a single droplet. If two different targets are amplified within a single droplet, the subsequent linking and amplification produces a concatenated amplicon. This unique concatenated product manifests as a new, distinct length in capillary electrophoresis. This concatenated amplicon not only reflects a sum of the lengths of the individual amplicons but also their specific fluorescent labels or mass tags. This produces a uniquely identifiable signal, whether in terms of length, color, or mass, that allows for the detection of co-occurrence within the same compartment. Therefore, this method enables the capture of co-occurrence information through multiple readouts, such as capillary electrophoresis, fluorescence, and mass spectrometry. PACE offers a flexible and precise mechanism for capturing co-occurrence information within individual compartments. The creation of concatenated amplicons that are uniquely identifiable allows for highly specific and informative analysis of complex samples. This capability further extends the utility of the disclosed methods in various fields requiring detailed co-occurrence information, from microbial ecology to virology and cell biology.

[0102] In some embodiments, the amplifying generates amplification products that do not include initial target sequences to which probes bound. In some embodiments, the probeset comprises molecular inversion probes that generate amplicons of a looping region with a length that is controlled independently of a target region to which the probes are bound. In standard PCR, the length of the amplicon can serve as an inherent label for identifying specific targets. This labeling scheme is often constrained by the target sequence itself, limiting the freedom to choose amplicon lengths that are most optimal for detection or differentiation purposes. Molecular inversion probes (MIPs) offer a solution to this limitation by enabling the decoupling of amplicon length from the target sequence. Using MIPs, one can design a looping probe set in which forward and reverse probes hybridize to specific regions on the target molecule. Once both probes are hybridized, amplification of the connecting loop is carried out. The resulting synthetic amplicons, while not sequence-related to the target itself, are nonetheless proportional to the target's concentration in the sample. This modular approach allows for amplicon lengths to be set independently of the target sequence. The advantages of this approach are manifold. Firstly, it allows for the fine-tuning and optimization of probe sets, thereby enhancing the assay's sensitivity and specificity. Secondly, this adaptability makes it possible to design assays for targets with varying sequences due to mutations, insertions, or deletions. Lastly, because the MIP -based strategy focuses on amplifying a loop region determined by the hybridized probes, it allows targeting of much shorter regions than would otherwise be possible, providing a more flexible and robust approach to detect conserved regions or regions less likely to be affected by mutations. Other probe and amplification strategies may also be implemented that provide a degree of decoupling between target and amplicon length and sequence.

[0103] In some embodiments, the analyzing comprises quantitative sequencing methods, nanodrop measurements, capillary electrophoresis, mass spectrometry, two-dimensional separation methods, or a combination thereof. In select embodiments, the analyzing comprises analysis of amplification products by capillary electrophoresis.

[0104] In some embodiments, the analysis comprises separation by length and detection of fluorescence properties. In some embodiments, amplicon peak length value is used to determine target identify and peak height to determine target concentration in the sample (e.g., number of droplets that amplified).

[0105] In some embodiments, the analysis further comprises detection of mass tags. In some embodiments, mass tags are analyzed by a mass spectrometer after capillary electrophoresis amplicon separation.

[0106] In some embodiments, the method further comprises the step of analyzing mass of the amplification products after capillary electrophoresis to obtain information about the amplification product, such as their base composition and inclusion of mass-labeled nucleotides during amplification.

[0107] In some embodiments, amplification products are run at different total dilutions, e.g., on the capillary electrophoresis range, to increase dynamical range of quantitation.

[0108] In some embodiments, the methods comprise adding control targets to the compartments, analyzing the control targets by one or more of length, fluorescence, mass, chemical, base composition properties and determining concentration of the nucleic acid targets based on data transformation and amplification factors of control targets.

[0109] In select embodiments, capillary electrophoresis peak height data is normalized to provide accurate quantitation by including control targets in the sample at known concentrations and using the amplifications of the control targets to estimate total reaction efficiency and thereby correct for variances in reaction efficiency.

[0110] In some embodiments, amplification efficiency of each probe within the probeset is estimated or determined computationally or empirically and resultant estimates or determinations are used to normalize probeset data during the analyzing step.

[0111] Accurate concentration measurements, in some embodiments, hinge on the successful conversion of arbitrary peak amplitudes generated by capillary electrophoresis into real-world concentrations, such as in the PACE method. Traditionally in qPCR, this is accomplished through the use of a standard curve that runs alongside the assay. In the methods disclosed herein, similar normalization approaches can be implemented to achieve precise concentration measurements. One option is to run standard samples in parallel with the assay for all the primer sets. This creates a reference for converting the peak amplitudes observed during capillary electrophoresis into actual concentrations. An alternative approach is to integrate specific standards directly into the sample of interest by spiking them in. The advantage of this method is that it avoids the need to split the sample and allows for the direct measurement of reaction efficiency under conditions identical to those of the actual sample. Additionally, another layer of accuracy can be added by pre-characterizing each primer set for its amplification efficiency at the time of its construction. This information, intrinsically linked to the sequence of the primer set and the target it is intended to amplify, can be used during the analysis to adjust for any sequence-specific biases. Moreover, it is possible to combine these approaches to achieve robust normalization. Using both internal standards and primer efficiency data not only corrects for sequence-specific biases but also adjusts for reaction-specific variables, such as inhibition effects or pipetting errors. This comprehensive normalization strategy allows highly accurate concentration measurements, negating the need for sample splitting or running parallel assays. Therefore, these normalization techniques collectively enhance the robustness and efficiency of the disclosed methods, ensuring a reliable approach for comprehensive nucleic acid quantification.

[0112] In some embodiments, the methods facilitate sub -nucleotide separation for increased multiplexing e.g., sub -nucleotide resolution can be achieved using a combination of factors such as amplicon length, fluorescence, and mass tag properties. To further increase multiplexing capacity, even with a single reporter like fluorescence, consideration is given to the downstream analysis method’s capacity to resolve targets. Several methods facilitate this, including sequencing for target identification and microarrays for resolving targets based on their unique interactions, as described elsewhere herein.

[0113] In some embodiments, the methods utilize enhanced length-based separation, using capillary electrophoresis combined with other techniques, for sub-nucleotide separation. Standard capillary electrophoresis allows for single-base resolution, but sub-base resolution is achieved by conducting separation under conditions that sufficiently space individual lengths, allowing gradations between them. These gradations can be targeted with specific factors other than length that influence molecular migration. For example, probes can have attached anchors causing them to fractionate differently, even if they are of identical length. During amplification, one would use probes with differently anchored tags, allowing separation based on these anchors. For instance, between lengths of 100 and 101 bases, one could create 10 gradations controlled by 10 different anchoring tags, each slightly modifying the separation within this range. Adjusting the properties of the capillary electrophoresis allows resolution of these gradations and expansion of the resolution scope, enabling multiplexing from single base pair bins to finer, sub-base pair resolution bins.

[0114] Various options exist for the types of anchors used in this process, encompassing a wide range of molecules and chemical linkers. These include small molecules, lipids, various nucleic acids, nanoparticles, proteins, and more. In some embodiments, the amplification products can be labeled with a variety of any of the anchors described herein. Selection of the anchors is based on the separation method being used, e.g., capillary electrophoresis, and the ability of the anchor to fractionate within that assay when attached to a target nucleic acid. For example, each type of anchor imparts unique properties to the nucleic acids they are attached to, affecting their migration during separation. Small molecules, for example, can be designed to add minimal mass and size variations, subtly altering migration patterns. Lipids, being larger and more varied in structure, can create more significant differences in separation behavior. Similarly, nucleic acid modifications can introduce specific interactions or structural changes. Nanoparticles and proteins offer even more diverse possibilities. Nanoparticles can vary greatly in size, charge, and surface properties, all of which can significantly alter the separation of the tagged molecules. Proteins, with their complex structures and potential for specific binding interactions, can be used to create highly distinct separation patterns. The selection of anchor(s) will also depend on the specific requirements of the assay, such as the degree of resolution needed, the nature of the targets being separated, and the compatibility of the anchor with the overall method.

[0115] In select embodiments, chemical modifications, such as methylation or the addition of small molecular groups is utilized. These modifications slightly alter the mass and charge distribution of the nucleic acids, thus affecting their separation in a controlled manner. In select embodiments, short peptide sequences are attached to the primers. These peptides, with their known length and composition, are designed to exert a minimal yet detectable influence on the migration pattern of the nucleic acids. Incorporating Locked Nucleic Acids (LNAs) or other modified nucleotide bases into the primers offers an additional method. LNAs have a unique structure that locks them into a rigid conformation, subtly altering the physical properties of the nucleic acid and, consequently, its behavior during electrophoresis. In some embodiments, short oligonucleotide tags are used with distinct sequences or modifications. These tags, smaller than a full nucleotide, can still impart a unique migration signature to the nucleic acids. Any or all of the disclosed anchors can enhance the resolution capabilities of separation techniques (e.g., capillary electrophoresis) allowing for the precise differentiation of nucleic acid sequences that differ by less than the size of a single nucleotide.

[0116] In some embodiments, analyzing comprises reacting amplification products with a microarray printed with associated probes. In some embodiments, binding of amplification products to the associated probes yields a quantitative fluorescence signal for each probe that indicates concentration of associated amplification product.

[0117] While capillary electrophoresis is highly effective, the methods disclosed herein also offer flexibility in terms of other readout systems. Microarrays present a viable alternative for quantifying amplicons generated by a partitioned amplification reaction. In this microarray-based approach, instead of subjecting the amplicons to capillary electrophoresis, they are reacted with a marker array designed to be specific to the amplicons of interest. This strategy offers the advantage of potentially even greater multiplexing capabilities, as well as possibly increased sensitivity and simplicity. Importantly, this obviates the need for an expensive capillary electrophoresis machine, requiring only a microarray chip and an imaging system to capture the post-reaction results. In this setup, each probe set on the microarray would correspond to a specific amplicon. The intensity or brightness of the probe set would provide information on the number of target amplicons present, functioning as a quantification mechanism. Furthermore, the need for amplicons of varying lengths is eliminated in this approach; sequence hybridization on the microarray suffices for identifying each specific amplicon. Therefore, while capillary electrophoresis serves as an effective readout mechanism, the methodology can be adapted to other readout systems like microarrays without compromising its core benefits. This adaptability makes the disclosed methods even more versatile for nucleic acid quantification, capable of meeting varying requirements for multiplexing, sensitivity, and equipment.

[0118] The analysis may further include techniques which employ separations in more than one dimension. Traditionally, techniques like capillary electrophoresis provide a one-dimensional separation, where molecules are differentiated based on their electrophoretic properties. However, two-dimensional separation methods, such as those employed in Western and Northern blots, offer molecular separation along two distinct axes. This two-dimensional separation adds an additional layer of resolution, substantially increasing multiplexing potential. For example, one dimension separates molecules by size, while the other separates them based on other properties, like charge or binding affinity. In some embodiments, the analysis combines electrophoresis with isoelectric focusing (IEF) for the separation of molecules first by their charge in an electric field and then by their isoelectric points, where they have no net charge. Each distinct “bin” or spot in the two-dimensional separation matrix can be correlated with a specific amplicon or molecular species. Moreover, incorporating sub -nucleotide labeling techniques, which as described above subtly alter the migration patterns of nucleic acids, can provide even more nuanced separations within each dimension. In some embodiments, these advanced labeling strategies are combined with two-dimensional separation techniques to achieve a higher density of separations, thereby improving the resolution and expanding the capacity for multiplexed analyses and allowing for a more detailed and comprehensive understanding of complex molecular mixtures. This can be particularly advantageous in fields requiring the analysis of diverse biomolecules, such as genomics, proteomics, and molecular diagnostics.

[0119] The detection limit of the partition amplification reactions is limited by the sensitivity of the readout. For example, the detection limit of PACE is limited by the sensitivity of the capillary electrophoresis readout, which is based on the fluorescence of specific length bands of amplicons. To increase the overall sensitivity of the disclosed methods, for example when the detection limit is limited by the selected readout e.g., fluorescence of specific length bands of amplicons in capillary electrophoresis, a larger quantity of amplicons can be generated.

[0120] In some embodiments, the quantity of amplicons is increased by using larger compartments (e.g., droplets) in the amplification step. Larger droplets can encapsulate more of the target molecule, leading to the production of more amplicons per droplet. However, the size of the droplets is limited by the stability constraints of the droplet formation and reaction process. To overcome this limitation and further enhance sensitivity multiple droplet amplification steps are performed sequentially to achieve an arbitrary increase in sensitivity. For example, in the first droplet reaction, a target might yield a thousand amplicons, which may not yet be detectable, e.g., by capillary electrophoresis fluorescence. This initial batch of amplicons can then be re-encapsulated in new droplets and amplified a second time, potentially using nested primers or other specialized primer designs. This secondary amplification can increase the number of amplicons dramatically. For example, if each of the thousand initial amplicons is amplified by another factor of a thousand, this results in a net amplification of a million-fold from the original target to the final amplicon concentration. This process can be repeated multiple times, each cycle increasing the number of amplicons, thereby enhancing the detection sensitivity to potentially single-molecule levels. However, it is important to note that while this method preserves the linearity of the reaction, it may introduce a noise factor due to variations in droplet size poly dispersity, sample loss, or reaction efficiencies across multiple amplification steps. Despite these potential noise factors, the linearity between the final amplicon concentration and the original target concentration should remain approximately intact, allowing standard quantitation as described elsewhere herein, e.g., based on the peak height in capillary electrophoresis, to reliably indicate the concentration of targets. Thus, in some embodiments, the methods further comprise re-encapsulating the amplification products in at least one successive compartment and amplifying the amplification products to generate a successive round of amplification products with an identifiable signal prior to the analyzing.

[0121] In some embodiments, nucleic acid targets are derived from pathogens. In some embodiments, pathogens are one or more of: bacteria, virus, and fungi. In some embodiments, the probeset comprises probes sufficient to analyze at least 10 different pathogens simultaneously (e.g., at least 100, at least 1000, at least 10,000).

[0122] The application of the disclosed technology for microbial or viral detection offers substantial advantages, particularly in contexts where the sample contains a potentially large variety of pathogens at low concentrations. In hospital settings, for instance, instruments like the BioFire are often used to screen for a limited number of potential pathogens. While effective, these tests are qualitative and offer only presence-absence data, which can result in false positives. They are also expensive and necessitate splitting the sample to test for different pathogens, thereby reducing sensitivity and increasing the risk of errors. The unique features of the disclosed methods can address these shortcomings. First, the technology allows for the simultaneous testing of hundreds of pathogens in a single assay, obviating the need for sample splitting. This aspect is especially important for more comprehensive and sensitive detection, covering a broader range of pathogens than current methods permit. Second, the disclosed methods are quantitative, capable of distinguishing between highly abundant and less abundant pathogens. This feature can lead to more precise diagnoses by differentiating the actual cause of an illness from incidental microbial or viral presence. The ability of the disclosed methods to provide highly accurate and quantitative information with a very low limit of detection stands in stark contrast to the qualitative approaches used in qPCR assays. This higher level of specificity and sensitivity can significantly reduce false positives, which are common drawbacks of qualitative methods. Furthermore, the quantitative nature of the disclosed methods enables clinicians to better assess the severity of an infection, thereby guiding more effective treatment strategies. Cost is another compelling advantage of using the disclosed technology, especially in non-clinical settings such as agricultural and food testing, where budget constraints often dictate the feasibility of broad-scale pathogen screening. Unlike traditional methods, which may require expensive bespoke instruments, the disclosed methods can be implemented using high- throughput well plate systems, making it an economically viable alternative. In summary, the disclosed technology's ability to simultaneously test for multiple pathogens in a quantitative manner, coupled with its cost-effectiveness and high sensitivity, positions it as a highly valuable tool for microbial and viral detection in both clinical and non-clinical settings.

[0123] In some embodiments, analyzing comprises determining specific genetic features present in viruses or microbes in a sample by including a probeset specific for the features. In some embodiments, the features comprise one or more of 16S RNA sequences, sequences associated with virulence factors, and sequences associated with antibiotic resistance.

[0124] In some embodiments, nucleic acid targets are pathogens and the probeset comprises probes specific for host targets as controls or to provide normalization.

[0125] In some embodiments, sample is from a patient or subject screened for cancer or suspected of having cancer and wherein the probeset comprises a plurality of probes that bind to cancer-specific nucleic acid sequences. In some embodiments, cancer-specific nucleic acid sequences are one or more of a point mutation, a deletion, an insertion, a translocation, and a methylated base or sequences derived therefrom (e.g., bisulfite converted sequence). In some embodiments, cancer-specific nucleic acid sequences are specific for a particular type of cancer. In some embodiments, cancer-specific nucleic acid sequences are specific to a plurality of different types of cancer. In some embodiments, probeset comprises probes sufficient to analyze at least 10 different cancer-specific nucleic acid molecules (e.g., at least 100, at least 1000, at least 10,000).

[0126] In current healthcare settings, cancer diagnoses often occur when patients exhibit symptoms, which typically suggests the disease has progressed to a more advanced, and consequently, less treatable stage. There is a significant opportunity to improve outcomes by diagnosing cancer earlier in its development, and technologies that facilitate earlier detection are a crucial frontier in cancer research. The partitioned amplification technology disclosed herein is well-suited for this endeavor, drawing on the strengths of digital PCR technology, sensitivity, quantitative accuracy, and low rates of false positives, to offer a method of early cancer detection that is both reliable and precise. A notable feature of the disclosed technology is its ability to perform high multiplexing, which enables the simultaneous analysis of a vast array of targets. This capability can be harnessed to create a screening assay that covers a near-universal range of cancers. For example, by developing an assay incorporating 300-500 carefully chosen targets that represent the most common mutations associated with prevalent cancers such as lung, colorectal, breast, and prostate cancer, an assay is provided that can identify a substantial portion of these cancers at an early stage, radically improving the prospects for successful treatment. In some embodiments, this approach leverages the capabilities of cell-free DNA sequencing, a method that facilitates such early diagnoses but is currently limited by high costs. The disclosed technology offers a pathway to overcome this limitation, presenting a solution that is not only more economical but also simpler and faster, allowing it to become a standard routine test. By enabling the early diagnosis of a wide variety of cancers, the disclosed technology shifts the cancer diagnostic paradigm to a more proactive stance, thereby enhancing the efficacy of subsequent treatments and improving outcomes for patients globally.

[0127] In some embodiments, cancer-specific nucleic acid sequences reveal minimizing residual disease (MRD). In some embodiments, probes in the probeset are tailored to a specific patient based on cancer genomic sequencing data from the patient.

[0128] In the context of minimizing residual disease (MRD) where the imperative is to identify and quantify traces of disease remaining after treatment, there exists a pertinent role for advanced diagnostic technologies that marry both sensitivity and multiplexing capacity. Traditionally, the scope has been defined by either digital PCR, offering high sensitivity albeit with limited multiplexing abilities, or sequencing techniques which, while capable of high multiplexing, are confined by prohibitive costs and limited sensitivity. The disclosed technology leverages the merits of both established methods, thereby fostering a new paradigm in MRD assays. The intrinsic advantage of the disclosed technology lies in its fusion of digital PCR’s high sensitivity with an enhanced ability to multiplex, a synthesis that affords unprecedented precision in the detection process without escalating costs. Given that MRD assays often commence with a clear understanding of the genetic landscape of the individual’s disease, it becomes possible to formulate specific primers that are personally tailored to the patient, enhancing the assay’s precision. Current MRD strategies, like those utilizing Signatera technology, generally focus on a set of 16 patient-specific mutations identified through sequencing. Using the disclosed technology achieves not just a higher sensitivity but also a quantitative richness due to digital PCR’s innate benefits, eclipsing the capabilities of the existing sequencing techniques. A pivotal attribute of the disclosed technology in this scenario is its multiplexing prowess which enables the simultaneous screening for hundreds of mutations in the patient without incurring additional costs, a feat unattainable through traditional sequencing due to its restrictive costs associated with enhanced sensitivity. Furthermore, the utility of the disclosed technology extends to encompass the potential for regular monitoring of patients over time, a crucial aspect in MRD assessments. This is especially pertinent in the analysis of patients with various solid tumors; the technology’s heightened sensitivity coupled with its multiplexing capabilities make it adept at discerning the minutest traces of targeted agents in circulation, providing a nuanced view of the patient’s disease landscape over time. The disclosed technology thus stands as a viable cornerstone in the evolution of MRD assays, fostering a greater degree of personalized medicine through higher sensitivity, multiplexing capabilities, and costeffectiveness. It opens a pathway to not just enhanced cancer screening but also a diligent, ongoing analysis of MRD, harmonizing sensitivity with scope, and precision with affordability.

[0129] In some embodiments, analyzing comprises characterizing gene expression of the sample by estimated gene concentration of a plurality of genes of interest.

[0130] In some embodiments, analyzing comprises analysis of gene editing or gene editing materials to determine or quantify one or more of editing efficiency, editing yield, editing quality, or off-target editing. In some embodiments, the editing materials comprise cells, vector, nuclease, guides, or accessory proteins.

[0131] In some embodiments, the analyzing comprises analysis of one or more genetic features of a fetus, wherein the sample comprises one or more of maternal blood, vaginal swabs, or other maternal samples. In some embodiments, the one or more genetic features comprises aneuploidy, point mutations, insertions, deletions, translocations, or methylation status. The technology allows for the sensitive detection of a broad range of genetic features that can be indicative of various conditions in a fetus, including aneuploidy and other genetic abnormalities. The possibilities of what can be examined are expansive, including but not limited to, identifying copy number variants and pinpointing specific genetic mutations that may have arisen in the fetus or that the parents might carry. One of the significant advantages of using this method is the ability to conduct testing through non-invasive means such as using maternal circulation or even non-invasive vaginal swabs. This non-invasive approach stands as a substantial benefit, facilitating the collection of necessary data without undertaking more invasive procedures such as amniocentesis or chorionic villus sampling which carry with them a higher risk to both the fetus and the mother. Furthermore, the disclosed technology’s high sensitivity and accuracy enhance its capability to reliably detect a vast spectrum of genetic variances, making it a robust tool in prenatal diagnostics, potentially over time, giving detailed insights into the genetic health status of the fetus.

[0132] In some embodiments, the analyzing comprises detecting one or more mutations or other sequence variations in the target nucleic acids as compared to a wild-type sequence. For example, a common objective is to ascertain the presence of a genetic variant without necessarily distinguishing between specific types of variants utilizing methods which effectively differentiate between the wild-type sequence and any one of numerous potential variants (e.g., a sequence having one or more mutations / substitutions, insertions, deletions, or other sequence variations. In some embodiments, the methods use a blocking oligonucleotide or peptide which associates with the wild-type sequence and prevents amplification, thereby allowing amplification of only the variant sequences. Conversely, in the presence of a non-wild-type sequence (a variant), the blocking oligonucleotide does not bind, thereby allowing amplification to proceed.

[0133] Accordingly, blocking assays such as, for example, Locked Nucleic Acids (LNAs) and Peptide Nucleic Acids (PNAs) can be integrated with the disclosed methods. The blocking oligonucleotide or peptide binds selectively and tightly to the wild-type sequence, thereby inhibiting the amplification of the wild-type sequence, as the polymerase is unable to displace the blocking oligonucleotide. Designs of the blocking agents can be strategically aligned with design of the probeset, including the use of oligo flaps and non-duplex structures to enhance specificity while balancing melting temperatures and binding affinities to ensure that the blocking oligonucleotide remains firmly bound to the wild-type sequence. The presence of a sequence variant can then be detected through the analysis of the amplification products, e g., where the appearance of an associated peak signifies the variant's presence. This detection can be integrated with other methods described herein, such as ligation-based approaches, to provide a comprehensive and versatile system for variant detection.

[0134] In some embodiments, provided herein are methods for identifying an unknown nucleic acid in a sample, comprising: a) encapsulating nucleic acids from the sample in compartments under limiting dilution conditions with probes from a multiplexed probeset, wherein the probeset comprises specific, degenerate, and / or random probes; b) amplifying the nucleic acids with the probeset to generate amplification products in which the quantity of different amplification products exhibits sequence-specific bias, thereby generating an amplification length fingerprint at least partly unique to target nucleic acids within the sample due to unique sequence biases of the target nucleic acids; c) pooling the amplification products; d) analyzing the amplification products to detect the amplification length fingerprint; and e) comparing the amplification length fingerprint to the fingerprints of known targets, thereby allowing one to identify an unknown nucleic acid from said sample. In some embodiments, the comparing comprises projecting high dimensional fingerprint data onto a lower dimensional space (e.g., 2D space) to generate plots that are used for target identification by clustering and association. In some embodiments, the clustering and association comprises use of one or more of Principal Component Analysis (PCA), t-distributed Stochastic Neighbor Embedding (tsNE), or Uniform Manifold Approximation and Projection (UMAP). In some embodiments, the characteristic peaks in the fingerprint data are used to identify targets based on look-up tables of known peaks. In some embodiments, nanopore sequencing of the fingerprints is used to identify targets. In some embodiments, comparing comprises determination of identify of pathogens in the sample. In some embodiments, the comparing comprises determination of subsequences associated with specific genetic sequences or virulence factors associated with one or more pathogens. In some embodiments, the comparing comprises determination of one or more specific mutations present in nucleic acid in the sample.

[0135] The methods disclosed herein can be deployed in a randomized fashion to serve as a universal diagnostic tool capable of amplifying a broad array of targets in a sample. By employing universal probe sets in combination with a variety of amplification reactions like PCR, LAMP, or MDA, this approach generates amplicons of varying lengths. When analyzed through capillary electrophoresis or other length-measurement techniques, the outcome is a sample-specific signal. This unique 'fingerprint' generated randomly can be used for the identification of unknown pathogens or molecules without the need for sequencing. For instance, if one aims to identify an unknown virus in a sample, the virus can be encapsulated and subjected to randomized analysis. The resulting amplicon spectrum serves as a fingerprint that can be cross-referenced with fingerprint databases of known organisms to identify the unknown pathogen. The primary advantages of this approach are its generality, speed, and costeffectiveness. The method does not require sequencing, thus accelerating the diagnostic process and significantly reducing costs. Additionally, depending on the resolution of the system, it can identify a broad range of specific targets in a sample in a highly general way. This makes the disclosed methods a valuable approach for rapid and cost-effective diagnostics in diverse settings, from healthcare to food safety.

[0136] In some embodiments, provided herein are methods for quantitative targeted proteomics comprising: a) incubating a sample comprising proteins to be detected with reagents comprising affinity probes that bind to protein targets of interest; b) washing to recover proteins and their bound affinity probes; c) reacting proteins and their bound affinity probes with secondary affinity probes labeled with oligonucleotides specific to the secondary probes; d) washing to enrich target proteins and their dual bound affinity probes; and e) using the disclosed methods e.g., as described above or elsewhere herein) to quantitate dual bound target proteins (which may comprise from one to thousands of unique species) by quantitating oligonucleotides specific to the secondary affinity probes.

[0137] The methods disclosed offer a versatile platform for target protein analysis as well as target RNA and DNA analysis. This multi-omic capability enables the simultaneous analysis of DNA, RNA, and proteins from a variety of samples, including fresh and fixed tissue samples and even single cells.

[0138] In such embodiments, the sample is initially labeled with antibodies or other affinity probes that target one or more specific proteins of interest. Each antibody or other affinity probe include a unique oligonucleotide, thereby associating a unique oligonucleotide with each protein of interest. Largely, the binding of these affinity probes to their target proteins is proportional to the quantity of the protein present in the sample. Various techniques, such as fixation and permeabilization, can be employed to enhance the binding efficiency, thereby allowing for the detection of both surface and internal proteins within cells. Once the binding process is complete, the oligonucleotides attached to the antibodies or other affinity probes are recovered and subjected to analysis in the disclosed methods. In this process, probes are designed to target specific oligonucleotide tag sequences, generating amplicons of desired lengths corresponding to each oligonucleotide tag. This approach facilitates the multiplexed readout of proteins using the disclosed methods.

[0139] To integrate protein analysis with DNA and RNA analysis, DNA, RNA, and antibody / affinity probe oligonucleotide tag sequences are each recovered from the sample. The probeset includes probes targeting the desired DNA, RNA, and protein related target sequences. The total number of different targets across these nucleic acids is designed to fit within the multiplexing capacity. With the advanced multiplexing methods described herein, combined with techniques for generating a large number of compartments, it becomes feasible to detect hundreds of DNA, RNA, and protein targets simultaneously in a single reaction using the disclosed methods, facilitating comprehensive multi-omic readout of DNA, RNA, and proteins and offering a highly efficient alternative to sequencing-based approaches. This approach can be extended to single-cell analysis. By isolating individual cells into wells and performing DNA, RNA, and / or protein analysis as described, single-cell resolution can be achieved. To ensure single-cell sensitivity, enhanced sensitivity methods such as the use of larger droplets for greater amplicon production, or the implementation of re-encapsulating and successive amplification, as described elsewhere herein, can be employed.

[0140] The technology provided herein significantly expands upon the capabilities of the traditional digital ELISA process by substituting the enzyme labeling with oligonucleotide labeling. In this approach, the secondary antibody is labeled with a synthetic oligonucleotide, which can later pair with specific probes om the methods disclosed herein, paving the path for remarkably enhanced multiplexing capabilities. The choice of oligonucleotides as labels, as opposed to enzymes, is strategic in escalating the multiplexing capacity massively. While traditional ELISA could restrict one to analyzing a small number of different targets (generally as few as three) in a single solution due to the limitation in the variety of enzymes and their specific outputs, the technology provided herein leaps far ahead by enabling the simultaneous analysis of thousands of targets in a single solution. Leveraging the methods disclosed herein, which can detect thousands of distinct nucleic acid targets in a solution, researchers can selectively pair thousands of synthetic oligonucleotides with the corresponding secondary antibodies, thereby creating a vastly diverse library for high-throughput proteomics analysis.

[0141] In some embodiments, provided herein are methods for quantitative targeted protein detection comprising: a) labeling proteins present in a sample with unique aptamer sequences; and b) using the disclosed methods (e.g., as described above or elsewhere herein) to detect the aptamer sequences.

[0142] SomaLogic Aptamer Assay. In this strategy, proteins are labeled with aptamer probes, which are then leveraged to specify which proteins are present in a solution. Although traditionally the readout is done through sequencing, which is quite costly, utilizing the disclosed methods eliminates the need for sequencing, making it possible to detect thousands of distinct aptamers, and consequently the proteins they are bound to, in a cost-effective manner.

[0143] In some embodiments, provided herein are methods for quantitative targeted protein detection comprising: a) generating DNA concatenations that indicate presence of dual bound affinity probes to target proteins; and b) using the disclosed methods (e.g., as described above or elsewhere herein) to detect the DNA concatenations.

[0144] The goal of targeted proteomics is to selectively and sensitively detect specific proteins of interest present in a sample. The disclosed methods enhance the multiplexing and quantitation capacity in various proteomic assays such as digital ELISA, use SomaLogic aptamers, and Olink technology.

[0145] Olink Proximity Extension or Ligation Assay. Here, two antibodies bind to single proteins, facilitating the production of linked DNA molecules when they are in close proximity. These linked products, traditionally detected by expensive methods like qPCR or sequencing, can now be quantitatively analyzed through the disclosed methods, which allow enhanced accuracy, affordability, and multiplexing.

[0146] In some embodiments, a label of the present invention comprises a fluorescent label. In some embodiments, the fluorescent label comprises a label selected from the group comprising fluorescein and its derivatives, rhodamine and its derivatives, cyanine and its derivatives, coumarin and its derivatives, Cascade Blue and its derivatives, Lucifer Yellow and its derivatives, BOD IP Y and its derivatives, and the like. Exemplary fluorophores include indocarbocyanine (C3), indodicarbocyanine (C5), Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Texas Red, Pacific Blue, Oregon Green 488, Alexa fluor-355, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor-555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, JOE, Lissamine, Rhodamine Green, BODIPY, fluorescein isothiocyanate (FITC), carboxy- fluorescein (FAM), phycoerythrin, rhodamine, dichlororhodamine (dRhodamine), carboxy tetramethylrhodamine (TAMRA), carboxy-X-rhodamine (ROX), LIZ, VIC, NED, PET, SYBR, PicoGreen, RiboGreen, and the like. In some embodiments, the label is attached to a primer. In some embodiments, the label is attached to a nucleotide that may be incorporated into an amplicon during amplification.

[0147] In addition to traditional methods of multiplexing using multicolor fluorescence, excitation multiplexing can be utilized. Excitation multiplexing expands the number of possible indicators which can be used to differentiate amplification products in fluorescent analysis by utilizing fluorophores or dyes that emit in the same or similar wavelength range but have distinct excitation spectra. The same set of emission colors / spectra is reused, but they are paired with a different set of excitation wavelengths that can be activated with different excitations turned on. This pairing effectively creates additional excitation channels. By carefully selecting dye combinations that allow for this differentiation, it becomes possible to significantly enhance multiplexing capabilities. The fluorophores or dyes can include specific dyes, quantum dots, or fluorescent systems employing FRET (Fluorescence Resonance Energy Transfer) technology.

[0148] For example, excitation multiplexing expands the multiplexing capabilities beyond what is achievable with standard capillary electrophoresis systems, which typically utilize four different fluorescence channels. A conventional capillary electrophoresis machines, with four fluorescence channels, can easily detect 100 lengths per channel, yielding a total of 400 channels in the combined length and color space. However, with four emission channels and four excitation channels, one can achieve 16 unique channel combinations. If each of these channels can detect up to 100 different lengths, this approach allows for the quantification of up to 1,600 different targets in a single sample. By leveraging the unique properties of different fluorophores and their excitation spectra, excitation multiplexing offers a significant increase in the data density that can be obtained, e.g., from a single capillary electrophoresis run.

[0149] Droplet compartments can be made by any of a variety of methodologies (see e.g., Vladisavljevic et al., Production of uniform droplets using membrane, microchannel and microfluidic emulsification devices, Microfluid nanofluid (2012) 13: 151-178, herein incorporated by reference in its entirety). The invention covers a comprehensive array of methods for forming compartments on a sub-millimeter scale, including both microfluidic and non-microfluidic techniques. Bulk emulsification methods such as homogenization, vortexing, and jet emulsification are employed to form emulsions or other types of compartments. In homogenization, mechanical mixing is used to create stable emulsions, while vortexing utilizes high-speed rotary mixing to produce similar outcomes. Jet emulsification involves the use of liquid jets to create fine emulsions, allowing for precise control over compartment size. Alongside these, membrane emulsification methods are also utilized, which rely on microperforated membranes to facilitate the creation of small-scale compartments. These membranebased techniques offer a higher degree of control compared to bulk emulsification methods, making them suitable for specialized applications. Outside the realm of microfluidics, nonmicrofluidic methods like particle templating are also employed. In this approach, particles serve as templates around which compartments can form, offering yet another pathway for creating sub-millimeter compartments. Within the field of microfluidics, specialized techniques such as flow focusing, step emulsification, and T-junctions are also employed. These techniques allow for the formation of various types of compartments, such as water-in-oil single emulsions, waterin-oil-in-water double emulsions, and even more complex structures like quadruple and quintuple emulsions. In addition to these compartmentalization methods, micron-scale physical wells can be fabricated in plates or on chips. These methods bear resemblance to techniques used in digital PCR and facilitate the creation of either uniform or polydispersed emulsions. Nonuniformity in compartment size can be strategically used to extend the dynamic range of measurements, particularly when screening over different droplet sizes.

[0150] Distribution of droplet size (both in terms of average and standard deviation) influences the accuracy, reliability, and sensitivity of the method. For instance, when emulsions are generated through vortexing, the resulting droplets are typically small (around 15 microns). While this produces a high number of droplets, their small size means each droplet generates a small amount of amplicon. Consequently, detecting a specific nucleic acid target involves a sufficient number of that nucleic acid target across various droplets to accumulate a quantity of amplification products for detection. By controlling droplet or compartment sizes, larger droplets can be produced, which enhance sensitivity and lower limit of detection, and reduce poly dispersity, thereby improving the quantification accuracy. Since limiting dilution is key to enabling accurate quantification of all the desired nucleic acid targets simultaneously and over the desired dynamical range, a high enough drop number ensures limiting dilution for all nucleic acid targets for the desired dynamical range of measurement. For example, to measure 10 targets over a dynamical range of 3 decades approximately 1000 examples of each of the 10 molecules are used, thus at least 10,000 droplets. Since encapsulation is random, an increased number of drops (generally another 10-fold) facilitates sufficient numbers of empty drops so that co-encapsulation of multiple targets in one drop is minimized - resulting in at least 100,000 drops. Formulaically as shown below:

[0151] Nd = Nt * Ndyn * 1 / P where Nd is the desired number of drops, Nt is the number of targets being analyzed, Ndyn is the desired dynamical range for all targets, and P is the Poisson loading factor (for example, 10% loaded drops). Ultimately, the number of drops is balanced to be sufficient to obtain quantitation, but also control droplet size and effect limit of detection. Limiting dilution (Poisson loading) enables multiple targets to be measured independently because if you have multiple targets in the same drop, the resultant amplification will not scale proportionally to each target's concentration in the drop due to primer competition.

[0152] The invention also incorporates a variety of mass-tagging techniques, similar to fluorescence or mass cytometry (see e.g., Spitzer and Nolan, Cell (May 5, 2016) 165:780-791; Tanner et al., Cancer Immunol. Immunother. (2013) 62:955-965; Delgado-Gonzalez and Sanchez-Martin, Anal. Chem. (2021) 93:657-664; herein incorporated by reference in their entireties). A straightforward example of a mass tag is the use of Rare Earth elements, which provide unique mass spectra and can label a large number of antibodies simultaneously. This method is compatible with mass cytometry or CyTOF technologies. These tags can also be implemented in nucleic acid primers, offering flexibility in tagging approaches. For example, during the disclosed methods, amplicons with these mass tags can be produced. These amplicons can carry a fluorescent tag on one end and a mass tag on the other, allowing for a three- dimensional characterization of samples involving size, fluorescence, and mass.

[0153] The invention also incorporates the use of nucleases. Nuclease-based multiplexing enhances the detection capabilities of the disclosed methods, particularly in applications requiring the simultaneous detection of a vast array of targets. In most separate techniques, a limited number of length channels is available. To overcome the limitation of length space, different nucleases generate specific products based on location of the cleavage site within the amplification product.

[0154] In the disclosed methods amplification occurs in droplets at limiting dilution, which ensures that the height of each peak is directly proportional to the original concentration of the target. This linear relationship allows for the estimation of target concentration based on peak height. In implementing this technology, specific targets are associated with amplicons of distinct lengths. There are several ways to achieve this. The most direct method involves designing primers that bind to the target with a precise length separation, thus generating amplicons of desired lengths for each target. However, amplicons of varying lengths tend to amplify at different rates. For instance, shorter amplicons often amplify more efficiently than longer ones. This differential amplification rate can lead to issues such as the dropout of long amplicons.

[0155] An alternative strategy involves designing all amplicons to be of equal length initially and then digesting them with a nuclease to yield fragments of the desired lengths. This method has the advantage of ensuring similar amplification rates for all targets while still allowing differentiation based on length during capillary electrophoresis. A particularly efficient variation of this method employs ligation probes. In this approach, the ligation of the target is necessary to generate an amplicon. Amplification occurs using universal primers present in all droplets, contingent on the successful ligation. The nuclease digestion site is placed within a specific ligated product to generate linear amplicons of predetermined sizes. This results in initially equal-sized amplicons, which, upon nuclease digestion, yield fragments suitable for length-based differentiation.

[0156] In some embodiments, specific primers are integrated into a ligation probe system containing a nuclease site. Different nucleases digest the sample in separate reactions, creating two types of products for each nuclease digestion reaction: one where the amplification product is undigested (indicating the absence of a nuclease site for that specific nuclease), and the other where the amplicon is digested (indicating a match with the nuclease), producing a fragment of a desired identifiable length. In essence, each nuclease digestion reaction independently quantitates different amplicons for varying targets, with the digested lengths corresponding to specific targets as per the ligation probe design. This process can be repeated with different nucleases, each generating unique products. The association between separations and specific nucleases allows for the identification of products corresponding to that nuclease, as predetermined during the ligation probe design phase. The use of nuclease digestion sites in this manner significantly enhances the multiplexing power of the disclosed methods. For instance, using two nucleases recognizing different sequence sites can double the number of targets quantifiable simultaneously.

[0157] This multiplicative increase in multiplexing capability can be further combined with other methods, such as fluorescence, to achieve even higher levels of multiplexing. For example, using four colors of fluorescence and four different nucleases in a capillary electrophoresis-based method, facilitates multiplexing of 16 different reactions, allowing for the detection of up to 16,000 different targets in a single reaction (considering a typical capillary electrophoresis machine's capacity to differentiate a thousand lengths).

[0158] An additional method for analyzing the amplification products involves using sequencing. In this approach, the amplification products are sampled and sequenced in a randomized fashion. This sequencing determines the levels of various molecules present in the sample. Unlike other methods that might require length encoding or other forms of labeling, this technique identifies the specific sequences of the molecules. Computational alignments are utilized to identify and quantify the molecules present in the sample. Since the molecules are amplified in a partitioned manner during the amplification step, the counts obtained from sequencing should be proportional to the target concentration. This proportionality is analogous to the peak heights observed in a typical capillary electrophoresis reaction. Employing sequencing as an alternative has the advantage of requiring no additional labeling schemes. It leverages the inherent sequences of the targets for their identification, eliminating the need for external labels or markers, relying instead on the intrinsic genetic information of the targets for quantification and analysis. This approach can be particularly valuable in situations where the preservation of the native state of the nucleic acids is important or when the addition of external labels is impractical or undesirable.

[0159] Also provided herein are systems (e.g., instruments, software, and associated reagents and robotic) that conduct any of the methods described herein to analyze a sample suspected of containing one or more targets. In some embodiments, the system comprises a well plate processing component and is configured for simultaneously analyzing multiple targets in a plurality of different samples. For example, the reactions can be performed in well plates, then the plates prepared and processed with any of the separation techniques described herein which are compatible with well plates, e.g., capillary electrophoresis. This would permit the analysis of 96, 384, etc., samples in minutes to hours in a highly parallel process.

[0160] EXAMPLES

[0161] Data demonstrating self-assembled partitioning allows scalable generation of monodispersed emulsions for PACE.

[0162] A major strength of SAP is that, because the droplets are generated in parallel, the number formed scales with the size of the reaction, not emulsification time as in microfluidics. This allows emulsions comprising IM, 50M, and 250M droplets to be generated in 30 s (FIG. 2A). Additionally, because PTE is a local process governed by average shear energy, particle properties (size, elasticity, and adhesion), and fluid properties (viscosity, interfacial tension) (35), it is insensitive to container size and shape. As we show in preliminary experiments (60), it generates monodispersed droplets in PCR, Eppendorf, and falcon tubes (FIG. 2A) and 96, 384, and 1536 well plates (FIG. 2B). In addition to making the approach scalable, generating the emulsions in the wells in which the samples are stored is a significant simplification that mitigates target loss and contamination. The emulsions are uniform, with a size dictated by the templating particle diameter and taking 30 s regardless of total emulsion volume (FIG. 2C). Droplet compartmentalization overcomes reaction inhibition in unprocessed samples, simplifying workflows. qPCR is sensitive to inhibitory molecules that can skew amplification rates (74,75). Thus, care must be taken to remove inhibitors through sample purification. This adds steps, reduces turnaround time, and adds costs associated with the purification reagents. In contrast, dPCR is intrinsically robust to inhibition because inhibitory factors are sequestered in droplet subsets and do not inhibit other droplets (FIG. 3) (56,74,75). Consequently, while qPCR is inhibited when performed on crude lysate without nucleic acid purification, dPCR is not. It was demonstrated that dPCR can accurately quantitate SARS-CoV-2 viral targets in raw, unprocessed samples, removing the need for nucleic acid purification, and minimizing contamination (56) (FIG. 4). Because PACE is a variant of dPCR, this robustness extends to it.

[0163] PACE exceeds qPCR in sensitivity. Because dPCR cycles to endpoint, it is insensitive to reaction efficiency or when the exponential phase initiates; this obviates the need for standard curves and is the source of dPCR’s superior sensitivity. Because PACE can also cycle to endpoint (35), it is also insensitive to these factors and, thus, requires no standard curve and matches the sensitivity of dPCR. To demonstrate this, we have performed experiments in which we directly compared the quantitation of PACE with qPCR. We prepared a dilution series of SAR-CoV2 genomes over 5 decades, and analyzed them in duplicate with all three methods. When we plot the measured copy number for qPCR versus dPCR, we find they agree to ~20 molecules, at which point qPCR saturates and exhibits increased noise (FIG. 5, red points).

[0164] These concentrations correspond to Ct-values above 32 cycles, in line with observations from others that Ct values above this point often become unreliable (49,76,77). By contrast, with PACE, we match the dPCR results down to ~1 target per 10,000 molecules, the LOD of dPCR (FIG. 5, purple points). By exploiting PACEs scalability, we can increase the number of droplets to 10 million, thereby increasing sensitivity by lOx, precision 25x, and dynamic range to 7 decades.

[0165] Capillary electrophoresis allows sensitive quantitation of nucleic acids with singlenucleotide resolution.

[0166] Capillary electrophoresis is a mature technology with a history spanning decades, being the basis of the first widespread sequencing technology (38). Consequently, significant investment has gone into optimizing CE for the quantitation of nucleic acids of different length, the basis of the Sanger sequencing method. These instruments achieve single-nucleotide resolution and simultaneous measurements of identically sized, but distinctly labeled, amplicons (63). Additionally, sensitive fluorescence quantitation (39-41) combined with precision electrophoretic aspiration to load different amounts of sample into the capillary allow for measuring product concentrations across 8 decades.

[0167] Exemplary PACE methodology

[0168] To match existing instruments, 100,000 droplet reactions (5 decades) are employed using 85 pm droplets (400 pL) with 75 pm particles. To enable 10 million droplet reactions (7 decades), 27 pm droplets (10 pL) with 20 pm particles are used. Perfluorinated oils and surfactants afford excellent stability to thermal cycling over this range of sizes (35,78). Many targets of interest require lysis before they can be quantitated, including cells, viruses, and exosomes. Moreover, in some instances, it is desirable to encapsulate the full organism in one droplet, then quantify its nucleic acids. This is sometimes necessary to determine the intactness and viability of the target agent using a multiplexed PCR assay (79-82). Lysis before encapsulation can result in DNA or RNA degradation, confounding results. The challenge is that bulk emulsification requires all reagents be combined prior to encapsulation; addition of lysing agents at this step would pre-lyse organisms. In some embodiments, in-droplet lysis is employed using micellular transport. For an emulsion to be stable, the surfactant concentration in the carrier phase should be above the critical micelle concentration (83,84). Under these conditions, the carrier phase is laden with nano-scale surfactant micelles with a hydrophilic core. These micelles have been previously shown to allow the passive transport of small, hydrophobic, or amphiphilic molecules into the droplets by dissolving them in micelles in the carrier phase (85- 88). As we have shown in preliminary experiments, this allows lysing agents (water-soluble detergents) to be introduced to the droplets after they have been formed, to induce cell lysis in combination with enzymes. Even gentle surfactants compatible with reverse transcription and PCR are potent lysing agents when combined with the elevated temperatures of thermal cycling (89,90), providing an effective means by which to lyse a broad variety of organisms. For robust microbes, such as fungi, gram-negative bacteria, and archaea, cell wall degrading enzymes can also be included that are kept cold prior to encapsulation then activated upon heating.

[0169] Emulsion stability can be assessed optically. Micelle transport can be assessed by demulsifying the processed emulsion, then measuring surfactant concentrations using LC-MS and surface tension contact angle. Pre-lysis can be assessed optically with calcein stains (live / dead) by imaging the droplets prior to addition of the lysing agent. In-droplet lysis efficiency can be assessed by imaging the droplets after addition of the lysing agent to confirm dissolution of the cell.

[0170] A key feature of PACE is its ability to multiplex numerous targets in a single reaction without sample splitting. This enables numerous high value applications beyond reach with conventional dPCR. Any multiplexing strategy should pair a given target amplicon with a unique label. In PCR, this is done using TaqMan assays in which each probe is labeled with a unique dye. However, the visible light spectrum is small, and fluorescent dyes have broad emission spectra. Consequently, with TaqMan, it is challenging to multiplex beyond 6 colors, the current state of the art in dPCR (Bio-rad, Stilla) (28,29). In PACE, a length spectrum is used as a space in which to identify amplicons of different targets. Each target is associated with an amplicon of a specific length, with the height of the peak relating to its count. To assign each amplicon to a specific “length channel” we design the primers to span the requisite distance. This is accomplished using available automated primer designers that assess optimal locations for a given target, and check for undesirable primer-dimer and off-target amplifications based on consultation of available databases (Blast) and inter-library primer complementarity (91-93). While primer design is complex, much work has gone into developing algorithms for efficient primer design, with multiplexed sets applied to complex samples now achieving 20,000-plex, single-pot assays (94). As with any encoding scheme, the number of available channels in a spectrum depends on the size of the spectrum and width of the peaks. With CE, nucleic acids can be quantitated from tens of bases based to a kilobase (63). For PACE, the optimal range is 200- 500 bp, since this produces amplicons that amplify at similar yields (64), and can be separated with single nucleotide resolution via CE. Setting a conservative spacing of 5 bp between amplicons, this will allow 50 targets to be multiplexed in one reaction.

[0171] The successful implementation of our PACE assay in diagnosing infections benefits from the meticulous processing of various human specimens, including blood, sputum, and cerebrospinal fluid. Effective sample preparation is important for eliminating contaminants or inhibitory molecules that can interfere with the efficiency of the PCR amplification process. Initially, the biological samples are subjected to chemical lysis to facilitate the release of cellular contents. Post lysis, we will proceed with the extraction and purification of nucleic acids, a step that is pivotal in ensuring the robustness of our subsequent PACE assay. Commercially available kits such as QIAGEN's QIAamp DNA Mini Kit or Invitrogen's PureLink Genomic DNA Mini Kit can be employed, which are proven to be reliable for this purpose. Following the purification, the nucleic acids is primed for the PACE technology, to allow for their efficient amplification. PACE methodology for Pathogen Detection

[0172] Multiplexed testing for bacterial infections is important for a variety of serious health conditions, including sepsis, gastrointestinal infections, and infections of the central nervous system. For an infectious disease diagnostic to be most useful, it should diagnose infection by virtually any pathogen. Currently, confident diagnosis of these infections requires NGS to cover the necessary pathogen range and takes over a month. With PACE, we can target sufficient pathogens and obtain necessary quantitative information to shorten this process to under a day. In some embodiments, the PACE targets common pathogens found in cerebrospinal fluid specimens obtained via lumbar puncture. Each organism is targeted with multiple amplicons, to increase detection probability and provide some level of strain resolution. In some embodiments, 16S ribosomal targets are employed. Fungi are included via assays targeting the fungal internal transcribed spacer (ITS) sequence (98). Most universal ribosomal primers are constrained by the inherent trade-off between which conserved region of ribosomal DNA is used for primer annealing and the need to span ribosomal hyper-variable regions (HVRs) that provide the most species-resolving information (99). PACE overcomes this limitation by allowing detection of all nine HVRs, and other genomic loci, even when utilizing the most generalized bacteria-detecting primers. Further motivating the 16S and ITS sequences as focal points is the fact that most organisms carry multiple copies of the ribosomal DNA clusters interspersed in their genomes, enhancing sensitivity (100,101). The assay also includes antimicrobial resistance phenotypes, as ribosomal mutations are often associated with drug tolerance (102).

[0173] Primers are included for common causative CNS infection pathogens including Escherichia coli KI, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus agalactiae, Streptococcus pneumoniae, cytomegalovirus, enterovirus, herpes simplex virus 1, herpes simplex virus 2, human herpesvirus 6, human parechovirus, varicella zoster virus, Cryptococcus neoformans / gattii. Human Cytomegalovirus (HCMV) can also cause meningitis and is often present in CSF. Primers are also included for Cryptococcal disease diagnosis caused by closely related yeasts, C. neoformans and C. gattii, infecting the immunocompromised (HIV, transplant) and immunocompetent. This is important because delayed diagnosis of these infections is associated with poor clinical outcomes (110). Cryptococcal disease is often missed with qPCR techniques (111), with new disease, stressing the importance of more sensitive and accurate diagnostics.

[0174] Highly Multiplexed Digital PCR Assay for Detection of KMT2A Fusions in AML.

[0175] The goal of this assay is to enable the detection of KMT2A fusions in acute myeloid leukemia (AML) by employing the methods described herein. Its primary aim is to distinguish between mRNA molecules with and without KMT2A fusions. This method uses specific amplicons as indicators of KMT2A fusions, irrespective of the exact fusion breakpoint. It operates by generating a 100-base amplicon when the 5’ end of the KMT2A gene is present in the molecule and, thus, in the compartments. To detect if KMT2A is fused with another protein, a second primer / probe set targets approximately 80 common fusion partner proteins. These partners are each assigned to amplicons of distinct lengths, ranging from 100 to 179 bases. If both are present in the same droplet or compartment, splicing by overlap extension (SOEing) PCR is employed to join the KMT2A amplicon with those of the partner proteins. The resulting fused amplicons, varying from 200 to 279 bases, indicate the specific partner protein fused with KMT2A. In cases where KMT2A is not fused, no such product is formed. Capillary electrophoresis is used for the final analysis. This allows for the identification of the presence and specific lengths of fused amplicons. By examining these lengths and their associated peak heights, the assay accurately identifies the presence or absence of KMT2A fusions and quantifies their concentrations relative to unfused components, offering a simpler and more cost-effective alternative to conventional sequencing assays, since no sequencing is required.

[0176] Amplicon length independence.

[0177] Methods in which amplicon length are used to a identify a target amplicon can be challenging, utilizing primers designed to yield an amplicon of the desired length, necessitating precise spacing on the target. Selecting the most effective primers becomes challenging, particularly due to the structural or sequence characteristics of the target. Moreover, mutations altering the target's length can disrupt the amplicon's length, leading to misidentification. A solution to these challenges is to make the target length independent of the primer selection for detecting the target's presence, referred to herein as “length independence.” Length independence decouples amplicon length from the primer sequences used for detection. One such method utilizes ligation assays. In such embodiments, primers are strategically placed and ligated on the target, forming a linkage that triggers the generation of the desired amplicon. Notably, the target region is not included in this amplicon; its production depends solely on the presence of the linkage. Another strategy employs padlock probes, which form a loop-like structure when annealed to their target. The length of the resultant amplicon is determined by the loop size between the two primers, making it independent of the primer's location on the genome. Inverse nested PCR is an additional alternative. This technique begins by creating an initial amplicon incorporating flanking sequences, which are then amplified in a secondary PCR to produce amplicons of the desired length. This method further exemplifies the technology's capability for achieving length independence. These strategies enhance the versatility, flexibility, and efficiency of the disclosed methods in various diagnostic applications. Detecting microsatellite instabilities in colorectal cancer.

[0178] The ability of the disclosed methods to conduct highly multiplexed and quantitative analysis of target nucleic acids from a range of sample types, including fresh, frozen, or fixed tissues is extremely useful in diagnostics involving microsatellite instability (MSI), a condition characterized by changes in DNA length. Uniquely, the disclosed methods provide a means to not only accurately measure the number of repetitions within a DNA sequence but also offers single base pair resolution. For thorough MSI detection, a comprehensive panel of microsatellite markers is utilized, including: mononucleotide repeat markers, such as BAT-25, BAT -26, NR- 21, NR-24, and MONO-27 which have high sensitivity and specificity in MSI detection; and dinucleotide repeat markers, such as D2S123, D5S346, and D17S250 which facilitate robust assessment of MSI across various types of cancer. This marker panel facilitates the detection of MSI in diverse cancers, particularly focusing on gastric and colorectal cancers where MSI significantly influences prognosis and treatment strategies. The disclosed technology excels in measuring microsatellite length variations with single base pair accuracy and provides a quantitative estimate of the abundance of such targets in the sample. This level of precision is crucial for distinguishing between MSI-High (MSI-H), MSI-Low (MSI-L), and Microsatellite Stable (MSS) tumors, impacting patient prognosis and informing therapy selection. In a clinical setting, this assay emerges as a potent tool for cancer diagnosis and prognosis. It is instrumental in identifying patients who might benefit from specific treatments, such as immunotherapies for MSI-H tumors. The technology's high throughput and accuracy also render it ideal for large- scale screening, particularly in cancers linked to Lynch syndrome. From a research perspective, the technology paves new pathways for delving into the role of MSI in cancer biology. It enables detailed studies of MSI's impact across various cancer types, potentially leading to novel insights and advancements in the field.

[0179] Multiplexed Ligation-dependent Probe Amplification (MLPA) Optimization.

[0180] Multiplexed Ligation-dependent Probe Amplification (MLPA) serves as a robust platform for SNP detection and allows for the amplification of constant-length amplicons using universal primers across all targets. The process begins with the design of target-specific primers that cover the desired detection targets, being ligated as specific pairs when targets are present. Once the ligation step is completed, the ligated products are encapsulated into droplets and amplified using primers specific to the ligated probes. This approach allows a single primer pair to amplify a large number obligated products for different targets, by base pairing with designed “universal sequences” on the ligation probes. The ligation probes may be grouped into different subsets to utilize a different “universal primer pair” to amplify them so as to encode additional signals into the amplicons during the amplification step, such as the incorporation of different dyes, amplicon lengths, or sequences. For each target, the ligation can be designed to produce, for example, 200 base pair amplicons, achieved by ligating two 100 base pair oligos. This design facilitates generation of resulting amplicons that are of a uniform size, facilitating efficient and consistent amplification with minimal bias or dropout. The use of universal primers in this step is particularly advantageous, as it contributes to the high-efficiency amplification of amplicons, especially those in the 100 to 200 base pair range. Further enhancing the multiplexing capability of this assay is the integration of nuclease multiplexing. The ligation probes can be engineered to include specific nuclease digestion sites corresponding to, for example, four different nucleases. This integration allows for splitting of the amplified sample into separate nuclease reactions. Each reaction is then subjected to digestion with its corresponding nuclease, followed by separate analysis through capillary electrophoresis runs. The analysis can also use excitation multiplexing, involving the use of a combination of four-color fluorescence along with four excitation multiplexing channels. The integration of these channels with the nuclease multiplexing creates a system, capable of delivering a total of 4 excitations, 4 emulsions, and 4 nuclease channels, yielding a total of 64 total channels. With each channel having the capacity to detect up to 100 lengths, the assay achieves detection of up to 6400 different targets in a single reaction.

[0181] Single-Cell Multiomics Analysis Using PACE Combined with Flow Cytometry.

[0182] The disclosed methods can be used with multiomic analyses at the single-cell level. This is achieved by integrating the disclosed methods with flow cytometry, enabling comprehensive analysis of large numbers of individual cells. The process begins with the labeling of cells using oligo tagged antibodies and other messengers. These tags not only prepare the cells for subsequent analysis but also allow for the retention of crucial fluorescence information related to cell properties, such as protein content. Flow cytometry is then employed to analyze, sort, and isolate these labeled cells into individual wells. Each well may retain the fluorescence data corresponding to the cell's properties, thereby preserving information for further analysis. Once isolated, the cells undergo lysis to release their nucleic acids, rendering them accessible for analysis. This includes processing steps like reverse transcription and / or ligation, as necessary, to produce substrates for quantitation. The droplets are generated at a size conducive to single molecule sensitivity sufficient for single cell analysis, followed by the amplification reaction. This step results in the production of amplicons where the target identity is encoded using various methods, such as nuclease digestion sites, fluorescence excitation multiplexing, and specific amplicon lengths. The amplicons, produced within the droplets, are then recovered and subjected to capillary electrophoresis. The different omic readouts, encoded across various multiplexing channels, are then individually analyzed.

[0183] Utilizing this approach, highly comprehensive single-cell analyses essential in applications such as cell and gene therapy can be performed. Such applications often measure compositional phenotypes through surface protein analysis, assess the percentage of cells transduced with a vector, determine vector titers, and evaluate genome stability. The technology allows for the quantification of vector present within a single cell, aiding in the calculation of transduction percentages. Genome stability can be assessed by employing probes targeting various locations across the genome, thereby providing single-cell copy number variation (CNV) measurements, multiplexed with vector copy number data and compositional phenotypes based on oligo tagged surface proteins. Furthermore, off-targeting edits can be detected using a fusion assay, where a fusion product comprising the vector insert and adjacent nucleic acid at the desired insertion site is generated. Proper insertions yield a fused product of a specific length, identifiable in the capillary electrophoresis. Conversely, off-target insertions, not connected to the desired genomic junction, result in a distinct peak length, indicative of the vector without the fusion. Thus, the disclosed methods can concurrently measure various cell properties, typically requiring separate assessments, in a single assay. By combining the disclosed methods with cell isolation methods like flow cytometry, single cell analysis can offer increased detail and efficiency in multi omic cell profiling systems and methods.

[0184] High-Throughput Single-Cell Analysis Using PACE Technology.

[0185] As described above, the disclosed methods can be used as an alternative approach for high-throughput single-cell analysis, an area traditionally reliant on library preparation and sequencing. Instead of using sequence barcodes, length-encoded barcodes readable the disclose methods, e.g., with capillary electrophoresis, offer a streamlined and efficient alternative. The process begins with fixing and permeabilizing a population of cells for analysis. This preparation allows ligation probes to bind effectively to cellular DNA, RNA, and protein tags. Incorporation of isolation within hydrogels can further facilitate the openness of genomic DNA and efficient binding of ligation tags. Once prepared, ligation probes are applied en masse. These probes are designed to target specific DNA, RNA, or protein markers, creating a comprehensive library corresponding to various cells and markers of interest. After the ligation step, the probes are recovered and subjected to analysis. This involves digesting the cells and encapsulating their contents into droplets for amplification and analysis. For high-throughput analysis of a large number of cells, such as analyzing 10,000 cells with 10 markers each, the system requires a substantial number of channels. In this case, one would need 100,000 channels in total. To achieve this, the capillary electrophoresis's capability of providing 1,000 length channels can be expanded through a combination of 10 excitation multiplexing channels and 10 nuclease digestion site channels. This combination results in a total of 100,000 channels, spread across 10 different capillary electrophoresis lanes, thus providing the capacity for the analysis of 10,000 cells at 10 markers each.

[0186] Quantitative measurements for each marker are obtained based on peak heights in the capillary electrophoresis readouts. The nuclease digestion sites incorporated within the system play a key role, as they yield products that contribute to the 1,000 channels of length for each of the 10 different nucleases used. To ensure single molecule sensitivity, a sufficient number of droplets of an appropriate size are used, allowing for the accurate quantification of individual cellular components. In summary, this approach to high-throughput single-cell analysis leverages the unique capabilities of the disclosed technology, moving beyond traditional sequencing methods. By employing length-encoded barcodes and a sophisticated combination of multiplexing strategies, it facilitates the comprehensive and efficient analysis of thousands of single cells, along with multiple markers per cell, opening opportunities in fields requiring detailed cellular analysis, such as cellular heterogeneity studies, disease research, and personalized medicine.

Claims

CLAIMSWhat is claimed:

1. A method for analyzing target nucleic acids in a sample, comprising: encapsulating target nucleic acids in compartments under limiting dilution conditions with primers from a multiplexed primerset; amplifying target nucleic acids in the compartments to generate amplicons in a concentration proportional to the target nucleic acid concentration prior to amplification; separating amplicons by size; and matching amplicons to target nucleic acids of the same size.

2. The method of claim 1, wherein the amplifying step incorporates a detectable label in the amplicon.

3. The method of claim 2, wherein the detectable label is selected from a fluorophore, a mass tag, a molecular tag, a peptide, an antibody, a detectable nucleic acid sequence, a modified nucleotide, a small molecule, a lipid, a nanoparticle, or a combination thereof.

4. The method of claim 1, wherein the separating step comprises capillary electrophoresis.

5. The method of claim 2, wherein the separating step additionally comprises detection of the detectable label to distinguish amplicons of similar size but from different target nucleic acids.

6. A method for single-cell analysis, comprising loading a single cell into a compartment; creating an emulsion in the compartment; amplifying target nucleic acid in the compartment to create amplicons; and separating amplicons by capillary electrophoresis.

7. The method of claim 6, wherein the compartment is adapted to a parallel capillary electrophoresis reader.

8. The method of claim 6, wherein a detectable label is introduced during the amplifying step.

9. The method of claim 6, wherein a detectable label is introduced after the amplifying step.

10. The method of claim 8 or 9, wherein the detectable label is selected from the group consisting of a fluorophore, a mass tag, a molecular tag, a peptide, antibody, a detectable nucleic acid sequence, a modified nucleotide, a small molecule, a lipid, a nanoparticle, or a combination thereof.

11. The method of claim 6, wherein the reader is a microCE reader.

12. The method of claim 6, wherein the compartment is selected from the group consisting of a microwell or a microtiter plate well.

13. The method of claim 6, further comprising introducing surface probes to the cell to enable hashing to distinguish individual cells.

14. The method of claim 6, wherein the loading step comprises stochastic loading.

15. The method of claim 6, wherein the loading step comprises fluorescence-activated cell sorting.

16. The method of claim 6, further comprising associating a barcode oligonucleotide with each single cell.

17. The method of claim 16, further comprising attaching the barcode oligonucleotide to amplicons.

18. The method of claim 17, wherein the separating step comprises electrophoresis readout in which the barcode oligonucleotide length and the target nucleic acid are read as a combined length in order to distinguish cells.

19. The method of claim 16, wherein the cell is associated with the barcode oligonucleotide by co-encapsulating the cell with barcode-containing beads and wherein the barcode is integrated into the amplicon.

20. The method of claim 16, wherein the barcode oligonucleotide is attached to the cell prior to the creating step.

21. The method of claim 16, wherein amplicons from the same cell are of the same length.

22. The method of any of claim 1, wherein the amplifying step amplifies at least 5 different target nucleic acids.

23. The method of claim 1, wherein the amplifying step comprises a method selected from PCR, MDA, LAMP, RPA, and ligase chain reaction.

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