Methods and systems for single cell gene profiling

Abstract

To provide methods and systems for single cell gene profiling.SOLUTION: This disclosure provides methods and systems for single-cell analysis, including single-cell transcriptome analysis, of target cells without microfluidic devices. The disclosed methods involve the use of template particles to template the formation of monodisperse droplets to generally capture a single target cell from a population of cells in an encapsulation, derive a plurality of distinct mRNA molecules from the single target cell, and quantify the distinct mRNA molecules to generate an expression profile.SELECTED DRAWING: None

Description

Technical Field

[0001] Technical Field The present disclosure relates to methods and systems for single cell gene profiling.

Background Art

[0002] Background The complexity of biological systems requires many experiments to characterize them. High-throughput methods are often performed to reduce the number of individual experiments that need to be conducted. 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 with respect to the number of cells that can be assayed in a given experiment. As a result, high-throughput single cell systems are not accessible in many clinical and research facilities.

Summary of the Invention

Means for Solving the Problems

[0004] Summary The present disclosure provides methods and systems for single cell analysis (including single cell transcriptome analysis) of target cells without a microfluidic device. The methods and systems of the present invention generate an emulsion having template particles to separate individual target cells into monodisperse droplets. Nucleic acid molecules are released from the target cells inside 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 analysis workflow for identifying the expression profiles of millions of single cells with a single library preparation.

[0005] The method and system of the present invention use template particles to template the formation of monodisperse droplets and isolate target cells for gene profiling. The method includes the steps of combining template particles and target cells in a first fluid, adding a second fluid to the first fluid, shearing the fluids to simultaneously generate a plurality of monodisperse droplets, wherein each of the monodisperse droplets contains only one of the template particles and only one of the target cells. The method further includes the steps of lysing the target cells within the monodisperse droplets to release a plurality of separate mRNA molecules, and quantifying the plurality of separate mRNA molecules. The data generated by quantifying the mRNA is used to create an expression for each of the target cells. The method further includes the step of 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 provide a method for quantifying gene expression of target cells. The method includes the step of releasing mRNA from target cells inside monodisperse droplets. The mRNA can be reverse transcribed into cDNA and simultaneously barcoded. The barcoded cDNA is amplified to generate a plurality of barcoded amplicons. The amplicons can be sequenced by next-generation sequencing methods, and due to 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 cells.

[0007] After obtaining an expression profile from the target cells, the profile can be analyzed by comparing the profile with a reference or control profile to confirm information about the target cells. In other cases, the profile of the target cells can be compared with a profile derived from cells having a certain phenotype to determine whether the target cells share the characteristics of the phenotypic cells.

[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 combining a target cell with a plurality of template particles in a first fluid, adding a second fluid immiscible with the first fluid, and shearing the fluids to generate an emulsion containing monodisperse droplets each containing a target cell and a single template particle, thereby isolating a plurality of target cells. The method further includes releasing a plurality of mRNA molecules inside 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 can be amplified to generate a plurality of barcoded amplicons that can be traced back to the target cells. Optionally, the method may include sequencing the plurality of barcoded amplicons, for example, by next-generation sequencing methods, to generate sequence reads. The method may further include processing the sequence reads to generate an expression profile for each target cell, and using the data, for example, by performing gene clustering analysis to identify one or more cell types or cell states among the target cells.

[0009] In another aspect, the methods and systems of the present disclosure 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 creating a mixture of the population of cells and a plurality of template particles and an aqueous fluid, adding oil, and vortexing the mixture to produce an emulsion containing droplets each containing only one of the population of cells and a template particle, thereby separating the population of cells into droplets. The method further includes releasing mRNA from each of the cells inside 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. Characteristics of cancer can be the presence or absence of one or more gene transcripts associated with cancer. The methods disclosed herein can further include using the features to diagnose a subject having 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 having 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 disease state cells to the agent, and determining the gene expression of the second population of cells, and comparing the gene expression with the expression signature of the disease state to confirm the effectiveness of the agent against the disease based on an increase or suppression in the level of expression of one or more genes. In some embodiments, the therapeutic agent can be delivered to the second population of cells inside the droplet. For example, the agent can be associated with the template particles by tethering the agent to the outer surface of the template particles or packaging the agent inside a compartment of the template particles and releasing the agent from the template particles inside 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, as an aqueous phase fluid dispersed in a 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 for mixing the solutions. The methods of the present invention include the steps of combining cells and template particles in a first fluid, adding a second fluid, and shearing or agitating the first and second fluids. Preferably, the first fluid is an aqueous phase fluid and, in some embodiments, can include reagents selected from, for example, buffers, water, soluble 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 mixes, or combinations thereof).

[0012] The method and system of the present invention use template particles to isolate target cells, with the formation of monodisperse droplets as a template. Template particles according to aspects of the present invention can include, for example, hydrogels 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 can be shaped to provide enhanced affinity for the target cells. For example, the template particles can generally be spherical, but their shape can include flat surfaces, craters, grooves, protrusions, and other irregularities in a spherical shape that promote association with the target cells so as to increase the probability that the shape of the template particles templates droplets containing the target cells.

[0013] In some aspects, the method and system of the present invention provide template particles that include one or more internal compartments. The internal compartments can contain reagents or compounds that are releasable upon an external stimulus. Examples of reagents included by the template particles can include, for example, cell lysis reagents or nucleic acid synthesis reagents (e.g., polymerase). The external stimulus can be heat, osmotic pressure, osmotic pressure, or an enzyme. For example, in some cases, the method of the present invention includes the step of releasing reverse transcriptase directly inside 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 the step of releasing mRNA from a single target cell contained within a droplet. In some embodiments, the released mRNA binds to the poly-T sequence of a barcoded capture probe that is bound to a template particle via complementary base pairing. Alternatively, the released RNA binds to the gene-specific sequence of the barcoded capture probe. After the binding of the mRNA molecule to the capture probe, reverse transcriptase synthesizes cDNA, thereby creating a first strand that includes 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 a denaturing temperature). In the next step, a second strand primer that includes a random hexamer sequence anneals to the first strand to form a DNA-primer hybrid. DNA polymerase synthesizes a complementary second strand. Optionally, the second strand is amplified, e.g., by PCR, to generate a plurality of amplicons. The amplicons are analyzed to confirm 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 comprising a plurality of capture sequences specific for one or more target genes. A researcher following the instructions provided by the kit can use the template particles to assay the single cell expression of a specific target gene (e.g., an oncogene). The kit can enable single cell profiling according to the methods described throughout the present disclosure (e.g., in FIG. 1). The template particles can be custom designed for the specific needs of the user (e.g., designed to include capture probe sequences specific for a particular target gene (e.g., an oncogene)). The template particles can be transported inside a sample preparation tube, or a sample collection tube (e.g., a blood collection tube). The template particles can preferably be in a dried form. The kit can further include reagents (e.g., cell lysis reagents, and nucleic acid synthesis reagents).

[0016] In other aspects, the methods and systems of the present invention provide a method for collecting data regarding the transcriptome of a single cell. The method includes the steps of releasing a plurality of distinct mRNA molecules from a single cell inside a monodisperse droplet, collecting data regarding the transcriptome of the single cell, and sending the data to a computer. The computer can be connected to a sequencing device. The data corresponding to the transcriptome can further be stored after being sent, e.g., the data can be stored on a computer-readable medium retrievable from the computer. The data can be transmitted from the computer to a remote location, e.g., via the Internet. BRIEF DESCRIPTION OF THE DRAWINGS

[0017]

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Mode for Carrying Out the Invention

[0018] Detailed Description The present disclosure provides systems and methods for forming monodisperse droplets for separating single cells using template particles, preparing a library preparation thereof, and profiling the expression of the single cells. The method of the present disclosure uses the formation of monodisperse droplets as a template to generally capture a single target cell within a capsule, obtain a plurality of distinct RNAs from the single target cell, and prepare a library of nucleic acids that can be traced to the cell (from which the library of nucleic acids is obtained), and includes the use of template particles to quantify distinct RNAs and generate an expression profile of the single target cell. The method of the present invention can be used to prepare a library for single cell analysis of, for example, at least 100 cells, at least 1000 cells, at least 1,000,000 cells, at least 2,000,000 cells, or more cells from a single reaction tube.

[0019] Figure 1 diagrammatically shows a method 101 for single cell profiling. The method 101 includes a step 109 of combining template particles and target cells in a first fluid, and a step of adding a second fluid immiscible with the first fluid to the mixture. The first fluid is preferably an aqueous fluid. Any suitable order may be used, and in some cases, the tube may be provided including 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 form. The combining step 109 can include pipetting a sample containing cells and, for example, the aqueous fluid into a tube containing the template particles using a pipette, and then adding a second fluid (e.g., oil) that is immiscible.

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

[0021] The next step of method 101 is to lyse the target cells 123. Cell lysis 123 can be induced by stimuli such as lysis reagents, surfactants, or enzymes. Reagents that induce cell lysis can be provided by the template particles via internal compartments. In some embodiments, the step of lysing 123 includes heating the monodisperse droplets to a temperature sufficient to release the lysis reagent contained within the template particles into the monodisperse droplets. This achieves cell lysis 123 of the target cells, thereby releasing mRNA into the interior of the droplets containing the target cells.

[0022] After the step 123 of lysing the target cells inside the droplets, the mRNA is released and subsequently quantified 131. The step 131 of quantifying the mRNA generally requires synthesizing cDNA to generate a library containing cDNA with barcode sequences, such that each library sequence can 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 a plurality of barcoded capture sequences that hybridize to the target mRNA. After hybridization, cDNA is synthesized by reverse transcription. Reagents for reverse transcription can be provided in various forms and in various ways. In some cases, the reagents and reverse transcriptase are provided by the template particles. Once a library containing barcoded cDNA is generated, the cDNA can be amplified, for example, by PCR, to generate amplicons for sequencing. Sequence reads are processed according to the methods described herein to achieve quantification 131 of the mRNA.

[0023] In some scenarios, the target cells may include live cells obtained, for example, from a patient sample (tissue such as body fluid). The sample may include a fine needle aspirate, biopsy, or body fluid from the patient. When isolating from the sample, the cells can be processed, for example, by generating a single cell suspension with 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, fetal bovine serum or other naturally occurring factors are supplemented with an acceptable buffer at low concentration (vs. 5 - 25 mM). Convenient buffers include HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), phosphate buffer, lactate buffer, etc. The isolated cells can be collected in any suitable medium that maintains cell viability and usually has a serum cushion at the bottom of the collection tube. Various media are commercially available and can be used depending on the nature of the cells (e.g., 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., and frequently, fetal bovine serum is supplemented).

[0024] The methods and systems of the present invention use template particles to template the formation of monodisperse droplets and isolate single target cells. The template particles of the present disclosure and methods for preparing their targeted libraries 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 isolated fluid volumes surrounded by immiscible partitioning fluids and stabilized by temperature-insensitive surfactants.

[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 a hydrogel material (e.g., agarose, alginate, polyethylene glycol (PEG), polyacrylamide (PAA), acrylate, acrylamide / bisacrylamide copolymer matrix, and combinations thereof). After the formation of the template particles, they are sized to the desired diameter. In some embodiments, the sizing of the template particles is performed by microfluidic co-flow into an immiscible oil phase.

[0026] In some embodiments of the template particles, the variation in the diameter or maximum diameter of the template particles varies by 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, and 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 in diameter or maximum diameter.

[0027] The template particles may be porous or non-porous. In any suitable embodiment herein, the template particles may include microcompartments (also referred to herein as "internal compartments"), which may contain additional components and / or reagents (e.g., additional components and / or reagents that may be releasable into monodisperse droplets as described herein). The template particles may include a polymer, such as a hydrogel. The template particles generally have a diameter or maximum diameter ranging from about 0.1 to about 1000 μm. In some embodiments, the template particles have a diameter or maximum diameter of about 1.0 μm to 1000 μm (including both ends) (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 (including both ends)). In some embodiments, the template particles have a diameter or maximum diameter of about 10 μm to about 200 μm, such as about 10 μm to about 150 μm, about 10 μm to about 125 μm, or about 10 μm to about 100 μm.

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

[0029] In other scenarios, the template particles can be hydrogels. In certain embodiments, the hydrogels are selected from naturally-derived substances, synthetically-derived 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 polyanhydrides, poly(propylene fumarate) (PPF).

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

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

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

[0033] The production 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 can be incubated to enable the association of the plurality of template particles with the target cell. A portion of the plurality of template particles can become associated with the target cell. The mixture is then combined with a second fluid that is immiscible with the first fluid. The fluids and the mixture are then sheared such that a plurality of monodisperse droplets are generated in the reaction tube. The monodisperse droplets generated 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 in practicing the methods of the invention provided by the present disclosure, a substantial number of the monodisperse droplets generated contain a single template particle and a single target particle, although in some cases, some of the monodisperse droplets may contain no template particles or target cells at all, or more than one.

[0034] In some embodiments, to increase the chance of generating encapsulations (e.g., monodisperse droplets containing one template particle and one target cell), the template particles and target cells are combined in a certain 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 as described above can be in the range of 5:1 to 1,000:1, respectively. In other embodiments, the template particles and target cells are combined in a ratio of 10:1, respectively. In other embodiments, the template particles and target cells are combined in a ratio of 100:1, respectively. In other embodiments, the template particles and target cells are combined in a ratio of 1,000:1, respectively.

[0035] To generate a monodisperse emulsion, the method of the present disclosure includes the step of shearing a second mixture provided by combining a first mixture containing target particles and target cells and a second fluid immiscible with the first mixture. 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 shaking the second mixture with a bead beater. In some embodiments, the vortexing can be performed, for example, for 30 seconds or in the range of 30 seconds to 5 minutes. The application of sufficient shear force breaks up the second mixture into monodisperse droplets each encapsulating one of the plurality of template particles.

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

[0037] In practicing the methods described herein, the composition and properties of the monodisperse droplets (such as single emulsions and multiple emulsion droplets) can vary. As mentioned above, in certain scenarios, 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 a non-miscible phase carrier fluid (such as a fluorocarbon oil, a silicone oil, or a hydrocarbon oil), or vice versa. Thus, the droplets can include surface-stabilized emulsions, such as surfactant-stabilized single emulsions or surfactant-stabilized double emulsions. Any convenient surfactant that enables the desired reaction to be carried out in the droplets may be used. In other scenarios, the monodisperse droplets are not stabilized by surfactants.

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

[0039] In some embodiments, the template particle includes a number of internal compartments. The internal compartments of the template particle can be used to encapsulate reagents that can be induced to release a desired compound (e.g., a substrate for an enzymatic reaction) or to induce a particular outcome (e.g., lysis of an associated target cell). Reagents encapsulated within the compartments of the template particle can be reagents selected from, but not limited to, 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 the single target cell occurs within the monodisperse droplet and can be induced by stimuli such as heat, osmotic pressure, lytic reagents (e.g., DTT, β-mercaptoethanol), surfactants (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., lytic reagents, surfactants, enzymes) are compartmentalized 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 the solution containing the monodisperse droplet, if desired.

[0041] Figure 3 illustrates droplet 201 after lysis of the target cell. The illustrated droplet 201 contains template particles 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 particles include a plurality of capture probes. Generally, the capture probes of the present disclosure are oligonucleotides. In some embodiments, the capture probes are covalently attached to the material of the template particles (e.g., a hydrogel material) via acrylic bonds. In some embodiments, the capture probes are acrydite-modified on their 5' ends (linker regions). Generally, acrydite-modified oligonucleotides can be stoichiometrically incorporated into a hydrogel (e.g., polyacrylamide) using standard free radical polymerization chemistry, where the double bond in the acrydite group reacts with a compound containing other activated double bonds (e.g., acrylamide). Specifically, copolymerization of the acrydite-modified capture probes with acrylamide, including a crosslinking agent (e.g., N,N'-methylenebis), results in a crosslinked gel material containing covalently attached capture probes. In some other embodiments, the capture probes include acrylate-terminated hydrocarbon linkers, and when combined with the template particles, their binding to the template particles is induced.

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

[0044] Figure 4 illustrates the capture of mRNA 301. A template particle 201 is shown that includes a plurality of capture probes 401 schematically illustrated by the dashed curve. One of the capture probes 401 is shown in greater scale and in detail. The capture probe 401 preferably includes, from the 5'-end to the 3'-end, a linker region enabling a covalent bond with the template particle 201, a PR1 471 nucleotide sequence region including a universal primer nucleotide sequence, at least one barcode region B1 473 that may include an index 475 nucleotide sequence index, and / or a UMI, and the capture probe 201 further includes a capture nucleotide sequence including a poly-T nucleotide sequence. The released nucleic acid, i.e., the mRNA molecule 301 including 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, reverse transcriptase is used to perform a reverse transcription reaction to synthesize cDNA, whereby a first strand including the cDNA and the capture probe sequence is created.

[0045] Figure 5 illustrates the synthesis of cDNA to form the first strand 23. Reverse transcriptase (not shown) synthesizes cDNA from the mRNA hybridized to the poly-T sequence of the capture probe 401. After synthesis, the first strand 23 is formed, where the first strand 23 includes the cDNA and the capture probe 401 sequence. After synthesis, the mRNA molecule 301 - first strand 23 hybrid may be denatured (not shown) using any conventional method in the art (e.g., exposure to a denaturing temperature).

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

[0047] Amplification or nucleic acid synthesis generally, as used herein, refers to a method for making copies of nucleic acids by using thermal cycling to expose reactants to repeated cycles of heating and cooling to enable different temperature - dependent reactions (e.g., by polymerase chain reaction (PCR)). Any suitable PCR method known in the art can be used in connection with the methods described previously. Non - limiting examples of PCR reactions include real - time PCR, nested PCR, multiple 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, dNTP (a mix of nucleotides dATP, dCTP, dGTP, and dTTP), buffer, surfactant, and, if required, solvent, and appropriate enzymes (e.g., polymerase or reverse transcriptase). The polymerase used in the targeted library preparation method of the present disclosure can be a DNA polymerase and can be 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 can be, for example, Moloney murine leukemia virus (MMLV) reverse transcriptase, or maxima reverse transcriptase. In some embodiments, the general parameters of the reverse transcription reaction include incubation at 25 degrees for about 15 minutes and then incubation at 52 degrees 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] Figures 7-9 illustrate methods for sequence-specific amplification of mRNA according to certain aspects of the present disclosure.

[0050] Figure 7 illustrates a method for sequence-specific capture of mRNA 301. The template particle 201 includes a plurality of capture probes 401 schematically illustrated by a dashed curve. The capture probe 401 with characteristics includes, from the 5' end to the 3' end, a linker region enabling covalent bonding with the template particle 201, a PR1" region including a universal primer nucleotide sequence, at least one barcode region B1 that may include an index sequence, and / or a UMI, and the capture probe 401 further includes a capture sequence including a gene-specific sequence 26. Molecules of mRNA 301 released inside the monodisperse droplets and including a target sequence 481 complementary to the gene-specific sequence 26 of the capture probe bind to the gene-specific sequence 26 of the capture probe via complementary base pairing. The gene-specific sequence can include any target sequence, for example, a sequence corresponding to an oncogene.

[0051] For example, in some cases, the template particle 201 according to aspects of the present invention may include a capture probe having a particular sequence specific to a target gene (e.g., an oncogene). Some non-limiting examples of target genes that can 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, keratin, 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] FIG. 8 illustrates the synthesis of cDNA to form the first strand 23. Reverse transcriptase (not shown) synthesizes cDNA from mRNA that hybridizes to the gene-specific sequence of the capture probe 12. After hybridization of the target mRNA molecule 301 and the capture probe 12, a reverse transcription reaction is performed to synthesize cDNA and create the first strand 23. The first strand 23 includes the synthesized cDNA and the sequence of 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 a random hexamer sequence anneals to the complementary sequence of the first strand 23 to form a DNA-primer hybrid.

[0053] Figure 9 illustrates the amplification of the first strand 23 to generate the amplicon 25. In particular, after the formation of the 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 the complementary second strand 25, i.e., the amplicon 25. In the illustrated embodiment, the second strand primer 24 includes a "tail" region that does not hybridize to the first strand 23. In some embodiments, the tail region includes 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 a primer binding site, e.g., one or more positions (if any) of a complementary sequence and prime with respect to 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, indexes or index sequences, UMIs (which are unique, i.e., distinguishable from other barcode sequences, or index sequences, UMI sequences). The sequences can be of any suitable length sufficient to distinguish the barcode sequence, or index sequence, from other barcode sequences. The barcode sequence 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 nucleotides, or more nucleotides. In some embodiments, the barcode or index is predefined and randomly selected.

[0056] In some methods of the present invention, the barcode array may include unique molecular identifiers (UMIs). A UMI is a type of barcode that, together with its barcode, can be provided to a sample to make each nucleic acid molecule unique or nearly unique. 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, any nucleic acid molecule in the sample, together with its UMI, becomes unique or nearly unique.

[0057] UMIs are advantageous in that they can be used to correct errors (e.g., amplification bias or inaccurate base pairing during amplification) that they cause during amplification. For example, when using UMIs, since any nucleic acid molecule in the sample, together with its UMI, becomes unique or nearly unique, after amplification and sequencing, molecules with the same sequence refer to the same starting nucleic acid molecule, thereby potentially reducing amplification bias. Methods for error correction using UMIs are 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 embodiments, the method of the invention comprises combining template particles and target cells in a first fluid, adding a second fluid to the first fluid, shearing the fluids to simultaneously generate a plurality of monodisperse droplets each containing only one of the template particles and only one of the target cells, wherein the template particles preferably comprise one or more oligos useful in template switching oligo (TSO) embodiments. The method also preferably comprises 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, for example, by using template switching PCR (TS-PCR) as described 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 natural PCR primer sequences at polyadenylation sites (also known as poly(A) tails) and adds a second primer through the activity of murine leukemia virus reverse transcriptase. This method allows for the reading of complete cDNA sequences and can provide high yields from a single source, even from single cells containing 10 to 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 specific TSO. During first-strand synthesis, when the 5’ end of the mRNA template is reached, the terminal transferase activity of MMLV reverse transcriptase adds several additional nucleotides (mostly deoxycytidines) to the 3’ end of its newly synthesized cDNA strand. These bases can function as a TSO tethering site. 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 to the 5’ end of the TSO. By doing so, the resulting cDNA contains the complete 5’ end of the transcript, and the selected universal sequence is added to the reverse transcription product. This approach enables the entire pool of full-length transcripts to be efficiently amplified in a completely sequence-independent manner.

[0060] Figure 10 illustrates the capture of mRNA 301 according to the 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 add, for example, a common 5' sequence to the full-length cDNA used for downstream cDNA amplification. A template particle 201 containing a first capture probe 401 and a second capture probe 403 are shown. The first capture probe 401 preferably includes, from the 5' end to the 3' end, a linker region enabling covalent bonding to the template particle 201, a P5 511 nucleotide sequence region including a universal primer nucleotide sequence, at least one barcode 33, and a capture nucleotide sequence 22 including a polyT nucleotide sequence. The second capture probe 403 preferably includes a TSO 1009, a UMI 531, a second barcode 541, and a P7 543 nucleotide sequence region including a universal primer nucleotide sequence. The released nucleic acid, i.e., the mRNA molecule 301 containing a polyA sequence, binds to the polyT sequence 22 of the first capture probe 401 via complementary base pairing. After hybridization of the mRNA molecule 301 and the capture probe 401, TS-PCR is performed using reverse transcriptase, i.e., murine leukemia virus reverse transcriptase, to synthesize cDNA and thereby create the first strand. During TS-PCR amplification, when the 5' end of the mRNA template is reached, the terminal transferase activity of the reverse transcriptase adds several additional nucleotides (mostly deoxycytidine) to the 3' end of its new first strand.

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

[0062] One of ordinary skill in the art will recognize that the method described for any one of the template particle embodiments, capture probes, primer probes, second strand primers, universal amplification primers, barcodes, UMIs, TSOs, and any one of the embodiments of the targeted library preparation method of the present disclosure can be used in various combinations or embodiments of the method 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 strands of the present disclosure to enable a DNA synthesis reaction to generate an amplicon.

[0063] In a preferred embodiment, the step of quantifying the released mRNA includes sequencing, which can be performed by methods known in the art. See, for example, generally 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 techniques include traditional dideoxy sequencing reactions (Sanger method) using labeled terminators or primers, and gel separation on slabs or capillaries, or preferably, next generation sequencing methods. 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. Pat. 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] Conventional pipelines for processing sequencing data include generating FASTQ format files containing reads sequenced from a next generation sequencing platform, aligning these reads to an annotated reference genome, and quantifying gene expression. These steps are routinely performed using known computer algorithms that are recognized by those skilled in the art and can be used for the practice of the present invention. See, for example, Kukurba, Cold Spring Harb Protoc, 2015 (11):951-969, incorporated by reference.

[0065] After obtaining an expression profile from a single cell, the expression profile can be analyzed, for example, by comparing the profile with a reference or control profile to confirm 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 combining the heterogeneous cells with a plurality of template particles in a first fluid, adding a second fluid immiscible with the first fluid, and shearing the fluids to generate an emulsion containing monodisperse droplets each containing a single target cell and a single template particle, thereby isolating a plurality of single target cells from the heterogeneous cell population. 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 can include generating a plurality of amplicons of the mRNA molecules, where each of the amplicons includes a barcode or index sequence specific to the cell from which the mRNA molecules were obtained. Optionally, the method can include sequencing the plurality of barcoded amplicons, for example, by next-generation sequencing methods, to generate sequence reads for each of the amplicons. The method further includes processing the 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 methods and systems of the present disclosure 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 combining the population of cells with a plurality of template particles in a first fluid, adding a second fluid that is immiscible with the first fluid, and shearing the fluids to produce an emulsion containing monodisperse droplets each containing only one of the population of cells and a single template particle, thereby separating the population of cells taken from the biopsy into droplets. The method further includes releasing a plurality of mRNA molecules from each one of the isolated 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 having 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 efficacy of a therapeutic agent. The method includes separating a first population of diseased cells into monodisperse droplets having 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 disease state cells to the agent, and determining the expression level of at least one nucleic acid from at least one of the individual cells from the second population, and comparing the expression level from the individual cells from the second population with the disease state expression signature, thereby determining the efficacy of the agent against the disease. In some embodiments, the therapeutic agent can be delivered to the second population of cells inside the monodisperse droplets. For example, the agent can be associated with the template particles by attaching the agent to the outer surface of the template particles or packaging the agent inside the compartments of the template particles, such that the agent can be delivered to the cells contained inside the monodisperse droplets.

[0069] In any one of the embodiments of the targeted library preparation method of the present disclosure, the template particles further include a capture moiety. In some embodiments, the capture moiety acts to capture specific target particles, such as specific types of cells. In some embodiments, the capture moiety includes an acrylate-terminated hydrocarbon linker having 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 aptamers and antibodies. Embodiments of the capture moiety and methods thereof are disclosed in International Application WO2020069298A1, which is incorporated herein by reference. The present invention provides, for example, the following items. (Item 1) A method for single cell analysis, the 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 each containing only one of the template particles and only one of the target cells; 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. A method comprising the steps of. (Item 2) The method according to item 1, further comprising, after quantifying the plurality of distinct mRNA molecules, generating an expression profile for each of the single target cells. (Item 3) The method according to item 1, further comprising reverse transcribing the plurality of distinct mRNA molecules inside the droplet. (Item 4) The method according to item 3, wherein the first fluid is an aqueous fluid. (Item 5) The method according to item 4, wherein the second fluid contains oil. (Item 6) The method according to item 5, wherein the step of shearing the fluid includes using one of the operations of transferring with a vortexer or a pipette. (Item 7) The method according to item 6, wherein the template particles further comprise one or more compartments. (Item 8) The method according to item 7, wherein the one or more compartments contain a reagent selected from the group consisting of a lysis reagent, a nucleic acid synthesis reagent, or a combination thereof. (Item 9) The method according to item 8, wherein the nucleic acid synthesis reagent contains polymerase. (Item 10) The method according to item 9, wherein the reagent is released from the one or more compartments in response to an external stimulus. (Item 11) The template particles include a plurality of capture probes, and the capture probes a universal primer sequence; at least one barcode; and a capture sequence. The method according to item 1. (Item 12) The method according to item 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) The method according to item 12, wherein the mRNA binds to the template particle by hybridizing to the poly-T nucleotide sequence upon release from the single target cell. (Item 14) The method according to item 13, wherein the mRNA bound to the template particle is reverse transcribed to generate a first strand comprising cDNA and the barcode sequence. (Item 15) The method according to item 14, comprising the step of amplifying the first strand by PCR to generate an amplicon. (Item 16) The method according to item 13, wherein the mRNA bound to the template particle is reverse transcribed using TSO. (Item 17) The method according to item 15, wherein the step of quantifying the plurality of distinct mRNA molecules comprises sequencing the amplicon. (Item 18) The method according to item 15, wherein the rare cell type is a cancer cell. (Item 19) A kit for single cell profiling, the kit comprising: A tube containing template particles, wherein the template particles comprise a capture sequence and an internal compartment containing a reagent. A kit comprising. (Item 20) The kit according to item 19, wherein the reagent is a reverse transcriptase. (Item 21) The kit according to item 19, wherein the capture sequence is complementary to mRNA transcribed from an oncogene.

Claims

【Claim 1】 The invention described in the specification.

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