Molecular barcoding of nucleic acid targets in single particles mediated by capture magnetic beads and compositions therefor

Abstract

Embodiments of the invention provide methods of capturing molecular barcoding of nucleic acid targets of particles, e.g., cells or extracellular vesicles, mediated by magnetic beads. The methods include the following aspects: a) combining a sample comprising particles with capture magnetic beads comprising a capture moiety for the particles to produce a captured sample; b) partitioning the captured particles of the captured sample using a magnetic field application mediated partitioning protocol to produce partitioned captured particles, wherein the partitioned captured particles are in spatial proximity to bead-bound barcode nucleic acids comprising target binding regions; and c) lysing the partitioned captured particles such that nucleic acids released therefrom bind to the target binding regions to produce captured nucleic acids. Also provided are compositions, e.g., capture magnetic beads, including barcoded magnetic beads, as well as devices / systems and kits for practicing embodiments of the methods.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority under 35 U.S.C. § 119(e) of the filing date of U.S. Provisional Patent Application Serial No. 62 / 943,647, filed December 4, 2019. The disclosure of that application is incorporated herein by reference. BACKGROUND

[0003] The ability to detect and quantify specific nucleic acid and protein molecules in individual cells is critical to understanding the role of cellular diversity in development, health, and disease. Flow cytometry has become the standard technique for high-throughput detection of single-cell protein markers and has been widely applied in basic research and clinical diagnostics. In contrast, nucleic acid measurement methods, such as mRNA expression, are typically performed on bulk samples, obscuring the contribution of individual cells.

[0004] To characterize the complexity of cellular systems, there is a great need to develop methods, devices, and systems for monitoring the expression of a large number of genes in thousands of cells. Current technologies allow for the measurement of single-cell gene expression in a massively parallel manner (e.g., >10,000 cells) by linking cell-specific oligonucleotide barcodes to poly(A) mRNA molecules from single cells, where each single cell is co-localized with a barcoding reagent bead in a partition.

[0005] In addition to cells, there is growing interest in detecting nucleic acids in extracellular vesicles, such as exosomes and microvesicles. Extracellular vesicles, such as exosomes and microvesicles, are shed from almost all cell types and are widely distributed in blood and other body fluids. Multiple studies have demonstrated that various RNA types (including mRNA, miRNA, and IncRNA) enclosed within EVs can be transferred from donor cells to recipient cells and interfere with the latter’s gene expression. Turchinovich et al., “Trascriptome of Extracellular Vesicles: Sate-of-the-Art,” Front Immunol. (2019) 10:202. Thus, the characterization of extracellular vesicle nucleic acids is of interest. SUMMARY

[0006] While current technologies allow for the measurement of single-cell gene expression in a massively parallel manner (e.g., >10,000 cells), the inventors have identified certain shortcomings of the methods used to date. For example, currently used platforms can have difficulty partitioning small cells and vesicles with low sedimentation rates. In addition, currently used platforms can barcode and sequence many cells that are not of interest, leading to inefficient use of resources. Embodiments of the present invention address these and other needs.

[0007] Embodiments of the present application provide methods of molecular barcoding of nucleic acid targets of particles, such as cells or extracellular vesicles, mediated by capture magnetic beads. The methods comprise the following aspects: a) combining a sample comprising particles with capture magnetic beads comprising capture moieties for the particles to produce a captured sample; b) partitioning the captured particles of the captured sample using a magnetic field application mediated partitioning protocol to produce partitioned captured particles, wherein the partitioned captured particles are in spatial proximity to bead-bound barcode nucleic acids comprising target binding regions; and c) lysing the partitioned captured particles such that nucleic acids released therefrom bind to the target binding regions to produce captured nucleic acids. Compositions, such as capture magnetic beads, bead-bound barcoded magnetic beads, and devices / systems and kits for practicing embodiments of the methods are also provided.

[0008] Provided herein are methods of barcoding nucleic acids of particles. The methods comprise the following aspects: a) combining a sample with capture magnetic beads comprising capture moieties for particles of the sample to produce a captured sample; b) partitioning captured particles of the captured sample using an applied magnetic field-mediated partitioning protocol to produce partitioned captured particles, wherein the partitioned captured particles are in spatial proximity to bead-bound barcode nucleic acids comprising a target-binding region, e.g., an oligo-dT domain, a gene-specific domain, or a random sequence domain; and c) lysing the partitioned captured particles such that nucleic acids released therefrom bind to the target-binding region of the bead-bound barcode nucleic acids to produce captured nucleic acids. In some cases, the bead-bound barcode nucleic acids are tethered to the capture magnetic beads, while in other cases, the bead-bound barcode nucleic acids are tethered to a different bead that is barcoded than the capture magnetic beads. In some cases, the partitioned particles are partitioned into microwells. In some cases, the capture moieties comprise specific binding members, e.g., antibodies or binding fragments thereof. In some cases, the bead-bound barcode nucleic acids comprise, in addition to the target-binding region, one or more than one cell label domain, a unique unique molecular index domain, and a universal primer binding domain, e.g., wherein the bead-bound barcode nucleic acids have the following structure: Bead-5’-Universal Primer Binding Domain-Cell Label Domain-Unique Unique Molecular Index Domain-Target Binding Region-3’. The methods can be used to barcode nucleic acids from a variety of particles, e.g., biological particles, e.g., cells, as well as subcellular-sized particles, e.g., extracellular vesicles, platelets, vesicles, e.g., exosomes, or microvesicles. In some cases, the methods further comprise separating the captured nucleic acids from other components of the partitioned captured particles, e.g., by using an applied magnetic field. In some cases, the methods further comprise mixing the captured nucleic acids. In some cases, the methods further comprise subjecting the captured nucleic acids to cDNA synthesis reaction conditions to produce first-strand cDNA domains comprising the captured nucleic acids. In some cases, the methods further comprise producing an amplicon composition from the first-strand cDNA domains comprising the captured nucleic acids. In some cases, e.g., in embodiments in which the amplicon composition comprises a next-generation sequencing (NGS) library, the amplicon composition is produced from the first-strand cDNA domains comprising the captured nucleic acids using one or more than one round of amplification. In some cases, the amplicon composition comprises NGS adapters comprising the nucleic acids. In some cases, the methods further comprise sequencing the NGS library.

[0009] In some cases, the method comprises: a) combining a sample comprising particles with capture magnetic beads, the capture magnetic beads comprising: i) a barcode nucleic acid comprising a target binding region, and ii) a capture moiety that specifically binds to the particles, to produce a captured sample; b) partitioning the captured particles of the captured sample into microwells using a magnetic field application mediated partitioning protocol, to produce partitioned captured particles; lysing the partitioned captured particles such that nucleic acids released therefrom bind to the target binding region of the barcode nucleic acid to produce captured nucleic acids; subjecting the captured nucleic acids to conditions of a cDNA synthesis reaction to produce first strand cDNA domains comprising the captured nucleic acids; constructing an NGS library from the first strand cDNA domains comprising the captured nucleic acids; and sequencing the NGS library, thereby sequencing the nucleic acids of the target particles.

[0010] In some cases, the method further comprises using oligonucleotide-labeled cellular component binding reagents, for example, in applications where it is desirable to detect, e.g., quantify, one or more cellular components, e.g., surface proteins. The oligonucleotide-labeled cellular component binding reagents used in such embodiments include cellular component binding reagents, e.g., antibodies or binding fragments thereof, coupled to cellular component binding reagent specific oligonucleotides comprising an identifier sequence for the cellular component binding reagent, the identifier sequence associated with the cellular component binding reagent specific oligonucleotide. In such cases, the capture magnetic beads can comprise a moiety configured to capture, e.g., specifically bind, the cellular component binding reagent specific oligonucleotide domain. In this way, it is possible to combine gene expression analysis with protein expression, e.g., when it is desirable to perform a multi-omic analysis, e.g., a combined analysis of the transcriptome and proteome.

[0011] Also provided are capture magnetic beads useful for embodiments of the method. Embodiments of the capture magnetic beads include magnetic beads having a barcode nucleic acid stably bound to the magnetic bead and a capture moiety that specifically binds to the particles. In some cases, the capture moiety comprises an antibody or binding fragment thereof. In some cases, the barcode nucleic acid comprises, in addition to a target binding region (e.g., an oligo-dT domain, a gene specific domain, or a random sequence domain), one or more cell label domains, unique molecular index domains, and universal primer binding domains, e.g., where the bead-bound barcode nucleic acid has the following structure: bead-5’-universal primer binding domain-cell label domain-unique molecular index domain-target binding region-3’.

[0012] Also provided are devices useful for embodiments of the method. Embodiments of the device include: a) a substrate comprising 100 or more microwells, the microwells comprising a capture magnetic bead, the capture magnetic bead comprising: i) a barcode nucleic acid comprising a target binding region; ii) a capture moiety that specifically binds to the bead; and b) a flow cell in fluid communication with the substrate. In some cases, the capture moiety comprises an antibody or binding fragment thereof. In some cases, the barcode nucleic acid further comprises one or more cell label domains, unique molecular index domains, and universal primer binding domains in addition to the target binding region (e.g., an oligo-dT domain, a gene-specific domain, or a random sequence domain), e.g., wherein the bead-bound barcode nucleic acid has the following structure: Bead-5’-Universal Primer Binding Domain-Cell Label Domain-Unique Molecular Index Domain-Target Binding Region-3’.

[0013] Also provided are systems useful for embodiments of the method. Embodiments of the system include: a) a substrate comprising 100 or more microwells, the microwells comprising a capture magnetic bead, the capture magnetic bead comprising: i) a barcode nucleic acid comprising a target binding region; ii) a capture moiety that specifically binds to the particle; b) a flow cell in fluid communication with the substrate; and c) a flow controller, wherein the flow controller is configured to control fluid delivery to the flow cell; and in some cases is a magnetic field applicator, e.g., as described in detail below. In some cases, the capture moiety comprises an antibody or binding fragment thereof. In some cases, the barcode nucleic acid further comprises one or more cell label domains, unique molecular index domains, and universal primer binding domains in addition to the target binding region (e.g., an oligo-dT domain, a gene-specific domain, or a random sequence domain), e.g., wherein the bead-bound barcode nucleic acid has the following structure: Bead-5’-Universal Primer Binding Domain-Cell Label Domain-Unique Molecular Index Domain-Target Binding Region-3’.

[0014] Also provided are kits useful for embodiments of the method. Embodiments of the kit include: (a) a capture magnetic bead comprising a capture moiety that specifically binds to a target particle, and a barcode nucleic acid. In some cases, the capture magnetic bead comprises the barcode nucleic acid, while in other cases, the barcode nucleic acid is tethered to a barcoded bead that is separate from the capture magnetic bead. In some cases, the capture moiety comprises an antibody or binding fragment thereof. In some cases, the barcode nucleic acid further comprises one or more cell label domains, unique molecular index domains, and universal primer binding domains in addition to the target binding region (e.g., an oligo-dT domain, a gene-specific domain, or a random sequence domain), e.g., wherein the bead-bound barcode nucleic acid has the following structure: Bead-5’-Universal Primer Binding Domain-Cell Label Domain-Unique Molecular Index Domain-Target Binding Region-3’. In some cases, the kit further comprises a device comprising: (i) a substrate comprising 100 or more microwells; and (ii) a flow cell in fluid communication with the substrate.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the specification, drawings and claims are not meant to be limiting. Other embodiments can be used, and other changes can be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as described herein, and illustrated in the drawings, can be arranged, substituted, combined, separated, and designed in numerous different configurations, all of which are implicitly contemplated herein, and are expressly considered part of the present disclosure.

[0017] Figure 1A and Figure 1B A representative of a captured magnetic bead according to various embodiments of the application is provided.

[0018] Figure 2 A representative of a captured sample preparation according to embodiments of the application is provided.

[0019] Figure 3A and Figure 3B A representative of a partitioned captured particle according to embodiments of the application is provided.

[0020] Figures 4A-4C A representative of a partitioned captured particle according to embodiments of the application is provided.

[0021] Figure 5 A representative of a workflow using a system according to embodiments of the application is provided.

[0022] Figure 6 A graphical representation of a sequencing library preparation workflow that can be used in embodiments of the application is provided.

[0023] DEFINITIONS

[0024] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of the present disclosure, the following terms are defined with the following meanings.

[0025] The term “adapter” as used herein can refer to a sequence that facilitates amplification or sequencing of a related nucleic acid. The related nucleic acid can include a target nucleic acid. The related nucleic acid can include one or more spatial marker, target marker, sample marker, index marker, or barcode sequence (e.g., molecular marker). The adapter can be linear. The adapter can be a pre-adenylated adapter. The adapter can be double-stranded or single-stranded. One or more adapters can be located at the 5’ end or the 3’ end of a nucleic acid. When adapters at the 5’ end and 3’ end comprise known sequences, the known sequences can be identical or different sequences. An adapter located at the 5’ end and / or 3’ end of a polynucleotide is capable of hybridizing to one or more oligonucleotides immobilized on a surface. In some embodiments, an adapter can comprise a universal sequence. A universal sequence can be a region of nucleotide sequence that is common to two or more nucleic acid molecules. Two or more nucleic acid molecules can also have regions that are different in sequence. Thus, for example, a 5’ adapter can comprise identical and / or universal nucleic acid sequences, and a 3’ adapter can comprise identical and / or universal sequences. A universal sequence that can be present in different members of a plurality of nucleic acid molecules can allow replication or amplification of a plurality of different sequences using a single universal primer that is complementary to the universal sequence. Similarly, at least one, two (e.g., a pair of), or more universal sequences that can be present in different members of a collection of nucleic acid molecules can allow replication or amplification of a plurality of different sequences using at least one, two (e.g., a pair of), or more single universal primers that are complementary to the universal sequences. Thus, a universal primer includes a sequence that can hybridize to such a universal sequence. A molecule carrying a target nucleic acid sequence can be modified to have a universal adapter (e.g., a non-target nucleic acid sequence) attached to one or both ends of the different target nucleic acid sequence. The one or more universal primers attached to the target nucleic acid can be identical or different from each other.

[0026] An antibody as used herein can be a full-length (e.g., naturally occurring or formed from a normal immunoglobulin gene segment recombination process) immunoglobulin molecule (e.g., an IgG antibody) or an immunologically active (i.e., specifically binds) portion of an immunoglobulin molecule, such as an antibody fragment. In some embodiments, the antibody is a functional antibody fragment. For example, the antibody fragment can be a portion of an antibody, such as a F(ab’)2, Fab’, Fab, Fv, sFv, etc. The antibody fragment can bind to the same antigen recognized by the full-length antibody. The antibody fragment can include isolated fragments consisting of the variable regions of antibodies, such as “Fv” fragments consisting of the light and heavy chain variable regions and recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker (“scFv proteins”) in which the light and heavy chain variable regions are connected by a peptide linker. Exemplary antibodies can include, but are not limited to, antibodies to cancer cells, antibodies to viruses, antibodies that bind to cell surface receptors (e.g., CD8, CD34, and CD45), and therapeutic antibodies.

[0027] The term “associated” or “associated with” as used herein can mean that two or more species are identified as being in the same location at a certain time. The association can mean that two or more species are or were in similar containers. The association can be an informatics association. For example, digital information about two or more species can be stored and used to determine that one or more species were in the same location at a certain time. The association can also be a physical association. In some embodiments, two or more associated species are “tethered,” “attached,” or “fixed” to another or a common solid or semi-solid surface. The association can refer to a covalent or non-covalent method of attaching a label to a solid or semi-solid support, such as a bead. The association can be a covalent bond between a target and a label. The association can include hybridization between two molecules, such as a target molecule and a label.

[0028] The term “complementary” as used herein can refer to the ability of two nucleotides to pair precisely. For example, if the nucleotide at a given position on a nucleic acid is capable of hydrogen bonding with the nucleotide of another nucleic acid, then the two nucleic acids are complementary to each other at that position. Complementarity between two single-stranded nucleic acid molecules can be “partial,” in which only some of the nucleotides bind, or it can be complete when total complementarity exists between the single-stranded molecules. A first nucleotide sequence can be said to be “complementary” to a second sequence if the first nucleotide sequence is complementary to the reverse complement of the second sequence. The terms “complementary,” “complement,” and “reverse complement” as used herein can be used interchangeably. It will be appreciated from the disclosure that if a molecule can hybridize to another molecule, then it can be complementary to the molecule with which it is hybridizing.

[0029] As used herein, the term "digital counting" can refer to a method of estimating the number of target molecules in a sample. Digital counting can include the step of determining the number of unique labels associated with a target in a sample. This approach, which can be random in nature, transforms the problem of counting molecules into a series of yes / no digital questions about detecting a predefined set of labels.

[0030] As used herein, the term "label" or "labels" can refer to a nucleic acid code associated with a target within a sample. The label can be, for example, a nucleic acid label. The label can be a fully or partially amplifiable label. The label can be a fully or partially sequencable label. The label can be a portion of a natural nucleic acid that is recognized as distinct. The label can be of known sequence. The label can comprise a concatenation of nucleic acid sequences, such as a concatenation of natural and non-natural sequences. As used herein, the term "label" can be used interchangeably with the terms "index," "tag," or "label-tag." The label can convey information. For example, in various embodiments, the label can be used to determine a characteristic of a sample, a source of a sample, a characteristic of a cell, and / or a target.

[0031] As used herein, the term "non-depleting reservoir" can refer to a pool of barcodes (e.g., stochastic barcodes) composed of many different labels. The non-depleting reservoir can contain a large number of different barcodes such that when the non-depleting reservoir is associated with a pool of targets, each target can be associated with a unique barcode. The uniqueness of each labeled target molecule, relative to the multiplicity of labels, can be determined by the statistics of random selection and depends on the number of copies of the same target molecule in the collection. The size of the resulting labeled target molecules can be determined by the randomness of the barcoding process, and then by analyzing the number of barcodes detected, the number of target molecules present in the original collection or sample can be calculated. When the number of copies of the target molecule present is low compared to the number of unique barcodes, the labeled target molecules are highly unique (i.e., the probability of more than one target molecule being labeled with a given label is very low).

[0032] The term "nucleic acid" as used herein refers to a polynucleotide sequence or fragment thereof. A nucleic acid can comprise nucleotides. A nucleic acid can be exogenous or endogenous to a cell. A nucleic acid can exist in a cell-free environment. A nucleic acid can be a gene or fragment thereof. A nucleic acid can be DNA. A nucleic acid can be RNA. A nucleic acid can comprise one or more than one analog (e.g., altered backbone, sugar, or nucleobase). Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholino nucleic acid, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotide, cordycepin, 7-deazagtp, fluorophore (e.g., rhodamine or fluorescein attached to a sugar), thiol-containing nucleotide, biotin-linked nucleotide, fluorescent base analog, CpG island, methyl-7-guanosine, methylated nucleotide, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine. "Nucleic acid," "polynucleotide," "target polynucleotide," and "target nucleic acid" can be used interchangeably.

[0033] A nucleic acid can comprise one or more than one modification (e.g., base modification, backbone modification) to impart a new or enhanced characteristic (e.g., increased stability) to the nucleic acid. A nucleic acid can comprise a nucleic acid affinity tag. A nucleoside can be a base-sugar combination. The base portion of the nucleoside can be a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. A nucleotide can be a nucleoside further comprising a phosphate group covalently attached to the sugar portion of the nucleoside. For those nucleosides that contain a pentofuranosyl sugar, the phosphate group can be attached to the 2' hydroxyl, 3' hydroxyl, or 5' hydroxyl moiety of the sugar. When the phosphate groups are covalently attached to the 5' hydroxyl moieties of adjacent nucleosides, they form a straight chain as in a DNA. Alternatively, the phosphate groups can be covalently attached to the 3' hydroxyl moieties of adjacent nucleosides, forming a branched chain as in a RNA. Either chain can be further modified, e.g., by polyethylene glycol (PEG) moieties. The straight chain and branched chain nucleic acids can have the same functional properties as each other, or they can have different functional properties as a result of the different connections to the sugar.

[0034] Nucleic acids can comprise modified backbones and / or modified intemucleosidic linkages. Modified backbones can include those that retain a phosphorus atom and those that do not. Suitable modified nucleic acid backbones that include a phosphorus atom can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, 2'-0-aminoalkylphosphoramidates, phosphodiesterase covalent intemediates (PDIs), and those containing mixtures of phosphate and phosphorothioate intemucleosidic linkages, and those having inverted polarity wherein one or more intemucleosidic linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage.

[0035] Nucleic acids can comprise polynucleotide backbones formed from short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatom or heterocyclic intemucleoside linkages. These can include those having linkages (formed by sugars) of morpholino nucleic acid; siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones of mixed N, O, S and CH2 components.

[0036] Nucleic acids can include nucleic acid analogs. The term "analog" can be intended to include polynucleotides in which only furanose rings or both furanose rings and intemucleosidic linkages are replaced with non-furanose groups, only the furanose rings also being referred to as sugar surrogate analogs. Heterocyclic base portions or modified heterocyclic base portions can be retained for hybridization with a suitable target nucleic acid. One such nucleic acid can be a peptide nucleic acid (PNA). In a PNA, the sugar-backbone of a polynucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleotides can be retained and are bound directly or indirectly to the nitrogenous heteroatoms of the amide portion of the backbone. The backbone in a PNA compound can comprise two or more linked aminoethylglycine units, which gives the PNA an amide containing backbone. The nitrogenous heteroatoms of the amide portion of the backbone can be bound directly or indirectly to the heterocyclic base portions.

[0037] Nucleic acids can comprise morpholino nucleic acid backbone structures. For example, a nucleic acid can comprise a 6-membered morpholino ring in place of a ribose ring. In some embodiments, a phosphorodiamidate or other non-phosphodiester intemucleosidic linkage can replace a phosphodiester linkage.

[0038] Nucleic acids can comprise linked morpholino units (e.g., morpholino nucleic acids) having a heterocyclic base attached to a morpholino ring. Linking groups can link the morpholino monomer units in morpholino nucleic acids. Non-ionic morpholino-based oligomeric compounds have less undesirable interactions with cellular proteins. Morpholino-based polynucleotides can be non-ionic analogs of nucleic acids. A variety of compounds in the class of morpholino nucleic acids can be linked using different linking groups. Another class of polynucleotide analogs can be referred to as cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in nucleic acid molecules can be replaced by a cyclohexenyl ring. CeNA DMT-protected phosphoramidite monomers can be prepared and used for oligomeric compound synthesis using phosphoramidite chemistry. Incorporation of CeNA monomers into nucleic acid strands can increase the stability of DNA / RNA hybrids. CeNA oligoadenylates can form complexes with nucleic acid complements that are similar in stability to natural complexes. Other modifications can include locked nucleic acids (LNAs), in which the 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring, forming a 2'-C-methylene group, a 4'-C-methylene group, thereby forming a bicyclic sugar moiety. The linkage can be a methylene (-CH2), a group bridging the 2' oxygen atom and the 4' carbon atom, where n is 1 or 2. LNAs and LNA analogs can exhibit very high duplex thermal stabilities (Tm = +3 °C to +10 °C) with complementary nucleic acids, stability to 3'-exonucleolytic degradation, and good solubility.

[0039] Nucleic acids can also include nucleobase (often referred to simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases can include the purine bases (e.g., adenine (A) and guanine (G)) and pyrimidine bases (e.g., thymine (T), cytosine (C) and uracil (U)). Modified nucleobases can include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH3) uracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8-azadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Modified nucleobases can include tricyclic pyrimidine bases such as xanthocytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenoxazinocytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamp such as substituted phenoxazinocytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamp such as substituted phenoxazinocytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamp such as substituted phenoxazinocytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamp such as substituted phenoxazinocytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamp such as substituted phenoxazinocytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamp such as substituted

[0040] As used herein, the term“sample” can refer to a composition comprising a target. Suitable samples for analysis by the disclosed methods, devices, and systems include cells, tissues, organs, or organisms.

[0041] As used herein, the term“sample device” or“device” can refer to a device that can acquire a portion of a sample and / or place the portion on a substrate. Sample devices can refer to, for example, a fluorescence-activated cell sorter (FACS) machine, a cell sorter machine, a biopsy needle, a biopsy device, a tissue sectioning device, a microfluidic device, a cascade, and / or a microtome.

[0042] As used herein, the term“solid support” can refer to a discrete solid or semi-solid surface on which a plurality of barcodes (e.g., stochastic barcodes) can be attached. A solid support can comprise any type of solid, porous, or hollow sphere, spheroid, bearing, cylinder, or other similar construct composed of plastic, ceramic, metal, or polymeric material (e.g., hydrogel) that can have nucleic acids immobilized thereon (e.g., covalently or non-covalently). A solid support can include a discrete particle, which can be spherical (e.g., a microsphere) or have a non-spherical or irregular shape, such as a cube, cuboid, pyramid, cylinder, cone, ellipse, or disc, among others. The shape of a bead can be non-spherical. A plurality of solid supports spaced apart in an array can not comprise a substrate. A solid support can be used interchangeably with the term“bead.”

[0043] As used herein, the term“stochastic barcode” can refer to a polynucleotide sequence comprising a label of the disclosure. A stochastic barcode can be a polynucleotide sequence that can be used for stochastic barcode encoding. Stochastic barcodes can be used to quantify targets in a sample. Stochastic barcodes can be used to control errors that can arise after a label is associated with a target. For example, stochastic barcodes can be used to assess amplification or sequencing errors. A stochastic barcode associated with a target can be referred to as a stochastic barcode-target or a stochastic barcode-tag-target.

[0044] As used herein, the term“gene-specific stochastic barcode” can refer to a polynucleotide sequence comprising a label and a gene-specific target-binding region. A stochastic barcode can be a polynucleotide sequence that can be used for stochastic barcode encoding. Stochastic barcodes can be used to quantify targets in a sample. Stochastic barcodes can be used to control errors that can arise after a label is associated with a target. For example, stochastic barcodes can be used to assess amplification or sequencing errors. A stochastic barcode associated with a target can be referred to as a stochastic barcode-target or a stochastic barcode-tag-target.

[0045] As used herein, the term“stochastic barcode encoding” can refer to stochastic labeling (e.g., barcode encoding) of nucleic acids. Stochastic barcode encoding can utilize a recursive Poisson strategy to correlate and quantify labels associated with targets. As used herein, the term“stochastic barcode encoding” can be used interchangeably with“stochastic labeling.”

[0046] The term "target" as used herein can refer to a composition associated with a barcode (e.g., a stochastic barcode). Exemplary suitable targets for analysis by the disclosed methods, devices, and systems include oligonucleotides, DNA, RNA, mRNA, microRNA, tRNA, and the like. The target can be single-stranded or double-stranded. In some embodiments, the target can be a protein, peptide, or polypeptide. In some embodiments, the target is a lipid. "Target" as used herein can be used interchangeably with "species."

[0047] The term "reverse transcriptase" as used herein can refer to a group of enzymes with reverse transcriptase activity (i.e., catalyzing the synthesis of DNA from an RNA template). In general, such enzymes include, but are not limited to, retroviral reverse transcriptases, reverse transcriptase transposon reverse transcriptases, reverse plasmid reverse transcriptases, retroelement reverse transcriptases, bacterial reverse transcriptases, group II intron-derived reverse transcriptases, and mutants, variants, or derivatives thereof. Non-retroviral reverse transcriptases include non-LTR reverse transcriptase transposon reverse transcriptases, reverse plasmid reverse transcriptases, retroelement reverse transcriptases, and group II intron reverse transcriptases. Examples of group II intron reverse transcriptases include Lactococcus lactis LI. LtrB gene intron reverse transcriptase, Anabaena variabilis Tel4c intron reverse transcriptase, or Geobacillus stearothermophilus Gsl-IIC intron reverse transcriptase. Other types of reverse transcriptases can include many types of non-retroviral reverse transcriptases (i.e., retroelements, group II introns, and diversity-generating retroelements, etc.).

[0048] The terms "universal adaptor primer," "universal primer adaptor," or "universal adaptor sequence" are used interchangeably to refer to a nucleotide sequence that can be used to hybridize to a barcode (e.g., a stochastic barcode) to generate a gene-specific barcode. For example, a universal adaptor sequence can be a known sequence that is common to all barcodes used in the methods of the disclosure. For example, when labeling multiple targets using the methods of the disclosure, each target-specific sequence can be ligated to the same universal adaptor sequence. In some embodiments, more than one universal adaptor sequence can be used in the methods of the disclosure. For example, when labeling multiple targets using the methods of the disclosure, at least two target-specific sequences are ligated to different universal adaptor sequences. The universal adaptor primer and its complement can be contained in two oligonucleotides, where one oligonucleotide comprises a target-specific sequence and the other oligonucleotide comprises a barcode. For example, a universal adaptor sequence can be part of an oligonucleotide comprising a target-specific sequence to generate a nucleotide sequence that is complementary to a target nucleic acid. A second oligonucleotide comprising a barcode and the complement of the universal adaptor sequence can hybridize to the nucleotide sequence and generate a target-specific barcode (e.g., a target-specific stochastic barcode). In some embodiments, the sequence of the universal adaptor primer is different from the universal PCR primer used in the methods of the disclosure.

[0049] Overview of molecular barcoding of nucleic acid targets in single cells

[0050] As a foundational research method for the methods, devices, and systems of the present disclosure, a single-cell molecular barcoding assay is implemented on a large number of single cells using a deposition strategy. For example, by associating a single cell with a single barcoded bead having a barcoded nucleic acid tethered thereto, molecular targets from the single cell can be randomly labeled with a cell label (also referred to as a cell index, barcode, or tag) and a molecular label (also referred to as a molecular index, barcode, or tag), where each single barcoded bead comprises a plurality of attached random labels. The random labels attached to a given bead can be used to randomly label protein or nucleic acid targets from the associated cell. In some embodiments, the single cells are randomly distributed into a plurality of microwells (e.g., a microwell array). A combined library of a plurality of barcoded beads each comprising a plurality of tethered random barcode nucleic acid labels is also randomly distributed into the plurality of microwells such that a subset of the microwells comprises a single cell and a single bead. In some embodiments, the barcoded beads are deposited prior to the deposition of the cells. In other embodiments, the barcoded beads are deposited after the deposition of the cells. In embodiments, e.g., where the barcoded beads comprise a capture moiety for a target cell (e.g., described in more detail below), the beads and cells (or subcellular particles, e.g., vesicles) can be deposited simultaneously, e.g., as a bound complex of the bead and cell. The random labels comprising the cell and molecular barcodes can also comprise a target recognition region capable of attaching to, or hybridizing with, a molecular target, e.g., a nucleic acid molecule. The target molecules can be released from each cell, e.g., by lysing the cell, and then attached to, or hybridized with, the random labels on the corresponding barcoded bead. In certain embodiments, the target molecules are released from the cell by lysis, e.g., enzymatic lysis. In some embodiments, e.g., when the target molecules are mRNA molecules, the mRNA target molecules are hybridized to the random labels, the beads are recovered from the microwells, and pooled prior to performing reverse transcription, amplification, and sequencing reactions.

[0051] In some embodiments, the plurality of random labels attached to a given bead comprises a cell label that is the same for all random labels attached to that bead, while the cell labels of the plurality of random labels attached to different beads are different. In some embodiments, the plurality of random labels attached to a given bead comprises different kinds of molecular labels selected from a group comprising a particular number of unique molecular label sequences. In some embodiments, the plurality of random labels attached to a given bead can comprise the same target recognition region. In some embodiments, the plurality of random labels attached to a given bead can comprise two or more different target recognition regions.

[0052] In some embodiments, the cell label diversity of the bead library (i.e., the number of unique cell label sequences) is at least one or two orders of magnitude higher than the number of cells to be labeled, such that the probability of each cell pairing with a unique cell barcode is very high. For example, the probability of each cell pairing with a unique cell barcode can be greater than 80%, greater than 90%, greater than 95%, greater than 99%, greater than 99.9%, greater than 99.99%, or greater than 99.999%.

[0053] In some embodiments, the molecular label diversity of the plurality of randomly labeled molecules attached to the beads (i.e., the number of unique molecular label sequences) is at least one or two orders of magnitude higher than the estimated number of occurrences of the target molecule species to be labeled, such that the probability of each occurrence of a target molecule (e.g., an mRNA molecule) within a cell being uniquely labeled is also very high. For example, the probability of each occurring target molecule pairing with a unique molecular barcode can be greater than 80%, greater than 90%, greater than 95%, greater than 99%, greater than 99.9%, greater than 99.99%, or greater than 99.999%. In these embodiments, the number of occurrences of a target molecule species in each cell can be counted (or estimated) by determining the number of unique molecular label sequences attached to the target molecule sequences. In many embodiments, the determining step can be performed by sequencing an amplified library of the labeled target molecules (or their complements).

[0054] In some embodiments, the molecular label diversity of the plurality of randomly labeled molecules attached to the beads is comparable to or lower than the estimated number of occurrences of the target molecule species to be labeled, such that the likelihood of multiple occurrences of a given type of target molecule being labeled by more than one copy of a given molecular label is great. In these embodiments, the number of target molecules in each cell can be calculated using Poisson statistics from the number of unique molecular label sequences attached to the target molecule sequences.

[0055] In certain embodiments, the target molecules of interest are mRNA molecules expressed in single cells. Since cDNA copies of all or a portion of the polyadenylated mRNA molecules in each cell are covalently preserved on the surface of the corresponding bead, any selected gene transcripts can be subsequently analyzed. When the barcoded transcripts are sequenced and assigned to the original cell (based on the recognized cell label) and counted (based on the number of recognized unique molecular labels), the digital gene expression profile of each cell can be reconstructed. An exemplary description of this analytical approach can be found in Fan, et al., "Combinatorial Labeling of Single Cells for Gene Expression Cytometry", Science 347(6222):628; and Science 347(6222): 1258367.

[0056] The various elements summarized above are now reviewed in more detail.

[0057] Barcoding and digital counting

[0058] Quantifying small amounts of nucleic acids, such as messenger ribonucleic acid (mRNA) molecules, is of great clinical interest for determining, for example, which genes are expressed by cells at different stages of development or under different environmental conditions. However, determining the absolute number of nucleic acid molecules (e.g., mRNA molecules) can also be very challenging, especially when the number of molecules is very small. One method for determining the absolute number of molecules in a sample is digital polymerase chain reaction (PCR). Ideally, PCR produces identical copies of a molecule in each cycle. However, PCR can have drawbacks, such as each molecule replicating with a random probability that varies with the number of PCR cycles and the gene sequence, leading to amplification bias and inaccurate measurements of gene expression. Random barcodes with unique molecular labels (also referred to as molecular indices (MIs)) can be used for molecule number counting and to correct for amplification bias. For example, Precise TM determinations such as the Precise

[0059] Precise TM determinations can utilize a non-depleting pool of random barcodes that contains a large number of unique molecular labels, for example, 6561 to 65536, on poly(T) oligonucleotides to hybridize to all poly(A)-mRNAs in a sample during the RT step. The random barcodes can contain universal PCR primer sites. During the RT process, target gene molecules randomly react with the random barcodes. Each target molecule can hybridize to a random barcode, resulting in a complementary ribonucleotide acid (cDNA) molecule with a random barcode. After labeling, the cDNA molecules with random barcodes from microwells of a microwell plate, droplets, or other partitions of microwells can be pooled into a single tube for PCR amplification and sequencing, for example, by a next-generation sequencing protocol, for example, as described below. The number of reads, the number of random barcodes with unique molecular labels, and the number of mRNA molecules can be generated by analysis of the raw sequencing data.

[0060] Barcoding, e.g., stochastic barcoding, has been described, e.g., in Fu et al., Proc Natl Acad Sci U.S.A., 2011 May 31, 108(22):9026-31; U.S. Patent Application Publication No. US2011 / 0160078; Fu et al., Science, 2015 February 6, 347(6222): 1258367; U.S. Patent Application Publication No. US2015 / 0299784; PCT Application Publication No. WO2015 / 031691, the contents of each of these, including any supporting or supplemental information or material, are incorporated herein by reference in their entirety. In some embodiments, a barcode disclosed herein can be a stochastic barcode for stochastically labeling (e.g., barcoding, tagging) polynucleotide sequences of targets. A barcode can be referred to as a stochastic barcode if the ratio of the number of different barcode sequences of the stochastic barcode to the number of occurrences of any target to be labeled can be, or be about, 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11: 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1, 90: 1, 100: 1, or a value or a range between any two of these values. A target can be an mRNA species, which comprises mRNA molecules with identical or nearly identical sequences, antibody identifier sequences, etc. A barcode can be referred to as a stochastic barcode if the ratio of the number of different barcode sequences of the stochastic barcode to the number of occurrences of any target to be labeled is at least or at most 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11: 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1, 90: 1, or 100: 1. A barcode sequence in a stochastic barcode can be referred to as a molecular label.

[0061] A barcode, e.g., a stochastic barcode, can comprise one or more labels. Exemplary labels can include a universal label, a cell label, a barcode sequence (e.g., a molecular label), a sample label, a plate label, a spatial label, and / or a pre-spatial label. The spatial label, the dimension label, and the cell label can be in any order. In some embodiments, the order of the universal label, the spatial label, the dimension label, the cell label, and the molecular label is arbitrary. A barcode can comprise a target binding region. The target binding region can interact with a target (e.g., a target nucleic acid, an RNA, an mRNA, a DNA) in a sample. For example, the target binding region can comprise an oligo(dT) sequence that can interact with a poly(A) tail of an mRNA and / or a reagent-specific oligonucleotide that binds a cellular component, etc. In some cases, the labels (e.g., the universal label, the dimension label, the spatial label, the cell label, and the barcode sequence) of a barcode can be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more than 20 nucleotides.

[0062] A label, e.g., a cell label, can comprise a set of unique nucleic acid subsequences of a determined length, e.g., seven nucleotides per nucleic acid subsequence (equivalent to the number of bits used in some Hamming error correction codes), which can be designed to provide error correction capability. A set of error correction subsequences comprising seven nucleotide sequences can be designed such that any pair-wise combination of the set of sequences represents a determined “genetic distance” (or number of mismatched bases), e.g., the set of error correction subsequences can be designed to represent a genetic distance of three nucleotides. In such cases, inspection of the error correction sequences in a set of sequence data from a labeled target nucleic acid molecule (described more fully below) can allow detection or correction of amplification or sequencing errors. In some embodiments, the length of the nucleic acid subsequences used to generate the error correction code can vary, e.g., the length can be, or about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 31, 40, 50, or a value or range between any two of these values or ranges of values, nucleotides. In some embodiments, nucleic acid subsequences of other lengths can be used to generate error correction codes.

[0063] Universal label

[0064] The barcode can comprise one or more universal labels. In some embodiments, the one or more universal labels can be the same for all barcodes in a set of barcodes attached to a given solid support. In some embodiments, the one or more universal labels can be the same for all barcodes attached to a plurality of beads. In some embodiments, the universal label can comprise a nucleic acid sequence that is capable of hybridizing to a sequencing primer, and thus can be referred to as a universal primer binding domain. The sequencing primer can be used to sequence the barcode comprising the universal label. The sequencing primer (e.g., a universal sequencing primer) can comprise a sequencing primer associated with a high-throughput sequencing platform. In some embodiments, the universal label can comprise a nucleic acid sequence that is capable of hybridizing to a PCR primer. In some embodiments, the universal label can comprise a nucleic acid sequence that is capable of hybridizing to a sequencing primer and a PCR primer. The universal label nucleic acid sequence that is capable of hybridizing to a sequencing or PCR primer can refer to a primer binding site. The universal label can comprise a sequence that can be used to initiate transcription of the barcode. The universal label can comprise a sequence that can be used to extend the barcode or a region within the barcode. The universal label can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or about any value or range between any two of these values. For example, the universal label can comprise at least 10 nucleotides. The universal label can be at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. In some embodiments, a cleavable linker or a modified nucleotide can be part of the universal label sequence to enable cleavage of the barcode from the support.

[0065] Dimensional label

[0066] A barcode can comprise one or more dimensional markers. In some embodiments, a dimensional marker can comprise a nucleic acid sequence that provides dimensional information about the occurrence of the marker (e.g., a stochastic marker). For example, a dimensional marker can provide information about the time at which a target was barcoded. A dimensional marker can be correlated to the time at which a target was barcoded (e.g., stochastically barcoded) in a sample. A dimensional marker can be activated at the time of marking. Different dimensional markers can be activated at different times. A dimensional marker provides information about the order in which targets, groups of targets, and / or samples were barcoded. For example, a population of cells can be barcoded at the G0 phase of the cell cycle. At the G1 phase of the cell cycle, the cells can be pulsed again with barcodes (e.g., stochastic barcodes). At the S phase, the cells can be pulsed again with barcodes, and so on. The barcodes of each pulse (e.g., each phase of the cell cycle) can comprise different dimensional markers. In this way, the dimensional markers provide information about which targets were marked at which phase of the cell cycle. A dimensional marker can query many different biological times. Exemplary biological times include, but are not limited to, the cell cycle, transcription (e.g., transcription initiation), and transcript degradation. In another example, a sample (e.g., a cell, a population of cells) can be marked before and / or after a drug and / or treatment. Changes in the copy number of different targets can be indicative of the sample’s response to the drug and / or treatment.

[0067] A dimensional marker can be activatable. An activatable dimensional marker can be activated at a particular point in time. For example, an activatable marker can be structurally activated (e.g., not turned off). For example, an activatable dimensional marker can be reversibly activated (e.g., an activatable dimensional marker can be turned on and off). A dimensional marker can be reversibly activated, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times. A dimensional marker can be reversibly activated, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times. In some embodiments, a dimensional marker can be activated with fluorescence, light, a chemical event (e.g., cleavage, linking to another molecule, addition of a modification (e.g., pegylation, ubiquitin-like modification, acetylation, methylation, deacetylation, demethylation), a photochemical event (e.g., a photo-cage), and the introduction of a non-natural nucleotide.

[0068] In some embodiments, the dimensional marker can be the same for all barcodes (e.g., stochastic barcodes) attached to a given solid support (e.g., bead), but different for different solid supports (e.g., beads). In some embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% of the barcodes on the same solid support can comprise the same dimensional marker. In some embodiments, at least 60% of the barcodes on the same solid support can comprise the same dimensional marker. In some embodiments, at least 95% of the barcodes on the same solid support can comprise the same dimensional marker.

[0069] Up to 10 6 or more than 10 6 unique dimensional marker sequences can be represented in a plurality of solid supports (e.g., beads). The length of the dimensional marker can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a value or a range between any two of these values, nucleotides. The length of the dimensional marker can be at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides. The dimensional marker can comprise about 5 to about 200 nucleotides. The dimensional marker can comprise about 10 to about 150 nucleotides. The length of the dimensional marker can comprise about 20 to about 125 nucleotides.

[0070] Spatial label

[0071] The barcode can comprise one or more spatial markers. In some embodiments, the spatial marker can comprise a nucleic acid sequence that provides information about the spatial orientation of the target molecule associated with the barcode. The spatial marker can be associated with a coordinate in the sample. The coordinate can be a fixed coordinate. For example, the coordinate can be fixed with reference to a substrate. The spatial marker can reference a two-dimensional or three-dimensional grid. The coordinate can be fixed with reference to a landmark. The landmark can be identifiable in space. The landmark can be an imageable structure. The landmark can be a biological structure, e.g., an anatomical landmark. The landmark can be a cellular landmark, e.g., an organelle. The landmark can be a non-natural landmark, e.g., a structure with an identifiable identifier, e.g., a color code, a barcode, a magnetism, a fluorescence, a radioactivity, or a unique size or shape. The spatial marker can be associated with a physical partition (e.g., a well, a vessel, or a droplet). In some embodiments, multiple spatial markers are used together to encode one or more locations in space.

[0072] The spatial label can be the same for all barcodes attached to a given solid support (e.g., bead), but different for different solid supports (e.g., beads). In some embodiments, the percentage of barcodes of the same solid support comprising the same spatial label can be, or be about, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a value or a range between any two of these values. In some embodiments, the percentage of barcodes of the same solid support comprising the same spatial label can be at least or at most 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. In some embodiments, at least 60% of the barcodes on the same solid support can comprise the same spatial label. In some embodiments, at least 95% of the barcodes on the same solid support can comprise the same spatial label.

[0073] Up to 10 6 or more than 10 6 unique spatial label sequences can be represented in a plurality of solid supports (e.g., beads). The spatial label can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a value or a range between any two of these values, nucleotides in length. The spatial label can be at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. The spatial label can comprise about 5 to about 200 nucleotides. The spatial label can comprise about 10 to about 150 nucleotides. The spatial label can comprise a length of about 20 to about 125 nucleotides.

[0074] Cell label

[0075] A barcode (e.g., a random barcode) may contain one or more cell markers. In some embodiments, the cell marker may contain a nucleic acid sequence that provides information for determining which target nucleic acid originates from which cell, and may therefore be referred to as a cell marker domain. In some embodiments, the cell marker is the same for all barcodes attached to a given solid support (e.g., beads), but different for different solid supports (e.g., beads). In some embodiments, the percentage of barcodes containing the same cell marker on the same solid support may be, or about, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a value or range between any two of these values. In some embodiments, the percentage of barcodes containing the same cell marker on the same solid support may be, or about, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. For example, at least 60% of the barcodes on the same solid support may contain the same cell marker. As another example, at least 95% of the barcodes on the same solid support can contain the same cell markers.

[0076] Up to 10 can be expressed in multiple solid supports (e.g., beads). 6 or more than 10 6 The unique cellular marker sequence. The length of the cellular marker can be, or approximately, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or any two of these values ​​or a range of nucleotides. The length of the cellular marker can be at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides. For example, a cellular marker can contain approximately 5 to approximately 200 nucleotides. As another example, a cellular marker can contain approximately 10 to approximately 150 nucleotides. As another example, a cellular marker can contain approximately 20 to approximately 125 nucleotides.

[0077] Barcode sequence

[0078] A barcode may contain one or more barcode sequences. In some embodiments, the barcode sequence may contain a nucleic acid sequence that provides identification information about a specific type of target nucleic acid species that hybridizes with the barcode. The barcode sequence may also contain a nucleic acid sequence that provides a count (e.g., a rough approximation) of the target nucleic acid species that hybridize with the barcode (e.g., a target binding region) in a specific occurrence.

[0079] In some embodiments, different sets of barcode sequences are attached to a given solid support (e.g., a bead). In some embodiments, there can be or there can be about 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or a value or a range between any two of these values or ranges, unique molecular label sequences. For example, a plurality of barcodes can comprise about 6561 barcode sequences with different sequences. As another example, a plurality of barcodes can comprise about 65536 barcode sequences with different sequences. In some embodiments, there can be at least or at most 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 unique barcode sequences. Unique molecular label sequences can be attached to a given solid support (e.g., a bead).

[0080] The length of a barcode can vary in different embodiments. For example, the length of a barcode can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a value or a range between any two of these values, nucleotides. As another example, the length of a barcode can be at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides.

[0081] Molecular label

[0082] A barcode (e.g., a stochastic barcode) can comprise one or more molecular labels. A molecular label can comprise a barcode sequence. In some embodiments, a molecular label can comprise a nucleic acid sequence that provides identification information for a particular type of target nucleic acid species that hybridizes to the barcode. A molecular label can comprise a nucleic acid sequence that provides a count of a particular occurrence of a target nucleic acid species that hybridizes to the barcode (e.g., a target binding region).

[0083] In some embodiments, different sets of molecular labels are attached to a given solid support (e.g., a bead). In some embodiments, there can be or there can be about 10 2 , 103 one, 10 4 one, 10 5 one, 10 6 one, 10 7 one, 10 8 one, 10 9 one, or a value or a range between any two of these values. For example, the plurality of barcodes can comprise about 6561 molecular labels with different sequences. As another example, the plurality of barcodes can comprise about 65536 molecular labels with different sequences. In some embodiments, there can be at least or at most 10 2 one, 10 3 one, 10 4 one, 10 5 one, 10 6 one, 10 7 one, 10 8 one, or 10 9 unique molecular label sequences. Barcodes with unique molecular label sequences can be attached to a given solid support (e.g., a bead).

[0084] For stochastic barcoding with a plurality of stochastic barcodes, the ratio of the number of different molecular label sequences to the number of occurrences of any target can be, or be about, 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11: 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1, 90: 1, 100: 1, or a value or a range between any two of these values. The targets can be mRNA species comprising mRNA molecules with identical or nearly identical sequences. In some embodiments, the ratio of the number of different molecular label sequences to the number of occurrences of any target is at least or at most 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11: 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1, 90: 1, or 100: 1.

[0085] The length of a molecular marker can be, or approximately, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or any value or range between any two of these values. The length of a molecular marker can be at least, or at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides.

[0086] Target binding region

[0087] A barcode may contain one or more target-binding regions, such as capture probes (which may also be referred to as captured nucleic acids). In some embodiments, the target-binding region may hybridize with a target of interest. In some embodiments, the target-binding region may contain a nucleic acid sequence that specifically hybridizes with a target (e.g., a target nucleic acid, a target molecule, or a cellular nucleic acid to be analyzed), such as a nucleic acid sequence that hybridizes with a specific gene sequence. In some embodiments, the target-binding region may contain a nucleic acid sequence capable of attaching (e.g., hybridizing) to a specific location on a specific target nucleic acid. In some embodiments, the target-binding region may contain a nucleic acid sequence capable of specifically hybridizing with a single-stranded overhang of a restriction endonuclease site (e.g., an EcoRI sticky single-stranded overhang). The barcode can then be attached to any nucleic acid molecule containing a sequence complementary to the single-stranded overhang of the restriction site.

[0088] In some implementations, the target-binding region may contain a non-specific target nucleic acid sequence. A non-specific target nucleic acid sequence can refer to a sequence that can bind multiple target nucleic acids and is independent of the specific sequence of the target nucleic acid. For example, the target-binding region may contain a random multimeric sequence, a multimeric (dA) sequence, a multimeric (dT) sequence, a multimeric (dG) sequence, a multimeric (dC) sequence, or a combination thereof. For example, the target-binding region may be an oligomeric (dT) sequence (e.g., an oligomeric dT domain) that hybridizes to a multimeric (A) tail on an mRNA molecule. For example, mRNA molecules can be reverse transcribed using a reverse transcriptase, such as Moroni mouse leukemia virus (MMLV) reverse transcriptase, to produce a cDNA molecule with a multimeric (dC) tail. Barcodes may include target-binding regions with multimeric (dG) tails. When base pairing occurs between the poly(dG) tail of the barcode and the poly(dC) tail of the cDNA molecule, the reverse transcriptase switches the template strand from the cellular RNA molecule to the barcode and continues copying to the 5' end of the barcode. This process yields a cDNA molecule containing a barcode (e.g., a molecular marker) sequence at the 3' end.

[0089] A random multimer sequence can be, for example, a random dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, or higher multimer sequence of any length (wherein the target binding region can be referred to as a random sequence domain).

[0090] In some embodiments, the target binding region is the same for all barcodes attached to a given bead. In some embodiments, the target binding region of a plurality of barcodes linked to a given bead can comprise two or more than two different target binding sequences. The length of the target binding region can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a value or a range between any two of these values, nucleotides. The length of the target binding region can be at most about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 nucleotides.

[0091] In some embodiments, the target binding region can comprise an oligo(dT) (i.e., e.g., an oligo dT domain), which can hybridize to an mRNA comprising a poly(A) tail. The target binding region can be gene specific. For example, the target binding region can be configured to hybridize to a specific region of a target (e.g., wherein the target binding region is a gene specific domain).

[0092] The length of the target binding region can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, or a value or a range between any two of these values, nucleotides. The length of the target binding region can be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. The length of the target binding region can be about 5 to 30 nucleotides. When a barcode comprises a gene specific target binding region, the barcode can be referred to herein as a gene specific barcode.

[0093] The target-binding region can interact with a target in a sample. The target can be or comprise a ribonucleotide (RNA), a messenger RNA (mRNA), a microRNA, a small interfering RNA (siRNA), an RNA degradation product, an RNA each comprising a poly(A) tail, or any combination thereof. In some embodiments, the plurality of targets can include deoxyribonucleic acid (DNA).

[0094] In some embodiments, the target-binding region can comprise an oligo(dT) sequence, which can interact with a poly(A) tail of an mRNA. One or more labels of the barcode (e.g., a universal label, a dimension label, a spatial label, a cell label, and a barcode sequence (e.g., a molecular label)) can be separated from another or two remaining labels of the barcode by a spacer. For example, the spacer can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or more than 20 nucleotides. In some embodiments, none of the labels of the barcode are separated by a spacer.

[0095] Universal adaptor primer

[0096] The barcode can comprise one or more universal adapter primers. For example, a gene-specific barcode, such as a gene-specific stochastic barcode, can comprise a universal adapter primer. A universal adapter primer can refer to a nucleotide sequence that is universal across all barcodes. The universal adapter primer can be used to construct a gene-specific barcode. The universal adapter primer can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or a value or a range between any two of these values or ranges of nucleotides in length. The universal adapter primer can be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The universal adapter primer can be up to 5 to 30 nucleotides in length.

[0097] Linker

[0098] When a barcode comprises more than one type of label (e.g., more than one cell label or more than one barcode sequence, e.g., one molecular label), these labels can be interspersed with a linker label sequence. The linker label sequence can be at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 nucleotides in length. The linker label sequence can be at most 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 nucleotides in length. In some cases, the linker label sequence is 12 nucleotides in length. The linker label sequence can be used to facilitate synthesis of the barcode. The linker label can comprise an error correction (e.g., Hamming) code.

[0099] Solid support

[0100] In some embodiments, a barcode disclosed herein, e.g., a stochastic barcode, can be associated with a solid support. The solid support can be, e.g., a synthetic particle. In some embodiments, some or all of the barcode sequences, e.g., the molecular label of a stochastic barcode (e.g., a first barcode sequence) having a first plurality of barcodes on a solid support (e.g., a first solid support), differ by at least one nucleotide. The cell label of a barcode on the same solid support can be the same. The cell label of a barcode on different solid supports can differ by at least one nucleotide. For example, the first cell label having a first plurality of barcodes on a first solid support can have the same sequence, the second cell label having a second plurality of barcodes on a second solid support can have the same sequence. The first cell label having a first plurality of barcodes on a first solid support can differ by at least one nucleotide from the second cell label having a second plurality of barcodes on a second solid support. The cell label can be, e.g., about 5 to 20 nucleotides in length. The barcode sequence can be, e.g., about 5 to 20 nucleotides in length. The synthetic particle can be, e.g., a bead.

[0101] The bead can be, e.g., a silica gel bead, a controlled pore glass bead, a magnetic bead, a Dynabead, a dextran gel / agarose gel bead, a cellulose bead, a polystyrene bead, or any combination thereof. The bead can further comprise, e.g., the following materials: polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, agarose gel, cellulose, nylon, silicone, or any combination thereof.

[0102] In some embodiments, the bead can be a polymer bead, e.g., a deformable bead or a gel bead, functionalized with barcodes or stochastic barcodes (e.g., gel beads from 10X Genomics (Pleasanton CA)). In some embodiments, the gel bead can comprise a polymer-based gel. For example, the gel bead can be produced by encapsulating one or more polymer precursors into a droplet. Upon exposure of the polymer precursors to an accelerator (e.g., tetramethyl ethylene diamine (TEMED)), the gel bead can be produced.

[0103] In some embodiments, the particle can be degradable. For example, the polymer bead can dissolve, melt, or degrade, e.g., under desired conditions. The desired conditions can include environmental conditions. The desired conditions can cause the polymer bead to dissolve, melt, or degrade in a controlled manner. The gel bead can dissolve, melt, or degrade due to a chemical stimulus, a physical stimulus, a biological stimulus, a thermal stimulus, a magnetic stimulus, an electrical stimulus, a light stimulus, or any combination thereof.

[0104] The analyte and / or reagent, e.g., oligonucleotide barcode, can be coupled / fixed, e.g., to the inner surface of the gel bead (e.g., by the oligonucleotide barcode and / or the interior accessible to diffusion of the material used to produce the oligonucleotide barcode) and / or the outer surface of the gel bead or any other microcapsule described herein. The coupling / fixing can be through any form of chemical bond (e.g., covalent bond, ionic bond) or physical phenomenon (e.g., van der Waals force, dipole-dipole interaction, etc.). In some embodiments, the coupling / fixing of the reagent to the gel bead or any other microcapsule described herein can be reversible, e.g., through a labile moiety (e.g., through a chemical crosslinker, including those described herein). Upon application of a stimulus, the labile moiety can be cleaved, and the fixed reagent is released. In some embodiments, the labile moiety is a disulfide bond. For example, in the case where the oligonucleotide barcode is fixed to the gel bead through a disulfide bond, exposure of the disulfide bond to a reducing agent can cleave the disulfide bond and release the oligonucleotide barcode from the bead. The labile moiety can be included as part of the gel bead or microcapsule, as part of a chemical linker that links the reagent or analyte to the gel bead or microcapsule, and / or as part of the reagent or analyte. In some embodiments, at least one barcode of the plurality of barcodes can be fixed on the particle, partially fixed on the particle, enclosed in the particle, partially enclosed in the particle, or any combination thereof.

[0105] In some embodiments, the gel beads can comprise a plurality of different polymers, including but not limited to: polymers, thermosensitive polymers, photosensitive polymers, magnetic polymers, pH-sensitive polymers, salt-sensitive polymers, chemically sensitive polymers, polyelectrolytes, polysaccharides, peptides, proteins, and / or plastics. The polymers can include, but are not limited to, the following materials: poly(N-isopropylacrylamide) (PNIPAAm), poly(styrene sulfonate) (PSS), poly(allylamine) (PAAm), poly(acrylic acid) (PAA), poly(ethylene imine) (PEI), poly(diallyldimethylammonium chloride) (PDADMAC), poly(pyrrole) (PPy), poly(vinylpyrrolidone) (PVPON), poly(vinylpyridine) (PVP), poly(methacrylic acid) (PMAA), poly(methyl methacrylate) (PMMA), polystyrene (PS), poly(tetrahydrofuran) (PTHF), poly(phthalaldehyde) (PTHF), poly(hexyl viologen) (PHV), poly(L-lysine) (PLL), poly(L-arginine) (PARG), poly(lactic-co-glycolic acid) (PLGA)

[0106] A large number of chemical stimuli can be used to trigger the breakage, dissolution, or degradation of the beads. Examples of these chemical changes can include, but are not limited to, pH-mediated bead wall changes, bead wall disintegration by chemical cleavage of crosslinks, triggered bead wall depolymerization, and bead wall switch reactions. A large number of changes can also be used to trigger the breakage of the beads.

[0107] Volume or physical changes to the microcapsules by various stimuli also provide a number of advantages for designing capsules that release reagents. Volume or physical changes exist on the macroscopic scale, where bead breakage is the result of a stimulus-induced mechanical-physical force. These processes can include, but are not limited to, pressure-induced breakage, bead wall melting, or changes in bead wall porosity.

[0108] Biological stimuli can also be used to trigger the breakage, dissolution, or degradation of the beads. Generally, biological triggers are similar to chemical triggers, but many examples use biological molecules, or molecules common in living systems, such as enzymes, polypeptides, sugars, fatty acids, nucleic acids, and the like. For example, a bead can comprise a cross-linked polymer with a peptide that is susceptible to cleavage by a specific protease. More specifically, one example can include a microcapsule comprising a GFLGK peptide cross-link. When a biological trigger such as the protease Cathepsin B is added, the peptide cross-links of the shell wall are cleaved and the contents of the bead are released. In other cases, the protease can be heat activated. In another example, a bead comprises a shell wall comprising cellulose. The hydrolytic enzyme chitosan is added as a biological trigger to cleave the cellulose bonds, depolymerize the shell wall, and release its contents. Beads can also be induced to release their contents with the application of a thermal stimulus. Changes in temperature can cause a variety of changes in the beads. Changes in heat can cause the beads to melt, causing the bead wall to disintegrate. In other cases, heat can increase the internal pressure of the components inside the bead, causing the bead to rupture or explode. In other cases, heat can transform the bead into a shriveled, dehydrated state. Heat can also act on heat-sensitive polymers within the bead wall, causing the bead wall to break apart.

[0109] The inclusion of magnetic nanoparticles on the bead wall of a microcapsule can allow for the triggering of bead breakage as well as the guidance of bead formation into arrays. The devices of the present disclosure can comprise magnetic beads for any purpose. In one example, the addition of Fe3O4 nanoparticles to a bead comprising a polyelectrolyte triggers breakage upon the stimulus of an oscillating magnetic field.

[0110] Beads can be broken, dissolved, or degraded due to an electrical stimulus. Similar to the magnetic particles described in the previous section, electroactive beads can both trigger bead breakage as well as perform other functions, such as alignment in an electric field, electrical conductivity, or redox reactions. In one example, beads comprising electroactive materials align in an electric field, allowing for the controlled release of internal reagents. In other examples, an electric field can induce redox reactions within the bead wall, allowing for increased porosity.

[0111] Light stimuli can also be used to break the beads. Many light triggers are possible and can include systems using a variety of molecules, such as nanoparticles and chromophores that are able to absorb photons of a specific wavelength range. For example, metal oxide coatings can be used as capsule triggers. Ultraviolet irradiation of polyelectrolyte capsules coated with SiO2 can cause the bead wall to disintegrate. In another example, a light switch material such as an azobenzene group can be incorporated into the bead wall. Upon the application of ultraviolet or visible light, such chemicals undergo a reversible cis-trans isomerization upon the absorption of a photon. In this regard, the addition of a photon switch can cause the bead wall to disintegrate or become more porous upon the action of a light trigger.

[0112] Barcodes of the present disclosure can be associated with (e.g., attached to) a solid support (e.g., a bead). Barcodes associated with a solid support can each comprise a barcode sequence selected from at least 100 or 1000 barcode sequences having unique sequences. In some embodiments, different barcodes associated with a solid support can comprise barcodes having different sequences. In some embodiments, a percentage of barcodes associated with a solid support comprise the same cell label. For example, the percentage can be, or about, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a value or a range between any two of these values. As another example, the percentage can be a minimum of, or a maximum of, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. In some embodiments, barcodes associated with a solid support can have the same cell label. Barcodes associated with different solid supports can have different cell labels selected from at least 100 or 1000 cell labels comprising unique sequences.

[0113] Barcodes disclosed herein can be associated with (e.g., attached to) a solid support (e.g., a bead). In some embodiments, a plurality of targets in a sample can be barcoded with a solid support comprising a plurality of synthetic particles associated with a plurality of barcodes. In some embodiments, a solid support can comprise a plurality of synthetic particles associated with a plurality of barcodes. The spatial labels of a plurality of barcodes on different solid supports can differ by at least one nucleotide. For example, a solid support can comprise a plurality of barcodes in two or three dimensions. A synthetic particle can be a bead. A bead can be a silica gel bead, a controlled pore glass bead, a magnetic bead, a Dynabead, a dextran gel / agarose gel bead, a cellulose bead, a polystyrene bead, or any combination thereof. A solid support can comprise a polymer, a matrix, a hydrogel, a needle array device, an antibody, or any combination thereof. In some embodiments, a solid support can be free floating. In some embodiments, a solid support can be embedded in a semi-solid or solid array. A barcode can not be associated with a solid support. A barcode can be a single nucleotide. A barcode can be associated with a substrate.

[0114] The terms "tethered," "attached," and "immobilized" are used interchangeably herein and can refer to covalent or non-covalent means of linking a barcode to a solid support. Any of a variety of different solid supports can be used as a solid support to which pre-synthesized barcodes are attached or to which barcodes are synthesized in situ.

[0115] In some embodiments, the solid support is a bead. The bead can comprise one or more than one type of solid, porous, or hollow sphere, spheroid, bearing, cylinder, or other similar construct that can immobilize a nucleic acid (e.g., covalently or non-covalently). For example, the bead can be composed of plastic, ceramic, metal, polymeric material, or any combination thereof. The bead can be, or comprise, a discrete particle that is spherical (e.g., a microsphere) or has a non-spherical or irregular shape, such as a cube, cuboid, pyramid, cylinder, cone, oval, or disc, among others. In some embodiments, the shape of the bead can be non-spherical.

[0116] The bead can comprise a variety of materials, including but not limited to paramagnetic materials (e.g., magnesium, molybdenum, lithium, and tantalum), superparamagnetic materials (e.g., ferrite (Fe3O4; magnetite) nanoparticles), ferromagnetic materials (e.g., iron, nickel, cobalt, some alloys thereof, and some rare earth metal compounds), ceramic, plastic, glass, polystyrene, silica, methylstyrene, acrylic polymers, titanium, latex, agarose gel, hydrogel, polymer, cellulose, nylon, or any combination thereof.

[0117] In some embodiments, the bead (e.g., a label-attached bead) is a hydrogel bead. In some embodiments, the bead comprises a hydrogel.

[0118] Some embodiments disclosed herein comprise one or more than one particle (e.g., a bead). Each particle can comprise a plurality of oligonucleotides (e.g., barcodes). Each of the plurality of oligonucleotides can comprise a barcode sequence (e.g., a molecular label sequence), a cell label, and a target-binding region (e.g., an oligo(dT) sequence, a gene-specific sequence, a random multimer, or a combination thereof). The cell label sequence of each of the plurality of oligonucleotides can be the same. The cell label sequences of the oligonucleotides on different particles can be different, such that the oligonucleotides on different particles can be identified. The number of different cell label sequences can be different in different implementations. In some embodiments, the number of cell label sequences can be, or be about, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 7 8 9 9 ​​​​In some implementations, the number of cell marker sequences may be at least 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 10 7 10 8 One, or 10 9 In some embodiments, multiple particles comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more than 1000 particles include oligonucleotides with the same cell sequence. In some embodiments, the multiple particles comprising oligonucleotides with the same cell sequence comprise at most 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more than 10%. In some implementations, multiple particles do not contain the same cell marker sequence.

[0119] Multiple oligonucleotides on each particle can contain different barcode sequences (e.g., molecular markers). In some embodiments, the number of barcode sequences can be, or approximately, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 10 7 10 8 10 9The number of barcode sequences may be at least 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 10 7 10 8 One or 10 9 For example, at least 100 of a plurality of oligonucleotides contain different barcode sequences. As another example, in a single particle, at least 100, 500, 1000, 5000, 10000, 15000, 20000, 50000, or more than 50000 oligonucleotides contain different barcode sequences, values ​​or ranges between any two of these values. Some embodiments provide multiple particles containing barcodes. In some embodiments, the ratio of the presence (or copy number) of the target to be labeled to the different barcode sequences can be at least 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or less than 1:90. In some embodiments, each of the plurality of oligonucleotides further comprises a sample label, a universal label, or both. For example, the particles can be nanoparticles or microparticles.

[0120] The size of the beads can vary. For example, the diameter of the beads can range from 0.1 micrometers to 50 micrometers. In some embodiments, the diameter of the beads can be, or approximately, 0.1 micrometers, 0.5 micrometers, 1 micrometer, 2 micrometers, 3 micrometers, 4 micrometers, 5 micrometers, 6 micrometers, 7 micrometers, 8 micrometers, 9 micrometers, 10 micrometers, 20 micrometers, 30 micrometers, 40 micrometers, 50 micrometers, or any value or range between any two of these values.

[0121] The diameter of the bead can be related to the diameter of the basal pore. In some embodiments, the bead diameter can be 10% longer or shorter than the pore diameter, 20% longer or shorter than the pore diameter, or a value or range between any two of these values, or approximately 10% longer or shorter than the pore diameter, 20% longer or shorter than the pore diameter, 20% longer or shorter than the pore diameter, or a value or range between any two of these values. The bead diameter can also be related to the diameter of a cell (e.g., a single cell embedded in a basal pore). In some embodiments, the diameter of the bead may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% longer or shorter than the diameter of the pore, or at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% longer or shorter than the diameter of the pore. The diameter of the bead may be related to the diameter of a cell (e.g., a single cell embedded in a basal pore). In some implementations, the diameter of the bead may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or any two of these values ​​longer or shorter than the diameter of the cell, or approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or any two of these values ​​longer or shorter than the diameter of the cell. In some embodiments, the diameter of the bead may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% longer or shorter than the diameter of the hole, or at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% longer or shorter than the diameter of the hole.

[0122] Beads can be attached to and / or embedded in a substrate. Beads can be attached to and / or embedded in gels, hydrogels, polymers, and / or matrices. The spatial location of the beads in the substrate (e.g., gel, matrix, scaffold, or polymer) can be identified using spatial markings on a barcode present on the beads, which can be used as a location address.

[0123] Examples of beads may include, but are not limited to, streptavidin beads, agarose beads, magnetic beads, Microbeads Microbeads, antibody conjugated beads (e.g., anti-immunoglobulin microbeads), protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, and BcMag TM Carboxyl-terminated magnetic beads.

[0124] The beads can be associated with (e.g., impregnated with) quantum dots or fluorescent dyes to cause them to fluoresce in a fluorescent light channel or channels. The beads can be associated with iron oxide or chromium oxide to make them paramagnetic or ferromagnetic. The beads can be identifiable. For example, the beads can be imaged using a camera. The beads can have a detectable code associated with the beads. For example, the beads can include a barcode. The beads can change size, for example, due to swelling in an organic or inorganic solution. The beads can be hydrophobic. The beads can be hydrophilic. The beads can be biocompatible.

[0125] The solid supports (e.g., beads) can be visualized. The solid supports can include a visualizing tag (e.g., a fluorescent dye). The solid supports (e.g., beads) can be etched with an identifier. The identifier can be visualized by imaging the beads.

[0126] DETAILED DESCRIPTION

[0127] Embodiments of the present invention provide methods of molecular barcoding of nucleic acid targets of particles, e.g., cells or extracellular vesicles, mediated by capture magnetic beads. The method comprises the following aspects: a) combining a sample comprising particles with capture magnetic beads comprising a capture moiety for the particles to produce a captured sample; b) partitioning the captured particles of the captured sample using a partitioning protocol mediated by application of a magnetic field to produce partitioned captured particles, wherein the partitioned captured particles are in spatial proximity to bead-bound barcode nucleic acids comprising a target binding region; and c) lysing the partitioned captured particles such that nucleic acids released therefrom, and optionally cell component binding reagent specific oligonucleotides released therefrom, bind to the target binding region to produce captured nucleic acids. Compositions, e.g., capture magnetic beads, barcoded magnetic beads, and devices / systems and kits for practicing embodiments of the method are also provided.

[0128] Before the present invention is described in detail, it is to be understood that the invention is not limited to the particular embodiments described herein and as such can of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims.

[0129] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the application. The upper and lower limits of these smaller ranges can independently be included in the smaller ranges, and are also encompassed within the application, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of the included limits are also included in the application.

[0130] Certain ranges are presented herein with numerical values being presented with the term "about" before the numerical value. The term "about" is used herein to provide literal support for the exact number that it precedes, but also to encompass numbers that are close to and roughly the same as the exact number. In determining whether a number is close to or roughly the same as an exact number, it is presumed that a person of ordinary skill in the art is considering the context in which the number is used and that either the context or ordinary experience will indicate that either a closer or a more approximate value is required.

[0131] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, representative illustrative methods and materials are now described.

[0132] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date of this application. Nothing herein is to be construed as an admission that the present application is not entitled to antedate such publication by virtue of prior application. Further, the dates of publication provided can be different from the actual publication dates which can need to be independently confirmed.

[0133] It should be noted that, as used in this application and the appended claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. It should also be noted that the recitation of "single" or "only" in the drafting of a claim can exclude any optional element. Therefore, this statement is intended to function as an antecedent basis for such exclusive terminology when reciting elements of a claim or using "negative" limitations.

[0134] It will be apparent to those skilled in the art that each of the embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with any of the several embodiments without departing from the scope or spirit of the present application. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0135] For the grammatical flow of the functional recitations, it is expressly understood that the claims are not to be construed as being limited by the "means" or "steps" recited therein unless such claims expressly recite under 35 U.S.C. § 112 that the claim is so limited. The use of the terms "step" and "steps" does not imply that any particular step is required, or that the recited steps are to be performed in any particular order. Nor does it suggest that all of or even any of the recited steps are necessary to fully utilize, or that mechanisms employed to carry out such steps must be those specifically recited.

[0136] In further description of various aspects of the application, first various schemes and reagents / systems are reviewed in more detail, followed by an in-depth review of various methods of the application, and then a description of kit embodiments for practicing various embodiments of the methods.

[0137] Methods

[0138] As described above, methods are provided for magnetic bead mediated molecular barcoding of target nucleic acids from particles, e.g., cells or subcellular components, e.g., vesicles. Aspects of the method include combining a sample suspected to include particles of interest with one or more capture magnetic beads (including a library of capture magnetic beads), wherein the capture magnetic beads include a capture moiety for the particles to produce a captured sample. The sample contacted with the one or more capture magnetic beads can be any sample, e.g., a sample including one or more cells and other components, e.g., extracellular components, e.g., vesicles. In some embodiments, the sample can be a particle, e.g., a single cell or extracellular vesicle (e.g., microvesicle, exosome, or apoptotic body), a plurality of particles, e.g., cells and / or vesicles, a tissue sample, a tumor sample, a blood sample, etc. In some embodiments, the sample can comprise a mixture of cell types, e.g., normal cells, tumor cells, blood cells, B cells, T cells, maternal cells, fetal cells, etc., or a mixture of cells from different subjects, and subcellular components thereof, e.g., vesicles. In some cases, the sample is an aqueous sample that includes one or more types of particles, e.g., cells and / or vesicles, e.g., other components of subcellular size are present, e.g., vesicles are present. While the number of particles in a given aqueous sample used in a workflow embodiment of the application can vary, in some cases the number of particles in a given sample volume ranges from 0.0001 particles / mL to 10 billion particles / mL. When the target particles are rare, e.g., circulating tumor cells (CTCs), fetal cells in maternal blood, infectious bacterial or fungal cells, the number of particles in a given sample can range, e.g., from 0.0001 particles / mL to 100 particles / mL, e.g., from 0.01 particles / mL to 10 particles / mL. When the target particles are abundant, e.g., EVs, RBCs, the number of particles in a given volume can range from 10 million particles / mL to 10 billion particles / mL, e.g., from 100 million particles / mL to 10 billion particles / mL. The above provided concentrations can be lower or higher depending on dilution or concentration of the sample. A given concentration represents a concentration naturally present in the sample.

[0139] In some embodiments, the methods comprise barcoding nucleic acids from cells. In some cases, the methods comprise barcoding nucleic acids from particles of subcellular size, e.g., extracellular vesicles, where such particles of subcellular size can have a diameter of 1000 nm or less than 1000 nm. In some embodiments, the methods comprise barcoding nucleic acids from extracellular vesicles, where in some cases the extracellular vesicles have a diameter of 5 pm or less than 5 pm, e.g., 1 pm or less than 1 pm, where in some cases the extracellular vesicles have a diameter of 30 nm to 2500 nm, e.g., 30 nm to 1000 nm. In some cases, the particles are microvesicles (e.g., 100 to 100 nm in diameter). In some cases, the particles are exosomes (e.g., 30 nm to 150 nm in diameter).

[0140] For example, as described above, the sample is contacted with one or more capture magnetic beads, the capture magnetic beads comprising a capture moiety for a particle, e.g., a cellular or subcellular (e.g., vesicle) component of the sample. The capture magnetic beads refer to beads that are affected (i.e., move) in space by an applied magnetic field. Thus, they can be moved in space, e.g., by applying a magnetic field to the environment of the capture magnetic beads, e.g., from another magnet, to move from one location to another. The magnetic capture beads comprise a magnetic solid support having stably associated therewith, e.g., on a surface thereof, a capture moiety for a particle, e.g., a cellular or subcellular sample component, e.g., a vesicle. The beads can comprise one or more types of solid, porous, or hollow spheres, spheroids, bearings, cylinders, or other similar constructs that can have a capture moiety (e.g., covalently or non-covalently) immobilized thereon. For example, the beads can comprise a plastic, ceramic, metal, polymeric material, or any combination thereof. The beads can be, or comprise, discrete particles that are spherical (e.g., microspheres) or have a non-spherical or irregular shape, e.g., a cube, cuboid, pyramid, cylinder, cone, oval, or disc, etc. In some embodiments, the beads can be non-spherical in shape. Because the beads are magnetic, they can comprise a variety of materials, including but not limited to paramagnetic materials (e.g., magnesium, molybdenum, lithium, and tantalum), superparamagnetic materials (e.g., ferrite (Fe304; magnetite) nanoparticles), ferromagnetic materials (e.g., iron, nickel, cobalt, some alloys thereof, and some rare earth metal compounds), etc. The beads can also include a number of added materials, e.g., ceramic, plastic, glass, polystyrene, silica, methylstyrene, acrylic polymers, titanium, latex, agarose gel, agarose, hydrogel, polymer, cellulose, nylon, or any combination thereof.

[0141] Stabilization of the solid support with the capture magnetic beads is associated with a capture moiety for the particle, e.g., a cell or a vesicle. The capture moiety is a moiety that binds to a component of the particle (i.e., a determinant), e.g., a surface protein or other structure. The binding can be of any type, including specific or non-specific binding, in the broadest sense. In some cases, the capture moiety specifically binds to a determinant of the particle, e.g., a surface protein. Where the capture moiety specifically binds to a determinant of the particle, the capture moiety and the determinant have affinity for each other. The affinity between a pair of specifically binding members and the determinant to which they specifically bind can vary, and in some cases they can specifically bind to each other in the form of a binding complex with a KD (dissociation constant) of 10 -5 M or less than 10 -5 M, 10 -6 M or less than 10 -6 M, 10 -7 M or less than 10 -7 M, 10 -8 M or less than 10 -8 M, 10 -9 M or less than 10 -9 M, 10 -10 M or less than 10 -10 M, 10 -11 M or less than 10 -11 M, 10 -12 M or less than 10 -12 M, 10 -13 M or less than 10 -13 M, 10 -14 M or less than 10 -14 M, or 10 -15 M or less than 10 -15M represents the characterization (note that in certain embodiments, these values can apply to the interaction of other specific binding pairs mentioned elsewhere in this description). Any suitable determinant binding reagent can be used for the capture moiety, such as a protein binding reagent, an antibody or fragment thereof, an aptamer, a small molecule, a ligand, a peptide, an oligonucleotide, etc., or any combination thereof. For example, the capture moiety can comprise an antibody, such as an antibody specific for a specific moiety (e.g., a receptor) on a target, such as a cell or vesicle. The antibody can be a full-length (i.e., naturally occurring or formed by normal immunoglobulin gene segment recombination processes) immunoglobulin molecule (e.g., an IgG antibody) or an immunologically active (i.e., specifically binding) portion of an immunoglobulin molecule, such as an antibody fragment. The antibody fragment can be a portion of an antibody, such as a F(ab’)2, Fab’, Fab, Fv, sFv, etc. In some embodiments, the antibody fragment can bind to the same antigen recognized by the full-length antibody. The antibody fragment can include isolated fragments consisting of the antibody variable region segments, such as “Fv” fragments consisting of the light chain variable and heavy chain variable regions and recombinant single-chain polypeptide molecules in which the light and heavy chain variable regions are connected by a peptide linker (“scFv proteins”). Exemplary antibodies can include, but are not limited to, anti-cancer cell antibodies, anti-viral antibodies, antibodies that bind to cell surface receptors (CD8, CD34, and CD45), and therapeutic antibodies.

[0142] As described above, the capture moiety is stably associated with the surface of the solid support to which the magnetic beads are captured. By stably associated, it is meant that the surface position of the capture moiety relative to the solid support to which it is attached is fixed. The terms “fixed,” “tethered,” and “attached” are used interchangeably herein and can refer to covalent or non-covalent means of linking the capture moiety to the solid support.

[0143] Figure 1A A magnetic capture example is shown that includes a capture moiety. Figure 1A A capture magnetic bead 100 is shown that includes a magnetic bead 110 (e.g., as described above) having a surface stably associated with a capture moiety 120. As described above, the capture moiety can vary, with examples of capture moieties including, but not limited to, an antibody specific for a target particle (e.g., a cell or extracellular vesicle), biotin, avidin, or another molecule (e.g., conjugated with an antibody that binds to avidin, biotin, or other molecule complementary to the capture moiety stably associated with the bead).

[0144] Optionally, the capturing magnetic beads may also include barcoded nucleic acids bound thereto, for example, as described above. When present, the barcoded nucleic acid includes a target-binding region. As described above, the target-binding region may contain a nucleic acid sequence that specifically hybridizes to a target (e.g., a target nucleic acid, a target molecule, or a cellular nucleic acid to be analyzed), such as a nucleic acid sequence that hybridizes to a specific gene sequence. In some embodiments, the target-binding region may contain a nucleic acid sequence that can attach (e.g., hybridize) to a specific location on a specific target nucleic acid. In other cases, the target-binding region may be non-specific, such as an oligomeric dT domain. And in still other cases, the target-binding region may be random, such as a random hexamer, for example, where the target-binding region is a random sequence domain.

[0145] Figure 1B An example of a magnetic bead containing barcode-coded nucleic acids is shown. For example... Figure 1B As shown, the capturing magnetic bead 150 includes a magnetic bead 110 (e.g., as described above), the surface of which is stably bound to a capturing portion 120, which is tethered to the bound surface via a connector 152. The surface of the magnetic bead 110 is also tethered with bead-bound barcode nucleic acid 160. The bead binding the barcode nucleic acid has the following structure: bead (110)-5'-universal primer-binding domain (162)-cell marker domain (164)-unique molecular index domain (166)-target-binding region (168)-3', wherein these domains are as described above.

[0146] As described above, the sample is brought into contact with capturing magnetic beads, including multiple capturing magnetic beads, such as a library of capturing magnetic beads, to produce a captured sample. In any given scheme, the number of different beads in contact with a given sample can vary, and in some cases, the number is one or more, such as 10 or more, 50 or more, 100 or more, 1000 or more, 5000 or more, 10000 or more, 20000 or more. The magnetic beads in contact with a given sample can be the same or different, for example, in terms of the capturing portion or other components, such as a barcode, such that the group of capturing magnetic beads in contact with the sample, such as a library, includes multiple beads that differ from other beads in some characteristics, such as barcodes, capturing portions, etc.

[0147] The sample and beads are brought into contact with each other under conditions sufficient to allow the trapping portion of the beads to bind to their particles, thereby producing a trapping sample comprising a binding complex of particles such as cells or vesicles bound to the trapping magnetic beads. The conditions can be varied as long as the desired binding complex is produced. The resulting trapping sample comprises trapping particles, which are the binding complex consisting of particles such as cells or vesicles bound to the trapping magnetic beads. An example of trapping sample preparation is provided. Figure 2 As shown.Figure 2 As shown, sample 200 is combined with magnetic capture beads 210 to produce a captured sample 220, which consists of bound complexes of particles, e.g., cells, vesicles, etc., bound to the capture magnetic beads. Following preparation of the captured sample, the captured particles can optionally be separated from other components of the captured sample, e.g., unbound particles, unbound beads, etc.

[0148] Following production of the captured sample, the method includes the aspect of partitioning the captured particles of the captured sample using a magnetic field application mediated partitioning scheme to produce partitioned captured particles. Partitioning refers to the placement of the captured particles in small reaction chambers, which can be fluidic separation structures defined by solid materials, e.g., microwells, configured to hold the captured particles. In some embodiments of the disclosed methods, devices, and systems, a plurality of microwells are used that are randomly distributed on a substrate. In some embodiments, the plurality of microwells are distributed in an ordered pattern, e.g., an ordered array, on the substrate. In some embodiments, the plurality of microwells are distributed in a random pattern, e.g., a random array, on the substrate. The microwells can be fabricated in a variety of shapes and sizes. Suitable well geometries include, but are not limited to, cylindrical, elliptical, cubic, conical, hemispherical, rectangular, or polyhedral, e.g., a three-dimensional geometry comprising several planes, e.g., a cuboid, a hexagonal prism, an octagonal prism, an inverted triangular pyramid, an inverted quadrangular pyramid, an inverted pentagonal pyramid, an inverted hexagonal pyramid, or an inverted truncated pyramid. In some embodiments, non-cylindrical microwells, e.g., wells with elliptical or square footprints, can provide advantages in terms of being able to hold larger cells. In some embodiments, the upper and / or lower edges of the well walls can be rounded to avoid sharp corners, thereby reducing electrostatic forces that can arise at sharp edges or points due to electrostatic field concentration. Thus, the use of rounded corners can improve the ability to recover beads from the microwells. Microwell dimensions can be characterized in absolute dimensions. In some cases, the average diameter of the microwells can be from about 5 pm to about 100 pm. In other embodiments, the average of the microwell diameters is at least 5 pm, at least 10 pm, at least 15 pm, at least 20 pm, at least 25 pm, at least 30 pm, at least 35 pm, at least 40 pm, at least 45 pm, at least 50 pm, at least 60 pm, at least 70 pm, at least 80 pm, at least 90 pm, or at least 100 pm. In other embodiments, the average of the microwell diameters is at most 100 pm, at most 90 pm, at most 80 pm, at most 70 pm, at most 60 pm, at most 50 pm, at most 45 pm, at most 40 pm, at most 35 pm, at most 30 pm, at most 25 pm, at most 20 pm, at most 15 pm, at most 10 pm, or at most 5 pm. The volume of the microwells used in the methods of the present disclosure can be varied, in some cases from about 200 pm 3 to about 800,000 pm 3 In some embodiments, the microwell volume is at least 200 pm3 at least 500 μιη 3 at least 1000 μιη 3 at least 10,000 μιη 3 at least 25,000 μιη 3 at least 50,000 μιη 3 at least 100,000 μιη 3 at least 200,000 μιη 3 at least 300,000 μιη 3 at least 400,000 μιη 3 at least 500,000 μιη 3 at least 600,000 μιη 3 at least 700,000 μιη 3 or at least 800,000 μιη 3 In other embodiments, the microwell volume is at most 800,000 μιη 3 at most 700,000 μιη 3 at most 600,000 μιη 3 at most 500,000 μιη 3 at most 400,000 μιη 3 at most 300,000 μιη 3 at most 200,000 μιη 3 at most 100,000 μιη 3 at most 50,000 μιη 3 at most 25,000 μιη 3 at most 10,000 μιη 3 at most 1,000 μιη 3 at most 500 μιη 3 or at most 200 μιη 3 The number of microwells in a given device used in embodiments of the application can vary, with in some cases the number being 100 or more, for example 250 or more, for example, 500 or more, including 1000 or more, for example 5000 or more, for example, 10,000 or more, with in some cases the number being 15,000 or less, for example 12,500 or less. Suitable microwells for use in embodiments of the application are further described in PCT application serial number PCT / US2016 / 014612, publication number WO / 2016 / 118915, the disclosure of which is incorporated herein by reference.

[0149] In partitioning a captured sample, the captured particles can be positioned in the microwells using any convenient protocol. The present disclosure provides methods for contacting a captured sample with a partitioning domain in order to partition the sample. For example, a contained captured sample can be introduced into a structure, e.g., a microwell, to partition the sample. The captured sample can be contacted by, e.g., gravity flow, in which the captured particles can settle into the partitioning structure. In some cases, the captured sample is contacted with the microwells, e.g., by flowing it through the microwells, such that the captured particles are deposited into the microwells. The captured sample can be flowed through a flow cell in fluid communication with the microwells. Suitable protocols and systems for partitioning captured particles into microwells are described in microwells suitable for use in embodiments of the present invention are described in PCT Application Serial No. PCT / US2016 / 014612, Publication No. WO / 2016 / 118915, the disclosure of which is incorporated herein by reference.

[0150] In partitioning a captured sample, the captured sample is contacted with the microwells using an applied magnetic field, e.g., the captured sample is flowed through the microwells when a magnetic field is applied to the captured sample. The applied magnetic field is one that moves the captured particles from the captured sample toward the microwells, and depending on its orientation and the properties of the magnetic captured particles, is attractive or repulsive to the magnetic captured particles. For example, in the case of a magnetic field applied below the bottom of the microwells, the magnetic field can attract the magnetic captured particles. Alternatively, in the case of a magnetic field applied above the microwells, the magnetic field can repel the magnetic captured particles. Any convenient source can be used to apply the magnetic field, e.g., permanent magnets, electromagnets, etc. While the strength of the applied magnetic field can vary as desired, in some cases the strength is from 10 to 15,000 Gauss, e.g., 100 to 1,000 Gauss. Systems and methods of using the systems comprising one or more magnets and controllers thereof are described in PCT Application Serial No. PCT / US2016 / 014612, Publication No. WO / 2016 / 118915, which can be used in embodiments of the present invention, the disclosure of which is incorporated herein by reference.

[0151] Figure 3A A schematic representation of partitioning captured particles prepared from captured magnetic beads comprising bead-bound barcode nucleic acids, e.g., as shown in Figure 1B is shown in a microwell of a flow cell. As shown in Figure 3A is shown, under the influence of an applied electric field (as shown by magnet 330), a captured particle 310 comprising, e.g., a cell or extracellular vesicle particle, bound to a captured magnetic bead comprising bead-bound barcode nucleic acids, flows through a microwell array 320, which results in the captured particle being partitioned into a well, as shown in Figure 3B .

[0152] FIG. 4 provides a schematic representation of partitioning of capture particles prepared from capture magnetic beads that do not include bead-bound barcode nucleic acids, e.g., as described above, in micro-wells using a flow cell. As shown, capture particles 410, e.g., cells or extracellular vesicle particles, bound to capture magnetic beads that do not include bead-bound barcode nucleic acids, are flowed through a micro-well array 420 under the influence of an applied electric field, as shown by magnet 430, which results in the partitioning of the captured particles into wells, as shown. Figure 1A As shown, capture particles 410, e.g., cells or extracellular vesicle particles, bound to capture magnetic beads that do not include bead-bound barcode nucleic acids, are flowed through a micro-well array 420 under the influence of an applied electric field, as shown by magnet 430, which results in the partitioning of the captured particles into wells, as shown. Figure 4A As shown, capture particles 410, e.g., cells or extracellular vesicle particles, bound to capture magnetic beads that do not include bead-bound barcode nucleic acids, are flowed through a micro-well array 420 under the influence of an applied electric field, as shown by magnet 430, which results in the partitioning of the captured particles into wells, as shown. Figure 4B As shown, capture particles 410, e.g., cells or extracellular vesicle particles, bound to capture magnetic beads that do not include bead-bound barcode nucleic acids, are flowed through a micro-well array 420 under the influence of an applied electric field, as shown by magnet 430, which results in the partitioning of the captured particles into wells, as shown. Figure 4C As shown, capture particles 410, e.g., cells or extracellular vesicle particles, bound to capture magnetic beads that do not include bead-bound barcode nucleic acids, are flowed through a micro-well array 420 under the influence of an applied electric field, as shown by magnet 430, which results in the partitioning of the captured particles into wells, as shown.

[0153] For example, as described above, partitioning of a capture sample results in partitioned capture particles being in spatial proximity to bead-bound barcode nucleic acids, e.g., as described above, that include target binding regions. In some cases, the bead-bound barcode nucleic acids are stably bound to the capture magnetic beads. In such cases, a partition, e.g., a micro-well, can include only a capture particle because the bead component of the capture particle includes the barcode nucleic acids. In other cases, the bead-bound barcode nucleic acids are part of a separate bead from the capture magnetic beads, e.g., a bead-bound barcode as described above. In such cases, a partition, e.g., a micro-well, can include both a capture particle and a separate bead-bound barcode that includes the barcode nucleic acids. When the barcode is in proximity to the target of the capture particle, the target can hybridize to the barcode. The barcodes can be contacted in a non-depleting ratio so that each different target can bind to a different barcode of the present disclosure. To ensure efficient binding between the target and the barcode, the target can be cross-linked to the barcode.

[0154] As described above, after partitioning particles, e.g., cells and / or vesicles, the particles can be lysed to release target molecules so that the released target molecules, e.g., nucleic acids, can bind to the target binding regions of the barcode nucleic acids to produce captured nucleic acids. Particle lysis can be accomplished in a variety of ways, e.g., chemically or biochemically, osmotic shock, or by thermal lysis, mechanical lysis, or optical lysis. Particles can be lysed by the addition of a cell lysis buffer that includes a surfactant (e.g., SDS, lithium dodecyl sulfate, Triton X-100, Tween-20, or NP-40), an organic solvent (e.g., methanol or acetone), or a digestive enzyme (e.g., proteinase K, pepsin, or trypsin), or any combination thereof. To increase the binding of the target and the barcode, the diffusion rate of the target molecules can be altered by, e.g., lowering the temperature and / or increasing the viscosity of the lysis solution.

[0155] In some embodiments, filter paper can be used to lyse the sample. The filter paper can be soaked with lysis buffer on top of the filter paper. The filter paper can be applied to the sample with pressure, which can facilitate lysis of the sample and hybridization of the targets of the sample to the substrate.

[0156] In some embodiments, lysis can be performed by mechanical lysis, thermal lysis, optical lysis, and / or chemical lysis. Chemical lysis can include the use of a digestion enzyme, such as proteinase K, pepsin, and trypsin. Lysis can be performed by adding a lysis buffer to the substrate. The lysis buffer can comprise Tris HC1. The lysis buffer can comprise at least about 0.01 M, 0.05 M, 0.1 M, 0.5 M, or 1 M or greater than 1 M of Tris HC1. The lysis buffer can comprise at most about 0.01 M, 0.05 M, 0.1 M, 0.5 M, or 1 M or greater than 1 M of Tris HC1. The lysis buffer can comprise about 0.1 M of Tris HC1. The lysis buffer can have a pH of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10. The lysis buffer can have a pH of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10. In some embodiments, the lysis buffer has a pH of about 7.5. The lysis buffer can comprise a salt (e.g., LiCl). The concentration of the salt in the lysis buffer can be at least about 0.1 M, 0.5 M, or 1 M or greater than 1 M. The concentration of the salt in the lysis buffer can be at most about 0.1 M, 0.5 M, or 1 M or greater than 1 M. In some embodiments, the concentration of the salt in the lysis buffer is about 0.5 M. The lysis buffer can comprise a surfactant (e.g., SDS, lithium dodecyl sulfate, triton X, Tween, NP-40). The concentration of the surfactant in the lysis buffer can be at least about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7% or greater than 7%. The concentration of the surfactant in the lysis buffer can be at most about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7% or greater than 7%. In some embodiments, the concentration of the surfactant in the lysis buffer is about 1% lithium dodecyl sulfate. The time of use in the lysis method can depend on the amount of surfactant used. In some embodiments, the more surfactant used, the less time required for lysis. The lysis buffer can comprise a chelator (e.g., EDTA, EGTA). The concentration of the chelator in the lysis buffer can be at least about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, or 30 mM or greater than 30 mM. The concentration of the chelator in the lysis buffer can be at most about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, or 30 mM or greater than 30 mM. In some embodiments, the concentration of the chelator in the lysis buffer is about 10 mM. The lysis buffer can comprise a reducing agent (e.g., b-mercaptoethanol, DTT).The concentration of reducing agent in the lysis buffer can be at least about 1 mM, 5 mM, 10 mM, 15 mM, or 20 mM or greater than 20 mM. The concentration of reducing agent in the lysis buffer can be at most about 1 mM, 5 mM, 10 mM, 15 mM, or 20 mM or greater than 20 mM. In some embodiments, the concentration of reducing agent in the lysis buffer is about 5 mM. In some embodiments, the lysis buffer can comprise about 0.1 M Tris HC1, about pH 7.5, about 0.5 M LiCl, about 1% lithium dodecyl sulfate, about 10 mM EDTA, and about 5 mM DTT.

[0157] Lysis can be performed at a temperature of about 4°C, 10°C, 15°C, 20°C, 25°C, or 30°C. Lysis can be performed for about 1 minute, 5 minutes, 10 minutes, 15 minutes, or 20 minutes or greater than 20 minutes. The lysed cells can comprise at least about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, or 700,000 or more than 700,000 target nucleic acid molecules. The lysed cells can comprise at most about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, or 700,000 or more than 700,000 target nucleic acid molecules.

[0158] After the particles are lysed and the nucleic acid molecules are released therefrom, the nucleic acid molecules can be randomly associated with the barcodes co-localized to the solid support. The association can include hybridization of a target recognition domain of the barcode to a complementary portion of the target nucleic acid molecule (e.g., an oligo(dT) of the barcode can interact with a poly(A) tail of the target). The assay conditions used for hybridization (e.g., buffer pH, ionic strength, temperature, etc.) can be selected to promote the formation of specific, stable hybrids. In some embodiments, the nucleic acid molecules released from the lysed cells can be associated with (e.g., hybridized to) a plurality of probes on the substrate. When the probes comprise oligo(dT), mRNA molecules can hybridize to the probes and be reverse transcribed. The oligo(dT) portion of the oligonucleotide can serve as a primer for first strand synthesis of a cDNA molecule, e.g., when placed under DNA synthesis reaction conditions, resulting in a first strand cDNA domain comprising the captured nucleic acid.

[0159] Attachment can also include ligation of a target recognition region of a barcode and a portion of a target nucleic acid molecule. For example, the target binding region can comprise a nucleic acid sequence capable of specifically hybridizing to a restriction site single-stranded overhang (e.g., an EcoRI cohesive end single-stranded overhang). The assay procedure can also include treating the target nucleic acid with a restriction endonuclease (e.g., EcoRI) to generate a restriction site single-stranded overhang. The barcode can then be ligated to any nucleic acid molecule comprising a sequence complementary to the restriction site single-stranded overhang. A ligase enzyme (e.g., T4 DNA ligase) can be used to ligate the two fragments together.

[0160] In some cases, the method further comprises using oligonucleotide-labeled cellular component binding reagents, e.g., where quantitative analysis of one or more cellular components, e.g., surface proteins, is desired. The oligonucleotide-labeled cellular component binding reagents used in the described embodiments comprise a cellular component binding reagent, e.g., an antibody or binding fragment thereof, coupled to a cellular component binding reagent-specific oligonucleotide comprising an identifier sequence for the cellular component binding reagent, which binds to the cellular component binding reagent-specific oligonucleotide. In such cases, the capture magnetic beads can comprise a nucleic acid configured to capture, e.g., a domain that specifically binds to the cellular component binding reagent-specific oligonucleotide. In this way, gene expression analysis can be combined with protein expression, e.g., where combined analysis of the transcriptome and proteome is desired. In such cases, the method can comprise preparing a capture sample using oligonucleotide-labeled cellular component binding reagents, and then providing capture of the cellular component binding reagent-specific oligonucleotides released from the captured, partitioned cells. Further details regarding the use of oligonucleotide-labeled cellular component binding reagents can be found in U.S. Published Patent Application Nos. US20180267036 and US20200248263; the disclosures of which are incorporated herein by reference.

[0161] In some cases, the method further comprises separating the captured nucleic acids from other components of the partitioned capture particles. Recovery of the solid support-based attached target barcode molecule collection can be performed by using magnetic beads and an externally applied magnetic field.

[0162] If desired, a given workflow can include a pooling step in which a product composition, e.g., consisting of captured nucleic acids, synthesized first-strand cDNA, or synthesized double-stranded cDNA, is combined or pooled with a product composition obtained from one or more additional samples, e.g., particles, e.g., cells and / or vesicles. In some cases, the pooling step is performed after the step of hybridizing between the barcode nucleic acids and the target nucleic acids, e.g., as described above. The number of different product compositions produced from different samples, e.g., cells, can vary in these embodiments, with the number being in the range of 2 to 1,000,000, e.g., 3 to 200,000, including 4 to 100,000, e.g., 5 to 50,000, in some cases, with the number being in the range of 100 to 10,000, e.g., 1,000 to 5,000. Prior to or after mixing, the product composition can be amplified by polymerase chain reaction (PCR) or other methods, e.g., as described in detail below.

[0163] After the target barcode molecules are pooled together, all other processing can be performed in a single reaction vessel. Other processing can include, for example, reverse transcription reactions, amplification reactions, cleavage reactions, dissociation reactions, and / or nucleic acid extension reactions. Other processing reactions can be performed in micro-wells, that is, without first mixing the labeled target nucleic acid molecules from multiple cells.

[0164] The present disclosure provides methods for producing target-barcode conjugates using any convenient protocol, e.g., reverse transcription or nucleotide extension (e.g., sample indexing oligonucleotides or cell component = binding reagent specific oligonucleotides). The target-barcode conjugates can comprise a barcode and a complement of all or part of a target nucleic acid (i.e., a barcode-encoded cDNA molecule, e.g., a stochastic barcode-encoded cDNA molecule). Reverse transcription of the relevant RNA molecule can be performed by the addition of reverse transcriptase and a reverse transcription primer. The reverse transcription primer can be an oligo(dT) primer, a random hexanucleotide primer, or a target-specific oligonucleotide primer. The oligo(dT) primer can be 12 to 18 nucleotides in length, or can be about 12 to 18 nucleotides in length, and binds to the endogenous poly(A) tail at the 3' end of mammalian mRNA. The random hexanucleotide primer can bind to mRNA at multiple complementary sites. The target-specific oligonucleotide primer typically selectively primes mRNA of interest.

[0165] In some embodiments, a labeled RNA molecule can be produced by reverse transcription of an mRNA molecule by the addition of a reverse transcription primer. In some embodiments, the reverse transcription primer is an oligo(dT) primer, a random hexanucleotide primer, or a target-specific oligonucleotide primer. Typically, oligo(dT) primers are 12 to 18 nucleotides in length and bind to the endogenous poly(A) tail at the 3' end of mammalian mRNA. Random hexanucleotide primers can bind to mRNA at multiple complementary sites. Target-specific oligonucleotide primers are typically prepared selectively for mRNA of interest.

[0166] In some embodiments, the target is a cDNA molecule. For example, an mRNA molecule can be reverse transcribed using a reverse transcriptase, such as Moloney murine leukemia virus (MMLV) reverse transcriptase, to produce a cDNA molecule with a poly(dC) tail. The barcode can include a target-binding region with a poly(dG) tail. When the poly(dG) tail of the barcode and the poly(dC) tail of the cDNA molecule base pair, the reverse transcriptase switches template strands from the cellular RNA molecule to the barcode and continues copying to the 5' end of the barcode. By doing so, a cDNA molecule is obtained that includes the barcode (e.g., molecular label) sequence at the 3' end of the cDNA molecule.

[0167] Reverse transcription can occur repeatedly to produce multiple labeled cDNA molecules. The methods disclosed herein can comprise performing at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 reverse transcription reactions. The methods can comprise performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 reverse transcription reactions.

[0168] One or more nucleic acid amplification reactions can be performed to generate multiple copies of the labeled target nucleic acid molecules. Amplification can be performed in a multiplexed manner, in which multiple target nucleic acid sequences are amplified simultaneously. The amplification reaction can be used to add sequencing adaptors to the nucleic acid molecules. If present, the amplification reaction can include amplification of at least a portion of the sample label. The amplification reaction can include amplification of at least a portion of the cell label and / or barcode sequence (e.g., molecular label). The amplification reaction can include amplification of at least a portion of the sample tag, cell label, spatial label, barcode sequence (e.g., molecular label), target nucleic acid, or combinations thereof. The amplification reaction comprises amplification of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 100%, or a value or a range between any two of these values, of the nucleic acids. The method can further comprise performing one or more cDNA synthesis reactions to generate one or more cDNA copies of the target-barcode molecules comprising the sample label, cell label, spatial label, and / or barcode sequence (e.g., molecular label).

[0169] In some embodiments, amplification can be performed using polymerase chain reaction (PCR). As described herein, PCR can refer to a reaction that amplifies specific DNA sequences in vitro by simultaneous primer extension of complementary DNA strands. As described herein, PCR can include derivative forms of the reaction, including but not limited to RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, digital PCR, and assembly PCR.

[0170] Amplification of the labeled nucleic acids can comprise non-PCR based methods. Examples of non-PCR based methods include, but are not limited to, multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, rolling circle amplification, or circle to circle amplification. Other non-PCR based amplification methods include multiple cycles of DNA-dependent RNA polymerase-driven RNA transcription amplification or RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets, ligase chain reaction (LCR), and Qbeta replicase (Qbeta) method, amplification methods using palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using restriction endonucleases hybridize primers to nucleic acid sequences, cleave resulting duplexes prior to extension reaction and amplification, strand displacement amplification using nucleic acid polymerases that lack 5' exonuclease activity, rolling circle amplification, and reticulate branching amplification (RAM). In some embodiments, amplification does not generate circular transcripts.

[0171] In some embodiments, the methods disclosed herein further comprise performing a polymerase chain reaction on the labeled nucleic acids (e.g., labeled-RNA, labeled-DNA, labeled-cDNA) to generate labeled amplicons (e.g., randomly labeled amplicons). The labeled amplicons can be double stranded molecules. The double stranded molecules can comprise double stranded RNA molecules, double stranded DNA molecules, or RNA molecules hybridized to DNA molecules. One or both strands of the double stranded molecules can comprise sample labels, spatial labels, cell labels, and / or barcode sequences (e.g., molecular labels). The labeled amplicons can be single stranded molecules. The single stranded molecules can comprise DNA, RNA, or a combination thereof. The nucleic acids of the present disclosure can comprise synthetic or altered nucleic acids. Accordingly, the methods can comprise generating an amplicon composition from the first strand cDNA domains comprising the captured nucleic acids.

[0172] The amplification can comprise the use of one or more unnatural nucleotides. The unnatural nucleotides can comprise photo-labile or triggerable nucleotides. Examples of unnatural nucleotides can include, but are not limited to, peptide nucleic acids (PNA), morpholino nucleic acids, and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threose nucleic acids (TNA). The unnatural nucleotides can be added to one or more cycles of the amplification reaction. The addition of the unnatural nucleotides can be used to identify the product as a specific cycle or time point in the amplification reaction.

[0173] Performing one or more amplification reactions can comprise using one or more primers. The one or more primers can comprise, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more than 15 nucleotides. The one or more primers can comprise at least 1, 2, 3, 4, 5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more than 15 nucleotides. The one or more primers can comprise fewer than 12 to 15 nucleotides. The one or more primers can anneal to at least a portion of the plurality of labeled targets (e.g., randomly labeled targets). The one or more primers can anneal to the 3’ end or 5’ end of the plurality of labeled targets. The one or more primers can anneal to an internal region of the plurality of labeled targets. The internal region can be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, or 1000 nucleotides from the 3’ end of the plurality of labeled targets. The one or more primers can comprise a fixed set of primers. The one or more primers can comprise at least one or more custom primers. The one or more primers can comprise at least one or more control primers. The one or more primers can comprise at least one or more gene-specific primers.

[0174] The one or more primers can comprise a universal primer. The universal primer can anneal to a universal primer binding site. The one or more custom primers can anneal to a first sample label, a second sample label, a spatial label, a cell label, a barcode sequence (e.g., a molecular label), a target, or any combination thereof. The one or more primers can comprise a universal primer and a custom primer. The custom primer can be designed to amplify one or more targets. The targets can comprise a subset of all nucleic acids in one or more samples. The targets can comprise a subset of all labeled targets in one or more samples. The one or more primers can comprise at least 96 or more than 96 custom primers. The one or more primers can comprise at least 960 or more than 960 custom primers. The one or more primers can comprise at least 9600 or more than 9600 custom primers. The one or more custom primers can anneal to two or more different labeled nucleic acids. The two or more different labeled nucleic acids can correspond to one or more genes.

[0175] Any amplification scheme can be used in the methods of the present disclosure. For example, in one scheme, a first round of PCR can amplify molecules attached to beads using gene-specific primers and primers to universal Illumina sequencing primer 1 sequence. A second round of PCR can amplify the first PCR product using nested gene-specific primers flanked by Illumina sequencing primer 2 sequence and primers opposite the universal Illumina sequencing primer 1 sequence. A third round of PCR adds P5 and P7 and sample index, converting the PCR product to an Illumina sequencing library. Using 150 bp x 2 sequencing can reveal cell label and barcode sequence (e.g., molecular label) on read 1, gene on read 2, and sample index on index 1 read.

[0176] In some embodiments, chemical cleavage can be used to remove the nucleic acid from the substrate. For example, a chemical group or modified base present in the nucleic acid can be used to facilitate its removal from the solid support. For example, an enzyme can be used to remove the nucleic acid from the substrate. For example, a restriction endonuclease digestion can be used to remove the nucleic acid from the substrate. For example, a uracil-d-glycosylase (UDG) treatment can be used to remove the nucleic acid from the substrate that comprises dUTP or ddUTP. For example, an enzyme that performs nucleotide excision, such as a base excision repair enzyme, such as apurinic-apyrimidinic endonuclease, can be used to remove the nucleic acid from the substrate. In some embodiments, a photocleavable and light can be used to remove the nucleic acid from the substrate. In some embodiments, a cleavable linker can be used to remove the nucleic acid from the substrate. For example, the cleavable linker can comprise at least one of biotin / avidin, biotin / streptavidin, biotin / neutravidin, Ig-protein A, a photo-labile linker, an acid or base-labile linker group, or an aptamer.

[0177] When the probe is a gene-specific probe, the molecule can be hybridized to the probe and reverse transcribed and / or amplified. In some embodiments, after the nucleic acid has been synthesized (e.g., reverse transcribed), it can be amplified. Amplification can be performed in a multiplexed manner, where multiple target nucleic acid sequences are amplified simultaneously. Amplification can add sequencing adaptors to the nucleic acid.

[0178] In some embodiments, amplification can be performed on a substrate, e.g., using bridge amplification. The cDNA can be homopolymer tailed in order to generate compatible ends for bridge amplification using oligo(dT) probes on the substrate. In bridge amplification, the primer complementary to the 3' end of the template nucleic acid can be the first primer covalently attached to a solid particle per pair. When the sample containing the template nucleic acid is contacted with the particle and a single thermal cycle is performed, the template molecule can anneal to the first primer and the first primer is extended in the forward direction by the addition of nucleotides, forming a double-stranded molecule consisting of the template molecule and the newly formed DNA strand complementary to the template. In the heating step of the next cycle, the double-stranded molecule can be denatured, releasing the template molecule from the particle, and leaving the complementary DNA strand attached to the particle through the first primer. In the annealing phase of the subsequent annealing and extension steps, the complementary strand can hybridize to the second primer, which is complementary to the segment of the complementary strand at the position removed from the first primer. This hybridization can cause the complementary strand to form a bridge between the first and second primers, fixed by covalent bonds to the first primer and by hybridization to the second primer. In the extension phase, the second primer can be extended in the reverse direction by the addition of nucleotides in the same reaction mixture, thereby converting the bridge to a double-stranded bridge. The next cycle then begins, and the double-stranded bridge can be denatured to produce two single-stranded nucleic acid molecules, each attached to the particle surface at one end by the first and second primers, respectively, and each unattached at the other end. In the annealing and extension steps of the second cycle, each strand can hybridize to another complementary primer that has not been used before on the same particle to form new single-stranded bridges. The two previously unused primers, after hybridization, stretch to convert the two new bridges to a double-stranded bridge.

[0179] The amplification reaction can comprise amplifying at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% of the plurality of nucleic acids.

[0180] The amplification of the labeled nucleic acids can comprise a PCR-based method or a non-PCR-based method. The amplification of the labeled nucleic acids can comprise exponential amplification of the labeled nucleic acids. The amplification of the labeled nucleic acids can comprise linear amplification of the labeled nucleic acids. Amplification can be performed using polymerase chain reaction (PCR). PCR can refer to a reaction that amplifies specific DNA sequences in vitro by simultaneous primer extension of complementary DNA strands. PCR can comprise derivative forms of the reaction including, but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, digital PCR, suppression PCR, semi-suppression PCR, and assembly PCR.

[0181] In some embodiments, amplification of the labeled nucleic acids comprises a non-PCR based method. Examples of non-PCR based methods include, but are not limited to, multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, rolling circle amplification, or circle-to-circle amplification. Other non-PCR based amplification methods include multiple cycles of DNA-dependent RNA polymerase driven RNA transcription amplification or RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets, ligase chain reaction (LCR), and Qbeta replicase (Qbeta) method, use of palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using restriction endonucleases, amplification methods that hybridize primers to nucleic acid sequences, cleave the resulting duplex prior to extension and amplification, strand displacement amplification using nucleic acid polymerases that lack 5' exonuclease activity, rolling circle amplification, and ramification amplification (RAM).

[0182] In some embodiments, the methods disclosed herein further comprise performing a nested polymerase chain reaction on the amplified amplicons (e.g., targets). The amplicons can be double-stranded molecules. The double-stranded molecules can comprise double-stranded RNA molecules, double-stranded DNA molecules, or RNA molecules hybridized to DNA molecules. One or both strands of the double-stranded molecules can comprise sample tag or molecular identifier labels. Alternatively, the amplicons can be single-stranded molecules. The single-stranded molecules can comprise DNA, RNA, or a combination thereof. The nucleic acids of the present invention can comprise synthetic or altered nucleic acids.

[0183] In some embodiments, the method comprises repeating amplification of the labeled nucleic acids to produce a plurality of amplicons. The methods disclosed herein comprise performing at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amplification reactions. Alternatively, the method comprises performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amplification reactions.

[0184] Amplification can further comprise adding one or more control nucleic acids to one or more samples comprising a plurality of nucleic acids. Amplification can further comprise adding one or more control nucleic acids to the plurality of nucleic acids. The control nucleic acids can comprise control labels.

[0185] The amplification can comprise the use of one or more non-natural nucleotides. The non-natural nucleotides can comprise light-labile and / or triggerable nucleotides. Examples of non-natural nucleotides can include, but are not limited to, peptide nucleic acids (PNAs), morpholino nucleic acids, and locked nucleic acids (LNAs), as well as glycol nucleic acids (GNAs) and threose nucleic acids (TNAs). The non-natural nucleotides can be added to one or more cycles of the amplification reaction. The addition of non-natural nucleotides can be used to identify the product as a specific cycle or time point in the amplification reaction.

[0186] Performing one or more amplification reactions can comprise the use of one or more primers. The one or more primers can comprise at least one or more oligonucleotides. The one or more oligonucleotides can comprise at least 7 to 9 nucleotides. The one or more oligonucleotides can comprise less than 12 to 15 nucleotides. The one or more primers can anneal to at least a portion of the plurality of labeled nucleotides. The one or more primers can anneal to the 3’ end or 5’ end of the plurality of labeled nucleic acids. The one or more primers can anneal to an internal region of the plurality of labeled nucleic acids. The internal region can be at a distance of at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, or 1000 nucleotides from the 3’ end of the plurality of labeled nucleic acids. The one or more primers can comprise a fixed set of primers. The one or more primers can comprise at least one or more custom primers. The one or more primers can comprise at least one or more control primers. The one or more primers can comprise at least one or more housekeeping gene primers. The one or more primers can comprise universal primers. The universal primers can anneal to a universal primer binding site. The one or more custom primers can anneal to the first sample tag, the second sample tag, the molecular identifier tag, the nucleic acid, or a product thereof. The one or more primers can comprise universal primers and custom primers. The custom primers can be designed to amplify one or more target nucleic acids. The target nucleic acids can comprise a subset of all nucleic acids in the one or more samples. In some embodiments, the primers are probes attached to the array of the present disclosure.

[0187] In some implementations, barcoding multiple targets in a sample (e.g., random barcoding) also includes generating an index library of barcoded targets (e.g., randomly barcoded targets) or barcoded fragments of targets. The barcode sequences of different barcodes (e.g., molecular markers of different random barcodes) can be different from each other. Generating an index library of barcoded targets includes generating multiple index polynucleotides from multiple targets in the sample. For example, for an index library containing barcoded targets of a first index target and a second index target, the labeled region of the first index polynucleotide can differ from the labeled region of the second index polynucleotide by approximately, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or any two of these values ​​or a range of nucleotides. In some embodiments, generating an index library of barcoded targets includes contacting multiple targets, such as mRNA molecules, with multiple oligonucleotides, said oligonucleotides including a poly(T) region and a labeled region; performing first-strand synthesis using reverse transcriptase to produce single-stranded labeled cDNA molecules, each labeled cDNA molecule containing a cDNA region and a labeled region, wherein the multiple targets include at least two different sequences of mRNA molecules and the multiple oligonucleotides include at least two different sequences of oligonucleotides. Generating an index library of barcoded targets may further include amplifying single-stranded labeled cDNA molecules to produce double-stranded labeled cDNA molecules, and performing nested PCR on the double-stranded labeled cDNA molecules to produce labeled amplicons. In some embodiments, the method may include generating amplicons labeled with adaptor tags.

[0188] Barcoding (e.g., random barcoding) can include tagging individual nucleic acid (e.g., DNA or RNA) molecules using nucleic acid barcodes or tags. In some implementations, it involves adding DNA barcodes or tags to cDNA molecules, since they are generated from mRNA. Nested PCR can be performed to reduce PCR amplification bias. Adaptors can be added for sequencing using, for example, next-generation sequencing (NGS). Sequencing results can be used to determine cellular markers, molecular markers, and nucleotide fragment sequences of one or more target copies.

[0189] In some embodiments, the provided method further includes placing the prepared expression library, for example, the amplicon composition generated as described above, into an NGS protocol. This protocol can be implemented on any suitable NGS sequencing platform. NGS sequencing platforms of interest include, but are not limited to, those mentioned above. Provided sequencing platforms (e.g., HiSeq) TM MiSeq TM and / or NextSeq TMSequencing System); Ion Torrent TM ( e.g., Ion PGM TM and / or Ion Proton TM Sequencing System); Pacific Biosciences (e.g., PACBIO RS II Sequel Sequencing System); Life Technologies TM ( e.g., SOLiD Sequencing System); Oxford Nanopore (e.g., Minion); Roche (e.g., 454 GS FLX+ and / or GS Junior Sequencing System); or any other sequencing platform of interest. NGS protocols will vary depending on the other NGS sequencing system used. Detailed protocols for sequencing, e.g., can also include amplification (e.g., solid-phase amplification), sequencing amplicons, and analyzing sequencing data can be obtained from the manufacturer of the NGS sequencing system used.

[0190] Devices and systems

[0191] Disclosed herein are devices and systems useful in practicing embodiments of the application. In some embodiments, a device comprises: a) a substrate comprising: i) at least 100 microwells, and ii) a plurality of capture particles and / or barcoded beads, wherein the plurality of at least 100 microwells each comprise a single capture particle optionally with a single barcoded bead (e.g., when the capture magnetic beads do not comprise a barcoded nucleic acid), and b) a flow cell in fluid communication with the substrate. In some embodiments, there is also at least one inlet port and at least one outlet port, wherein the at least one inlet port and at least one outlet port are in fluid communication with the flow cell through a fluidic channel, wherein the at least one inlet port and at least one outlet port are capable of directing a flow of fluid through the flow cell to contact the microwells with the fluid. In some embodiments, the device further comprises a valve that prevents fluid flow within the device unless a pipette tip is inserted into the pipette tip interface of the conical feature.

[0192] Also disclosed herein are systems comprising: a) a device comprising: i) a substrate comprising at least 100 microwells; ii) a flow cell in fluid communication with the substrate; and iii) at least one inlet port and at least one outlet port, wherein the at least one inlet port and at least one outlet port are capable of directing a flow of fluid through the flow cell to contact the microwells with the fluid; and b) a flow controller; wherein the flow controller is configured to control delivery of fluid.

[0193] In some embodiments, the system further comprises a fluid, wherein the fluid comprises a particle (e.g., cell and / or vesicle) sample, a bead suspension, an assay reagent, or any combination thereof. In some embodiments, the device is a removable, consumable component of the system. In some embodiments, the cell sample and bead suspension are dispensed or injected into the device directly by a user. In some embodiments, the beads and assay reagent in addition to the cell sample are pre-installed in the device. In some embodiments, the flow controller is configured to inject the fluid into the flow cell separately from the air injection. In some embodiments, the system further comprises a distribution mechanism for enhancing uniform distribution of cells and beads in the at least 100 microwells, wherein the distribution mechanism is selected from the group consisting of rocking, shaking, swirling, circulating flow, low frequency agitation, and high frequency agitation, or any combination thereof. In some embodiments, the system further comprises a cell lysis mechanism, e.g., ultrasonication of cells using a high frequency piezoelectric transducer. In some embodiments, the system further comprises a temperature controller for maintaining a user-specific temperature, or for ramping between two or more user-specific temperatures over two or more user-specific time intervals. In some embodiments, the system further comprises a magnetic field controller for generating a magnetic field gradient for eluting beads from the at least 100 microwells or for transporting beads through the device. In some embodiments, the system further comprises an imaging system configured to capture and process images of all or a portion of the at least 100 microwells, wherein the imaging system further comprises an illumination subsystem, an imaging subsystem, and a processor. In some embodiments, the imaging system is configured to perform brightfield, darkfield, fluorescent, or quantitative phase imaging. In some embodiments, the imaging system is configured to provide real-time image analysis capabilities, and wherein the real-time image analysis is used to control the distribution mechanism to enhance uniform distribution of cells or beads in the at least 100 microwells to achieve a pre-determined cell or bead distribution. In some embodiments, the pre-determined cell and bead distribution refers to at least 10% of the microwells containing a single cell and a single bead simultaneously. In some embodiments, the pre-determined cell and bead distribution refers to at least 25% of the microwells containing a single cell and a single bead simultaneously. In some embodiments, the system further comprises a selection mechanism, wherein information obtained from the processed images is used to identify a subset of cells that exhibit one or more specific characteristics, and the selection mechanism is configured to include or exclude the subset of cells from subsequent data analysis. In some embodiments, the selection mechanism comprises physically removing beads co-localized with cells of the identified subset of cells from the at least 100 microwells. In some embodiments, the selection mechanism comprises physically encapsulating beads co-localized with cells of the identified subset of cells from the at least 100 microwells.In some embodiments, the selection mechanism comprises using dual-coded beads, wherein each individual bead is optically encoded and encoded by an attached oligonucleotide cell label, sequencing the cell label attached to the bead that co-localizes with the cells of the identified subset of cells, generating a list of sequence data that includes or excludes other analyses. In some embodiments, the flow controller is configured to deliver a first test compound into at least 100 microwells at a first time and to deliver a cell lysis reagent into at least 100 microwells at a second time. In some embodiments, the first time and the second time are the same. In some embodiments, the one or more specific features are selected from the group consisting of cell size, cell shape, live cells, dead cells, a specific range of intracellular pH, a specific range of membrane potential, a specific intracellular calcium level, one or more specific cell surface markers present, and expression of one or more specific genetic markers.

[0194] There can also be software saved in a computer readable medium that is programmed to perform one or more of the following sequence data analysis steps: a) decoding or demultiplexing of sample barcodes, cell barcodes, molecular barcodes, and target sequence data; b) automatic clustering of cell labels to compensate for amplification or sequencing errors, wherein sequence data is collected as a library of randomly labeled target oligonucleotide molecules; c) alignment of sequence data to known reference sequences; d) determining the number of reads per gene in each cell, and the number of unique transcript molecules per gene in each cell; e) statistical analysis to predict confidence intervals to determine the number of transcript molecules per gene in each cell; and f) statistical analysis to cluster cells or identify rare cell subpopulations based on gene expression data.

[0195] There can also be software saved in a computer readable medium that is programmed to perform the following image processing and instrument control steps: a) detecting microwells in one or more images of a plurality of microwells; b) detecting microwells containing single cells in one or more images of a plurality of microwells; c) detecting microwells containing two or more cells in one or more images of a plurality of microwells and determining the number of cells in each detected microwell; d) detecting microwells containing single beads in one or more images of a plurality of microwells; e) detecting microwells containing two or more beads in one or more images of a plurality of microwells and determining the number of beads in each detected microwell; f) determining the number of microwells containing single cells after performing step (b); g) determining the number of microwells containing single beads after performing step (d); and h) determining the number of microwells containing single cells and single beads after performing steps (b) and (d), wherein the numbers determined in steps (f) to (h) are used to control an apparatus configured to distribute cells and beads through a plurality of microwells.

[0196] Software stored in a computer-readable medium may also exist, which is programmed to perform the following image processing and instrument control steps: a) detecting a subset of cells exhibiting one or more specific features in one or more images of a plurality of microwells, wherein a subset of microwells contains cells; b) determining the location of the microwells containing the subset of cells; and c) using the location determined in step (b) to control a selection device configured to exclude the subset of cells from subsequent sequence data analysis.

[0197] In some embodiments, the selection device in step c) is configured to include only a subset of cells in subsequent sequence data analysis. In some embodiments, one or more images of the plurality of micropores are selected from bright-field images, dark-field images, fluorescence images, luminescent images, and phosphorescent images. In some embodiments, the software further includes the use of one or more algorithms selected from Canny edge detection methods, Canny-Deriche edge detection methods, Sobel operator methods, first-order gradient detection methods, second-order differential edge detection methods, phase coherence edge detection methods, intensity thresholding methods, intensity clustering methods, intensity histogram-based methods, generalized Hough transform, circular Hough transform, Fourier transform, fast Fourier transform, wavelet analysis, and autocorrelation analysis. In some embodiments, the one or more specific features are selected from cell size, cell shape, live cells, dead cells, specific ranges of intracellular pH, specific ranges of membrane potential, specific intracellular calcium levels, one or more present specific cell surface markers, and the expression of one or more specific genetic markers.

[0198] PCT application PCT / US2016 / 014612, publication number WO / 2016 / 118915 describes, for example, the system described above and the method of using the system that can be used in embodiments of the present invention, the disclosure of which is incorporated herein by reference.

[0199] Figure 5 The illustration shows an example of a workflow according to an embodiment of the present invention, which uses, for example, a system as described above, which can serve as a Rhapsody. TM Single-cell analysis systems (Becton, Dickinson, and the Company) are commercially available. For example... Figure 5 As shown, the capture particles contain cells bound to the capture magnetic beads and bead-bound barcode nucleic acids. The capture particles are divided into micropores in an array using an applied magnetic field, for example, as... Figure 3A and Figure 3B As shown. The cells containing the segmented capture particles are then lysed, allowing the released mRNA to hybridize with the barcoded nucleic acids captured on the beads. The beads are then recovered from the wells using an applied magnetic field to generate sequencing libraries, such as...Figure 6 The cells are then sequenced, e.g., using a next generation sequencing platform, e.g., as described above.

[0200] Flow cytometer

[0201] In some embodiments, the methods comprise isolating, obtaining, and / or enriching cells of interest. Isolating, obtaining, and / or enriching cells of interest have been described in U.S. Patent Application No. 2016 / 0244828, the entire contents of which are incorporated by reference herein. For example, the methods comprising isolating, obtaining, and / or enriching cells of interest can be performed using a flow cytometer. In some embodiments, the flow cytometer utilizes fluorescence-activated cell sorting.

[0202] Flow cytometry is a valuable method of cell analysis and isolation. As such, it has a wide range of diagnostic and therapeutic applications. Flow cytometry utilizes a fluid stream straight line of isolated cells so that they can pass through a detection device in a single file. Individual cells can be distinguished according to their position in the fluid stream and the presence of a detectable marker or moiety (e.g., a fluorophore on an oligonucleotide bound by a cellular component binding reagent). The cells flow through a focused interrogation point, where at least one laser directs a laser beam to a focal point within the channel. The sample fluid stream containing the cells is hydrodynamically focused to a very small core diameter by flowing a sheath stream around the sample stream at a very high volumetric rate. The small core diameter can be less than 10 microns, 20 microns, 30 microns, 40 microns, 50 microns, 60 microns, 70 microns, 80 microns, 90 microns, 100 microns, 110 microns, 120 microns, 130 microns, 140 microns, 150 microns, 160 microns, 170 microns, 180 microns, 190 microns, 200 microns, or a value or a range between any two of these values. The volumetric rate of the sheath stream is at least 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, or 1000 times the volumetric rate of the sample, or a value or a range between any two of these values. This results in a very fast velocity of the focused cell straight line, on the order of meters per second. So the residence time of each cell at the excitation point is very limited, e.g., less than 1 microsecond, 2 microseconds, 3 microseconds, 4 microseconds, 5 microseconds, 6 microseconds, 7 microseconds, 8 microseconds, 9 microseconds, 10 microseconds, 20 microseconds, 30 microseconds, 40 microseconds, 50 microseconds, 60 microseconds, 70 microseconds, 80 microseconds, 90 microseconds, 100 microseconds, or a value or a range between any two of these values. After the cell passes through the interrogation point, the cell cannot be redirected back to the interrogation point because the flow velocity of the straight line cannot be reversed.

[0203] Flow cytometers are tools that can characterize cells based on optical parameters such as light scatter and fluorescence. In a flow cytometer, cells in a fluid suspension pass through a detection zone where they are exposed to excitation light, typically from one or more lasers, and the light scatter and fluorescence properties of the cells are measured. The cells or components thereof are often labeled with fluorescent dyes to facilitate detection. Multiple different cells or components can be detected simultaneously by labeling them with spectrally distinct fluorescent dyes. In some implementations, the analyzer contains multiple photodetectors, one for each scatter parameter to be measured and one for each different dye to be detected. The data obtained includes a signal measured for each light scatter parameter and fluorescence emission.

[0204] Separation of biological cells is achieved by adding a sorting or collection capability to the flow cytometer. Cells in the separation stream that are detected to have one or more desired characteristics are removed by mechanical or electrical means to be individually separated from the sample stream. This flow sorting method has been used to sort different types of cells, separate sperm carrying X and Y chromosomes for breeding, sort chromosomes for genetic analysis, and isolate a particular organism from a complex biological population.

[0205] A common flow sorting technique utilizes droplet sorting, where the fluid stream containing the linearly separated cells is broken into droplets, and the droplets containing the cells of interest are electrically charged and deflected by an electric field into a collection tube. Droplet sorting systems are capable of forming droplets at a rate of about 100 droplets / second, 500 droplets / second, 1000 droplets / second, 2000 droplets / second, 3000 droplets / second, 4000 droplets / second, 5000 droplets / second, 6000 droplets / second, 7500 droplets / second, 10000 droplets / second, 20000 droplets / second, 30000 droplets / second, 40000 droplets / second, 50000 droplets / second, 60000 droplets / second, 75000 droplets / second, 100000 droplets / second, 200000 droplets / second, 300000 droplets / second, 400000 droplets / second, 500000 droplets / second, 600000 droplets / second, 750000 droplets / second, 1000000 droplets / second, or a value or range between any two of these values, through a nozzle having a diameter of less than 1000 microns, 750 microns, 600 microns, 500 microns, 400 microns, 300 microns, 200 microns, 100 microns, 75 microns, 60 microns, 50 microns, 40 microns, 30 microns, 20 microns, 10 microns, 5 microns, 2 microns, 1 micron, or a value or range between any two of these values. Droplet sorting requires that the droplets break from the stream at a fixed distance from the tip of the nozzle. This distance is typically on the order of a few millimeters from the tip of the nozzle, and can be maintained by vibrating the tip of the nozzle at a predefined frequency to maintain an undisturbed flow of fluid.

[0206] Linearly separated cells in the stream can be characterized as they pass through a viewing point located below the tip of the nozzle. Cells are identified as meeting about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 75, about 100, about 200, about 300, about 400, about 500, about 600, about 750 criteria, or a value or range between any two of these values, or greater than 1000 criteria, and the time it takes for the cell to reach the point of impact and the time for the point of impact to break from the stream is predictable. A brief electrical charge can be applied to the fluid stream before the droplet containing the selected cell breaks from the stream, and then grounded immediately after the droplet breaks. The droplet that is sorted remains charged when it breaks from the fluid stream, while all other droplets are uncharged. The charged droplet deviates from the downward trajectory of the other droplets under the influence of the electric field and collects in the sample tube. The uncharged droplets fall directly into the drain.

[0207] Flow cytometers can also include means for recording the measured data and analyzing the data. For example, a computer connected to the detection electronics can be used for data storage and analysis. For example, the data can be stored in tabular form, with each row corresponding to data for one cell and columns corresponding to each measured parameter. Data from a flow cytometer is stored using a standard file format, such as the "FCS" file format, to facilitate analysis of the data using separate programs and / or machines. Using current methods of analysis, data is often displayed in two dimensions ("2D") for ease of visualization, but other methods of visualizing multi-dimensional data can also be used.

[0208] Parameters measured using a flow cytometer typically include the excitation light scattered by the cell in a primarily forward direction, referred to as forward scatter ("FSC"), the excitation light scattered by the cell in a primarily sideways direction, referred to as side scatter ("SSC"), the light emitted by fluorescent molecules in one or more channels (ranges of frequencies) of the spectrum, referred to as FL1, FL2, etc., or the fluorescent dyes primarily measured in that channel. Different cell types can be identified by the scatter parameters and the fluorescent emissions produced by various cellular proteins labeled with dyes.

[0209] Fluorescence-activated cell sorting is a specialized type of flow cytometer. It provides a method for sorting a heterogeneous mixture of cells into two or more vessels or wells of a microtiter plate, one cell at a time, based on specific light scattering and fluorescent characteristics of each cell. It records the fluorescent signal from individual cells and physically separates cells of particular interest. The acronym FACS is a trademark owned by Becton Dickinson.

[0210] The cell suspension is placed near the center of a narrow, fast flowing liquid stream. The stream is configured so that there is a large separation (Poisson distribution) between the average of the cells relative to their diameters. A vibration mechanism causes the stream to break up into single liquid droplets. The system is adjusted so that the likelihood of more than one cell being present in a droplet is very low. Before the stream breaks up into droplets, it passes through one or more laser cross-sections where the fluorescent characteristics of each cell of interest are measured. If the cells are to be collected, an electrical charge is applied to the flow cell for a period of time during which one or more droplets are formed and broken off from the stream. These charged droplets then fall into a target container according to the charge on the droplet via an electrostatic deflection system.

[0211] Non-limiting examples of suitable cell sorting sorting devices include BD FACSJazz TM Cell Sorter, BD FACSseq TM Cell Sorter, or any other cell sorter, including Bio-Rad Laboratories, Inc. (Hercules, CA) S3e TM Cell Sorter, Sony Biotechnology Inc. (San Jose, CA) SH800 Cell Sorter, Beckman Coulter Inc. (Brea, CA) MoFlo TM XDP Cell Sorter.

[0212] In some embodiments, the method utilizes tagging and staining of cell surface markers for cell sorting. In some embodiments, the method utilizes tagging of cell surface markers for magnetic depletion of cells of interest, interfering cells, and debris. In some embodiments, the method comprises determining one or more of a patient's genotype based on the sequence information obtained; determining a patient's phenotype based on the sequence information obtained; determining one or more genetic mutations of a patient based on the sequence information; predicting a patient's susceptibility to one or more diseases. At least one of the one or more diseases is a cancer or a genetic disease.

[0213] FACS can use antibodies to targets to detectable cell markers to label and stain cell surface markers, thereby sorting cells. Antibodies include monoclonal and polyclonal antibodies coupled to fluorophores. Detectable cell markers include cell surface markers of interest. In some embodiments, magnetic depletion can be based on labeling cell surface markers with magnetic beads with antibodies including monoclonal and polyclonal antibodies, conjugated to their non-target cell surface of interest, interfering with cells and / or debris. Non-limiting examples of cell surface markers include CD surface markers, growth factor / cytokine, chemokine receptors, nuclear receptors, and other receptors. Examples of cell surface markers include, but are not limited to, ALCAM, CD166, ASGR1, BCAM, BSG, CD147, CD14, CD19, CD2, CD200, CD127 BV421, CD25 BB515, CD161 PE, CD45RA PerCP-Cy TM 5.5, CD15S AF647, CD4 APC-H, CD4, CD25, CD127, CD45RA, CD15S, CD161, CD3, EpCAM, CD44, and Her2 / Neu. Examples of growth factor / cytokine receptors, chemokine receptors include ACVR1B, ALK4, ACVR2A, ACVR2B, BMPR1A, BMPR2, CSF1R, MCSFR, CSF2RB, EGFR, EPHA2, EPHA4, EPHB2, EPHB4, and ERBB2. Examples of nuclear receptors include androgen receptor, CAR, ERa, ERp, ESRRA, ESRRB, ESRRG, FXR, glucocorticoid receptor, LXR-a, LXR-b, PPARA, PPARD, PPARG, PXR, SXR, estrogen receptor beta, progesterone receptor, RARA, RARB, RARG, RORA, RXRA, RXRB, THRA, THRB, and vitamin D3 receptor. Examples of other receptors include AGER, APP, CLEC12A, MICL, CTLA4, FOLR1, FZD1, FRIZZLED-1, KLRB1A, LRPAP1, NCR3, NKP30, OLR1, PROCR, PTPN1, SOX9, SCARB2, TACSTD2, TREM1, TREM2, TREML1, and VDR.

[0214] Compositions and kits

[0215] Aspects of the application also include kits and compositions useful in practicing the various methods of the application. Compositions include, for example, capture magnetic beads as described above. In some cases, capture magnetic beads can include, for example, barcoded nucleic acids as described above.

[0216] A kit of the invention can include, for example, capture magnetic beads as described above. Where the capture magnetic beads do not include barcoded nucleic acids, the kit can also include, for example, barcoded beads as described above. The kit can also include one or more additional components for practicing the method embodiments. For example, the kit can include one or more of primers, polymerase (e.g., a thermostable polymerase with hot start properties or similar properties, reverse transcriptase, etc.), dsDNAase, exonuclease, dNTPs, metal cofactor, one or more nuclease inhibitors (e.g., RNAse inhibitors and / or DNAse inhibitors), one or more molecular crowding reagents (e.g., polyethylene glycol, etc.), one or more enzyme stability components (e.g., DTT), stimulus responsive polymers, or any other desired kit components, such as devices, e.g., solid supports, containers, cartridges, such as tubes, beads, plates, microfluidic chips, etc., as described above. The components of the kit can be present in separate containers, or multiple components can be placed in one container.

[0217] In addition to the components described above, the subject kits can also include (in certain embodiments) instructions for practicing the subject methods. These instructions can be present in the subject kits in a variety of forms, one or more of which can be present in the kit. One form in which these instructions can be present is as printed information on a suitable medium or substrate, e.g., as printed information on one or more papers, envelopes, boxes, containers, etc. on which is printed the information, in the packaging of the kit, in a package insert, in a label attached to the device, etc. Another form in which these instructions can be present is as electronic

[0218] While the appended claims set forth the dimensions and features required for practicing the disclosed subject matter, they are not meant to limit but rather to elucidate the disclosure. The skilled artisan will readily recognize numerous modifications and substitutions obvious in view of the above disclosure without undue experimentation. Accordingly, the appended claims are intended to cover all such modifications and substitutions as fall within the true spirit and scope of the disclosed subject matter.

[0219] 1. A method of barcoding nucleic acids of particles, the method comprising:

[0220] a) combining a sample with capture magnetic beads comprising a capture moiety for particles of the sample to produce a captured sample;

[0221] b) partitioning the captured particles of the captured sample using a magnetic field application mediated partitioning scheme to produce partitioned captured particles, wherein the partitioned captured particles are in spatial proximity to bead-bound barcoded nucleic acids comprising a target binding region; and

[0222] c) lysing the partitioned captured particles such that nucleic acids released therefrom bind to the target binding region of the bead-bound barcoded nucleic acids to produce captured nucleic acids.

[0223] 2. The method of clause 1, wherein the bead-bound barcode nucleic acids are tethered to the capture magnetic beads.

[0224] 3. The method of clause 1, wherein the bead-bound barcode nucleic acids are tethered to barcoded beads that are different from the capture magnetic beads.

[0225] 4. The method of any of the preceding clauses, wherein the partitioned particles are partitioned into microwells.

[0226] 5. The method of any of the preceding clauses, wherein the capture moiety comprises a specific binding member.

[0227] 6. The method of clause 5, wherein the specific binding member comprises an antibody or binding fragment thereof.

[0228] 7. The method of any of the preceding clauses, wherein the bead-bound barcode nucleic acids further comprise a cell label domain.

[0229] 8. The method of any of the preceding clauses, wherein the bead-bound barcode nucleic acids further comprise a unique molecular index domain.

[0230] 9. The method of any of the preceding clauses, wherein the bead-bound barcode nucleic acids further comprise a universal primer binding domain.

[0231] 10. The method of any of clauses 1-6, wherein the bead-bound barcode nucleic acids comprise the structure: bead-5’-universal primer binding domain-cell label domain-unique molecular index domain-target binding region-3’.

[0232] 11. The method of any of the preceding clauses, wherein the target binding region comprises an oligo-dT domain, a gene-specific domain, or a random sequence domain.

[0233] 12. The method of any of the preceding clauses, wherein the particles comprise subcellular-sized particles.

[0234] 13. The method of clause 12, wherein the subcellular-sized particles comprise vesicles.

[0235] 14. The method of any of the preceding clauses, wherein the particles comprise cells.

[0236] 15. The method of any of the preceding clauses, wherein the method further comprises separating the captured nucleic acids from other components of the partitioned captured particles, e.g., separating the oligonucleotide-labeled cellular component binding reagent from the nucleic acids.

[0237] 16. The method of clause 15, wherein the separating comprises use of an applied magnetic field.

[0238] 17. The method of any of the preceding clauses, wherein the method further comprises pooling the captured nucleic acids.

[0239] 18. The method of any of the preceding clauses, wherein the method further comprises subjecting the captured nucleic acids to cDNA synthesis reaction conditions to produce first strand cDNA domains comprising the captured nucleic acids.

[0240] 19. The method of clause 18, wherein the method further comprises producing an amplicon composition from the first strand cDNA domains comprising the captured nucleic acids.

[0241] 20. The method of clause 19, wherein the amplicon composition is produced from the first strand cDNA domains comprising the captured nucleic acids using one or more rounds of amplification.

[0242] 21. The method of clause 19 or clause 20, wherein the amplicon composition comprises a next generation sequencing (NGS) library.

[0243] 22. The method of clause 21, wherein the amplicon composition comprises a NGS adaptor comprising a nucleic acid.

[0244] 23. The method of clause 21 or clause 22, wherein the method further comprises next generation sequencing NGS library.

[0245] 24. A method of sequencing nucleic acids of a particle, the method comprising:

[0246] a) combining a sample comprising a particle with a capture magnetic bead, the capture magnetic bead comprising:

[0247] i) a barcode nucleic acid comprising a target binding region, and

[0248] ii) a capture moiety that specifically binds to the particle;

[0249] to produce a captured sample;

[0250] b) partitioning the captured particles of the captured sample into microwells using a magnetic field application mediated partitioning scheme to produce partitioned captured particles;

[0251] c) lysing the partitioned captured particles such that nucleic acids released therefrom bind to the target binding domain of the barcode nucleic acid to produce captured nucleic acids;

[0252] d) subjecting the captured nucleic acids to cDNA synthesis reaction conditions to produce first strand cDNA domains comprising the captured nucleic acids;

[0253] e) constructing a NGS library from the first strand cDNA domains comprising the captured nucleic acids; and

[0254] f) sequencing the NGS library, thereby sequencing the nucleic acids of the target particles.

[0255] 25. The method of clause 24, wherein the particles comprise subcellular-sized particles.

[0256] 26. The method of clause 25, wherein the subcellular-sized particles comprise vesicles.

[0257] 27. The method of any of the preceding clauses, wherein the particles comprise cells.

[0258] 28. The method of clause 24, wherein the method further comprises separating the captured nucleic acids from other components of the partitioned captured particles.

[0259] 29. The method of clause 28, wherein the separating comprises using an applied magnetic field.

[0260] 30. The method of any of clauses 24-29, wherein the method further comprises mixing the captured nucleic acids prior to subjecting the captured nucleic acids to conditions of a cDNA synthesis reaction.

[0261] 31. The method of any of clauses 24-30, wherein the NGS library is produced from the first strand cDNA domains comprising the captured nucleic acids using one or more than one round of amplification.

[0262] 32. The method of clause 31, wherein the amplicon composition comprises NGS adapters comprising the nucleic acids.

[0263] 33. The method of any of clauses 24-32, wherein the capture moiety comprises an antibody or binding fragment thereof.

[0264] 34. The method of any of clauses 24-33, wherein the barcode nucleic acid further comprises a cell label domain.

[0265] 35. The method of any of clauses 24-34, wherein the barcode nucleic acid further comprises a unique molecular index domain.

[0266] 36. The method of any of clauses 24-35, wherein the barcode nucleic acid further comprises a universal primer binding domain.

[0267] 37. The method of any of clauses 24-33, wherein the barcode nucleic acid comprises the structure: bead-5’-universal primer binding domain-cell label domain-unique molecular index domain-target binding region-3’.

[0268] 38. The method of any one of Clauses 24-37, wherein the target-binding region comprises an oligo-dT domain, a gene-specific domain, or a random sequence domain.

[0269] 39. The method of any one of Clauses 24-38, wherein partitioning comprises using a flow cell.

[0270] 40. The method of Clause 39, wherein the flow cell comprises a substrate.

[0271] 41. A capture magnetic bead comprising a barcode nucleic acid and a capture moiety that specifically binds to a particle.

[0272] 42. The capture magnetic bead of any one of Clauses 41, wherein the capture moiety comprises an antibody or binding fragment thereof.

[0273] 43. The method of any one of Clauses 41-42, wherein the barcode nucleic acid further comprises a cell label domain.

[0274] 44. The capture magnetic bead of any one of Clauses 41-43, wherein the barcode nucleic acid further comprises a unique molecular index domain.

[0275] 45. The method of any one of Clauses 41-44, wherein the barcode nucleic acid further comprises a universal primer binding domain.

[0276] 46. The capture magnetic bead of any one of Clauses 41-42, wherein the barcode nucleic acid comprises the structure: bead-5’-universal primer binding domain-cell label domain-unique molecular index domain-target-binding region-3’.

[0277] 47. The capture magnetic bead of any one of Clauses 41-46, wherein the target-binding region comprises an oligo-dT domain, a gene-specific domain, or a random sequence domain.

[0278] 48. An apparatus comprising:

[0279] a) a substrate comprising 100 or more microwells, the microwells comprising a capture magnetic bead, the capture magnetic bead comprising:

[0280] i) a barcode nucleic acid comprising a target-binding region, and

[0281] ii) a capture moiety that specifically binds to a particle; and

[0282] b) a flow cell in fluid communication with the substrate.

[0283] 49. The apparatus of Clause 48, wherein the capture moiety comprises an antibody or binding fragment thereof.

[0284] 50. The device of any one of Clauses 48-49, wherein the barcode nucleic acid further comprises a cell label domain.

[0285] 51. The device of any one of Clauses 48-50, wherein the barcode nucleic acid further comprises a unique molecular index domain.

[0286] 52. The method of any one of Clauses 48-51, wherein the barcode nucleic acid further comprises a universal primer binding domain.

[0287] 53. The device of any one of Clauses 48-49, wherein the barcode nucleic acid comprises the structure: bead-5’-universal primer binding domain-cell label domain-unique molecular index domain-target binding region-3’.

[0288] 54. The device of any one of Clauses 48-53, wherein the target binding region comprises an oligo-dT domain, a gene-specific domain, or a random sequence domain.

[0289] 55. A system comprising:

[0290] a) a substrate comprising 100 or more microwells, the microwells comprising a capture magnetic bead, the capture magnetic bead comprising:

[0291] i) a barcode nucleic acid comprising a target binding region, and

[0292] ii) a capture moiety that specifically binds to a particle;

[0293] b) a flow cell in fluid communication with the substrate; and

[0294] c) a flow controller; wherein the flow controller is configured to control delivery of fluid to the flow cell.

[0295] 56. The system of Clause 55, wherein the capture moiety comprises an antibody or binding fragment thereof.

[0296] 57. The system of any one of Clauses 55-56, wherein the barcode nucleic acid further comprises a cell label domain.

[0297] 58. The system of any one of Clauses 55-57, wherein the barcode nucleic acid further comprises a unique molecular index domain.

[0298] 59. The method of any one of Clauses 55-58, wherein the barcode nucleic acid further comprises a universal primer binding domain.

[0299] 60. The system of any one of Clauses 55-56, wherein the barcode nucleic acid comprises the structure: bead-5'-universal primer binding domain-cell label domain-unique molecular index domain-target binding region-3'.

[0300] 61. The system of any one of Clauses 55-60, wherein the target binding region comprises an oligo-dT domain, a gene-specific domain, or a random sequence domain.

[0301] 62. A kit comprising

[0302] (a) a capture magnetic bead comprising a capture moiety that specifically binds to a target particle; and

[0303] (b) a barcode nucleic acid.

[0304] 63. The kit of Clause 62, wherein the capture magnetic bead comprises the barcode nucleic acid.

[0305] 64. The kit of Clause 62, wherein the barcode nucleic acid is tethered to a barcoded bead that is separate from the capture magnetic bead.

[0306] 65. The kit of any one of Clauses 62-64, wherein the kit further comprises:

[0307] a device comprising:

[0308] (i) a substrate comprising 100 or more microwells; and

[0309] (ii) a flow cell in fluid communication with the substrate.

[0310] 66. The kit of any one of Clauses 62-65, wherein the capture moiety comprises an antibody or binding fragment thereof.

[0311] 67. The kit of any one of Clauses 62-66, wherein the barcode nucleic acid further comprises a cell label domain.

[0312] 68. The kit of any one of Clauses 62-67, wherein the barcode nucleic acid further comprises a unique molecular index domain.

[0313] 69. The kit of any one of Clauses 62-68, wherein the barcode nucleic acid further comprises a universal primer binding domain.

[0314] 70. The kit of any one of Clauses 62-63, wherein the barcode nucleic acid comprises the structure: bead-5'-universal primer binding domain-cell label domain-unique molecular index domain-target binding region-3'.

[0315] 71. The kit of any one of Clauses 62 to 70, wherein the target-binding region comprises an oligo-dT domain, a gene-specific domain, or a random sequence domain.

[0316] In at least some of the previously described embodiments, one or more elements used in one embodiment can be used interchangeably in another embodiment, unless such substitution is not technically feasible. Those skilled in the art will appreciate that various other omissions, additions and modifications can be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and alterations are intended to be within the scope of the subject matter as defined by the appended claims.

[0317] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations can be explicitly set forth herein for sake of clarity. It is noted that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Any inclusion of "or" in this document is meant to encompass both "and / or" as well as "and / or" unless the context clearly dictates otherwise.

[0318] Those skilled in the art will appreciate that, in general, the terms used herein, and especially in the appended claims (e.g., the body of the appended claims), are intended to be interpreted broadly such that the terms "comprises", "comprising", "includes", "including" and the like are not intended to be limiting (e.g., when using such terms, the compositions or methods that include the recited elements, integers, steps, etc. are not meant to be more limited than those elements, integers, steps, etc. listed). Those skilled in the art will further appreciate that the terms "consisting essentially of" and "consisting of" are intended to be interpreted in accordance with the broadest possible interpretation of the appended claims in light of the specification as a whole and in accordance with the doctrine of equivalents. For example, to help understand, the following appended claims can contain introductory phrases "at least one" and "one or more" to introduce claim statements. However, the use of such phrases should not be interpreted as implying that the claim statements introduced by "one" are limited to any particular claim that limits to an embodiment containing only one such statement, even if the same claim includes the introductory phrases "one or more" or "at least one" (e.g., an unqualified element is to be interpreted as "at least one" or "one or more"). Moreover, even if a specific number of introduced claim statements is explicitly recited, those skilled in the art will recognize that such recitations are to be interpreted to mean at least the recited number (e.g., a simple recitation of "two statements" without further modifiers means at least two statements, or two or more statements). Furthermore, where those like the convention "at least one of A, B, and C, etc." are used, generally such structures are intended to be understood in the sense that they are intended to be understood by those of skill in the art (e.g., "a system having at least one of A, B, or C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Where those like the convention "at least one of A, B, or C, etc." are used, generally such structures are intended to be understood in the sense that they are intended to be understood by those of skill in the art (e.g., "a system having at least one of A, B, or C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art will further appreciate that almost any disjunctive word or phrase presenting two or more alternative terms, whether in descriptions, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, one of the terms, or both terms. For example, the phrase "A or B" would be understood to include the possibilities of "A" or "B" or "A and B."

[0319] Furthermore, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0320] Those skilled in the art will understand that, in any event, such as in providing a written description, all scopes disclosed herein include any and all possible subscopes and combinations thereof. Any listed scope can be readily identified as sufficient to describe and capable of breaking down the same scope into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope discussed herein can be readily decomposed into a lower third, a middle third, and an upper third, etc. Those skilled in the art will also understand that all language, such as “at most,” “at least,” “greater than,” “less than,” etc., includes the listed numbers and refers to a scope that can subsequently be decomposed into subscopes as described above. Finally, as those skilled in the art will understand, a scope includes each individual member. Thus, for example, a group having 1 to 3 clauses refers to a group having 1, 2, or 3 clauses. Similarly, a group having 1 to 5 clauses refers to a group having 1, 2, 3, 4, or 5 clauses, and so on.

[0321] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The aspects and embodiments disclosed herein are for illustrative purposes and not intended to be limiting; their true scope and spirit are indicated by the appended claims.

[0322] Therefore, the foregoing merely illustrates the principles of the invention. It should be understood that those skilled in the art will be able to design various arrangements, although not explicitly described or shown herein, which embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language listed herein are primarily intended to aid the reader in understanding the principles of the invention and the inventors' contributions to the field, and should be interpreted as not being limited to the specific examples and conditions described above. Moreover, all statements herein that enumerate the principles, aspects, and embodiments of the invention and their specific examples are intended to include their structural and functional equivalents. Furthermore, these equivalents are intended to include both currently known equivalents and future-developed equivalents, i.e., any element developed to perform the same function, regardless of its structure. Furthermore, nothing disclosed herein is intended for public viewing, whether or not such disclosures are explicitly listed in the claims.

[0323] Accordingly, the scope of the present application is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope of the present application is intended to be embodied by the appended claims. In the claims, the term "comprises" or "comprising," or the like, are used in the inclusive and open ended sense of "has, or having, ingredients, steps, or components" and do not exclude the presence of unrelated items. Further, the terms "a" or "an" shall not be construed as limiting when they are used in a claim section. The terms "first," "second," "third," "fourth," and the like in the description and in the claims, if any, are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is intended that the logie of the steps recited in the claims is not limited to the specific order in which the steps are recited. Any process or method steps described in the specification can be performed in any order that is practicable unless otherwise stated.

Claims

1. A method for barcoding nucleic acids in particles, the method comprising: a) Combining a sample with capture magnetic beads containing a capture portion for sample particles to produce a capture sample, wherein a barcode nucleic acid containing a target binding region is tethered to the capture magnetic beads; b) Use a magnetic field-mediated partitioning scheme to partition the captured particles in the captured sample to produce partitioned captured particles; as well as c) The fragmented capture particles are cleaved so that the released nucleic acids bind to the target binding region of the bead-bound barcode nucleic acid to produce captured nucleic acids.

2. The method of claim 1, wherein the segmented capture particles are segmented into micropores.

3. The method according to any one of the preceding claims, wherein the capturing portion comprises a specific binding member.

4. The method according to claim 1, wherein the bead-bound barcode nucleic acid further comprises a cell marker domain, a unique molecular index domain, and a universal primer binding domain.

5. The method according to claim 1, wherein the target binding region comprises an oligodT domain, a gene-specific domain, or a random sequence domain.

6. The method of claim 1, wherein the particles comprise subcellular particles.

7. The method of claim 6, wherein the subcellular particles comprise vesicles.

8. The method of claim 1, wherein the method further comprises separating the captured nucleic acid from other components of the divided captured particles.

9. The method of claim 8, wherein separation comprises using an applied magnetic field.

10. The method of claim 1, wherein the capturing magnetic bead comprises a barcoded nucleic acid and a capturing portion that specifically binds to the particle.

11. The method of claim 1, wherein the method is implemented by an apparatus comprising: a) A substrate comprising 100 or more micropores, wherein the micropores contain trapping magnetic beads, the trapping magnetic beads comprising: i) Barcode nucleic acids containing target-binding regions, and ii) the capture portion that specifically binds to particles; and b) A flow cell in fluid communication with the substrate.

12. The method of claim 1, wherein the method is implemented by an apparatus comprising: a) A substrate comprising 100 or more micropores, wherein the micropores contain trapping magnetic beads, the trapping magnetic beads comprising: i) Barcode nucleic acids containing target-binding regions, and ii) The capture portion that specifically binds to particles; b) A flow cell in fluid communication with the substrate; and c) Flow controller; where... The flow controller is configured to control the delivery of fluid to the flow tank.

13. The method of claim 1, wherein the method is implemented by a kit comprising: The capturing magnetic beads contain a capturing portion that specifically binds to the target particle and barcode nucleic acid.

Images