Single cell sequencing

Abstract

To provide single cell sequencing.SOLUTION: The disclosure provides methods and systems of analyzing single cells by simultaneously separating the cells into monodisperse droplets and tagging each nucleic acid molecule derived from the cells with a barcode unique to each droplet. The methods and systems combine template particles with a plurality of single cells in a tube, generate in the tube monodisperse droplets encapsulating only one of the template particles and only one of the single cells, release nucleic acid molecules from the single cells, and provide each nucleic acid molecule with a barcode unique to the respective droplet. The nucleic acid molecules can then be analyzed by any known method, for example, by sequencing the nucleic acid molecules.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to methods and systems for single cell analysis. [Background technology]

[0002] background A major challenge in treating cancer is the difficulty and expense of detecting cancer cells among healthy cells at the early stages, when cancer treatment is most effective. Identifying cells and cellular components that exist in small proportions against a background of more general cellular material is a significant diagnostic challenge. Many solutions to this problem have been proposed. For example, single-cell sequencing has been proposed as a method for identifying cancer cells that exist in small proportions in a sample. Traditional methods for isolating single cells use flow cytometry and droplet microfluidics to separate one single cell at a time. However, these methods require complex equipment that is expensive and difficult to use. Furthermore, because each cell must be processed individually, such methods are speed-limited and require long periods (often several days) to separate cancer cells from surrounding cells. This limitation is particularly problematic in early cancer detection, when the proportion of cancer cells in a sample is at its lowest. Furthermore, such methods are particularly difficult to use by clinicians, as samples must be sent to facilities capable of handling such equipment, further increasing the time and expense required to study cancer diagnosis, resulting in early cancer detection being unaffordable and inaccessible to a large proportion of cancer patients. Summary of the Invention [Means for solving the problem]

[0003] Summary of the Invention The present invention provides methods and systems for single-cell analysis that significantly reduce the complexity and cost of single-cell sequencing and early cancer detection. The methods of the present invention simultaneously isolate single cells in a sample, rather than one at a time, by encapsulating each cell into individual monodisperse droplets with a barcode unique to each droplet. These barcodes can then be provided to nucleic acid molecules released from each single cell, and once sequenced, the nucleic acid molecules can be traced back to the droplet. Because each droplet encapsulates only a single cell, the nucleic acid molecules thereby provide genotypic information about the cell. By simultaneously isolating cells into droplets, rather than individually, and tagging nucleic acid molecules with a barcode unique to each droplet, these methods enable the isolation of cancer cells from a sample within hours rather than days, providing faster and earlier cancer detection. Furthermore, the methods of the present invention are performed without the need for complex and expensive machinery required by microfluidic cell separation techniques, dramatically reducing the cost of single-cell analysis and early cancer detection.

[0004] Furthermore, the methods of the present invention provide an approach that is easily scalable from small to large sample volumes and can be automated. By reducing the complexity of single-cell analysis, the methods and systems of the present invention allow clinicians to prepare samples for single-cell analysis themselves, further reducing the cost of early cancer detection. This dramatic reduction in the cost and time required for single-cell analysis greatly expands the population available for early cancer detection.

[0005] The present invention is achieved in part by combining a template particle and a plurality of single cells in a tube, and simultaneously generating a plurality of monodisperse droplets in the tube, each encapsulating exactly one of the template particles and exactly one of the single cells. Nucleic acids are released from the single cells, and each nucleic acid molecule in the droplet is provided with a barcode unique to the droplet. Each nucleic acid molecule can then be analyzed by any known method, for example, by sequencing the nucleic acid. The nucleic acid molecule can be any nucleic acid molecule, including DNA and / or RNA.

[0006] The method of the present invention simultaneously separates single cells by combining the template particle and the single cell in a first fluid, adding a second fluid to the first fluid, and shearing the fluid to simultaneously generate multiple monodisperse droplets containing only one of the template particles and only one of the single cells. The method of releasing nucleic acid molecules from the single cells further includes lysing the single cells within the monodisperse droplets to release nucleic acid molecules simultaneously tagged with a barcode unique to each droplet, thereby allowing identification of nucleic acid molecules from any droplet.

[0007] In such a method, the first fluid and the second fluid may be immiscible. For example, the first fluid may comprise an aqueous phase fluid, and / or the second fluid may comprise an oil. The first fluid may comprise, for example, a buffer, salt, lytic enzyme (e.g., proteinase k) and / or other lysis reagent (e.g., Triton X-100, Tween®-20, IGEPAL, or a combination thereof), a nucleic acid synthesis reagent (e.g., a nucleic acid amplification reagent or a reverse transcription mix, or a combination thereof), a reagent selected from the group consisting of fluorocarbon oil, silicone oil, or hydrocarbon oil, or a combination thereof. Shearing the fluid may include vortexing, shaking, flicking, stirring, pipetting, or any known method for mixing a solution.

[0008] The droplets produced by the methods of the present invention are monodisperse and encapsulate only one of the template particles and only one of the single cells. Advantageously, the template particles can each provide a barcode unique to that template particle. Because each droplet contains only one template particle and one single cell, the template particle thereby provides a barcode that is also unique to each droplet and, therefore, unique to the cell encapsulated in each droplet.

[0009] The template particle may comprise any known particle that can be used to form the monodisperse droplets and may advantageously provide a unique barcode for each droplet. The template particle may be a hydrogel, such as 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 particle may be shaped to provide enhanced affinity for the single cell. For example, the template particle may be generally spherical, but its shape may include features such as flat surfaces, craters, grooves, protrusions, and other irregularities in the spherical shape that promote association with single cells, increasing the probability that the shape of the template particle will template monodisperse droplets containing single cells.

[0010] Furthermore, the template particle may further comprise one or more compartments. For example, the one or more compartments may comprise one or more of a lysis reagent, a nucleic acid synthesis reagent, the barcode specific to each droplet, or a combination thereof. For example, when PCR is desired, it may be advantageous for the nucleic acid synthesis reagent to comprise a polymerase. When a reagent is used, the reagent may be released from the one or more compartments in response to an external stimulus.

[0011] The template particle may also include a plurality of capture probes, each of which may include one or more of a primer sequence, the barcode unique to each droplet, a unique molecular identifier (UMI), and / or a capture sequence. The primer sequence may include a binding site that hybridizes to a complementary sequence (if present) on any nucleic acid molecule released from a cell and includes a sequence predicted to provide an initiation site for a reaction (e.g., an extension or polymerization reaction). Because the capture probe includes a barcode unique to each droplet, the capture probe may be used to tag nucleic acid molecules released from a single cell with the barcode. A capture sequence may be used in the capture probe to target a gene-specific nucleotide sequence.

[0012] Tubes for single-cell analysis of the present invention can be selected based on the volume of the sample from which cells need to be separated and / or based on the number of cells to be separated. For example, the tube can be a single large tube (e.g., a conical centrifuge tube (e.g., Falcon® as sold by Corning Inc., Corning, New York)), e.g., a tube with a volume of less than 40 mL. The tube can also be a well (e.g., a standard 96-sample well kit). The tube can also be a centrifuge, microcentrifuge, or PCR tube (e.g., those sold by Eppendorf®, Hamburg, Germany). Such tubes can be, for example, between 0.1 and 6 mL.

[0013] For any given tube, sample preparation for sequencing can be completed in less than one day, and advantageously, in less than three hours. Furthermore, sample preparation within each tube can be completed in as little as about five minutes or as little as about two minutes, which is in contrast to microfluidic cell preparations, which often require three days for sample preparation, and is even more advantageous than previous emulsion-based preparations, which required at least additional steps and time to barcode each nucleic acid molecule.

[0014] Methods for sequencing nucleic acid molecules are well known, and the present invention can include any known method for nucleic acid sequencing, such as Sanger sequencing or next-generation sequencing. In the method of the present invention, the sequencing step includes detecting mutations (e.g., cancer mutations). Advantageously, the present invention allows for the detection of mutations with a frequency of less than 0.05% in cells in a sample. Thus, the present invention allows for the detection of cancer cells from small amounts of DNA, for example, less than 10 ng of DNA. The method of the present invention can be applied to any known human sample. For example, the above method can be easily applied to the analysis of tumors taken from a subject (e.g., from a tumor biopsy). By nucleic acid sequencing, cancer cells can be identified from a biopsy, and a patient can be diagnosed with cancer. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows monodisperse droplets according to one aspect of the present invention. [Figure 2] FIG. 2 shows a micrograph of a monodisperse droplet containing a flat-faceted template particle according to the embodiment of FIG. [Figure 3] FIG. 3 shows a schematic diagram of a monodisperse droplet containing an internal compartment. [Figure 4] FIG. 4 shows a schematic diagram of a monodisperse droplet containing an internal compartment. [Figure 5] FIG. 5 shows a schematic diagram of monodisperse droplets after release of nucleic acid molecules from single cells. [Figure 6]FIG. 6 shows a schematic diagram of a method for single cell analysis according to some aspects of the present disclosure. [Figure 7] FIG. 7 shows a schematic diagram of monodisperse droplets after release of nucleic acid molecules from single cells. [Figure 8] FIG. 8 shows a schematic diagram of monodisperse droplets with capture probes. [Figure 9] FIG. 9 illustrates a method for single cell analysis according to another aspect of the present disclosure. [Figure 10] FIG. 10 shows a schematic diagram of monodisperse droplets after release of nucleic acid molecules from single cells and the distribution of the template particles. [Figure 11] FIG. 11 shows a schematic diagram of monodisperse droplets with capture probes. [Figure 12] FIG. 12 shows a representative diagram of a capture probe. [Figure 13] FIG. 13 shows a representative diagram of the first complementary strand synthesis. [Figure 14] FIG. 14 shows a representative diagram of second complementary strand synthesis. [Figure 15] FIG. 15 shows a schematic diagram of monodisperse droplets in a tube. [Figure 16] FIG. 16 shows a schematic diagram of a method for bursting monodisperse droplets. DETAILED DESCRIPTION OF THE INVENTION

[0016] Detailed Description The present invention provides a method and system for analyzing single cells by combining template particles and multiple single cells in a tube and simultaneously generating multiple monodisperse droplets in the tube, each encapsulating exactly one of the template particles and exactly one of the single cells. Nucleic acids are released from the single cells, and each nucleic acid molecule in the droplets is provided with a barcode unique to the droplet. Each nucleic acid molecule can then be analyzed by any known method, for example, by sequencing the nucleic acid. The nucleic acid molecules can be any nucleic acid molecule, including DNA and / or RNA.

[0017] The barcode can be any group of nucleotide or oligonucleotide sequences that are distinguishable from other barcodes within the group. The droplets encapsulating the template particles and single cells provide each nucleic acid molecule released from the single cell with the same barcode derived from the group of barcodes. The barcode provided by each droplet is unique to that droplet and distinguishable from barcodes provided to nucleic acid molecules by any other droplets. Once sequenced, the nucleic acid molecule can be traced back to the droplet and thereby to each single cell using the barcode sequence. The barcode can be of any appropriate length sufficient to distinguish the barcode from other barcodes. For example, the barcode can be 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 length. The barcodes may be predefined, degenerate, and / or randomly selected.

[0018] Barcodes can be added to nucleic acid molecules by "tagging" the nucleic acid molecules with the barcode. Tagging can be performed using any known method for barcode addition, such as direct ligation of a barcode to one or more of the ends of each nucleic acid molecule. Nucleic acid molecules can be end-repaired, for example, to allow for direct or blunt-end ligation of the barcode. Barcodes can also be added to nucleic acid molecules via first-strand synthesis or second-strand synthesis, for example, using capture probes, as described herein below.

[0019] The template particles may include any known particle that can be used to form the monodisperse droplets, and may advantageously provide a unique barcode for each droplet. Template particles for single-cell analysis utilize the particle-templated emulsification technique previously described in Hatori et al., Anal. Chem., 2018 (90):9813-9820 (incorporated by reference). Most frequently, micron-scale beads (e.g., hydrogels) are used to define an isolated fluid volume surrounded by an immiscible partitioning fluid and stabilized by a temperature-insensitive surfactant.

[0020] 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 the desired diameter to capture and specifically tag cells. For example, the size of the template particles can be adjusted by microfluidic co-flow into an immiscible oil phase.

[0021] Template particles can vary in size. Variation can be limited, for example, so that at least 50% or more of the template particles, for example, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more, can vary in diameter or maximum diameter by less than 10 times, for example, less than 5 times, less than 4 times, less than 3 times, less than 2 times, less than 1.5 times, less than 1.4 times, less than 1.3 times, less than 1.2 times, less than 1.1 times, less than 1.05 times, or less than 1.01 times.

[0022] Advantageously, the absorbency of the template particles of the present disclosure can be increased by storing them in dehydrating conditions, generally with the intention of reducing their volume before using them in the methods of the present disclosure for single-cell analysis. Advantageously, shrinking the template particles allows for control of the shape and size of the template particles to capture cells and barcode released nucleic acid molecules, e.g., with a barcode unique to each droplet. For example, dehydration of the template particles can be achieved by storing them in a buffer with a high osmolarity (i.e., polyethylene glycol) to promote shrinkage. Alternatively, the template particles can be dehydrated with ethanol. Shrinkage can occur upon application of an external stimulus (e.g., heat). For example, the template particles can be advantageously encapsulated in a fluid by shear, followed by application of heat, causing the template particles to shrink in size. Some other examples of drying approaches include, but are not limited to, heating, drying under vacuum, freeze-drying, and supercritical drying. The dried template particles can also be combined with a fluid but still retain their shape and structure as independent, often spherical, gel particles. The dried template particles can be combined with an appropriate fluid, causing a portion of the fluid to be absorbed by the template particles. The porosity of the template particles can also be varied to allow at least one of a plurality of cells to be absorbed into the template particles when combined with the appropriate fluid. Any convenient fluid can be used that allows the desired absorption to occur in the template particles.

[0023] The template particle is advantageously a very small, generally spherical particle. The template particle may be porous or non-porous. The template particle may also comprise microcompartments or internal compartments, which may advantageously comprise additional components and / or reagents (for example, additional components and / or reagents that may be releasable into monodisperse droplets). Advantageously, the template particle may comprise a microcompartment that comprises the barcode specific to each droplet for use in tagging nucleic acid molecules released from single cells within the droplet.

[0024] Template particles for such uses may comprise polymers such as hydrogels. Template particles generally range in diameter or maximum diameter from about 0.1 to about 1000 μm. Template particles have 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). Template particles may have 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.

[0025] Cells analyzed by the present invention 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, HBSS (Hank's Balanced Salt Solution)), and in certain cases, supplemented with fetal bovine serum or other naturally occurring factors at low concentrations (generally 5-25 mM) along with an acceptable buffer. Convenient buffers include HEPES, phosphate buffer, lactate buffer, and the like. Separated cells may be collected in any suitable medium that maintains cell viability, typically with a cushion of serum at the bottom of the collection tube. Various media are commercially available and may be used depending on the nature of the cells (e.g., dMEM, HBSS, DPBS, RPMI, IMDM (Iscove's Medium), and the like, frequently supplemented with fetal bovine serum). Preferably, the cell is a mammalian cell, such as a human cell.

[0026] The composition and properties of the template particles can vary depending on the single-cell analysis being performed. For example, the template particles can be microgel particles, which are micron-scale spheres of a gel matrix. The microgels are composed of hydrophilic polymers (including alginate or agarose) that dissolve in water. The microgels can also be composed of lipophilic microgels.

[0027] The template particle can also be a hydrogel (e.g., a hydrogel derived from a naturally-derived material, a synthetic material, or a combination 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 (NIPAM), and polyanhydride, poly(propylene fumarate) (PPF).

[0028] The template particles may further advantageously comprise a substance that provides the template particles with a positive or increased positive surface charge. Such substances may be, but are not limited to, polylysine or polyethyleneimine, or a combination thereof. This may increase the chance of association between the template particles and, for example, cells, which generally have mostly negatively charged membranes.

[0029] Other strategies aimed at increasing the chance of template particle-cell association include creating specific template particle geometries. For example, 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.

[0030] Any one of the above strategies and methods, or a combination thereof, can be used in implementing the template particles and methods for single-cell analysis of the present disclosure. Methods for generating template particles and template particle-based encapsulation are described in International Patent Publication WO 2019 / 139650, which is incorporated herein by reference.

[0031] Fabrication of template particle-based encapsulation for single-cell analysis can include combining a single cell and multiple template particles in a first fluid to provide a mixture in a reaction tube. The mixture can be incubated to allow the multiple template particles to associate with the single cell. A portion of the multiple template particles can become associated with the single cell. The mixture is then combined with a second fluid that is immiscible with the first fluid. The fluid and mixture are then sheared to generate multiple monodisperse droplets in the reaction tube. The generated monodisperse droplets contain (i) at least a portion of the mixture, (ii) a single template particle that provides the droplet with a unique barcode, and (iii) a single cell. It should be noted that, while a substantial number of the generated monodisperse droplets contain a single template particle and a single cell, in some cases, some of the monodisperse droplets may contain zero or more than one template particle or cell. In such cases, monodisperse droplets containing no cells, or more than one cell, no template particle, and / or more than one template particle (and thus more than one barcode) may be excluded from further analysis.

[0032] FIG. 1 shows monodisperse droplets according to one aspect of the invention. The illustrated monodisperse droplet 10 includes a template particle 1, a single cell 3. The illustrated template particle includes a flat facet 2, providing a barcode molecule unique to the droplet. FIG. 2 shows a micrograph of a flat-faceted template particle 1 according to one embodiment of FIG. 1. Each monodisperse droplet 1 in FIG. 2 includes a unique barcode that is distinguishable from barcodes used by other droplets. In some embodiments, the first fluid is an aqueous-phase fluid and the second fluid is an oil, such as a fluorocarbon oil, a silicone oil, or a hydrocarbon oil, or a combination thereof.

[0033] To increase the chance of generating an encapsulation (e.g., a monodisperse droplet 10 containing one template particle 1 and one single cell 3), the template particles and cells can be combined in a ratio where there are more template particles than cells. For example, the ratio of template particles to cells combined in the mixture can range from 5:1 to 1,000:1, respectively. The template particles and cells can also be combined in ratios of 10:1, 100:1, or 1000:1, respectively.

[0034] The step of shearing the second mixture to produce the monodisperse emulsion 10 is provided by combining a first mixture containing template particles and cells with a second fluid immiscible with the first mixture. Any suitable method can 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. 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 encapsulating one of the template particles.

[0035] Generating the template particle-based monodisperse droplets may require shearing two liquid phases. For example, the mixture may be an aqueous phase containing a reagent selected from, for example, a buffer, salt, lytic enzymes (e.g., proteinase k) and / or other lytic reagents (e.g., Triton X-100, Tween-20, IGEPAL, bm 135, or a combination thereof), a nucleic acid synthesis reagent (e.g., a nucleic acid amplification reagent), or a combination thereof. The fluid may be a continuous phase and may be an immiscible oil (e.g., a fluorocarbon oil, a silicone oil, or a hydrocarbon oil, or a combination thereof). The fluid may advantageously contain a reagent such as a surfactant (e.g., octylphenol ethoxylate and / or octylphenoxypolyethoxyethanol), a reducing agent (e.g., DTT, β-mercaptoethanol, or a combination thereof).

[0036] In practicing the methods as described herein, the composition and properties of the monodisperse droplets (e.g., single emulsion and multiple emulsion droplets) can be varied. Advantageously, a surfactant can be used to stabilize the droplets 10. The monodisperse droplets described herein can be prepared as emulsions, e.g., 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, e.g., 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.

[0037] 3 is a schematic diagram of a single monodisperse droplet according to another aspect of the present invention. The monodisperse droplet 10 shown includes a template particle 1 and a single cell 3. The template particle 3 includes crater-like depressions 2, and in the illustrated embodiment, the single cell 3 is associated with one of the crater-like depressions 2. The single cell 3 further includes at least one internal compartment 4.

[0038] As described above, the template particle can include multiple internal compartments 4. The internal compartments 4 of the template particle 1 can be used to encapsulate reagents that can be triggered to release a desired compound (e.g., a substrate for an enzymatic reaction) or induce a particular outcome (e.g., lysis of associated single cells 3). The reagents encapsulated within compartments 4 of the template particle can be reagents selected from, but are 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.

[0039] The interior compartment 4 can also be used to encapsulate a barcode unique to the template particle 1, and thus the droplet 10. When nucleic acid molecules are released from the cell 3, they are then tagged with the droplet-specific barcode provided by the template particle 1. Once sequenced, each nucleic acid molecule can be identified along with its source template particle 1, droplet 10, and cell 3.

[0040] Figure 4 shows a schematic diagram of another embodiment of one of a plurality of monodisperse droplets 10. The depicted monodisperse droplet 10 in Figure 4 includes a template particle 1 and a single cell 3. The depicted template particle 1 is generally spherical, and in the depicted embodiment, the single cell 3 is associated with the template particle 1. The template particle 1 further includes an internal compartment 4, which contains a reagent (such as a lysis reagent). The internal compartment 4 can also be used to encapsulate a barcode unique to the template particle 1, and thus to the droplet 10.

[0041] 5 shows a schematic diagram of the monodisperse droplets after an external stimulus 6. After the external stimulus 6 is applied, lysis reagents are activated and released, lysing the template particles 1 8 and lysing the single cells 9, while the monodisperse droplets 10 remain intact as indicated by the intact encapsulation shell 5. In some embodiments, the external stimulus 6 can be heat or osmotic pressure. Within the droplets, each nucleic acid molecule is tagged with a barcode unique to the droplet, and the droplets remain intact to allow each nucleic acid molecule to be tagged.

[0042] Release of nucleic acid molecules from single cells can include lysis of the single cells within the monodisperse droplets 10. Lysis 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. As shown in FIG. 3, one or more of the reagents (e.g., lysis reagents, detergents, enzymes) can be compartmentalized within the template particle 14. In other embodiments, one or more of the reagents are present in a mixture. In some other embodiments, one or more of the reagents are added to a solution containing the monodisperse droplets 10, if desired.

[0043] The methods of the present invention generally relate to the analysis and sequencing of barcoded nucleic acid molecules from single cells. The methods involve releasing nucleic acid molecules from isolated single cells 3 within monodisperse droplets 10, tagging each nucleic acid molecule with a barcode unique to the monodisperse droplet, and then sequencing the nucleic acid molecules. The sequencing step can analyze genomic regions of interest, such as oncogenes. Thus, PCR amplification of products derived from nucleic acid molecules released by single cells can be used to determine the cell's genotype for a given genetic mutation, such as a mutation associated with cancer. Genes and mutations of interest may 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, M UC1 / 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. For example, identifying the gene or mutation of interest can provide information that the cell into which the nucleic acid molecule is released has a cancer genotype or is a cancer cell. Because each nucleic acid molecule is tagged with a barcode unique to the droplet and the single cell from which it was released, any gene or mutation of interest can be traced back to the droplet and single cell, thereby allowing for the identification of the cancer genotype in the cell.For RNA or mRNA sequencing, the sequencing step can include first preparing a cDNA library from barcoded RNA via reverse transcription, and sequencing the cDNA. RNA sequencing can advantageously allow for the quantification of gene expression within the single cell, and can be used to identify characteristics of the single cell that can be used, for example, to make a diagnosis, predict prognosis, or determine drug efficacy. Reverse transcription of cDNA molecules from RNA can be performed either within the droplet, or after the barcoded RNA molecules are released from each droplet.

[0044] Reverse transcription can be carried out using, without limitation, dNTPs (a mix of nucleotides dATP, dCTP, dGTP, and dTTP), a buffer, a detergent, or, if needed, a solvent, and an appropriate enzyme (e.g., a polymerase or reverse transcriptase). The polymerase used can be a DNA polymerase and can be selected from Taq DNA polymerase, Phusion polymerase (as provided by Thermo Fisher Scientific (Waltham, Massachusetts)), or Q5 polymerase. Nucleic acid amplification reagents are commercially available and can be purchased, for example, from New England Biolabs (Ipswich, MA, USA). The reverse transcriptase used in the targeted library preparation method of the present disclosure can be, for example, maxima reverse transcriptase. In some embodiments, the 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.

[0045] Sequencing of nucleic acid molecules can be performed by methods known in the art. For example, see 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 molecule sequencing techniques include classical dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in slabs or capillaries, or preferably, next generation sequencing methods. For example, sequencing can be performed according to the techniques described in U.S. Patent Application 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 of which is incorporated by reference).

[0046] 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).

[0047] The present invention provides a method for identifying rare cells (e.g., cancer cells) from a heterogeneous cell population during early cancer detection. The method involves 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 cell and a single template particle. The method further includes releasing a nucleic acid molecule from each of the single cells within the monodisperse droplets, tagging each nucleic acid molecule with a barcode unique to the monodisperse droplet, and sequencing the nucleic acid molecule. The method enables the detection of cancer cells in both small and large samples, where each cell is simultaneously separated and the nucleic acid molecule is tagged to the single cell from which the nucleic acid molecule was released, while the rare single cell is within the sample.

[0048] For example, the method allows for the analysis of 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 taken 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 nucleic acid molecule from each of the separated single cells contained in the monodisperse droplets, tagging each nucleic acid molecule with a barcode unique to the droplet, and sequencing the nucleic acid molecule to identify one or more genotypic characteristics of the tumor. Advantageously, the unique barcode is provided to the droplets by the template particle. The disclosed methods further include using the genotype to diagnose a subject with cancer or to diagnose the stage of cancer, or to devise a treatment plan based, for example, on the number of cells identified as cancerous cells.

[0049] Nucleic acid molecules can be advantageously amplified before sequencing. Amplification can include the use of thermal cycling to create copies of nucleic acids by exposing the reaction to repeated cycles of heating and cooling, allowing for 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 previously described methods. Non-limiting examples of PCR reactions include real-time PCR, nested PCR, multiplex PCR, quantitative PCR, or touchdown PCR. Notably, each amplified copy of the nucleic acid molecule contains a droplet-specific barcode to identify the droplet and cell from which the nucleic acid molecule is released.

[0050] The template particle may also contain multiple capture probes. Typically, the capture probes are oligonucleotides. The capture probes may be attached to the template particle material via a covalent acrylic bond. The capture probes may be 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 other activated double bond-containing compounds (e.g., acrylamide). Specifically, copolymerization of the acrydite-modified capture probes with acrylamide, including a crosslinker (e.g., N,N'-methylenebis), results in a crosslinked gel material containing covalently attached capture probes. The capture probes may also contain acrylate-terminated hydrocarbon linkers, which, when combined with the template particle, cause their binding to the template particle.

[0051] The capture probe may include one or more of a primer sequence, the barcode unique to each droplet, a unique molecular identifier (UMI), and / or a capture sequence.

[0052] The primer sequence may contain a binding site, for example, the primer sequence is expected to hybridize to a complementary sequence (if present) on any nucleic acid molecule released from a cell and provide a starting site for a reaction (e.g., an extension or polymerization reaction). The primer sequence may also be a "universal" primer sequence, i.e., a sequence that is complementary to a nucleotide sequence that is very common in a particular set of nucleic acid fragments. The primer sequence that can be used may be the P5 and P7 primers provided by Illumin, Inc. (San Diego, California). The primer sequence may also allow the capture probe to bind to a solid support (e.g., a template particle).

[0053] By providing each droplet with a capture probe containing the unique barcode, the capture probe can be used to tag nucleic acid molecules released from single cells with the barcode. This process (discussed further hereinbelow) can involve hybridizing the nucleic acid molecule to the capture probe, followed by an amplification or reverse transcription reaction.

[0054] A unique molecular identifier (UMI) is a type of barcode that can be provided to nucleic acid molecules in a sample to make each nucleic acid molecule, along with its barcode, unique or nearly unique. This is achieved by adding, for example, ligating, one or more UMIs to the end of each nucleic acid molecule, so that any two previously identical nucleic acid molecules, along with their UMIs, are unlikely to have the same sequence. By selecting an appropriate number of UMIs, every nucleic acid molecule in the sample, along with its UMI, will be unique or nearly unique. One strategy for achieving this is to provide a sample of nucleic acid molecules with a number of UMIs in excess of the number of starting nucleic acid molecules in the sample. In this way, each starting nucleic acid molecule is provided with a different UMI, so that each molecule is unique along with its UMI. However, the number of UMIs provided can be as small as the number of identical nucleic acid molecules in the original sample. For example, if six or fewer nucleic acid molecules in a sample are likely to be identical, as few as six different UMIs can be provided, regardless of the number of starting nucleic acid molecules.

[0055] 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.Methods for error correction using UMI are described in Karlsson et al., 2016, "Counting Molecules in cell-free DNA and single cells RNA", Karolinska Institutet, Stockholm, Sweden (<https: / / openarchive.ki.se / xmlui / handle / 10616 / 45053> and is incorporated herein by reference).

[0056] The capture sequence used in the capture probe is advantageously for targeting a gene-specific nucleotide sequence, such as a nucleotide sequence known to be associated with a particular cancer genotype or phenotype. In such a method, the target nucleic acid sequence, if present, binds to the template particle by hybridizing to the capture sequence upon release from the single cell.

[0057] 6 is a schematic diagram of a monodisperse droplet. The illustrated monodisperse droplet 10 includes a template particle 1 and a single cell 3. The illustrated template particle 1 is generally spherical and may include multiple capture probes 12. The probes advantageously include a barcode unique to the droplet. Reagents (e.g., lysis reagent 11) are present within the monodisperse droplet 10.

[0058] Figure 7 shows the monodisperse droplets of Figure 6 after an external stimulus 6. After the stimulus 6, a lysis reagent 11 is activated, lysing the single cell 3 while the encapsulation, i.e., monodisperse droplet 10 (shown with intact encapsulation shell 5) and template particle 1, remain intact. Upon lysis of the single cell 3, nucleic acid molecules 15 previously contained within the cell are released into the monodisperse droplet 10. Some of the released nucleic acid molecules 15 associate with some of the capture probes 12, as shown in Figure 8. Advantageously, the capture probes may include a barcode unique to the monodisperse droplet and can be used to tag the nucleic acid molecules with the barcode.

[0059] The nucleic acid molecules hybridized to the capture probe can be released, for example, by dissolving the template particle or by using a reducing agent to reduce the dithiol oligonucleotide modification in the capture probe linker region. Subsequent steps (e.g., amplification) following the association of the nucleic acid molecule and the capture probe can be performed either inside the encapsulation or in bulk. When a step is performed in bulk, an aqueous solution containing a medium inside the encapsulation is generated upon disruption of the encapsulation. Any reagents (e.g., lysis reagents or nucleic acid synthesis reagents) can be supplied in bulk, provided during the creation of partitions (e.g., present in the first mixture), compartmentalized within the template particle, or a combination thereof.

[0060] 9 shows a schematic diagram of a method for single-cell analysis according to another aspect of the present disclosure. A single monodisperse droplet 10 from among multiple monodisperse droplets is shown, containing a template particle 1 and a target cell 3. The template particle 1 contains multiple capture probes 12. As noted above, each capture probe may contain a barcode unique to the droplet. Reagents (e.g., lysis reagent 11) are present within the monodisperse droplet 10.

[0061] Figure 10 shows the monodisperse droplets of Figure 9 after the stimulus 6. After the external stimulus 6, the lysis reagent 11 is activated, lysing the single cell 3 and dissolving the template particle 1, while the monodisperse droplet 10 remains intact (shown is the encapsulation shell 5). As the template particle 1 dissolves, the capture probe 12 is released therefrom. Upon lysis of the single cell 3, the nucleic acid molecules 15 previously contained within the single cell 3 are released. Some of the released nucleic acid molecules 15 associate with the capture probe 12, as shown in Figure 11. Even in the absence of the template particle, the capture probe may contain a barcode unique to the monodisperse droplet and can be used to tag the nucleic acid molecule with the barcode.

[0062] 12 illustrates a method for barcoding nucleic acids with capture probes according to certain aspects of the present disclosure. As illustrated, the template particle 1 includes multiple capture probes 12, which are diagrammatically illustrated by dashed curves. One of the capture probes 12 is characterized on a larger scale and in more detail. The capture probe 12 preferably includes, from its 5' to 3' end, a linker region capable of covalently bonding to the template particle 1, a "PR1" nucleotide sequence region including a primer nucleotide sequence, at least one UMI, a barcode 201 ("BRCD") unique to the droplet, and a capture nucleotide sequence 22 including a sequence complementary to the nucleic acid molecule.

[0063] Figure 13 shows a released nucleic acid molecule 15, optionally including a tail sequence. The nucleic acid molecule binds to a capture probe of complementary sequence 22 of Figure 12 via complementary base pairing. For RNA molecules, the poly-A tail of the RNA molecule can be used to bind the RNA molecule to the capture probe, for example, by using a capture probe with a poly-T sequence. After hybridization of the nucleic acid molecule 15 and the capture probe 12, a polymerase (or, in the case of RNA, a reverse transcriptase) is used to generate a first complementary strand 23. For analysis of RNA, the first complementary strand can be a cDNA strand. The first strand 23 includes a complement to the nucleic acid molecule and the barcode sequence 201. The nucleic acid molecule 15-first complementary strand 23 hybrid can be denatured (not shown) using any method conventional in the art, such as exposure to denaturing temperatures.

[0064] Figure 14 shows the composite of Figure 13, in which a second strand primer 24 containing a random hexamer sequence anneals to the first strand 23 to form a DNA-primer hybrid. A DNA polymerase is used to synthesize a second complementary strand 25, complementary to the first strand. The second complementary strand contains the sequence of the released nucleic acid molecule and a barcode unique to the droplet. Upon denaturation from the first complementary strand, the second complementary strand may be sequenced, and the sequence of the barcode may be used to identify the droplet and cell from which the nucleic acid molecule was released.

[0065] Methods using one or more ligation tagging and capture probe tagging of nucleic acid molecules with droplet-specific barcodes and / or UMIs can be performed.

[0066] The complement of the nucleic acid when aligned does not need to be perfect; a stable duplex can contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can empirically consider many variables, including, for example, the length of the oligonucleotide, the percentage concentration of cytosine and guanine bases in the oligonucleotide, ionic strength, and the occurrence of mismatched base pairs, to determine duplex stability.

[0067] 15 and 16 show schematic diagrams of a method for bursting monodisperse droplets 10 according to aspects of the present disclosure. The monodisperse droplets 10 are shown as circles in a fluid at the bottom of a test tube. The fluid containing the encapsulated material is overlaid with a reagent such as a high-salt buffer (middle layer) and a breaker reagent (top layer). The high-salt buffer may include β-mercaptoethanol and / or DTT, or other reducing reagents. The breaker reagent may include perfluorooctanol (PFO). Incubation of the fluid with the high-salt buffer and breaker reagent is preferably performed on ice.

[0068] Figure 16 shows the monodisperse droplets of Figure 15 after mixing, for example, by vortexing, shearing 18, and / or spinning 19. The monodisperse droplets 10 are disrupted, forming two phases 20, 21, aqueous and oil. Depending on the type of oil used, the oil layer can be the bottom or top layer. The template particles and any associated nucleic acids reside in the aqueous layer.

[0069] Nucleic acid molecules (including nucleic acid molecules bound to capture probes, released nucleic acid molecules, or amplified nucleic acid molecules) can be bound to streptavidin-coated magnetic beads. For example, streptavidin-coated magnetic beads bound to biotin-labeled oligonucleotides containing a bait sequence can be used. The bait sequence can be complementary to the primer sequence of the nucleic acid molecule. This can be, for example, one or more UMIs, droplet-specific barcodes on the released nucleic acid molecules. The streptavidin-coated magnetic beads containing the bait sequence can then be incubated with the nucleic acid molecule to allow hybridization of complementary sequences. The nucleic acid molecule can first be incubated with a biotin-labeled oligonucleotide containing a bait sequence, where the bait sequence is complementary to one or more barcodes of the nucleic acid molecule to allow hybridization of complementary sequences. After incubation, streptavidin-coated magnetic beads are added to the nucleic acid molecule / biotin-labeled oligonucleotide mixture and further incubated to allow streptavidin-biotin binding. The incubation step can be performed on ice.

[0070] Alternatively, generic nucleic acid capture beads can be used (e.g., polystyrene beads surrounded by a layer of magnetite and / or carboxyl molecules, e.g., beads with a similar surface characteristic of SPRI beads). SPRI beads are described by Deangelis et al. (1995) "Solid-phase reversible immobilization" (SPRI). for the isolation of PCR products,” Nucleic Acids Res. 23(22):4742-3 (incorporated by reference).

[0071] The template particle used in the present invention may further comprise a capture moiety. This capture moiety acts to capture specific cells, for example, specific types of cells. This capture moiety may comprise an acrylate-terminated hydrocarbon linker with a biotin terminus. This capture moiety may be bound to a target-specific capture element, for example, an aptamer and / or an antibody. Examples of capture moieties and methods thereof are disclosed in PCT Application No. PCT / US2019 / 053426 (incorporated herein by reference).

[0072] As mentioned above, the tube can be selected based on the volume of the sample from which cells need to be separated and / or based on the number of cells to be separated. For example, the tube can be a single large tube (e.g., a conical centrifuge tube (e.g., Falcon® as sold by Corning Inc., Corning, New York)), e.g., a tube with a volume of less than 40 mL. Such a tube can be advantageous when the number of cells to be analyzed is between 100,000 and 1 million cells or more than 1 million cells. This method is useful for analyzing cells for targeted coverage of heterogeneous cell types and investigating pathways in complex tissues, e.g., in the detection of cancer in mixed cell populations.

[0073] The tubes may also be wells (e.g., a standard 96-sample well kit). The wells may be part of a microplate with multiple wells (each used as a tube). The microplate may contain any number of wells (e.g., 6 to 1536 wells) as desired. Advantageously, the microplate may contain 96 wells. Wells may be advantageous when the number of cells to be analyzed is about 100 cells. This method is useful for deep profiling of homogeneous cells under different conditions (e.g., early cancer detection at tumor sites).

[0074] The tubes can also be centrifuge, microcentrifuge, or PCR tubes (e.g., those sold by Eppendorf® (Hamburg, Germany)). Such tubes can be, for example, between 1 and 6 mL and can be advantageous when the number of cells to be analyzed is approximately 10,000 cells. This method is useful for deep profiling of heterogeneous cell populations (e.g., in early cancer detection in mixed cell populations).

[0075] As described above, because cells are simultaneously encapsulated in monodisperse droplets, the method of the present invention can be easily scaled to analyze any number of cells. For example, a tube can be selected to analyze at least 1 million cells, at least 2 million cells, at least 10 million cells, at least 100 million cells, or 200 million cells, or more. Furthermore, because cells are simultaneously encapsulated in any tube and for any number of cells, sample preparation for sequencing can be completed within one day, or even within three hours. Furthermore, sample preparation in each tube can be completed in as little as about 5 minutes or about 2 minutes.

[0076] Primers and / or reagents can be added to the monodisperse droplets after they are formed in the tube. Primers and / or reagents can be added in one step or more than one step. For example, the primers can be added in two or more steps, three or more steps, four or more steps, or five or more steps. Regardless of whether the primers are added in one step or more than one step, they can be added after, before, or simultaneously with the addition of the lysis agent. If added before or after the addition of the lysis agent, PCR primers can be added in a step separate from the addition of the lysis agent.

[0077] References References and citations to other documents (e.g., patents, patent applications, patent publications, journals, books, articles, web content) are made throughout this disclosure. All such documents are incorporated herein by reference in their entirety for all purposes.

[0078] equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the foregoing embodiments should be considered in all respects as illustrative rather than limiting on the invention described herein. The scope of the invention is, therefore, indicated by the appended claims, rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are accordingly intended to be embraced therein. The present invention provides, for example, the following items. (Item 1) 1. A method for single cell sequencing, the method comprising: combining the template particles and a plurality of single cells in a tube; simultaneously generating a plurality of monodisperse droplets in the tube, each encapsulating exactly one of the template particles and exactly one of the single cells; Releasing nucleic acid molecules from the single cells and providing each nucleic acid molecule within the droplet with a barcode that is unique to the droplet; and sequencing the nucleic acid molecule; A method that encompasses (Item 2) 2. The method of claim 1, wherein the nucleic acid molecule is a DNA molecule. (Item 3) Item 10. The method of claim 1, wherein the barcode unique to the droplet is provided to the droplet by the template particle encapsulated by the droplet. (Item 4) Item 4. The method of item 3, wherein the template particle further comprises one or more compartments. (Item 5) 5. The method of claim 4, wherein the one or more compartments comprise: (1) a reagent selected from the group consisting of a lysis reagent, a nucleic acid synthesis reagent, or a combination thereof; (2) the barcode unique to the droplet; and / or (3) a unique molecular identifier (UMI). (Item 6) 6. The method according to item 5, wherein the nucleic acid synthesis reagent comprises a polymerase or a reverse transcriptase. (Item 7) 7. The method of claim 6, wherein the reagent, barcode, or UMI is released from the one or more compartments in response to an external stimulus. (Item 8) The template particle is Primer sequences; a barcode unique to said droplet; Unique molecular identifier (UMI); and capture sequence, 4. The method of claim 3, comprising a plurality of capture probes comprising one or more of: (Item 9) 9. The method of claim 8, wherein the capture sequence is a gene-specific nucleotide sequence. (Item 10) 9. The method of claim 8, wherein the nucleic acid molecule, upon release from the single cell, binds to the template particle by hybridizing to the capture sequence. (Item 11) 11. The method of claim 10, wherein the nucleic acid molecule is tagged with the barcode and / or UMI unique to the droplet by first and / or second strand synthesis. (Item 12) The steps of combining the template particles, generating droplets, and releasing the nucleic acid molecules from the cells include: combining the template particle and the single cell 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 single cells; and lysing each of the single cells contained within the monodisperse droplets to release a plurality of nucleic acid molecules; 12. The method according to any one of items 1 to 11, comprising: (Item 13) Item 13. The method of item 12, wherein the first fluid and the second fluid are immiscible. (Item 14) Item 13. The method of item 12, wherein the first fluid comprises an aqueous phase fluid. (Item 15) Item 15. The method of claim 14, wherein the second fluid comprises oil. (Item 16) 13. The method of claim 12, wherein the step of shearing the fluid comprises vortexing, shaking, flicking, stirring, or pipetting. (Item 17) 13. The method of claim 12, further comprising amplifying the nucleic acid molecule by PCR prior to the sequencing step. (Item 18) 18. The method according to any one of items 1 to 17, further comprising, after the sequencing step, identifying, among the plurality of cells, cells having a gene or mutation associated with cancer. (Item 19) 19. The method of claim 18, wherein the mutation has a frequency of less than 0.05% among cells in the sample. (Item 20) 19. The method of claim 18, wherein the cancer mutation is detected from an amount of DNA or cDNA less than 10 ng before any amplification step. (Item 21) 21. The method according to any one of items 1 to 20, wherein the tube is a conical tube for centrifugation. (Item 22) 22. The method according to item 21, wherein the plurality of single cells is between 100,000 and 1 million cells. (Item 23) 21. The method according to any one of items 1 to 20, wherein the tube is a well, and the well is part of a microplate. (Item 24) 24. The method of claim 23, wherein the plurality of single cells in the well is about 100 cells. (Item 25) 21. The method of any one of items 1 to 20, wherein the tube is a centrifuge, microcentrifuge, or polymerase chain reaction (PCR) tube. (Item 26) 26. The method of claim 25, wherein the plurality of single cells is about 10,000 cells. (Item 27) 2. The method of claim 1, wherein the plurality of single cells is at least 1 million cells, at least 2 million cells, at least 10 million cells, at least over 100 million cells, or 200 million cells, or more cells. (Item 27) 28. The method of any one of items 21 to 27, wherein the steps of combining template particles, generating droplets, and releasing nucleic acid molecules are completed within 3 hours.

Claims

[Claim 1] The invention described in the present specification.

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