Methods and systems for single cell genetic profiling

Abstract

The present disclosure provides methods and systems for single-cell analysis of target cells (including single-cell transcriptome analysis) without microfluidic devices. The disclosed methods involve the use of template particles to template the formation of monodisperse droplets to generally capture within an encapsulation a single target cell from a population of cells, obtain a plurality of distinct mRNA molecules from the single target cell, and quantify the distinct mRNA molecules to generate an expression profile.

Description

[Technical Field]

[0001] Technical Field The present disclosure relates to methods and systems for single-cell genetic profiling. [Background technology]

[0002] background The complexity of biological systems requires many experiments to characterize them. High-throughput methods are often implemented to reduce the number of individual experiments that need to be performed. Unfortunately, methods for high-throughput analysis of single cells are limited by the costs associated with isolating single cells and preparing libraries.

[0003] Methods for isolating single cells generally require microfluidic devices that are complex to use and expensive to operate. Furthermore, because cells are processed individually, microfluidic devices are inherently limited in the number of cells that can be assayed in a given experiment. As such, high-throughput single-cell systems are unavailable in many clinical and research facilities. Summary of the Invention [Means for solving the problem]

[0004] Abstract The present disclosure provides methods and systems for single-cell analysis of target cells (including single-cell transcriptome analysis) without microfluidic devices. The methods and systems of the present invention generate emulsions with template particles to separate individual target cells into monodisperse droplets. Nucleic acid molecules are released from the target cells within the monodisperse droplets and quantified to generate an expression profile for each of the target cells. This approach provides an inexpensive and scalable massively parallel analytical workflow for confirming the expression profiles of millions of single cells with a single library preparation.

[0005] The present method and system uses template particles to template the formation of monodisperse droplets to isolate target cells for genetic profiling. The method includes combining template particles and target cells in a first fluid, adding a second fluid to the first fluid, and shearing the fluid to simultaneously generate a plurality of monodisperse droplets, each of which contains only one of the template particles and only one of the target cells. The method further includes lysing the target cells within the monodisperse droplets to release a plurality of distinct mRNA molecules and quantifying the plurality of distinct mRNA molecules. Data generated by quantifying the mRNA is used to generate an expression profile for each of the target cells. The method further includes processing the expression profile to identify characteristics of the target cells that can be used, for example, to make a diagnosis, predict a prognosis, or determine drug efficacy.

[0006] The method and system of the present invention provides a method for quantifying gene expression in target cells. The method involves releasing mRNA from target cells within monodisperse droplets. The mRNA can be reverse transcribed into cDNA and simultaneously barcoded. The barcoded cDNA is amplified to generate multiple barcoded amplicons. The amplicons can be sequenced by next-generation sequencing, and because of the barcode, each sequence read can be traced back to the target cell. The sequence reads are processed by a specific computer algorithm to generate an expression profile for the target cell.

[0007] After an expression profile is obtained from a target cell, the profile can be analyzed by comparing the profile to a reference or control profile to ascertain information about the target cell. In other cases, the profile of a target cell can be compared to a profile derived from a cell with a certain phenotype to determine whether the target cell shares characteristics of cells of that phenotype.

[0008] In one aspect, the methods and systems of the present invention provide a method for identifying the presence of rare cells in a heterogeneous cell population. The method includes isolating a plurality of target cells by combining the target cells and a plurality of template particles in a first fluid, adding a second fluid immiscible with the first fluid, and shearing the fluid to generate an emulsion containing monodisperse droplets containing the target cells and single template particles. The method further includes releasing a plurality of mRNA molecules within the droplets containing the target cells and quantifying the plurality of mRNA molecules. The quantifying step may include reverse transcribing the mRNA into barcoded cDNA. The barcoded cDNA may be amplified to generate a plurality of barcoded amplicons that can be traced back to the target cells. In some cases, the method may include sequencing the plurality of barcoded amplicons to generate sequence reads, for example, by next-generation sequencing. The method may further include processing the sequence reads to generate an expression profile for each target cell, and using the data by, for example, performing gene clustering analysis to identify one or more cell types or cell states among the target cells.

[0009] In another aspect, the disclosed methods and systems provide a method for analyzing a heterogeneous tumor biopsy taken from a subject. The method includes obtaining a biopsy from a patient and isolating a population of cells from the biopsy. The method further includes separating the population of cells into droplets by creating a mixture of the population of cells, a plurality of template particles, and an aqueous fluid, adding oil, and vortexing the mixture to produce an emulsion containing droplets, each droplet containing only one of the population of cells and the template particles. The method further includes releasing mRNA from each of the cells within the droplets and performing transcriptome analysis on one or more genes. Analysis of one or more genes can be used to identify one or more characteristics of cancer. The cancer characteristic can be the presence or absence of one or more gene transcripts associated with cancer. The methods disclosed herein can further include using the characteristics to diagnose a subject with cancer and devise a treatment plan.

[0010] In some aspects, the methods and systems of the present invention provide a method for determining the potential effectiveness of a therapeutic agent. The method includes separating a first population of diseased cells into droplets containing template particles and determining gene expression from at least one of the diseased cells, thereby generating an expression signature of the disease state. The method further includes exposing a second population of diseased cells to a drug, determining gene expression of the second population of cells, and comparing the gene expression to the disease state expression signature to confirm the effectiveness of the drug for the disease based on an increased or suppressed level of expression of one or more genes. In some embodiments, the therapeutic agent can be delivered to the second population of cells within the droplet. For example, the drug can be associated with the template particle by tethering the drug to the outer surface of the template particle or by packaging the drug within a compartment of the template particle and releasing the drug from the template particle within the droplet.

[0011] In certain aspects, the methods and systems of the present invention provide a method for separating cells into droplets. The droplets can be prepared as an emulsion, for example, an aqueous phase fluid dispersed in an immiscible phase carrier fluid (e.g., fluorocarbon oil, silicone oil, or hydrocarbon oil), or vice versa. Generally, the droplets are formed by shearing two liquid phases. The shearing step can include any one of vortexing, shaking, flicking, stirring, pipetting, or any other similar method to mix the solution. The method of the present invention includes combining cells and template particles in a first fluid, adding a second fluid, and shearing or stirring the first and second fluids. Preferably, the first fluid is an aqueous phase fluid, which in some embodiments may comprise reagents selected from, for example, buffers, water, lytic enzymes (e.g., proteinase k) and / or other lysis reagents (e.g., Triton X-100, Tween®-20, IGEPAL, or combinations thereof), nucleic acid synthesis reagents (e.g., nucleic acid amplification reagents or reverse transcription mix, or combinations thereof).

[0012] The methods and systems of the present invention use template particles to template the formation of monodisperse droplets and isolate target cells. Template particles according to aspects of the present invention may comprise, for example, a hydrogel selected from agarose, alginate, polyethylene glycol (PEG), polyacrylamide (PAA), acrylate, acrylamide / bisacrylamide copolymer matrix, azide-modified PEG, polylysine, polyethyleneimine, and combinations thereof. In certain cases, the template particles may be shaped to provide enhanced affinity for target cells. For example, the template particles may be generally spherical, but their shape may include features such as flat surfaces, craters, grooves, protrusions, and other irregularities in the spherical shape that promote association with the target cells, increasing the probability that the shape of the template particle will template droplets containing the target cells.

[0013] In some aspects, the methods and systems of the present invention provide template particles containing one or more internal compartments. The internal compartments may contain reagents or compounds that can be released upon external stimuli. The reagents contained by the template particles may include, for example, cell lysis reagents or nucleic acid synthesis reagents (e.g., polymerases). The external stimuli may be heat, osmotic pressure, or enzymes. For example, in some cases, the methods of the present invention include releasing reverse transcriptase directly into the droplets containing mRNA.

[0014] In some aspects, the methods and systems of the present invention provide a library preparation method for analyzing the transcriptome of a single cell. The method includes releasing mRNA from a single target cell contained within a droplet. In some embodiments, the released mRNA binds to a poly-T sequence of a barcoded capture probe attached to a template particle via complementary base pairing. Alternatively, the released RNA binds to a gene-specific sequence of the barcoded capture probe. After binding of the mRNA molecule to the capture probe, a reverse transcriptase synthesizes cDNA, thereby creating a first strand containing the cDNA and the capture probe sequence. The mRNA molecule-first strand hybrid is then denatured using any method known in the art (e.g., exposure to denaturing temperatures). In the next step, a second strand primer containing a random hexamer sequence anneals to the first strand to form a DNA-primer hybrid. A DNA polymerase synthesizes a complementary second strand. Optionally, the second strand is amplified, for example, by PCR, to generate multiple amplicons. The amplicons are analyzed to ascertain the expression profile of the single cell.

[0015] In certain aspects, the present disclosure provides a kit for single-cell profiling according to the methods of the present invention. The kit includes template particles containing multiple capture sequences specific to one or more genes of interest. Researchers following the instructions provided with the kit can use the template particles to assay single-cell expression of specific genes of interest (e.g., oncogenes). The kit can enable single-cell profiling according to the methods described throughout this disclosure (e.g., in FIG. 1). The template particles can be custom designed for the user's specific needs (e.g., designed to include capture probe sequences specific to a particular gene of interest (e.g., an oncogene)). The template particles can be transported within a sample preparation tube or a sample collection tube (e.g., a blood collection tube). The template particles can preferably be in a dry format. The kit can further include reagents (e.g., cell lysis reagents and nucleic acid synthesis reagents).

[0016] In another aspect, the method and system of the present invention provide a method for collecting data on the transcriptome of a single cell. The method comprises releasing a plurality of distinct mRNA molecules from a single cell into monodisperse droplets, collecting data on the transcriptome of the single cell, and transmitting the data to a computer. The computer can be connected to a sequencing device. The data corresponding to the transcriptome can also be stored after being sent, for example, the data can be stored on a computer-readable medium that can be retrieved from the computer. The data can be transmitted from the computer to a remote location, for example, via the Internet. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 illustrates a method for single cell profiling. [Figure 2] FIG. 2 illustrates a droplet according to one aspect of the present invention. [Figure 3]FIG. 3 illustrates the droplet after lysis of the target cells. [Figure 4] FIG. 4 illustrates the capture of mRNA. [Figure 5] FIG. 5 illustrates the synthesis of cDNA to form the first strand. [Figure 6] FIG. 6 illustrates first strand amplification to generate an amplicon. [Figure 7] FIG. 7 illustrates a method for sequence-specific capture of mRNA. [Figure 8] FIG. 8 illustrates the synthesis of cDNA to form the first strand. [Figure 9] FIG. 9 illustrates first strand amplification to generate an amplicon. [Figure 10] FIG. 10 illustrates the capture of mRNA according to a TSO embodiment. [Figure 11] FIG. 11 shows the first strand after TS-PCR amplification. DETAILED DESCRIPTION OF THE INVENTION

[0018] Detailed Description The present disclosure provides systems and methods for using template particles to form monodisperse droplets for isolating single cells, preparing library preparations thereof, and profiling the expression of the single cells. The disclosed methods involve using template particles to generally capture single target cells within encapsulations using the formation of monodisperse droplets as a template, obtain multiple distinct RNAs from the single target cells, prepare a library of nucleic acids that can be traced to the cells (from which the library of nucleic acids is obtained), and quantify the distinct RNAs to generate an expression profile of the single target cells. The methods of the present invention can be used to prepare libraries for single-cell analysis of, for example, at least 100 cells, at least 1,000 cells, at least 1,000,000 cells, at least 2,000,000 cells, or more cells from a single reaction tube.

[0019] FIG. 1 diagrammatically illustrates a method 101 for single-cell profiling. Method 101 includes combining template particles and target cells in a first fluid 109 and adding a second fluid immiscible with the first fluid to the mixture. The first fluid is preferably an aqueous fluid. While any suitable order can be used, in some cases, a tube can be provided containing the template particles. The tube can be any type of tube (e.g., a sample preparation tube sold under the trade name Eppendorf or a blood collection tube sold under the trade name Vacutainer). The template particles can be in a dried format. Combining 109 can include using a pipette to pipette a sample containing cells and, for example, the aqueous fluid into a tube containing template particles, and then adding a second immiscible fluid (e.g., oil).

[0020] The method 101 then includes shearing 115 the fluid to generate monodisperse droplets (i.e., droplets). Preferably, the shearing step includes vortexing the tube containing the fluid by forcing the tube over a vortexer. After the vortexing step 115, a plurality (e.g., thousands, tens of thousands, hundreds of thousands, millions, two million, ten million, or more) of aqueous partitions are formed essentially simultaneously. The vortexing step causes the fluid to partition into a plurality of monodisperse droplets. A substantial portion of the droplets contain a single template particle and a single target cell. Droplets containing more than one or no template particle or target cell can be removed, destroyed, or otherwise ignored.

[0021] The next step in method 101 is lysing 123 the target cells. Cell lysis 123 can be induced by a stimulus such as, for example, a lytic reagent, a detergent, or an enzyme. The reagent that induces cell lysis can be provided by the template particle via an internal compartment. In some embodiments, lysing 123 includes heating the monodisperse droplets to a temperature sufficient to release a lytic reagent contained within the template particle into the monodisperse droplets. This achieves cell lysis 123 of the target cells, thereby releasing mRNA into the droplets containing the target cells.

[0022] After lysing the target cells within the droplets (step 123), mRNA is released and subsequently quantified (step 131). Quantifying the mRNA (step 131) generally requires synthesizing cDNA to generate a library containing cDNAs with barcode sequences, allowing each library sequence to be traced back to the single cell from which the mRNA was obtained. In a preferred embodiment, the template particles isolated with the mRNA contain multiple barcoded capture sequences that hybridize with the target mRNA. After hybridization, cDNA is synthesized by reverse transcription. Reagents for reverse transcription can be provided in various formats and in various ways. In some cases, the reagents and reverse transcriptase are provided by the template particles. Once a library containing barcoded cDNAs is generated, the cDNAs can be amplified, for example, by PCR, to generate amplicons for sequencing. Sequence reads are processed according to the methods described herein to achieve mRNA quantification (step 131).

[0023] In some aspects, the target cells may include, for example, viable cells obtained from a patient sample (tissue, such as a bodily fluid). The sample may include a fine needle aspirate, biopsy, or bodily fluid from the patient. Upon isolation from the sample, the cells may be processed, for example, by generating a single-cell suspension in an appropriate solution. Such solutions are generally balanced salt solutions (e.g., normal saline, PBS, Hank's balanced salt solution, etc.), and in certain cases, supplemented with fetal bovine serum or other naturally occurring factors at low concentrations (e.g., 5-25 mM) along with an acceptable buffer. Convenient buffers include HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), phosphate buffer, lactate buffer, etc. Separated cells may be collected in any appropriate medium that maintains cell viability, typically with a cushion of serum at the bottom of the collection tube. Various media are commercially available and can be used depending on the nature of the cells, including Dulbecco's modified Eagle's medium (dMEM), Hank's balanced salt solution (HBSS), phosphate buffered saline (PBS), Dulbecco's phosphate buffered saline (dPBS), Roswell Park Memorial Institute medium (RPMI), Iscove's medium, etc., frequently supplemented with fetal bovine serum.

[0024] The methods and systems of the present invention use template particles to template the formation of monodisperse droplets to isolate single target cells. The disclosed template particles and methods for targeted library preparation utilize the particle-templated emulsification technique previously described in Hatori et al., Anal. Chem., 2018 (90):9813-9820, which is incorporated by reference. Essentially, micron-scale beads (e.g., hydrogels) or "template particles" are used to define an isolated fluid volume surrounded by an immiscible partitioning fluid and stabilized by a temperature-insensitive surfactant.

[0025] The template particles of the present disclosure can be prepared using any method known in the art. Generally, the template particles are prepared by combining hydrogel materials (e.g., agarose, alginate, polyethylene glycol (PEG), polyacrylamide (PAA), acrylate, acrylamide / bisacrylamide copolymer matrix, and combinations thereof). After the template particles are formed, they are sized to a desired diameter. In some embodiments, the size of the template particles is adjusted by microfluidic co-flow into an immiscible oil phase.

[0026] In some embodiments of the template particles, the variation in diameter or maximum diameter of the template particles is such that at least 50% or more of the template particles, e.g., 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more, vary in diameter or maximum diameter by less than 10-fold, e.g., less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, less than 1.5-fold, less than 1.4-fold, less than 1.3-fold, less than 1.2-fold, less than 1.1-fold, less than 1.05-fold, or less than 1.01-fold.

[0027] The template particle may be porous or non-porous. In any suitable embodiment herein, the template particle may include microcompartments (also referred to herein as "internal compartments"), which may include additional components and / or reagents (e.g., additional components and / or reagents that may be releasable into monodisperse droplets as described herein). The template particle may include a polymer, e.g., a hydrogel. The template particle generally ranges in diameter or maximum diameter from about 0.1 to about 1000 μm. In some embodiments, the template particle has a diameter or maximum diameter of about 1.0 μm to 1000 μm (inclusive) (e.g., 1.0 μm to 750 μm, 1.0 μm to 500 μm, 1.0 μm to 250 μm, 1.0 μm to 200 μm, 1.0 μm to 150 μm, 1.0 μm to 100 μm, 1.0 μm to 10 μm, or 1.0 μm to 5 μm (inclusive)). In some embodiments, the template particle has a diameter or maximum diameter of about 10 μm to about 200 μm, e.g., about 10 μm to about 150 μm, about 10 μm to about 125 μm, or about 10 μm to about 100 μm.

[0028] In carrying out the methods described herein, the composition and properties of the template particles can be varied. For example, in certain aspects, the template particles can be microgel particles, which are micron-scale spheres of gel matrix. In some embodiments, the microgel is composed of a hydrophilic polymer (including alginate or agarose) that dissolves in water. In other embodiments, the microgel is composed of a lipophilic microgel.

[0029] In other aspects, the template particle can be a hydrogel. In certain embodiments, the hydrogel is selected from naturally occurring substances, synthetically occurring substances, and combinations thereof. Examples of hydrogels include, but are not limited to, collagen, hyaluronan, chitosan, fibrin, gelatin, alginate, agarose, chondroitin sulfate, polyacrylamide, polyethylene glycol (PEG), polyvinyl alcohol (PVA), acrylamide / bisacrylamide copolymer matrix, polyacrylamide / poly(acrylic acid) (PAA), hydroxyethyl methacrylate (HEMA), poly(N-isopropylacrylamide) (PNIPAM), and polyanhydride, poly(propylene fumarate) (PPF).

[0030] In some embodiments, the template particles of the present disclosure further comprise a substance that provides the template particles with a positive or increased positive surface charge. Such substances can be, but are not limited to, polylysine or polyethyleneimine, or a combination thereof. This can increase the chance of association between the template particles and, for example, cells, which generally have mostly negatively charged membranes.

[0031] Other strategies can be used to increase the chances of template particle-target cell association, including creating specific template particle geometries. For example, in some embodiments, the template particle may have a generally spherical shape, but the shape may include features such as flat surfaces, craters, grooves, protrusions, and other irregularities in the spherical shape.

[0032] Any one of the above strategies and methods, or a combination thereof, can be used in the implementation of the template particles and methods of the present disclosure for the targeted library preparation thereof. Methods for the generation of template particles and template particle-based encapsulation were described in International Patent Publication WO 2019 / 139650, which is incorporated herein by reference.

[0033] The preparation of template particle-based encapsulation for single-cell expression profiling involves combining a target cell and a plurality of template particles in a first fluid to provide a mixture in a reaction tube. The mixture may be incubated to allow the plurality of template particles to associate with the target cells. Some of the plurality of template particles may become associated with the target cells. The mixture is then combined with a second fluid that is immiscible with the first fluid. The fluid and the mixture are then sheared to generate a plurality of monodisperse droplets in the reaction tube. The generated monodisperse droplets contain (i) at least a portion of the mixture, (ii) a single template particle, and (iii) a single target particle. It should be noted that, while a substantial number of the generated monodisperse droplets contain a single template particle and a single target particle in practicing the methods provided by the present disclosure, in some cases, some of the monodisperse droplets may contain zero or more than one template particle or target cell.

[0034] In some embodiments, to increase the chance of generating encapsulation (e.g., monodisperse droplets containing one template particle and one target cell), the template particles and target cells are combined at a fixed ratio, where there are more template particles than target cells. For example, the ratio of template particles to target cells 213 combined in the mixture described above can range from 5:1 to 1,000:1, respectively. In other embodiments, the template particles and target cells are combined at a ratio of 10:1, respectively. In other embodiments, the template particles and target cells are combined at a ratio of 100:1, respectively. In other embodiments, the template particles and target cells are combined at a ratio of 1000:1, respectively.

[0035] To produce a monodisperse emulsion, the disclosed method involves shearing a first mixture containing target particles and target cells and a second mixture formed by combining the first mixture with an immiscible second fluid. Any suitable method or technique can be utilized to apply sufficient shear force to the second mixture. For example, the second mixture can be sheared by flowing the second mixture through a pipette tip. Other methods include, but are not limited to, shaking the second mixture with a homogenizer (e.g., a vortexer) or a bead beater. In some embodiments, vortexing can be performed for, for example, 30 seconds, or for a period ranging from 30 seconds to 5 minutes. The application of sufficient shear force breaks the second mixture into monodisperse droplets that encapsulate one of the template particles.

[0036] In some aspects, generating the template particle-based monodisperse droplets requires shearing two liquid phases. The mixture is an aqueous phase and, in some embodiments, contains reagents selected from, for example, buffers, salts, lytic enzymes (e.g., proteinase k) and / or other lytic reagents (e.g., Triton X-100, Tween-20, IGEPAL, bm 135, or combinations thereof), nucleic acid synthesis reagents (e.g., nucleic acid amplification reagents or reverse transcription mixes, or combinations thereof). The fluid is a continuous phase and can be an immiscible oil (e.g., fluorocarbon oil, silicone oil, or hydrocarbon oil, or combinations thereof). In some embodiments, the fluid can contain reagents such as surfactants (e.g., octylphenol ethoxylate and / or octylphenoxypolyethoxyethanol), reducing agents (e.g., DTT, β-mercaptoethanol, or combinations thereof).

[0037] In practicing the methods described herein, the composition and properties of the monodisperse droplets (e.g., single emulsion and multiple emulsion droplets) can be varied. As mentioned above, in certain aspects, surfactants can be used to stabilize the droplets. The monodisperse droplets described herein can be prepared as emulsions, for example, as an aqueous phase fluid dispersed in an immiscible phase carrier fluid (e.g., a fluorocarbon oil, a silicone oil, or a hydrocarbon oil), or vice versa. Thus, the droplets can comprise surface-stabilized emulsions, for example, surfactant-stabilized single emulsions or surfactant-stabilized double emulsions. Any convenient surfactant that allows the desired reaction to occur in the droplets can be used. In other aspects, the monodisperse droplets are not stabilized by a surfactant.

[0038] 2 illustrates a droplet 201 according to one aspect of the invention. The droplet 201 shown is only one of a plurality of monodisperse droplets produced by shearing a mixture according to the methods of the invention. The droplet 201 includes a template particle 207 and a single target cell 213. The illustrated template particle 207 includes a crater-like depression 231 to facilitate capture of the single cell 213. The template particle 231 further includes an interior compartment 211 for delivering one or more reagents to the droplet 201 upon stimulation.

[0039] In some embodiments, the template particle comprises multiple internal compartments. The internal compartments of the template particle can be used to encapsulate reagents that can be triggered to release desired compounds (e.g., substrates for enzymatic reactions) or induce a specific outcome (e.g., lysis of associated target cells). The reagents encapsulated within the compartments of the template particle can be, but are not limited to, reagents selected from buffers, salts, lytic enzymes (e.g., proteinase k), other lytic reagents (e.g., Triton X-100, Tween-20, IGEPAL, bm 135), nucleic acid synthesis reagents, or combinations thereof.

[0040] Lysis of single target cells occurs within the monodisperse droplets and can be induced by stimuli such as heat, osmotic pressure, lysis reagents (e.g., DTT, β-mercaptoethanol), detergents (e.g., SDS, Triton X-100, Tween-20), enzymes (e.g., proteinase k), or combinations thereof. In some embodiments, one or more of the reagents (e.g., lysis reagent, detergent, enzyme) are partitioned within the template particle. In other embodiments, one or more of the reagents are present in the mixture. In some other embodiments, one or more of the reagents are added to a solution containing the monodisperse droplets, if desired.

[0041] 3 illustrates a droplet 201 after lysis of a target cell. The droplet 201 shown contains a template particle 207 and released mRNA 301. The method of the present invention quantifies the amplification products of the released mRNAs 301, preferably by sequencing.

[0042] In preferred embodiments, the template particle contains multiple capture probes. Generally, the capture probes of the present disclosure are oligonucleotides. In some embodiments, the capture probes are attached to the template particle's material (e.g., a hydrogel material) via a covalent acrylic bond. In some embodiments, the capture probes are acrydite-modified at their 5' ends (linker regions). Generally, acrydite-modified oligonucleotides can be stoichiometrically incorporated into hydrogels (e.g., polyacrylamide) using standard free radical polymerization chemistry, where the double bond in the acrydite group reacts with another compound containing an activated double bond (e.g., acrylamide). Specifically, copolymerization of the acrydite-modified capture probe with acrylamide, including a crosslinker (e.g., N,N'-methylenebis), results in a crosslinked gel material containing the covalently attached capture probe. In some other embodiments, the capture probe comprises an acrylate-terminated hydrocarbon linker, and combining the capture probe with a template particle causes them to bind to the template particle.

[0043] 4-6 show exemplary methods for non-specific amplification of mRNA according to certain aspects of the present disclosure. In particular, the methods rely on the presence of a polyA tail at the 3' end of mRNA for non-specific capture of mRNA.

[0044] Figure 4 illustrates the capture of mRNA 301. A template particle 201 is shown containing multiple capture probes 401, which are diagrammatically represented by dashed curves. One of the capture probes 401 is characterized on a larger scale and in more detail. The capture probe 401 preferably includes, from its 5' to 3' end, a linker region capable of covalently binding to the template particle 201, a PR1 471 nucleotide sequence region containing a universal primer nucleotide sequence, at least one barcode region B1 473, which may contain an index 475 nucleotide sequence index, and / or a UMI. The capture probe 201 further includes a capture nucleotide sequence containing a poly-T nucleotide sequence. The released nucleic acid, i.e., the mRNA molecule 301 containing a poly-A sequence, binds to the poly-T sequence 22 of the capture probe via complementary base pairing. After hybridization of the mRNA molecule 301 and the capture probe 401, a reverse transcriptase is used to perform a reverse transcription reaction to synthesize cDNA, thereby creating a first strand comprising the cDNA and the capture probe sequence.

[0045] 5 illustrates the synthesis of cDNA to form first strand 23. Reverse transcriptase (not shown) synthesizes cDNA from mRNA that is hybridized to the poly-T sequence of capture probe 401. After synthesis, first strand 23 is formed, where first strand 23 comprises cDNA and capture probe 401 sequences. After synthesis, the mRNA molecule 301-first strand 23 hybrid can be denatured (not shown) using any method conventional in the art, such as exposure to denaturing temperatures.

[0046] 6 illustrates the amplification of a first strand to generate an amplicon. In particular, after formation of first strand 23, a second strand primer 24 containing a random sequence (e.g., a random hexamer) anneals to the first strand 23 to form a DNA-primer hybrid. A DNA polymerase is used to synthesize a complementary second strand 25, i.e., an amplicon. In the illustrated embodiment, the second strand primer 24 contains a "tail" region that does not hybridize to first strand 23. In some embodiments, the tail region contains a second universal primer sequence. The second strand 25 can be further amplified by PCR to generate multiple amplicons, which can be quantified by DNA sequencing.

[0047] Amplification or nucleic acid synthesis, as used herein, generally refers to the method of using thermal cycling to make copies of nucleic acid by exposing reaction material to repeated cycles of heating and cooling, allowing different temperature-dependent reactions (for example, by polymerase chain reaction (PCR)).Any suitable PCR method known in the art can be used in conjunction with the method described above.Non-limiting examples of PCR reaction include real-time PCR, nested PCR, multiplex PCR, quantitative PCR, TS-PCR or touchdown PCR.

[0048] The term "nucleic acid amplification reagent" or "reverse transcription mix" includes, but is not limited to, dNTPs (a mix of nucleotides dATP, dCTP, dGTP, and dTTP), buffer, detergent, or, if necessary, solvent, and appropriate enzymes (e.g., polymerase or reverse transcriptase). The polymerase used in the targeted library preparation method of the present disclosure may be a DNA polymerase, selected from, but not limited to, Taq DNA polymerase, Phusion polymerase, or Q5 polymerase. The reverse transcriptase used in the targeted library preparation method of the present disclosure may be, for example, Moloney murine leukemia virus (MMLV) reverse transcriptase or maxima reverse transcriptase. In some embodiments, general parameters for the reverse transcription reaction include incubation at 25°C for about 15 minutes, followed by incubation at 52°C for about 90 minutes. Nucleic acid amplification reagents are commercially available and can be purchased, for example, from New England Biolabs (Ipswich, MA, USA) or Clonetech.

[0049] 7-9 illustrate methods for sequence-specific amplification of mRNA according to certain aspects of the present disclosure.

[0050] FIG. 7 illustrates a method for sequence-specific capture of mRNA 301. The template particle 201 includes multiple capture probes 401, which are depicted schematically by dashed curves. The characterized capture probes 401 include, from their 5' to 3' ends, a linker region capable of covalently binding to the template particle 201, a PR1" region containing a universal primer nucleotide sequence, at least one barcode region B1, which may contain an index sequence, and / or a UMI. The capture probes 401 further include a capture sequence containing a gene-specific sequence 26. Molecules of mRNA 301 released within monodisperse droplets and containing a target sequence 481 complementary to the gene-specific sequence 26 bind to the gene-specific sequence 26 of the capture probe through complementary base pairing. The gene-specific sequence may include any sequence of interest, for example, a sequence corresponding to an oncogene.

[0051] For example, in some cases, template particles 201 according to aspects of the present invention may include a capture probe having a certain sequence specific to a gene of interest (e.g., an oncogene). Some non-limiting examples of genes of interest that may be assayed include, but are not limited to: BAX, BCL2L1, CASP8, CDK4, ELK1, ETS1, HGF, JAK2, JUNB, JUND, KIT, KITLG, MCL1, MET, MOS, MYB, NFKBIA, EGFR, Myc, EpCAM, NRAS, PIK3CA, PML, PRKCA, RAF1, RARA, REL, ROS1, RUNX1, SRC, STAT3, CD45, cytokeratin, CEA, CD133, HER2, CD44, CD49f, CD146, MUC1 / 2, ABL1, AKT1, APC , ATM, BRAF, CDH1, CDKN2A, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR2, FGFR3, FLT3, GNAS, GNAQ, GNA11, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, STK11, TP53, VHL, and ZHX2.

[0052] 8 illustrates the synthesis of cDNA to form first strand 23. A reverse transcriptase (not shown) synthesizes cDNA from mRNA that is hybridized to the gene-specific sequence of capture probe 12. After hybridization of the target mRNA molecule 301 and the capture probe 12, a reverse transcription reaction is carried out to synthesize cDNA and create first strand 23. The first strand 23 contains the sequence of the synthesized cDNA and the capture probe 401. The target mRNA molecule-first strand hybrid is then denatured (not shown) using methods conventional in the art, and a second strand primer 24 containing random hexamer sequences anneals to the complementary sequence of the first strand 23 to form a DNA-primer hybrid.

[0053] 9 illustrates the amplification of first strand 23 to generate amplicon 25. In particular, after formation of first strand 23, second strand primer 24, which comprises a random sequence (e.g., a random hexamer), anneals to the first strand 23 to form a DNA-primer hybrid. A DNA polymerase is used to synthesize a complementary second strand 25, i.e., amplicon 25. In the illustrated embodiment, the second strand primer 24 comprises a "tail" region that does not hybridize to the first strand 23. In some embodiments, the tail region comprises a second universal primer sequence.

[0054] According to aspects of the present disclosure, the term "universal primer sequence" generally refers to a primer sequence that is predicted to hybridize (base pair) to one or more positions (if any) of a primer binding site, e.g., a complementary sequence, and prime a nucleic acid fragment. In some embodiments, the universal primer sequences used in connection with the methods of the present invention are P5 and P7.

[0055] The term barcode region can include any number of barcodes, indices, or index sequences, UMIs (which are unique, i.e., distinguishable from other barcode, index, or UMI sequences). The sequences can be of any suitable length sufficient to distinguish the barcode or index sequence from other barcode sequences. A barcode or index sequence can have a length of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides. In some embodiments, the barcodes or indices are predefined and randomly selected.

[0056] In some methods of the present invention, the barcode sequence can include a unique molecular identifier (UMI). A UMI is a type of barcode that can be provided to a sample to make each nucleic acid molecule unique or nearly unique together with its barcode. This can be achieved by adding one or more UMIs to one or more capture probes of the present invention. By selecting an appropriate number of UMIs, every nucleic acid molecule in the sample, together with its UMI, becomes unique or nearly unique.

[0057] UMI is advantageous in that it can be used to correct the errors (such as amplification bias or incorrect base pairing during amplification) that occur during amplification.For example, when using UMI, every nucleic acid molecule in a sample becomes unique or nearly unique together with its UMI, so after amplification and sequencing, molecules with identical sequences can be considered to refer to the same starting nucleic acid molecule, thereby reducing amplification bias.The method for error correction using UMI is described in Karlsson et al., 2016, "Counting Molecules in cell-free DNA and single cells RNA", Karolinska Institutet, Stockholm, Sweden (incorporated herein by reference).

[0058] In certain aspects, the method of the present invention includes combining template particles and target cells in a first fluid, adding a second fluid to the first fluid, and shearing the fluid to simultaneously generate a plurality of monodisperse droplets containing only one of the template particles and only one of the target cells, where the template particle preferably contains one or more oligos useful in template-switching oligo (TSO) embodiments. The method also preferably includes lysing each of the single target cells contained within the monodisperse droplets to release a plurality of distinct mRNA molecules; and quantifying the plurality of distinct mRNA molecules by using template-switching PCR (TS-PCR), for example, as discussed in U.S. Patent No. 5,962,272, which is incorporated herein by reference. TS-PCR is a method of reverse transcription and polymerase chain reaction (PCR) amplification that relies on the natural PCR primer sequence at the polyadenylation site (also known as the poly(A) tail) and adds a second primer through the activity of murine leukemia virus reverse transcriptase. This method allows for the complete cDNA sequence to be read and can result in high yields from a single source, even a single cell containing 10-30 picograms of mRNA.

[0059] TS-PCR generally relies on the unique properties of Moloney murine leukemia virus (MMLV) reverse transcriptase and the use of a unique TSO. During first-strand synthesis, upon reaching the 5' end of the mRNA template, the terminal transferase activity of MMLV reverse transcriptase adds several additional nucleotides (mostly deoxycytidines) to the 3' end of the newly synthesized cDNA strand. These bases can function as TSO anchoring sites. After base pairing between the TSO and the added deoxycytidine stretch, the reverse transcriptase "switches" the template strand from the cellular RNA to the TSO and continues replication toward the 5' end of the TSO. By doing so, the resulting cDNA contains the complete 5' end of the transcript, and a selected universal sequence is added to the reverse transcription product. This approach allows for efficient amplification of entire pools of full-length transcripts in a completely sequence-independent manner.

[0060] Figure 10 illustrates the capture of mRNA 301 according to a TSO embodiment. The TSO 1009 is an oligo that hybridizes to non-templated C nucleotides added by reverse transcriptase during reverse transcription. The TSO can, for example, add a common 5' sequence to full-length cDNA for downstream cDNA amplification. A template particle 201 is shown, including a first capture probe 401 and a second capture probe 403. The first capture probe 401 preferably includes, from its 5' to 3' end, a linker region that enables covalent attachment to the template particle 201, a P5 511 nucleotide sequence region that includes a universal primer nucleotide sequence, at least one barcode 33, and a capture nucleotide sequence 22 that includes a poly-T nucleotide sequence. The second capture probe 403 preferably includes TSO 1009, a UMI 531, a second barcode 541, and a P7 543 nucleotide sequence region that includes a universal primer nucleotide sequence. The released nucleic acid, i.e., mRNA molecule 301 containing a polyA sequence, binds to the polyT sequence 22 of the first capture probe 401 through complementary base pairing. After hybridization of the mRNA molecule 301 and the capture probe 401, TS-PCR is performed using a reverse transcriptase, i.e., murine leukemia virus reverse transcriptase, to synthesize cDNA, thereby creating a first strand. During TS-PCR amplification, once the 5' end of the mRNA template is reached, the terminal transferase activity of the reverse transcriptase adds a few additional nucleotides (mostly deoxycytidine) to the 3' end of the new first strand.

[0061] 11 shows the first strand 23 after TS-PCR amplification. The first strand 23 includes an additional nucleotide that can function as a TSO anchoring site 34. The TSO anchoring site 34 can hybridize to TSO 1009; after base pairing between the TSO and the TSO anchoring site 34, the reverse transcriptase "switches" the template strand from the cellular RNA to the TSO and continues replication toward the 5' end of the TSO. In doing so, the resulting cDNA contains the complete 5' end of the transcript and sequence derived from the second capture probe 403. After synthesis of the first strand 23, the first strand 23, including capture probes 401, 403, can be released either by cleaving the covalent bond linking the capture probes 401, 403 to the surface of the template particle 201 or by melting the template particle 201, for example, by heat.

[0062] Those skilled in the art will recognize that any one of the above template particle embodiments, capture probes, primer probes, second-strand primers, universal amplification primers, barcodes, UMIs, TSOs, and any one of the targeted library preparation methods described in the present disclosure can be used in various combinations or embodiments of the present invention. For example, any one of the second-strand primers or primer probes described herein can be used to prime any one of the first-strand primers of the present disclosure to enable a DNA synthesis reaction to generate an amplicon.

[0063] In preferred embodiments, quantifying released mRNA comprises sequencing, which can be carried out by methods known in the art.For example, generally see Quail et al., 2012, A tale of three next-generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers, BMC Genomics 13:341.Nucleic acid sequencing technology includes the traditional dideoxy sequencing reaction (Sanger method) that uses labeled terminator or primer, and gel separation in slab or capillary, or preferably, next-generation sequencing method. For example, sequencing can be performed according to the techniques described in U.S. Patent Publication Nos. 2011 / 0009278, 2007 / 0114362, 2006 / 0024681, 2006 / 0292611, U.S. Patent Nos. 7,960,120, 7,835,871, 7,232,656, 7,598,035, 6,306,597, 6,210,891, 6,828,100, 6,833,246, and 6,911,345 (each incorporated by reference).

[0064] The conventional pipeline for processing sequencing data includes the steps of generating FASTQ format file that contains the reads that are sequenced from next-generation sequencing platform, aligning these reads with annotated reference genome, and quantifying gene expression.These steps are routinely carried out by using known computer algorithms that those skilled in the art will recognize and can be used to implement the present invention.For example, see Kukurba, Cold Spring Harb Protoc, 2015 (11):951-969 (incorporated by reference).

[0065] After an expression profile is obtained from a single cell, the expression profile can be analyzed, for example, by comparing the profile to a reference or control profile to ascertain information about the single target cell. See, for example, generally, Efroni, Genome Biology, 2015; and Stahlberg, Nucleic Acids Research, 2011, 39(4)e24 (each of which is incorporated by reference).

[0066] In one aspect, the methods and systems of the present invention provide a method for identifying rare cells from a heterogeneous cell population. The method includes isolating a plurality of single target cells from the heterogeneous cell population by combining the heterogeneous cells and a plurality of template particles in a first fluid, adding a second fluid immiscible with the first fluid, and shearing the fluid to generate an emulsion containing monodisperse droplets each containing a single target cell and a single template particle. The method further includes releasing a plurality of mRNA molecules from each of the single target cells contained within the monodisperse droplets and quantifying the plurality of mRNA molecules. The quantifying step may include generating a plurality of amplicons of the mRNA molecules, each of which contains a barcode or index sequence unique to the cell from which the mRNA molecule was obtained. In some cases, the method may include sequencing the plurality of barcoded amplicons, for example, by next-generation sequencing, to generate sequence reads for each of the amplicons. The method may further include processing sequence reads associated with single cells of the heterogeneous cell population to generate an expression profile for each of the single cells, and using the data, for example, by performing gene clustering analysis to identify one or more cell types or cell states.

[0067] In another aspect, the disclosed methods and systems provide a method for analyzing a heterogeneous tumor biopsy taken from a subject. The method includes obtaining a biopsy from a patient and isolating a population of cells from the biopsy. The method further includes separating the population of cells from the biopsy into droplets by combining the population of cells and a plurality of template particles in a first fluid, adding a second fluid immiscible with the first fluid, and shearing the fluid to produce an emulsion containing monodisperse droplets, each containing only one of the population of cells and a single template particle. The method further includes releasing a plurality of mRNA molecules from each of the separated single cells contained within the monodisperse droplets, performing transcriptome analysis on one or more genes of the single cells, and using the transcriptome data to identify one or more characteristics of the tumor. The identified characteristics can be the presence or absence of one or more gene transcripts associated with cancer. The methods disclosed herein further include using the characteristics to diagnose a subject with cancer, or to diagnose the stage of cancer, or to devise a treatment plan.

[0068] In some aspects, the methods and systems of the present invention provide a method for determining the potential effectiveness of a therapeutic agent. The method includes separating a first population of diseased cells into monodisperse droplets containing template particles and determining the expression level of at least one nucleic acid from at least one of the diseased cells, thereby generating a disease state expression signature. The method further includes exposing a second population of diseased cells to a drug and determining the expression level of at least one nucleic acid from at least one individual cell from the second population, and comparing the expression level from each cell from the second population with the disease state expression signature, thereby determining the effectiveness of the drug for the disease. In some embodiments, the therapeutic agent can be delivered to the second population of cells within the monodisperse droplets. For example, the drug can be associated with the template particle by tethering the drug to the outer surface of the template particle or packaging the drug within a compartment of the template particle, such that the drug can be delivered to cells contained within the monodisperse droplets.

[0069] In any one of the embodiments of the targeted library preparation method of the present disclosure, the template particle further comprises a capture moiety. In some embodiments, the capture moiety acts to capture a specific target particle, such as a specific type of cell. In some embodiments, the capture moiety comprises an acrylate-terminated hydrocarbon linker with a biotin terminus. In some embodiments, the capture moiety is bound to a target-specific capture element. In some embodiments, the target-specific capture element is selected from an aptamer and an antibody. Embodiments of the capture moiety and method thereof are disclosed in International Application WO2020069298A1 (incorporated herein by reference). The present invention provides, for example, the following items. (Item 1) 1. A method for single cell analysis, said method comprising: combining template particles and target cells in a first fluid; adding a second fluid to the first fluid; shearing the fluid to simultaneously generate a plurality of monodisperse droplets comprising exactly one of the template particles and exactly one of the target cells; lysing each single target cell contained within said monodisperse droplets to release a plurality of distinct mRNA molecules; and quantitating said plurality of distinct mRNA molecules; A method that encompasses (Item 2) 2. The method of claim 1, further comprising generating an expression profile for each of the single target cells after quantifying the plurality of distinct mRNA molecules. (Item 3) 2. The method of claim 1, further comprising reverse transcribing the plurality of distinct mRNA molecules within the droplets. (Item 4) 4. The method of claim 3, wherein the first fluid is an aqueous fluid. (Item 5) 5. The method of claim 4, wherein the second fluid comprises oil. (Item 6) 6. The method of claim 5, wherein shearing the fluid comprises using one of a vortexer or a pipetting operation. (Item 7) Item 7. The method of item 6, wherein the template particle further comprises one or more compartments. (Item 8) 8. The method of claim 7, wherein the one or more compartments contain a reagent selected from the group comprising a lysis reagent, a nucleic acid synthesis reagent, or a combination thereof. (Item 9) 9. The method of claim 8, wherein the nucleic acid synthesis reagent comprises a polymerase. (Item 10) 10. The method of claim 9, wherein the reagent is released from the one or more compartments in response to an external stimulus. (Item 11) The template particle comprises a plurality of capture probes, the capture probes comprising: Universal primer sequence; At least one barcode; and capture sequence, The method according to item 1, comprising: (Item 12) 12. The method of claim 11, wherein the capture sequence is selected from one of a poly-T nucleotide sequence, a gene-specific nucleotide sequence, or a random nucleotide sequence. (Item 13) Item 13. The method of item 12, wherein the mRNA, upon release from the single target cell, binds to the template particle by hybridizing to the poly-T nucleotide sequence. (Item 14) The mRNA bound to the template particle contains the cDNA and the barcode sequence. 14. The method of claim 13, wherein the first strand is reverse transcribed to produce a first strand comprising the (Item 15) 15. The method of claim 14, comprising amplifying the first strand by PCR to generate an amplicon. (Item 16) 14. The method of claim 13, wherein the mRNA bound to the template particles is reverse transcribed using TSO. (Item 17) 16. The method of claim 15, wherein the step of quantifying the plurality of distinct mRNA molecules comprises sequencing the amplicon. (Item 18) 16. The method of item 15, wherein the rare cell type is a cancer cell. (Item 19) 1. A kit for single cell profiling, said kit comprising: a tube containing a template particle, the template particle comprising a capture sequence and an internal compartment containing a reagent; Kit including: (Item 20) 20. The kit of item 19, wherein the reagent is a reverse transcriptase. (Item 21) 20. The kit of item 19, wherein the capture sequence is complementary to mRNA transcribed from an oncogene.

Claims

1. 1. A method for single cell analysis, said method comprising: combining template particles and target cells in a first fluid, each of the template particles comprising: a plurality of first capture probes, each of the first capture probes comprising a universal primer sequence, at least one first barcode, and a capture sequence selected from the group consisting of a poly-T nucleotide sequence, a gene-specific nucleotide sequence, and a random nucleotide sequence; a plurality of second capture probes, each of said second capture probes comprising a template switching oligo (TSO) and at least one second barcode; a process comprising: adding a second fluid to the first fluid; shearing the first fluid and the second fluid to simultaneously generate a plurality of monodisperse droplets comprising exactly one of the template particles and exactly one of the target cells; lysing each single target cell contained within said monodisperse droplets to release a plurality of distinct mRNA molecules; and quantitating said plurality of distinct mRNA molecules; A method that encompasses

2. 10. The method of claim 1, further comprising generating an expression profile for each of the single target cells after quantifying the plurality of distinct mRNA molecules.

3. 10. The method of claim 1, further comprising reverse transcribing the plurality of distinct mRNA molecules within the droplet.

4. The method of claim 3 , wherein the first fluid is an aqueous fluid.

5. The method of claim 4 , wherein the second fluid comprises oil.

6. 6. The method of claim 5, wherein shearing the first and second fluids comprises using one of a vortexer or a pipetting operation.

7. The method of claim 6 , wherein the template particle further comprises one or more interior compartments within the template particle.

8. 8. The method of claim 7, wherein the one or more internal compartments contain a reagent selected from the group comprising a lysis reagent, a nucleic acid synthesis reagent, or a combination thereof within the one or more internal compartments.

9. The method of claim 8 , wherein the nucleic acid synthesis reagent comprises a polymerase.

10. 10. The method of claim 9, wherein the reagent is released from the one or more internal compartments in response to an external stimulus.

11. 2. The method of claim 1, wherein the mRNA, upon release from the single target cell, binds to the template particle by hybridizing to the poly-T nucleotide sequence.

12. 12. The method of claim 11, wherein the mRNA bound to the template particle is reverse transcribed to generate a first strand comprising cDNA and the first barcode.

13. 13. The method of claim 12, comprising amplifying the first strand by PCR to generate an amplicon.

14. The method of claim 11 , wherein the mRNA bound to the template particle is reverse transcribed using the TSO.

15. 14. The method of claim 13, wherein quantifying the plurality of distinct mRNA molecules comprises sequencing the amplicons.

16. 14. The method of claim 13, wherein the rare cell type is a cancer cell.

17. A kit for use in the method of claim 1, comprising: a tube containing the template particle, the template particle comprising the plurality of first and second capture probes and an internal compartment within the template particle comprising a reagent within the internal compartment; Kit including:

18. 18. The kit of claim 17, wherein the reagent is a reverse transcriptase enzyme.

19. 18. The kit of claim 17, wherein the capture sequence is complementary to mRNA transcribed from an oncogene.

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