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] This application is a divisional application of Chinese Patent Application No. 202080095421.0, filed on November 16, 2020, entitled "Molecular barcode encoding of nucleic acid targets in single particles mediated by magnetic beads and a composition therefor".

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 943647, filed on December 4, 2019. The disclosure of that application is incorporated herein by reference. Technical Field

[0003] This application relates to the field of high-throughput detection technology, and more specifically to molecular barcode encoding of nucleic acid targets in single particles mediated by magnetic beads and compositions thereof. Background Technology

[0004] The ability to detect and quantify specific nucleic acid and protein molecules in single cells is crucial for 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 biomarkers and is widely used in basic research and clinical diagnostics. In contrast, nucleic acid measurements, such as mRNA expression, are typically performed on large numbers of samples, thus masking the contribution of individual cells.

[0005] To characterize the complexity of cellular systems, there is a great need to develop methods, devices, and systems for monitoring the expression of large numbers of genes in thousands of cells. Current technologies allow for the measurement of gene expression in single cells in a large-scale 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 colocalized with barcode reagent beads between septa.

[0006] Beyond cells, there is growing interest in detecting nucleic acids within extracellular vesicles, such as exosomes and microvesicles. Extracellular vesicles, including exosomes and microvesicles, detach from almost all cell types and are widely distributed in blood and other bodily fluids. Several studies have demonstrated that various RNA types (including mRNA, miRNA, and lncRNA) embedded within EVs can transfer from donor cells to recipient cells and interfere with gene expression in the latter. (Turchinovich et al., “Trascriptome of Extracellular Vesicles: Sate-of-the-Art,” Front Immunol. (2019) 10: 202). Therefore, characterizing nucleic acids within extracellular vesicles is of great interest. Summary of the Invention

[0007] While current technology allows for the measurement of gene expression in single cells 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 may struggle to segment small cells and vesicles with low sedimentation rates. Furthermore, current platforms may barcode and sequence many cells of little interest, leading to inefficient resource utilization. Embodiments of the present invention address these and other needs.

[0008] Embodiments of the present invention provide a method for molecular barcoding of nucleic acid targets of particles, such as cells or extracellular vesicles, mediated by capture magnetic beads. The method includes: a) combining a sample containing particles with capture magnetic beads containing a capture portion for the particles to produce a capture sample; b) dividing the capture particles of the capture sample using a magnetic field-mediated partitioning scheme to produce partitioned capture particles, wherein the partitioned capture particles are spatially adjacent to bead-bound barcoded nucleic acids containing target-binding regions; and c) cleaving the partitioned capture particles such that the nucleic acids released therefrom bind to the target-binding regions to produce captured nucleic acids. Compositions, such as capture magnetic beads, comprising barcoded magnetic beads, and apparatus / systems and kits for carrying out embodiments of the method are also provided.

[0009] This article provides a method for barcoding nucleic acids in particles. The method includes: a) combining a sample with capture magnetic beads containing a capture portion for sample particles to generate a captured sample; b) dividing the capture particles of the captured sample using a magnetic field-mediated partitioning scheme to generate partitioned capture particles, wherein the partitioned capture particles are spatially adjacent to bead-bound barcoded nucleic acids containing a target-binding region, such as an oligo-dT domain, a gene-specific domain, or a random sequence domain; and c) cleaving the partitioned capture particles such that the released nucleic acids bind to the target-binding region of the beads bound to the barcoded nucleic acids to generate captured nucleic acids. In some cases, the bead-bound barcoded nucleic acids are tethered to the capture magnetic beads, while in others, the beads connecting the barcoded nucleic acids are tethered to barcoded beads different from the capture magnetic beads. In some cases, the partitioned particles are partitioned into microwells. In some cases, the capture portion contains a specific binding member, such as an antibody or a binding fragment thereof. In some cases, in addition to the target-binding region, the bead-bound barcoded nucleic acid also includes one or more cell marker domains, unique molecular index domains, and universal primer-binding domains. For example, the bead binding the barcoded nucleic acid has the following structure: bead-5'-universal primer-binding domain-cell marker domain-unique molecular index domain-target-binding region-3'. The method can be used to barcode nucleic acids from a variety of particles, such as biological particles, for example, cells, and subcellular particles, such as extracellular vesicles, platelets, vesicles, such as exosomes or microvesicles. In some cases, the method further includes, for example, separating the captured nucleic acid from other components of the segmented captured particle by applying a magnetic field. In some cases, the method also includes mixing the captured nucleic acid. In some cases, the method further includes placing the captured nucleic acid under cDNA synthesis reaction conditions to produce a first-strand cDNA domain containing the captured nucleic acid. In some cases, the method further includes generating an amplicon composition from the first-strand cDNA domain containing the captured nucleic acid. In some cases, such as embodiments where the amplicon composition comprises a next-generation sequencing (NGS) library, the amplicon composition is generated from a first-strand cDNA domain containing captured nucleic acids using one or more rounds of amplification. In some cases, the amplicon composition comprises an NGS adaptor containing nucleic acids. In some cases, the method also includes sequencing the NGS library.

[0010] In some cases, the method includes: a) combining a sample containing particles with capture magnetic beads, the capture magnetic beads comprising: i) a barcoded nucleic acid containing a target binding region, and ii) a capture portion specifically binding to the particles, to generate a capture sample; b) partitioning the capture particles of the capture sample into microwells using a magnetic field-mediated partitioning scheme to generate partitioned capture particles; cleaving the partitioned capture particles such that the nucleic acid released therefrom binds to the target binding region of the barcoded nucleic acid to generate captured nucleic acid; placing the captured nucleic acid under conditions of a cDNA synthesis reaction to generate a first-strand cDNA domain containing the captured nucleic acid; constructing an NGS library from the first-strand cDNA domain containing the captured nucleic acid; and sequencing the NGS library to sequence the nucleic acid of the target particles.

[0011] In some cases, the method further includes the use of oligonucleotide-labeled cell component binding reagents, for example, in applications where it is necessary to detect, for example, quantify, one or more cell components, such as surface proteins. The oligonucleotide-labeled cell component binding reagents used in this embodiment include cell component binding reagents, such as antibodies or binding fragments thereof, conjugated to a cell component binding reagent-specific oligonucleotide, said cell component binding reagent-specific oligonucleotide containing an identifier sequence associated with the cell component binding reagent-specific oligonucleotide. In this case, the capturing magnetic beads may include those configured to capture, for example, specifically bind to the cell component binding reagent-specific oligonucleotide domain. In this way, protein expression can be analyzed in conjunction with gene expression analysis, for example, when multi-omics analysis is required, such as a combined transcriptome and proteome analysis.

[0012] Capture magnetic beads are also provided for implementations of this method. Implementations of capture magnetic beads include magnetic beads having a barcoded nucleic acid stably bound to the magnetic bead and a capture portion that specifically binds to the particle. In some cases, the capture portion contains an antibody or a binding fragment thereof. In some cases, the barcoded nucleic acid, in addition to a target-binding region (e.g., an oligo-dT domain, a gene-specific domain, or a random sequence domain), includes one or more cell marker domains, unique molecular index domains, and universal primer-binding domains; for example, the bead-bound barcoded nucleic acid has the following structure: bead-5'-universal primer-binding domain-cell marker domain-unique molecular index domain-target-binding region-3'.

[0013] An apparatus for use in embodiments of the method is also provided. Embodiments of the apparatus include: a) a substrate comprising 100 or more microwells, the microwells comprising capture magnetic beads, the capture magnetic beads comprising: i) a barcoded nucleic acid containing a target-binding region; ii) a capture portion specifically binding to the beads; and b) a flow cell in fluid communication with the substrate. In some cases, the capture portion comprises an antibody or a binding fragment thereof. In some cases, in addition to the target-binding region (e.g., an oligo-dT domain, a gene-specific domain, or a random sequence domain), the barcoded nucleic acid also includes one or more cell marker domains, unique molecular index domains, and universal primer-binding domains, for example, wherein the bead-binding barcoded nucleic acid has the following structure: bead-5'-universal primer-binding domain-cell marker domain-unique molecular index domain-target-binding region-3'.

[0014] A system for implementation of the method is also provided. Implementations of the system include: a) a substrate comprising 100 or more microwells, the microwells comprising capture magnetic beads, the capture magnetic beads comprising: i) a barcode nucleic acid containing a target binding region; ii) a capture portion specifically binding to the particle; b) a flow cell in fluid communication with the substrate; and c) a flow controller configured to control fluid delivery to the flow cell; and in some cases, a magnetic field applicator, for example, as detailed below. In some cases, the capture portion comprises an antibody or a binding fragment thereof. In some cases, in addition to the target binding region (e.g., an oligo-dT domain, a gene-specific domain, or a random sequence domain), the barcode nucleic acid also includes one or more cell marker domains, unique molecular index domains, and universal primer binding domains, for example, wherein the bead-bound barcode nucleic acid has the following structure: bead-5'-universal primer binding domain-cell marker domain-unique molecular index domain-target binding region-3'.

[0015] A kit for implementation of this method is also provided. Implementation of the kit includes: (a) a capture magnetic bead comprising a capture portion that specifically binds to a target particle, and a barcoded nucleic acid. In some cases, the capture magnetic bead comprises a barcoded nucleic acid, while in others, the barcoded nucleic acid is tethered to a barcoded bead separate from the capture magnetic bead. In some cases, the capture portion comprises an antibody or a binding fragment thereof. In some cases, in addition to the target binding region (e.g., an oligo-dT domain, a gene-specific domain, or a random sequence domain), the barcoded nucleic acid also includes one or more cell marker domains, unique molecular index domains, and universal primer binding domains, for example, wherein the bead-bound barcoded nucleic acid has the following structure: bead-5'-universal primer binding domain-cell marker domain-unique molecular index domain-target binding region-3'. In some cases, the kit also includes a device comprising: (i) a substrate comprising 100 or more microwells; and (ii) a flow cell in fluid communication with the substrate. Attached Figure Description

[0016] In the following detailed description, reference is made to the accompanying drawings, which form a part thereof. In the drawings, like symbols generally identify like components unless the context otherwise requires. The illustrative embodiments described in the specification, drawings, and claims are not intended to be limiting. Other embodiments may be used, and other modifications may be made, without departing from the spirit or scope of the subject matter described herein. It will be readily understood that the various aspects of this disclosure, as described herein and illustrated in the figures, can be arranged, replaced, combined, separated, and designed in a variety of different constructions, all of which are expressly contemplated herein and form part of this disclosure.

[0017] Figure 1A and Figure 1B Representatives of capturing magnetic beads according to various embodiments of the present invention are provided.

[0018] Figure 2 A representative method for preparing a capture sample according to an embodiment of the present invention is provided.

[0019] Figure 3A and Figure 3B A representative of the captured particles divided according to an embodiment of the present invention is provided.

[0020] Figure 4 ( Figure 4 A to Figure 4 C) Provides a representative of the captured particles divided according to an embodiment of the present invention.

[0021] Figure 5 A representative workflow using a system according to an embodiment of the present invention is provided.

[0022] Figure 6 A diagram illustrating a sequencing library preparation workflow that can be used in embodiments of the present invention is provided.

[0023] definition

[0024] Unless otherwise defined, the 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 pertains. See, for example, 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 the purposes of this disclosure, the following terms are defined as follows.

[0025] As used herein, the term "adaptor" can refer to a sequence that facilitates the amplification or sequencing of a related nucleic acid. The related nucleic acid may include a target nucleic acid. The related nucleic acid may include one or more spatial markers, target markers, sample markers, index markers, or barcode sequences (e.g., molecular markers). The adaptor may be linear. The adaptor may be a pre-adenylated adaptor. The adaptor may be double-stranded or single-stranded. One or more adaptors may be located at the 5' or 3' end of the nucleic acid. When adaptors at the 5' and 3' ends contain known sequences, the known sequences may be the same or different sequences. Adaptors located at the 5' and / or 3' ends of a polynucleotide are capable of hybridizing with one or more oligonucleotides immobilized on a surface. In some embodiments, the adaptor may contain a universal sequence. The universal sequence may be a nucleotide sequence region common to two or more nucleic acid molecules. Two or more nucleic acid molecules may also have regions with different sequences. Thus, for example, the 5' adaptor may contain the same and / or universal nucleic acid sequence, and the 3' adaptor may contain the same and / or universal sequence. A universal sequence that can be present in different members of multiple nucleic acid molecules allows for the replication or amplification of multiple different sequences using a single universal primer complementary to the universal sequence. Similarly, at least one, two (e.g., a pair), or more than two universal sequences that can be present in different members of a set of nucleic acid molecules allow for the replication or amplification of multiple different sequences using at least one, two (e.g., a pair), or more than two single universal primers complementary to the universal sequence. Therefore, universal primers include sequences that can hybridize with such universal sequences. Molecules carrying target nucleic acid sequences can be modified to attach universal adaptors (e.g., non-target nucleic acid sequences) to one or both ends of different target nucleic acid sequences. One or more universal primers attached to the target nucleic acid can provide sites for universal primer hybridization. The one or more universal primers attached to the target nucleic acid can be the same or different from each other.

[0026] The antibodies used herein may be full-length immunoglobulin molecules (e.g., naturally occurring or formed from recombination processes of normal immunoglobulin gene fragments) (e.g., IgG antibodies) or immunologically active (i.e., specifically binding) portions of immunoglobulin molecules, such as antibody fragments. In some embodiments, the antibody is a functional antibody fragment. For example, the antibody fragment may be a portion of an antibody, such as 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 may include a separate fragment composed of antibody variable regions, such as an "Fv" fragment composed of light and heavy chain variable regions and a recombinant single-chain polypeptide molecule ("scFv protein") wherein the light and heavy chain variable regions are linked by peptide linkers. Exemplary antibodies may include, but are not limited to, antibodies against cancer cells, antibodies against viruses, antibodies that bind to cell surface receptors (e.g., CD8, CD34, and CD45), and therapeutic antibodies.

[0027] As used herein, the terms “related” or “affected” can refer to two or more species identified as being in the same location at a given time. Relatedness can mean that two or more species are in or have been in similar containers. Relatedness can be informatics-related. 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 given time. Relatedness can also be physical-related. In some embodiments, two or more related species are “tethered,” “attached,” or “fixed” to another or a common solid or semi-solid surface. Relatedness can refer to a covalent or non-covalent method of attaching a marker to a solid or semi-solid support, such as a bead. Relatedness can be a covalent bond between a target and a marker. Relatedness can include hybridization between two molecules (e.g., a target molecule and a marker).

[0028] The term "complementary" as used herein can refer to the ability of two nucleotides to pair precisely. For example, if a nucleotide at a given position on a nucleic acid can be linked to a nucleotide of another nucleic acid via a hydrogen bond, then the two nucleic acids are complementary to each other at that position. Complementarity between two single-stranded nucleic acid molecules can be "partial," where only some nucleotides bind, or it can be completely complementary when there is complete complementarity between the single-stranded molecules. If the first nucleotide sequence is complementary to the second nucleotide sequence, then the first nucleotide sequence can be said to be "complementary" to the second sequence. If the first nucleotide sequence is complementary to the second sequence in reverse order (i.e., the nucleotides are in the opposite order), then the first nucleotide sequence can be said to be "anti-complementary" to the second sequence. The terms "complementary," "complementary," and "anti-complementary" as used herein are interchangeable. It can be understood from the publication that if a molecule can hybridize with another molecule, then it can be complementary to the molecule that is hybridizing.

[0029] The term "numerical counting" as used in this article can refer to a method for estimating the number of target molecules in a sample. Numerical counting may include steps to determine the number of unique markers associated with a target in the sample. This research approach, which can be stochastic in nature, transforms the molecule counting problem from locating and identifying identical molecules into a series of yes / no numerical questions about detecting a predefined set of markers.

[0030] As used herein, the term "tag" or "multiple tags" can refer to a nucleic acid code associated with a target within a sample. A tag can be, for example, a nucleic acid tag. A tag can be a fully or partially amplifiable tag. A tag can be a fully or partially sequenceable tag. A tag can be part of a distinct natural nucleic acid. A tag can be a known sequence. A tag can contain a linker of nucleic acid sequences, such as a linker of natural and non-natural sequences. The term "tag" as used herein may be used interchangeably with the terms "index," "tag," or "tag-tag." A tag can convey information. For example, in various embodiments, a tag can be used to determine the characteristics of a sample, the source of the sample, the characteristics of the cells, and / or the target.

[0031] The term "non-consumable reservoir" as used in this paper can refer to a pool of barcodes consisting of many different labels (e.g., random barcodes). A non-consumable reservoir can contain a large number of different barcodes, such that when the non-consumable reservoir is associated with a target pool, each target is likely associated with a unique barcode. The uniqueness of each labeled target molecule relative to the diversity of labels can be determined by randomly selected statistical data and depends on the copy number of the same target molecule in the set. The size of the resulting labeled target molecules can be determined by the randomness of the barcode encoding process, and the number of target molecules present in the original set or sample can be calculated by analyzing the number of detected barcodes. Labeled target molecules are highly unique when the ratio of the number of copies of existing target molecules to the number of unique barcodes is low (i.e., the probability that more than one target molecule is labeled by a given label is very low).

[0032] As used herein, the term "nucleic acid" refers to a polynucleotide sequence or a fragment thereof. Nucleic acids may contain nucleotides. Nucleic acids may be exogenous or endogenous to the cell. Nucleic acids may exist in a cell-free environment. Nucleic acids may be genes or fragments thereof. Nucleic acids may be DNA. Nucleic acids may be RNA. Nucleic acids may contain one or more analogs (e.g., modified backbone, sugar, or nucleobase). Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acids, heteronucleic acids, morpholine nucleic acids, locked nucleic acids, glycol nucleic acids, threonine nucleic acids, dideoxynucleotides, cordycepin, 7-deazon-GTP, fluorophores (e.g., rhodamine or fluorescein linked to sugars), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, piracetamidine, and woyoside. The terms "nucleic acid," "polynucleotide," "target polynucleotide," and "target nucleic acid" are used interchangeably.

[0033] Nucleic acids can contain one or more modifications (e.g., base modifications, backbone modifications) to give them new or enhanced characteristics (e.g., improved stability). Nucleic acids can contain nucleic acid affinity tags. Nucleosides can be base-sugar combinations. The base moiety of a nucleoside can be a heterocyclic base. The two most common types of such heterocyclic bases are purines and pyrimidines. Nucleotides can also include a phosphate group covalently linked to the sugar moiety of the nucleoside. For nucleosides containing pentamuryl sugars, the phosphate group can be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. In the formation of nucleic acids, phosphate groups can covalently link adjacent nucleosides to form linear polymeric compounds. In turn, the ends of this linear polymeric compound can also link to form cyclic compounds; however, linear compounds are generally preferred. Furthermore, linear compounds can have internal nucleotide base complementarity and therefore may fold in some way to produce fully or partially double-stranded compounds. Within nucleic acids, the phosphate group is often referred to as the internucleotide backbone that forms the nucleic acid. The linking bond or backbone can be a 3' to 5' phosphodiester bond.

[0034] Nucleic acids may contain a modified backbone and / or modified nucleotide linkages. Modified backbones may include those that retain phosphorus atoms and those that do not. Suitable modified nucleic acid backbones containing phosphorus atoms may include, for example, thiophosphates, chiral thiophosphates, dithiophosphates, phosphate triesters, aminoalkyl phosphate triesters, methyl phosphonates, and other alkyl phosphonates, such as 3'-alkylphosphonate esters, 5'-alkylphosphonate esters, chiral phosphonates, hypophosphonates, aminophosphates including 3'-aminoaminophosphates and aminoalkylaminophosphates, diaminophosphates, thioaminophosphates, alkyl thiophosphonates, alkyl thiophosphate triesters, selenophosphates, and boron phosphates, which have normal 3' to 5' bonds, 2' to 5' bonds, and those with reverse polarity, wherein one or more nucleotide linkages are 3' to 3', 5' to 5', or 2' to 2' bonds.

[0035] Nucleic acids can contain polynucleotide backbones formed by short-chain alkyl or cycloalkyl nucleoside interstices, mixed heteroatom and alkyl or cycloalkyl nucleoside interstices, or one or more short-chain heteroatoms or heterocyclic nucleoside interstices. These can include those with morpholine nucleic acid linkages (partially formed by the sugar moiety of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; riboacetyl backbones; backbones containing alkenes; aminosulfonate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones that combine N, O, S, and CH2 components.

[0036] Nucleic acids can include nucleic acid analogs. The term "analyte" may be intended to include polynucleotides in which only the furanose ring or the bonds between the furanose ring and the nucleotide are replaced by non-furanose groups; those replacing only the furanose ring may also be called sugar-substituted analogs. The heterocyclic base moiety, or the modified heterocyclic base moiety, 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 the polynucleotide can be replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleotide can be retained and directly or indirectly bound to the aza-nitrogen atom of the amide moiety of the backbone. The backbone in a PNA compound may contain two or more linked aminoethylglycine units, giving the PNA an amide-containing backbone. The heterocyclic base moiety can be directly or indirectly bound to the aza-nitrogen atom of the amide moiety of the backbone.

[0037] Nucleic acids can contain a morpholine nucleic acid backbone structure. For example, nucleic acids can contain a 6-membered morpholine ring that replaces the ribose ring. In some embodiments, the internucleotide bond between diaminophosphate or other non-phosphodiester nucleosides can replace the phosphodiester bond.

[0038] Nucleic acids can contain linked morpholino units (e.g., morpholinonucleotides) with heterocyclic bases linked to a morpholino ring. Linking groups can connect the morpholino monomeric units within morpholinonucleotides. Nonionic morpholino-based oligomers exhibit fewer undesirable interactions with cellular proteins. Morpholino-based polynucleotides can be nonionic analogs of nucleic acids. Various compounds in the morpholinonucleotide class can be linked using different linking groups. Another class of polynucleotide analogs can be called cyclohexenyl nucleic acids (CeNA). The furanose ring, normally present in nucleic acid molecules, can be replaced by a cyclohexenyl ring. CeNA DMT-protected phosphorusamide monomers can be prepared using phosphorusamide chemical synthesis and used in oligomer synthesis. Incorporating CeNA monomers into nucleic acid chains can increase the stability of DNA / RNA hybrids. CeNA oligoadenylates can form complexes with nucleic acid complements exhibiting similar 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-formaldehyde bond and a 4'-C-formaldehyde bond, thus forming a bicyclic sugar moiety. This bond can be a methylene (-CH2) group, or a group bridging the 2' oxygen atom and the 4' carbon atom, where n is 1 or 2. LNAs and LNA analogs exhibit very high double-strand thermal stability (Tm = +3℃ to +10℃), stability against 3'-exonuclease degradation, and good solubility with complementary nucleic acids.

[0039] Nucleic acids may also include nucleobase (usually referred to simply as "bases") modifications or substitutions. The "unmodified" or "natural" nucleobases used in this article may include purine bases (e.g., adenine (A) and guanine (G)) and pyrimidine bases (e.g., thymine (T), cytosine (C), and uracil (U)). Modified nucleobases may include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl derivatives of adenine and guanine and other alkyl derivatives, 2-propyl derivatives of adenine and guanine and other alkyl derivatives, 2-thiouracil, 2-thiothymidine and 2-thiocytosine, 5-halouracil and 5-halocytosine, 5-propynyl (-C=C-CH3)uracil and 5-propynyl (-C=C-CH3)cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, 6-azocytosine and 6-azothymidine, 5-Uracil (pseudouracil), 4-thiouracil, 8-halogenated adenine and guanine, 8-aminoadenine and guanine, 8-mercaptoadenine and guanine, 8-thioalkyladenine and guanine, 8-hydroxyadenine and guanine and other 8-substituted adenine and guanine, 5-halogenated uracil and cytosine, especially 5-bromouracil and cytosine, 5-trifluoromethyluracil and cytosine and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazoguanine and 7-deazoadenine and 3-deazoguanine and 3-deazoadenine. Modified nucleobases may include tricyclic pyrimidines, for example, phenanthrene. Azincytosine (1H-pyrimidinyl(5,4-b)(1,4)benzo[]) Phenothiazine-2(3H)-one), phenthiazine cytidine (1H-pyrimidino(5,4-b)(1,4)benzothiazine-2(3H)-one), G-shaped clips, such as substituted phen... Acinocytosides (e.g., 9-(2-aminoethoxy)-H-pyrimidino(5,4-(b)(1,4))benzo[] Azine-2(3H)-one), phenothiazine cytidine (1H-pyrimidinyl(5,4-b)(1,4)benzothiazine-2(3H)-one), G-type clips such as substituted phenanthrene Acinocytosides (e.g., 9-(2-aminoethoxy)-H-pyrimidino(5,4-(b)(1,4)benzo) Azine-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indole-2-one), pyridoindole cytidine (H-pyrido(3',2':4,5)pyrrolo[2,3-d]pyrimido-2-one).

[0040] As used herein, the term "sample" can refer to a composition containing a target. Suitable samples for analysis using the disclosed methods, apparatus, and systems include cells, tissues, organs, or organisms.

[0041] As used herein, the term "sample apparatus" or "device" can refer to an apparatus that can acquire a portion of a sample and / or place that portion on a substrate. Sample apparatus can refer to, for example, a fluorescence activated cell sorting (FACS) machine, a cell sorter, a biopsy needle, a biopsy apparatus, a tissue sectioning apparatus, a microfluidic apparatus, a leaflet 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 multiple barcodes (e.g., random barcodes) may be attached. Solid supports may comprise any type of solid, porous or hollow sphere, ball, bearing, cylinder, or other similar structure composed of plastic, ceramic, metallic, or polymeric materials (e.g., hydrogels) on which nucleic acids may be immobilized (e.g., covalently or non-covalently). Solid supports may include discrete particles that can be spherical (e.g., microspheres) or have non-spherical or irregular shapes, such as cubes, cuboids, pyramids, cylinders, cones, ellipses, or disks. Beads may be non-spherical. Multiple solid supports spaced apart in an array may not contain a substrate. The term "solid support" is used interchangeably with the term "bead."

[0043] As used herein, the term "random barcode" can refer to a polynucleotide sequence containing a tag of the present disclosure. A random barcode can be a polynucleotide sequence used for encoding. Random barcodes can be used to quantify a target in a sample. Random barcodes can be used to control for errors that may occur after a tag is associated with a target. For example, random barcodes can be used to assess amplification or sequencing errors. A target-associated random barcode can be referred to as a random barcode-target or a random barcode-tag-target.

[0044] The term "gene-specific random barcode" as used in this article can refer to a multinucleotide sequence containing a marker and a gene-specific target binding region. A random barcode can be any multinucleotide sequence that can be encoded using random barcodes. Random barcodes can be used to quantify targets in a sample. Random barcodes can be used to control for errors that may occur after a marker is associated with a target. For example, random barcodes can be used to assess amplification or sequencing errors. Target-associated random barcodes can be referred to as random barcode-target or random barcode-tag-target.

[0045] As used in this paper, the term "random barcode encoding" can refer to the random labeling of nucleic acids (e.g., barcode encoding). Random barcode encoding can utilize a recursive Poisson strategy to correlate and quantify target-related labels. The term "random barcode encoding" as used in this paper is interchangeable with "random labeling."

[0046] As used herein, the term "target" can refer to a composition associated with a barcode (e.g., a random barcode). Exemplary suitable targets analyzed by the disclosed methods, apparatus, and systems include oligonucleotides, DNA, RNA, mRNA, microRNA, tRNA, etc. Targets 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. The term "target" as used herein may be used interchangeably with "type".

[0047] As used herein, the term "reverse transcriptase" can refer to a group of enzymes possessing reverse transcriptase activity (i.e., catalyzing the synthesis of DNA from an RNA template). Generally, such enzymes include, but are not limited to, retroviral reverse transcriptases, retrotransposon reverse transcriptases, retroplasmid reverse transcriptases, retrotranscripton reverse transcriptases, bacterial reverse transcriptases, class II intron-derived reverse transcriptases, and their mutants, variants, or derivatives. Non-retroviral reverse transcriptases include non-LTR retrotransposon reverse transcriptases, retroplasmid reverse transcriptases, retrotranscripton reverse transcriptases, and class II intron reverse transcriptases. Examples of class II intron reverse transcriptases include the intron reverse transcriptase of the *Lactococcus lactis* LI.LtrB gene, the intron reverse transcriptase of *Synechococcus thermophilus* TeI4c, or the intron reverse transcriptase of *Bacillus stearothermophilus* GsI-IIC. Other types of reverse transcriptases can include many types of non-retroviral reverse transcriptases (i.e., retrotranscriptons, class II introns, and reverse transcription elements that produce diversity).

[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 hybridized with a barcode (e.g., a random barcode) to produce 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 this disclosure. For example, when labeling multiple targets using the methods of this 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 this disclosure. For example, when labeling multiple targets using the methods of this disclosure, at least two target-specific sequences are ligated to different universal adaptor sequences. A universal adaptor primer and its complement can be contained in two oligonucleotides, one containing the target-specific sequence and the other containing the barcode. For example, a universal adaptor sequence can be a portion of an oligonucleotide containing the target-specific sequence to produce a nucleotide sequence complementary to the target nucleic acid. A second oligonucleotide containing a complementary sequence to both the barcode and the universal adaptor sequence may hybridize with the nucleotide sequence to produce a target-specific barcode (e.g., a target-specific random barcode). In some embodiments, the sequence of the universal adaptor primer differs from the universal PCR primers used in the methods of this disclosure.

[0049] Overview of Molecular Barcoding of Nucleic Acid Targets in Single Cells

[0050] The fundamental research method for the methods, apparatus, and systems disclosed herein utilizes a deposition strategy to achieve single-cell molecular barcode encoding determination of a large number of single cells. For example, by associating a single cell with a single barcoded bead tethered to a barcoded nucleic acid, molecular targets from the single cell can be randomly labeled with both cellular markers (also referred to as cell indexes, barcodes, or tags) and molecular markers (also referred to as molecular indexes, barcodes, or tags), wherein each single barcoded bead contains multiple attached random markers. The random markers attached to a given bead can be used to randomly label protein or nucleic acid targets from the associated cell. In some embodiments, single cells are randomly distributed into multiple microwells (e.g., a microwell array). A combined library of barcoded beads, each containing multiple tethered random barcoded nucleic acid markers, is also randomly distributed into multiple microwells, such that a subset of the microwells contains both single cells and single beads. In some embodiments, the barcoded beads are deposited prior to cell deposition. In other embodiments, the barcoded beads are deposited after cell deposition. In some embodiments, for example, the barcoded beads include a capture portion for target cells (as described in more detail below), and the beads and cells (or subcellular particles, such as vesicles) can be deposited simultaneously, for example, as a bead-cell binding complex. The random markers containing both cell and molecular barcodes may also include target recognition regions capable of attaching to or hybridizing with molecular targets, such as nucleic acid molecules. Target molecules can be released from each cell, for example, by lysing the cells, and then attached to or hybridized with the corresponding barcoded bead. In some embodiments, the target molecules are released from the cells by lysis, for example, enzymatic lysis. In some embodiments, for example, when the target molecule is an mRNA molecule, the beads are recovered from the microwells after hybridization with the random markers and collected before reverse transcription, amplification, and sequencing reactions.

[0051] In some embodiments, the plurality of random markers attached to a given bead comprises cellular markers that are identical for all random markers attached to that bead, while the cellular markers of the plurality of random markers attached to different beads are different. In some embodiments, the plurality of random markers attached to a given bead comprises different types of molecular markers selected from a set of unique molecular marker sequences comprising a specific number of such sequences. In some embodiments, the plurality of random markers attached to a given bead may comprise the same target recognition region. In some embodiments, the plurality of random markers attached to a given bead may comprise two or more distinct target recognition regions.

[0052] In some implementations, the cell marker diversity (i.e., the number of unique cell marker sequences) of the pearl library is at least one or two orders of magnitude higher than the number of cells to be labeled, making the probability of each cell matching a unique cell barcode very high. For example, the probability of each cell matching 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 diversity of molecular markers (i.e., the number of unique molecular marker sequences) associated with multiple random markers attached to the beads is at least one or two orders of magnitude higher than the estimated number of occurrences of the target molecule species to be labeled, making the probability that each occurrence of the intracellular target molecule (e.g., mRNA molecule) is uniquely labeled very high. For example, the probability that each occurrence of the target molecule is paired 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 target molecule species occurring in each cell can be counted (or estimated) by determining the number of unique molecular marker sequences attached to the target molecule sequence. In many embodiments, the determination step can be performed by sequencing an amplified library of the labeled target molecule (or its complementary sequence).

[0054] In some implementations, the molecular marker diversity of the multiple random markers attached to the beads is comparable to or low compared to the estimated number of occurrences of the target molecule species to be labeled, making it highly likely that a given type of target molecule occurring multiple times will be labeled with more than one copy of a given molecular marker. In these implementations, the number of target molecules per cell can be calculated using Poisson statistics based on the number of unique molecular marker sequences attached to the target molecule sequence.

[0055] In some implementations, the target molecule of interest is an mRNA molecule expressed in a single cell. Since cDNA copies of all or part of the polyadenylated mRNA molecule in each cell are covalently stored on the surface of the corresponding beads, arbitrarily selected gene transcripts can then be analyzed. A digital gene expression profile for each cell can be reconstructed when the barcoded transcripts are sequenced and assigned to the original cells (based on identified cell markers) and counted (based on the number of identified unique molecular markers). 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] Now, let's summarize the various elements outlined above in more detail.

[0057] Barcodes and digital counters

[0058] Quantifying small amounts of nucleic acids, such as messenger RNA (mRNA) molecules, is clinically significant for identifying genes expressed by cells at different developmental stages or under different environmental conditions. However, determining the absolute number of nucleic acid molecules (e.g., mRNA molecules) can 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 the same copy of the molecule in each cycle. However, PCR has drawbacks, such as the fact that each molecule replicates with a random probability that varies with the PCR cycle and the gene sequence, leading to amplification bias and inaccurate gene expression measurements. Random barcodes with unique molecular markers (also known as molecular indexes (MIs)) can be used for molecule counting and to correct for amplification bias. For example, Precise... TM Random barcode encoding, such as assays (Cellular Research, Inc. (Palo Alto, CA)), can correct for biases induced by PCR and library preparation steps by tagging mRNA with molecular markers (ML) during reverse transcription (RT).

[0059] Precise TM The assay can utilize a non-consumable pool of random barcodes containing a large number of unique molecular markers on poly(T) oligonucleotides, such as 6561 to 65536, to hybridize with all poly(A)-mRNAs in the sample during the RT step. The random barcodes can contain universal PCR primer sites. During RT, target gene molecules react randomly with the random barcodes. Each target molecule can hybridize with a random barcode, resulting in complementary ribonucleotide (cDNA) molecules with random barcodes. After labeling, cDNA molecules with random barcodes from the wells of a microplate, droplets, or other partitions can be pooled into a single tube for PCR amplification and sequencing, for example, using a next-generation sequencing protocol, as described below. The number of reads, the number of random barcodes with unique molecular markers, and the number of mRNA molecules can be determined by analyzing the raw sequencing data.

[0060] Barcode encoding, such as random barcode encoding, has been described, for example, in Fu et al., Proc Natl Acad Sci U.SA, 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; and PCT Application Publication No. WO2015 / 031691, the contents of each of which, including all of any supporting or supplementary information or material, are incorporated herein by reference. In some embodiments, the barcode disclosed herein may be a random barcode for randomly marking (e.g., barcode, tag) a target polynucleotide sequence. A barcode can be called a random barcode if the ratio of the number of different barcode sequences to the number of occurrences of any target to be labeled is, or approximately, 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 any two of these values ​​or a range thereof. The target can be a type of mRNA, including mRNA molecules with the same or nearly identical sequences, antibody identifier sequences, etc. A barcode can be called a random barcode if the ratio of the number of different barcode sequences 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. The barcode sequences in a random barcode can be called molecular markers.

[0061] A barcode, such as a random barcode, may contain one or more markers. Exemplary markers may include universal markers, cell markers, barcode sequences (e.g., molecular markers), sample markers, plate markers, spatial markers, and / or pre-spatial markers. Spatial markers, dimensional markers, and cell markers may be in any order. In some embodiments, the order of universal markers, spatial markers, dimensional markers, cell markers, and molecular markers is arbitrary. The barcode may contain a target-binding region. The target-binding region may interact with a target in the sample (e.g., target nucleic acid, RNA, mRNA, DNA). For example, the target-binding region may contain an oligomeric (dT) sequence that may interact with poly(A) tails of mRNA and / or reagent-specific oligonucleotides that bind cellular components. In some cases, barcode markers (e.g., universal markers, dimensional markers, spatial markers, cellular markers, and barcode sequences) 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 nucleotides.

[0062] Taglets, such as cell tags, can contain unique sets of nucleic acid subsequences of a defined length, for example, each nucleic acid subsequence containing seven nucleotides (equivalent to the number of bits used in some Hamming error-correcting codes), which can be designed to provide error correction capabilities. Error-correcting subsequence sets containing seven nucleotide sequences can be designed such that any pairwise combination of sequences in this set represents a defined “genetic distance” (or the number of mismatched bases); for example, the error-correcting subsequence set can be designed to represent a genetic distance of three nucleotides. In this case, examining the error-correcting sequences (described more fully below) in a set of labeled target nucleic acid molecule sequence data can allow the detection or correction of amplification or sequencing errors. In some embodiments, the length of the nucleic acid subsequence used to generate the error-correcting code can be varied; for example, the length can be, or approximately, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 31, 40, 50 nucleotides, or a value or range between any two of these values. In some implementations, nucleic acid subsequences of other lengths can be used to generate error-correcting codes.

[0063] General tags

[0064] A barcode may contain one or more universal markers. In some embodiments, one or more universal markers may be the same for all barcodes in a group of barcodes attached to a given solid support. In some embodiments, one or more universal markers may be the same for all barcodes attached to multiple beads. In some embodiments, the universal marker may contain a nucleic acid sequence capable of hybridizing with sequencing primers, and may therefore be referred to as a universal primer-binding domain. Sequencing primers may be used to sequence barcodes containing universal markers. Sequencing primers (e.g., universal sequencing primers) may contain sequencing primers associated with a high-throughput sequencing platform. In some embodiments, the universal marker may contain a nucleic acid sequence capable of hybridizing with PCR primers. In some embodiments, the universal marker may contain a nucleic acid sequence capable of hybridizing with both sequencing primers and PCR primers. The universal marker nucleic acid sequence capable of hybridizing with sequencing or PCR primers may be a primer binding site. The universal marker may contain a sequence that can be used to initiate barcode transcription. The universal marker may contain a sequence that can be used to extend the barcode or a region within the barcode. The length of a universal marker can be, or approximately, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides, or any two or more of these values. For example, a universal marker can contain at least 10 nucleotides. The length of a universal 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. In some embodiments, a cleavable linker or modified nucleotide can be part of the universal marker sequence to allow the barcode to be cleaved from the support.

[0065] Dimension Marking

[0066] A barcode can contain one or more dimension markers. In some implementations, the dimension markers can contain nucleic acid sequences that provide dimensional information about the markers that appeared (e.g., random markers). For example, a dimension marker can provide information about when a target was barcoded. The dimension marker can be correlated with the time of barcode encoding (e.g., random barcode encoding) in the sample. Dimension markers can be activated at the time of barcode encoding. Different dimension markers can be activated at different times. Dimension markers provide information about the order in which targets, target groups, and / or samples were barcoded. For example, a cell population can be barcoded during the G0 phase of the cell cycle. During the G1 phase of the cell cycle, cells can be pulsed again with barcodes (e.g., random barcodes). During the S phase, cells can be pulsed again with barcodes, and so on. The barcode for each pulse (e.g., each phase of the cell cycle) can contain different dimension markers. In this way, the dimension markers provide information about which targets were labeled at which phase of the cell cycle. Dimension markers can query many different biological time periods. Exemplary biological timeframes include, but are not limited to, the cell cycle, transcription (e.g., transcription initiation), and transcript degradation. In another example, samples (e.g., cells, cell populations) may be labeled before and / or after drug and / or treatment. Changes in copy numbers of different targets can indicate a sample's response to drug and / or treatment.

[0067] Dimensional markers can be activated. Activation of an activatable dimensional marker can be achieved at specific time points. 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). Dimensional markers can be reversibly activated, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times. Dimensional markers 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, dimensional markers can be activated by fluorescence, light, chemical events (e.g., cleavage, linking to another molecule, addition of modifications (e.g., PEGylation, ubiquitination, acetylation, methylation, deacetylation, demethylation), photochemical events (e.g., photocages), and the introduction of non-natural nucleotides.

[0068] In some embodiments, the dimension markers may be the same for all barcodes (e.g., random barcodes) attached to a given solid support (e.g., beads), 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 may contain the same dimension markers. In some embodiments, at least 60% of the barcodes on the same solid support may contain the same dimension markers. In some embodiments, at least 95% of the barcodes on the same solid support may contain the same dimension markers.

[0069] Up to 10 can be expressed in multiple solid supports (e.g., beads). 6 Species or more than 10 6 A unique dimensional marker sequence for a species. The length of the dimensional marker can be, or about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or any two of these values ​​or a range thereof. 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 contain about 5 to about 200 nucleotides. The dimensional marker can contain about 10 to about 150 nucleotides. The length of the dimensional marker can contain about 20 to about 125 nucleotides.

[0070] Spatial markers

[0071] A barcode may contain one or more spatial markers. In some embodiments, the spatial markers may contain nucleic acid sequences that provide information about the spatial orientation of the target molecule associated with the barcode. The spatial markers may be associated with coordinates in a sample. The coordinates may be fixed coordinates. For example, the coordinates may be fixed to a reference substrate. The spatial markers may reference a two-dimensional or three-dimensional grid. The coordinates may be fixed to a reference landmark. The landmark can be identifiable in space. The landmark can be an imageable structure. The landmark can be a biological structure, such as an anatomical landmark. The landmark can be a cellular landmark, such as an organelle. The landmark can be a non-natural landmark, such as a structure with an identifiable identifier, such as a color code, barcode, magnetism, fluorescence, radioactivity, or a unique size or shape. The spatial markers may be associated with physical partitions (e.g., pores, containers, or droplets). In some embodiments, multiple spatial markers are used together to encode one or more locations in space.

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

[0073] Up to 10 can be expressed in multiple solid supports (e.g., beads). 6 One or more 10 6 A unique spatial marker sequence. The length of the spatial 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 these values. The length of the spatial 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. A spatial marker can contain approximately 5 to approximately 200 nucleotides. A spatial marker can contain approximately 10 to approximately 150 nucleotides. A spatial marker can contain approximately 20 to approximately 125 nucleotides in length.

[0074] Cell markers

[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 groups of barcode sequences are attached to a given solid support (e.g., beads). In some embodiments, there may be 10 or more. 2 10 3 10 4 10 5 10 6 10 7 10 8 10 9 A unique molecular marker sequence, or a value or range between any two of these values. For example, multiple barcodes can contain approximately 6561 barcode sequences with different sequences. As another example, multiple barcodes can contain approximately 65536 barcode sequences with different sequences. In some implementations, there can be at least or at most 10 2 10 3 10 4 10 5 10 6 10 7 10 8 One or 10 9 A unique barcode sequence. A unique molecular marker sequence can be attached to a given solid support (e.g., beads).

[0080] The length of a barcode can vary in different implementations. For example, the length of a barcode 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. 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 markers

[0082] A barcode (e.g., a random barcode) may contain one or more molecular markers. The molecular marker may include a barcode sequence. In some embodiments, the molecular marker 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 molecular marker may also contain a nucleic acid sequence that provides a count of specific target nucleic acid species that hybridize with the barcode (e.g., a target binding region).

[0083] In some embodiments, different groups of molecular markers are attached to a given solid support (e.g., beads). In some embodiments, there may be or about 10 2 103 10 4 10 5 10 6 10 7 10 8 10 9 A unique molecular marker sequence, or a value or range between any two of these values. For example, multiple barcodes can contain approximately 6561 molecular markers with different sequences. As another example, multiple barcodes can contain approximately 65536 molecular markers 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 One or 10 9 A unique molecular marker sequence. A barcode with a unique molecular marker sequence can be attached to a given solid support (e.g., beads).

[0084] For random barcode encoding using multiple random barcodes, the ratio of the number of different molecular marker sequences to the number of occurrences of any target can be, or approximately, 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 any two of these values ​​or a range thereof. Targets can be mRNA species containing mRNA molecules with the same or nearly identical sequences. In some implementations, the ratio of the number of different molecular marker 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] Random multimer sequences can be, for example, random dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, or multimer sequences of any length (where the target binding region can be called the random sequence domain).

[0090] In some implementations, the target binding region is identical for all barcodes attached to a given bead. In some implementations, the target binding regions of multiple barcodes linked to a given bead may contain two or more different target binding sequences. The length of the target binding region may 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 the target binding region may be at most approximately 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 nucleotides.

[0091] In some implementations, the target-binding region may include oligo(dT) domains (i.e., oligo-dT domains) that can hybridize with mRNA containing polyadenylate ends. The target-binding region may be gene-specific. For example, the target-binding region may be configured to hybridize with a specific region of a target (e.g., where the target-binding region is a gene-specific domain).

[0092] The length of the target binding region can be, or approximately, 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, or any value or range between any two of these values. The target binding region can be at least 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 long. The length of the target binding region can be approximately 5 to 30 nucleotides. When a barcode contains a gene-specific target binding region, it is referred to herein as a gene-specific barcode.

[0093] The target-binding region can interact with a target in the sample. The target can be or contains ribonucleotides (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA each containing a poly(A) tail, or any combination thereof. In some embodiments, multiple targets may include deoxyribonucleic acid (DNA).

[0094] In some embodiments, the target-binding region may contain an oligomeric (dT) sequence that can interact with the polymeric (A) tail of the mRNA. One or more markers of the barcode (e.g., universal markers, dimensional markers, spatial markers, cellular markers, and barcode sequences (e.g., molecular markers)) may be separated from another or two remaining markers of the barcode by spacer bases. For example, the spacer bases may 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 barcode markers are separated by spacer bases.

[0095] Universal adapter primers

[0096] Barcodes can contain one or more universal adaptor primers. For example, gene-specific barcodes, such as gene-specific random barcodes, can contain universal adaptor primers. A universal adaptor primer can refer to a nucleotide sequence that is common across all barcodes. Universal adaptor primers can be used to construct gene-specific barcodes. The length of a universal adaptor primer can be, or approximately, 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 nucleotides, or any value or range between these values. Universal adaptor primers can be at least 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 long. The length of universal adaptor primers can range from 5 to 30 nucleotides.

[0097] connector

[0098] When a barcode contains more than one type of marker (e.g., more than one cellular marker or more than one barcode sequence, such as a molecular marker), these markers can be interspersed with adapter marker sequences. The length of the adapter marker sequence can be at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides. The length of the adapter marker sequence can be at most 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides. In some cases, the length of the adapter marker sequence is 12 nucleotides. Adapter marker sequences can be used to facilitate barcode synthesis. Adapter markers can contain error-correcting (e.g., Hamming) codes.

[0099] solid support

[0100] In some embodiments, the barcodes disclosed herein, such as random barcodes, may be associated with a solid support. The solid support may be, for example, a synthetic particle. In some embodiments, some or all of the barcode sequences, such as the molecular markers of random barcodes (e.g., a first barcode sequence) having multiple barcodes (e.g., a first plurality of barcodes) on the solid support, differ by at least one nucleotide. Cellular markers of barcodes on the same solid support may be identical. Cellular markers of barcodes on different solid supports may differ by at least one nucleotide. For example, a first cell marker having a first plurality of barcodes on a first solid support may have the same sequence, and a second cell marker having a second plurality of barcodes on a second solid support may have the same sequence. The first cell marker having a first plurality of barcodes on a first solid support and the second cell marker having a second plurality of barcodes on a second solid support may differ by at least one nucleotide. The length of the cell marker may be, for example, about 5 to 20 nucleotides. The length of the barcode sequence may be, for example, about 5 to 20 nucleotides. The synthetic particle may be, for example, a bead.

[0101] The beads can be, for example, silicone beads, controlled-pore glass beads, magnetic beads, Dynabead, dextran / agarose gel beads, cellulose beads, polystyrene beads, or any combination thereof. The beads may also contain materials such as: polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymers, titanium, latex, agarose gel, cellulose, nylon, silicone, or any combination thereof.

[0102] In some embodiments, the beads may be polymer beads, such as deformable beads or gel beads, functionalized with barcodes or random barcodes (e.g., gel beads from 10X Genomics (Pleasanton CA)). In some embodiments, the gel beads may contain a polymer-based gel. For example, gel beads can be generated by encapsulating one or more polymer precursors into droplets. Gel beads can also be generated by exposing the polymer precursor to an accelerator (e.g., tetramethylethylenediamine (TEMED)).

[0103] In some embodiments, the particles may be biodegradable. For example, polymer beads may dissolve, melt, or degrade under desired conditions. Desired conditions may include environmental conditions. These conditions may cause the polymer beads to dissolve, melt, or degrade in a controlled manner. Gel beads may dissolve, melt, or degrade due to chemical, physical, biological, thermal, magnetic, electrical, or light stimulation, or any combination thereof.

[0104] Analytes and / or reagents, such as oligonucleotide barcodes, can be coupled / immobilized to the inner surface of gel beads (e.g., the interior accessible via diffusion of the oligonucleotide barcode and / or the material used to generate it) and / or the outer surface of the gel beads or any other microcapsules described herein. Coupling / immobilization can be achieved through any form of chemical bond (e.g., covalent, ionic) or physical phenomenon (e.g., van der Waals forces, dipole-dipole interactions, etc.). In some embodiments, the coupling / immobilization of the reagent to the gel beads or any other microcapsules described herein can be reversible, for example, through an unstable portion (e.g., through a chemical crosslinking agent, including those described herein). When a stimulus is applied, the unstable portion can be cleaved, releasing the immobilized reagent. In some embodiments, the unstable portion is a disulfide bond. For example, in the case where the oligonucleotide barcode is immobilized on the gel beads via disulfide bonds, exposing the disulfide bonds to a reducing agent can cleave the disulfide bonds and release the oligonucleotide barcode from the beads. The unstable portion may include as part of a gel bead or microcapsule, as part of a chemical linker connecting a reagent or analyte to the gel bead or microcapsule, and / or as part of the reagent or analyte. In some embodiments, at least one of a plurality of barcodes may be affixed to the particle, partially affixed to the particle, enclosed in the particle, partially enclosed in the particle, or any combination thereof.

[0105] In some embodiments, the gel beads may comprise a variety 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 may 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(ethyleneimine) (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), and polylactic-co-glycolic acid copolymer (PLGA).

[0106] Numerous chemical stimuli can be used to trigger bead breakage, dissolution, or degradation. Examples of these chemical changes include, but are not limited to, pH-mediated bead wall changes, bead wall disintegration caused by chemical cleavage of cross-links, triggered bead wall depolymerization, and bead wall switching reactions. Numerous changes can also be used to trigger bead breakage.

[0107] The ability to induce volume or physical changes in microcapsules through various stimuli also offers significant advantages for designing capsules that release reagents. These volume or physical changes occur on a macroscopic scale, where bead rupture is the result of stimulus-induced mechano-physical forces. These processes can include, but are not limited to, pressure-induced rupture, bead wall melting, or changes in bead wall porosity.

[0108] Biostimuli can also be used to trigger the breakage, dissolution, or degradation of beads. Typically, biotriggers are similar to chemical triggers, but many instances use biomolecules, or molecules common in living systems, such as enzymes, peptides, sugars, fatty acids, and nucleic acids. For example, beads can contain peptide cross-linked polymers sensitive to cleavage by specific proteases. More specifically, one example could include microcapsules containing GFLGK peptide cross-links. When a biotrigger, such as the protease cathepsin B, is added, the peptide cross-links in the shell wall are cleaved, and the contents of the bead are released. In other cases, the protease can be thermally activated. In another example, beads contain shell walls containing cellulose. The hydrolase chitosan is added as a biotrigger to cleave cellulose bonds, depolymerize the shell wall, and release its contents. The release of their contents can also be induced by the application of thermal stimulation. Changes in temperature can cause a variety of changes in the beads. Thermal changes can cause the beads to melt, causing the bead walls to disintegrate. In other cases, heat can increase the internal pressure of the bead's internal components, causing the bead to rupture or explode. In still other cases, heat can transform the beads into a shrunken, dehydrated state. Heat can also act on the heat-sensitive polymer inside the bead wall, causing the bead wall to break.

[0109] Incorporating magnetic nanoparticles into the bead walls of microcapsules allows for triggering bead rupture and guiding bead formation into an array. The apparatus of this disclosure can include magnetic beads for either purpose. In one example, the addition of Fe3O4 nanoparticles to beads containing a polyelectrolyte, stimulated by an oscillating magnetic field, triggers rupture.

[0110] Beads can break, dissolve, or degrade due to electrical stimulation. Similar to the magnetic particles described in the previous section, electrosensitive beads can both trigger bead breakage and perform other functions, such as alignment, conductivity, or redox reactions in an electric field. In one example, beads containing electrosensitive materials are arranged in an electric field, thereby controlling the release of internal reagents. In other examples, the electric field can induce redox reactions within the bead walls, thereby increasing porosity.

[0111] Photostimulation can also be used to break beads. Many phototriggers are possible and can include systems using a variety of molecules, such as nanoparticles and chromophores capable of absorbing photons in specific wavelength ranges. For example, metal oxide coatings can be used as capsule triggers. Ultraviolet irradiation of a SiO2-coated polyelectrolyte capsule can cause the bead wall to disintegrate. In another example, photo-switching materials, such as azophenyl groups, can be incorporated into the bead wall. When ultraviolet or visible light is applied, such chemicals undergo reversible cis-trans isomerization upon absorbing photons. In this respect, the addition of a photon switch can cause the bead wall to disintegrate or become more porous under the action of a phototrigger.

[0112] The barcodes disclosed herein may be associated with (e.g., attached to) a solid support (e.g., a bead). Each barcode associated with the solid support may contain a barcode sequence selected from at least 100 or 1000 barcode sequences having a unique sequence. In some embodiments, different barcodes associated with the solid support may contain barcodes with different sequences. In some embodiments, a certain percentage of the barcodes associated with the solid support contain the same cell marker. For example, the percentage may be, or about, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a value or range between any two of these values. As another example, the percentage may be at least or at most 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. In some embodiments, the barcodes associated with the solid support may have the same cell marker. Barcodes associated with different solid supports may have different cell markers, selected from at least 100 or 1000 cell markers containing unique sequences.

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

[0114] The terms “tethering,” “attachment,” and “fixation” used in this document are used interchangeably and can refer to covalent or non-covalent methods of attaching barcodes to solid supports. Any variety of solid supports can be used as attachments for pre-synthesized barcodes or as solid supports for in-situ solid-phase synthesis of barcodes.

[0115] In some embodiments, the solid support is a bead. Beads can comprise one or more types of solids, porous or hollow spheres, spheres, bearings, cylinders, or other similar structures capable of immobilizing nucleic acids (e.g., covalent or non-covalent). For example, beads can be made of plastic, ceramic, metal, polymer materials, or any combination thereof. Beads can be, or comprise, discrete particles that are spherical (e.g., microspheres) or have non-spherical or irregular shapes, such as cubes, cuboids, pyramids, cylinders, cones, ellipses, or disks. In some embodiments, the shape of the beads can be non-spherical.

[0116] Beads can contain 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), ceramics, plastics, glass, polystyrene, silica, methylstyrene, acrylic polymers, titanium, latex, agarose gel, hydrogel, polymers, cellulose, nylon, or any combination thereof.

[0117] In some embodiments, the beads (e.g., beads with attached markers) are hydrogel beads. In some embodiments, the beads contain hydrogel.

[0118] Some embodiments disclosed herein include one or more particles (e.g., beads). Each particle may contain multiple oligonucleotides (e.g., barcodes). Each of the multiple oligonucleotides may contain a barcode sequence (e.g., a molecular marker sequence), a cell marker, and a target-binding region (e.g., an oligo(dT) sequence, a gene-specific sequence, a random multimer, or a combination thereof). The cell marker sequence of each of the multiple oligonucleotides may be identical. The cell marker sequences of oligonucleotides on different particles may be different, thereby allowing identification of oligonucleotides on different particles. The number of different cell marker sequences may vary in different embodiments. In some implementations, the number of cell marker 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, or 10 6 10 7 10 8 10 9 One, any two of these values ​​or a range thereof, or more than 10 9In 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 embodiments, the 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%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any two of these values ​​longer or shorter than the pore diameter, or approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any two of these values ​​longer or shorter than the pore diameter. The diameter of the bead can also be related to the diameter of a cell (e.g., a single cell embedded in a basal pore). In some embodiments, the bead diameter can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% longer or shorter than the pore diameter, or at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% longer or shorter than the pore diameter. The diameter of the bead can be related to the diameter of the cell (e.g., a single cell embedded in a basal pore). In some embodiments, the diameter of the bead can 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 implementations, 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, Dynabeads® microbeads, MACS® 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, oligomeric (dT)-conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, and BcMag™ carboxyl-terminated magnetic beads.

[0124] Beads can be associated with quantum dots or fluorescent dyes (e.g., impregnated with them) to make them fluoresce in one or more fluorescent light channels. Beads can be associated with iron oxide or chromium oxide to make them paramagnetic or ferromagnetic. Beads can be identifiable. For example, beads can be imaged using a camera. Beads can have a detectable code associated with them. For example, beads can contain barcodes. For example, beads can change size due to swelling in organic or inorganic solutions. Beads can be hydrophobic. Beads can be hydrophilic. Beads can be biocompatible.

[0125] Solid supports (e.g., beads) can be visualized. Solid supports can contain visual labels (e.g., fluorescent dyes). Solid supports (e.g., beads) can be etched with identifiers. Identifiers can be visualized by imaging the beads. Detailed Implementation

[0126] Embodiments of the present invention provide a method for molecularly barcoding nucleic acid targets of particles, such as cells or extracellular vesicles, mediated by capture magnetic beads. This method includes: a) combining a sample containing particles with capture magnetic beads comprising a particle-capturing portion to produce a capture sample; b) dividing the capture particles of the capture sample using a magnetic field-mediated partitioning scheme to produce partitioned capture particles, wherein the partitioned capture particles are spatially adjacent to bead-bound barcoded nucleic acids containing target-binding regions; and c) cleaving the partitioned capture particles such that the released nucleic acids, and optionally released cell components, bind reagent-specific oligonucleotides to the target-binding regions to produce captured nucleic acids. Compositions, such as capture magnetic beads, comprising barcoded magnetic beads, and apparatus / systems and kits for carrying out embodiments of the method are also provided.

[0127] Before describing the invention in more detail, it should be understood that the invention is not limited to the particular embodiments described, and therefore variations are possible. It should also be understood that the terminology used herein is for describing particular embodiments only and is not intended to be limiting, as the scope of the invention will be limited to the appended claims.

[0128] When a numerical range is provided, it should be understood that, unless the context explicitly specifies otherwise, every value between the upper and lower limits of the range and any other specified value or intermediate value within the range that falls within one-tenth of the lower limit unit is included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and also within this invention, subject to any specific exclusions within the specified range. Where the range includes one or both limitations, ranges excluding one or both limitations are also included in this invention.

[0129] Certain ranges are presented in this document as numerical values ​​beginning with the term “about”. The term “about” as used herein provides literal support for the precise number preceding it, as well as numbers that are close to or approximate to the number preceding it. In determining whether a number is close to or approximate to a specifically listed number, a close or approximate unlisted number may be substantially equal to a specifically listed number provided in the context in which it appears.

[0130] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, representative illustrative methods and materials are described hereafter.

[0131] All publications and patents referenced in this specification are incorporated herein by reference, as each individual publication or patent is specifically and individually indicated to be incorporated herein by reference, and is hereby incorporated by reference to disclose and describe methods and / or materials relating to the publication. References to any publication are for its disclosure prior to the application date and should not be construed as an admission that the invention is not entitled to precedence over that publication by virtue of a prior invention. Furthermore, the publication dates provided may differ from the actual publication dates, which require independent verification.

[0132] It should be noted that, unless the context requires otherwise, the singular form used herein and in the appended claims includes plural references. It should also be noted that the drafting of the claims may exclude any optional factors. Therefore, this statement is intended as a prior basis for the use of exclusive terms such as “solely” or “only” when stating elements of the claims or using the “negative” limitation.

[0133] It will be apparent to those skilled in the art upon reading this disclosure that each individual embodiment described and illustrated herein has discrete components and features, and these embodiments can be readily separated from or combined with any features of several other embodiments without departing from the scope or spirit of the invention. Any of the enumerated methods can be implemented in the order of the enumerated events or in any other logically possible order.

[0134] For the sake of grammatical fluency in functional interpretation, it is necessary to clearly understand that, unless expressly stated in accordance with 35 USC §112, claims should not be interpreted as necessarily limited in any respect to “apparatus” or “steps”, but should be given the full meaning and scope of the definitions provided under the principle of equivalence, in which case a claim expressly stated in accordance with 35 USC §112 will acquire the full legal effect of equivalence as provided in 35 USC §112.

[0135] In the further description of various aspects of the invention, the various schemes and reagents / systems herein are first reviewed in more detail, followed by an in-depth review of the various methods of the invention, as well as a description of reagent kit implementation schemes for practicing various embodiments of the methods.

[0136] method

[0137] As described above, a method for molecularly barcoding target nucleic acids from particles, such as cells or subcellular component molecules, such as vesicles, mediated by magnetic beads is provided. Aspects of this method include combining a sample suspected of containing at least the particle of interest with one or more capturing magnetic beads (including a capture magnetic bead library), wherein the capturing magnetic beads include a capturing portion for the particle to produce a captured sample. The sample contacted with one or more capturing magnetic beads can be any sample, for example, a sample comprising one or more cells and other components, such as extracellular components, such as vesicles. In some embodiments, the sample can be a particle, such as a single cell or extracellular vesicle (e.g., microvesicles, exosomes, or apoptotic bodies), multiple particles, such as cells and / or vesicles, tissue samples, tumor samples, blood samples, etc. In some embodiments, the sample can comprise a mixture of cell types, such as normal cells, tumor cells, blood cells, B cells, T cells, maternal cells, fetal cells, etc., or a mixture of cells from different experimental subjects, and their subcellular components, such as vesicles. In some cases, the sample is an aqueous sample comprising one or more types of particles, such as cells and / or vesicles, for example, the presence of other cellular and subcellular components, such as vesicles. While the number of particles in a given aqueous sample used in the workflow embodiments of the invention 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 target particles are scarce, such as circulating tumor cells (CTCs), fetal cells in maternal blood, infectious bacterial or fungal cells, the number of particles in a given sample can be in the range, for example, from 0.0001 particles / mL to 100 particles / mL, or for example, from 0.01 particles / mL to 10 particles / mL. When target particles are abundant, such as EVs, RBCs, the number of particles in a given volume can be from 10 million particles / mL to 10 billion particles / mL, for example, from 100 million particles / mL to 10 billion particles / mL. Depending on the dilution or concentration of the sample, the concentrations provided above can be lower or higher. A given concentration represents the concentration that is naturally present in the sample.

[0138] In some embodiments, these methods include barcoding nucleic acids derived from cells. In some cases, the method includes barcoding nucleic acids derived from subcellular-sized particles, such as extracellular vesicles, wherein such subcellular-sized particles may have a diameter of 1000 nm or less. In some embodiments, the method includes barcoding nucleic acids from extracellular vesicles, wherein in some cases, the extracellular vesicles have a diameter of 5 µm or less, for example, 1 µm or less, and in some cases, the extracellular vesicles have a diameter of 30 nm to 2500 nm, for example, 30 nm to 1000 nm. In some cases, the particles are microvesicles (e.g., with a diameter of 100 to 100 nm). In some cases, the particles are exosomes (e.g., with a diameter of 30 nm to 150 nm).

[0139] For example, as described above, the sample is in contact with one or more trapping magnetic beads, which contain trapping portions for particles, such as cellular or subcellular (e.g., vesicle) components of the sample. Trapping magnetic beads are beads that are affected (i.e., moved) by an applied magnetic field in space. Therefore, they can move in space, for example, by applying a magnetic field, for example, from another magnet, to the environment of the trapping magnetic beads to move them from one location to another. Magnetic trapping beads contain a magnetic solid support having trapping portions for particles, such as cellular or subcellular sample components, such as vesicles, associated with their stability, for example on their surface. Beads can contain one or more types of solids, porous or hollow spheres, spheres, bearings, cylinders, or other similar structures that can fix the trapping portions (e.g., covalent or non-covalent). For example, beads can contain plastic, ceramic, metal, polymer materials, or any combination thereof. Beads may be, or comprise, discrete particles that are spherical (e.g., microspheres) or have non-spherical or irregular shapes, such as cubes, cuboids, pyramids, cylinders, cones, ellipses, or disks. In some embodiments, the beads may be non-spherical. Because beads are magnetic, they may contain 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), etc. Beads may also include many additive materials, such as ceramics, plastics, glass, polystyrene, silica, methylstyrene, acrylic polymers, titanium, latex, agarose gel, agarose, hydrogels, polymers, cellulose, nylon, or any combination thereof.

[0140] Related to the stability of the solid support for the trapping magnetic beads is the trapping portion for the particle, such as a cell or vesicle. The trapping portion is the part that binds to the particle's components (i.e., determinants), such as surface proteins or other structures. The most generalized binding can be of any type, including specific or non-specific binding. In some cases, the trapping portion specifically binds to the particle's determinants, such as surface proteins. In cases where the trapping portion specifically binds to the particle's determinants, the trapping portion and the determinants have an affinity for each other. The affinity between a pair of specifically binding members and their specifically bound determinants can vary, and in some cases, they can specifically bind to each other as a binding complex, which can be expressed as 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 characterization (it should be noted that in some embodiments, these values ​​may be applied to the interactions of other specific binding pairs mentioned elsewhere in this description). Any suitable determinant binding agent may be used for the capture moiety, such as a protein binding agent, an antibody or a fragment thereof, an aptamer, a small molecule, a ligand, a peptide, an oligonucleotide, etc., or any combination thereof. For example, the capture moiety may contain an antibody, such as an antibody specific to a target, such as a specific portion on a cell or vesicle (e.g., a receptor). The antibody may be a full-length (i.e., naturally occurring or formed from a normal immunoglobulin gene fragment recombination process) 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 may be a portion of an antibody, such as F(ab')2, Fab', Fab, Fv, sFv, etc. In some embodiments, the antibody fragment may bind to the same antigen recognized by the full-length antibody. Antibody fragments may include isolated fragments composed of antibody variable regions, such as “Fv” fragments composed of light chain variable regions and heavy chain variable regions, and recombinant single-chain polypeptide molecules, wherein the light chain variable regions and heavy chain variable regions are linked by peptide linkers (“scFv proteins”). Exemplary antibodies may include, but are not limited to, anticancer cell antibodies, antiviral antibodies, antibodies that bind to cell surface receptors (CD8, CD34, and CD45), and therapeutic antibodies.

[0141] As described above, the capture portion is stably associated with the surface of the solid support to which the capture bead is attached. Stability-associated means that the position of the capture portion relative to the surface of the solid support to which it is attached is fixed. The terms “fixed,” “tethered,” and “attached” used herein are used interchangeably and can refer to covalent or non-covalent methods of attaching the capture portion to the solid support.

[0142] Figure 1A An example of magnetic trapping, including the trapping section, is shown. Figure 1A The diagram illustrates a trapping magnetic bead 100 comprising a magnetic bead 110 (e.g., as described above), the surface of which is stably bound to a trapping portion 120. As described above, the trapping portion can be varied, and examples of the trapping portion include, but are not limited to, antibodies, biotin, avidin, or other molecules specific to target particles (e.g., cells or extracellular vesicles) that bind to avidin, biotin, or other molecules complementary to the trapping portion that is stably bound to the bead.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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 trapping beads 210 to produce a trapping sample 220, which consists of a binding complex of particles such as cells and vesicles bound to the trapping magnetic beads. After the preparation of the trapping sample, the trapping particles can optionally be separated from other components of the trapping sample, such as unbound particles, unbound beads, etc.

[0147] After generating the captured sample, this method includes the following aspects: dividing the captured particles of the captured sample using a partitioning scheme mediated by an applied magnetic field to produce partitioned captured particles. Partitioning refers to placing the captured particles in a small reaction chamber, which can be a fluid separation structure such as micropores defined by a solid material, configured to accommodate the captured particles. In some embodiments of the disclosed methods, apparatus, and systems, a plurality of micropores randomly distributed on a substrate are used. In some embodiments, the plurality of micropores are distributed on the substrate in an ordered pattern, such as an ordered array. In some embodiments, the plurality of micropores are distributed on the substrate in a random pattern, such as a random array. Micropores can be fabricated in a variety of shapes and sizes. Suitable pore geometries include, but are not limited to, cylindrical, elliptical, cubic, conical, hemispherical, rectangular, or polyhedral shapes, such as three-dimensional geometries containing several planes, such as cuboids, hexagonal prisms, octagonal prisms, inverted triangular pyramids, inverted square pyramids, inverted pentagonal pyramids, inverted hexagonal pyramids, or inverted truncated pyramids. In some embodiments, non-cylindrical micropores, such as pores with elliptical or square overlays, may offer advantages in terms of the ability to accommodate larger cells. In some embodiments, the upper and / or lower edges of the pore walls may be rounded to avoid sharp corners, thereby reducing electrostatic forces that may occur at sharp edges or points due to electrostatic field concentration. Therefore, using rounded corners can improve the ability to recover beads from the micropores. The micropore size can be characterized by absolute dimensions. In some cases, the average diameter of the micropores can be from about 5 µm to about 100 µm. In other embodiments, the average micropore diameter is at least 5 µm, at least 10 µm, at least 15 µm, at least 20 µm, at least 25 µm, at least 30 µm, at least 35 µm, at least 40 µm, at least 45 µm, at least 50 µm, at least 60 µm, at least 70 µm, at least 80 µm, at least 90 µm, or at least 100 µm. In other embodiments, the average micropore diameter is at most 100 µm, at most 90 µm, at most 80 µm, at most 70 µm, at most 60 µm, at most 50 µm, at most 45 µm, at most 40 µm, at most 35 µm, at most 30 µm, at most 25 µm, at most 20 µm, at most 15 µm, at most 10 µm, or at most 5 µm. The volume of the micropores used in the method of the present invention can vary, and in some cases is about 200 µm. 3 Approximately 800,000 µm 3 In some implementations, the micropore volume is at least 200 µm.3 At least 500µm 3 At least 1000µm 3 At least 10000µm 3 At least 25000µm 3 At least 50000µm 3 At least 100,000 µm 3 At least 200,000 µm 3 At least 300,000 µm 3 At least 400,000 µm 3 At least 500,000 µm 3 At least 600,000 µm 3 At least 700,000 µm 3 Or at least 800,000 µm 3 In other embodiments, the micropore volume is at most 800,000 µm. 3 Up to 700,000µm 3 Up to 600,000µm 3 Up to 500,000µm 3 Up to 400,000µm 3 Up to 300,000µm 3 Up to 200,000µm 3 Up to 100,000µm 3 Up to 50000µm 3 Up to 25000µm 3 Up to 10000µm 3 Up to 1000µm 3 Up to 500µm 3 or at most 200µm 3 The number of micropores in a given device used in embodiments of the present invention can vary, wherein in some cases the number is 100 or more, for example 250 or more, for example 500 or more, including 1000 or more, for example 5000 or more, for example 10000 or more, for example 10000 or more, and in some cases the number is 15000 or less, for example 12500 or less. Suitable micropores for embodiments of the present invention are further described in PCT application PCT / US2016 / 014612, publication number WO / 2016 / 118915, the disclosure of which is incorporated herein by reference.

[0148] When partitioning a captured sample, any convenient method can be used to position the captured particles within the micropores. This disclosure provides methods for contacting the captured sample with a partitioning domain to partition the sample. For example, the contained captured sample can be introduced into a structure, such as micropores, to partition the sample. The captured sample can be contacted, for example, by gravity flow, where the captured particles can settle into the partitioning structure. In some cases, the captured sample is contacted with the micropores, for example, by flowing it through the micropores, causing the captured particles to deposit into the micropores. The captured sample can flow through a flow cell in fluid communication with the micropores. Suitable methods and systems for partitioning captured particles into micropores are described in micropores suitable for use in embodiments of the present invention, and are further described in PCT application PCT / US2016 / 014612, publication number WO / 2016 / 118915, the disclosure of which is incorporated herein by reference.

[0149] When segmenting the captured sample, an applied magnetic field is used to bring the captured sample into contact with the micropores; for example, the captured sample flows through the micropores when a magnetic field is applied. The applied magnetic field is one that moves the captured particles from the captured sample toward the micropores, and depending on its orientation and the properties of the magnetically captured particles, it may be attractive or repulsive to the magnetically captured particles. For example, if a magnetic field is applied below the bottom of the micropore, the magnetic field may attract magnetically captured particles. Alternatively, if a magnetic field is applied above the micropore, the magnetic field may repel magnetically captured particles. Any convenient source can be used to apply the magnetic field, such as permanent magnets, electromagnets, etc. Although the strength of the applied magnetic field can be varied as needed, in some cases the strength ranges from 10 Gauss to 15,000 Gauss, for example, from 100 Gauss to 1,000 Gauss. A system and a method of using the system are described in PCT application No. PCT / US2016 / 014612, Publication No. WO / 2016 / 118915. The system includes one or more magnets and their controllers, which can be used in embodiments of the present invention. The disclosure of the disclosed system is incorporated herein by reference.

[0150] Figure 3A A schematic representation of segmented capture particles prepared from capture magnetic beads, the capture magnetic beads comprising bead-bound barcode nucleic acids, such as... Figure 1B As shown, in the micropores of a flow cell. (As illustrated...) Figure 3A As shown, under the influence of an applied electric field (as shown by magnet 330), capture particles 310, including, for example, cellular or extracellular vesicle particles, bound to capture magnetic beads containing barcode nucleic acids, flow through a micropore array 320. This causes the capture particles to be divided into the pores, such as... Figure 3B As shown.

[0151] Figure 4A schematic diagram representing the segmentation of capture particles prepared from capture magnetic beads is provided, wherein the capture magnetic beads do not include bead-bound barcode nucleic acids, such as... Figure 1A As shown, the micropores are divided using a flow cell. Figure 4 As shown in Figure A, under the influence of an applied electric field (as shown by magnet 430), capture particles 410, including, for example, cellular or extracellular vesicle particles, bound to capture magnetic beads (excluding bead-bound barcode nucleic acids), flow through a micropore array 420. This causes the captured particles to be divided into pores, such as... Figure 4 As shown in B. Next, beads containing barcode nucleic acids are dispensed into the microwells, as shown in Figure B. Figure 4 As shown in C. Thus, some holes include both captured particles and beads with barcodes.

[0152] For example, the segmentation of the captured sample as described above results in the segmented captured particles being spatially close to, for example, a barcode nucleic acid bound to beads as described above, which includes a target-binding region. In some cases, the bead-bound barcode nucleic acid is stably bound to the captured magnetic beads. In this case, the segmentation, such as a microwell, may consist only of the captured particles because the bead component of the captured particles includes the barcode nucleic acid. In other cases, the bead-bound barcode nucleic acid is a separate bead portion distinct from the captured magnetic beads, for example, a barcode-bearing bead as described above. In this case, the segmentation, such as a microwell, may include the captured particles and separate barcode-bearing beads including the barcode nucleic acid. When the barcode is close to the target of the captured particles, the target can hybridize with the barcode. The barcode can be contacted at a non-consumable ratio, allowing each different target to bind to a different barcode of this disclosure. To ensure effective binding between the target and the barcode, the target can be cross-linked with the barcode.

[0153] As described above, after fragmenting particles such as cells and / or vesicles, the particles can be lysed to release target molecules, which, such as nucleic acids, can bind to the target-binding region of the barcode nucleic acid to produce captured nucleic acid. Particle lysis can be accomplished in various ways, such as chemical or biochemical methods, osmotic shock, or by thermal lysis, mechanical lysis, or optical lysis. Particles can be lysed by adding a cell lysis buffer, which includes surfactants (e.g., SDS, lithium dodecyl sulfate, Triton X-100, Tween-20, or NP-40), organic solvents (e.g., methanol or acetone), or digestive enzymes (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, for example, by decreasing the temperature and / or increasing the viscosity of the lysis buffer.

[0154] In some implementations, filter paper can be used to lyse the sample. The filter paper can be soaked in lysis buffer on top. The filter paper can be applied to the sample under pressure, which can promote sample lysis and hybridization of the sample's target with the substrate.

[0155] In some embodiments, lysis can be performed by mechanical lysis, thermal lysis, optical lysis, and / or chemical lysis. Chemical lysis may include the use of digestive enzymes such as proteinase K, pepsin, and trypsin. Lysis can be performed by adding a lysis buffer to the substrate. The lysis buffer may contain Tris HCl. The lysis buffer may contain 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 HCl. The lysis buffer may contain 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 HCl. The lysis buffer may contain about 0.1 M of Tris HCl. The pH of the lysis buffer may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10. The pH of the lysis buffer may be at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater than 10. In some embodiments, the pH of the lysis buffer is about 7.5. The lysis buffer may contain a salt (e.g., LiCl). The concentration of the salt in the lysis buffer may be at least about 0.1M, 0.5M, or 1M or greater than 1M. The concentration of the salt in the lysis buffer may be at most about 0.1M, 0.5M, or 1M or greater than 1M. In some embodiments, the concentration of the salt in the lysis buffer is about 0.5M. The lysis buffer may contain a surfactant (e.g., SDS, lithium dodecyl sulfate, Triton X, Tween, NP-40). The concentration of the surfactant in the lysis buffer may 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 up to 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 taken in the lysis method can depend on the amount of surfactant used. In some embodiments, the more surfactant used, the less time is required for lysis. The lysis buffer may contain a chelating agent (e.g., EDTA, EGTA). The concentration of the chelating agent 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. In some embodiments, the concentration of the chelating agent in the lysis buffer is approximately 10 mM. The lysis buffer may contain a reducing agent (e.g., β-mercaptoethanol, DTT).The concentration of the 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 the 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 the reducing agent in the lysis buffer is about 5 mM. In some embodiments, the lysis buffer may contain about 0.1 M Tris HCl, about pH 7.5, about 0.5 M LiCl, about 1% lithium dodecyl sulfate, about 10 mM EDTA, and about 5 mM DTT.

[0156] Lysis can be performed at temperatures of approximately 4°C, 10°C, 15°C, 20°C, 25°C, or 30°C. Lysis can last for approximately 1 minute, 5 minutes, 10 minutes, 15 minutes, or 20 minutes or longer. Lysed cells can contain at least approximately 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, or 700,000 or more than 700,000 target nucleic acid molecules. Lysed cells can contain up to approximately 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, or 700,000 or more than 700,000 target nucleic acid molecules.

[0157] After particle lysis and release of nucleic acid molecules therefrom, the nucleic acid molecules can randomly bind to barcodes on a colocalized solid support. Binding can include hybridization of the target recognition domain of the barcode with a complementary portion of the target nucleic acid molecule (e.g., the oligomeric (dT) portion of the barcode can interact with the polymeric (A) tail of the target). The analytical 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, nucleic acid molecules released from lysed cells can bind to multiple probes on the substrate (e.g., hybridize with probes on the substrate). When the probe contains oligomeric (dT) portions, mRNA molecules can hybridize with the probes and be reverse transcribed. The oligomeric (dT) portions of the oligonucleotides can serve as primers for first-strand synthesis of cDNA molecules, for example, when placed under DNA synthesis reaction conditions, producing a first-strand cDNA domain containing the captured nucleic acid.

[0158] Attachment can also involve linking a target-binding region of the barcode to a portion of the target nucleic acid molecule. For example, the target-binding region can contain a nucleic acid sequence capable of specifically hybridizing to a restriction site single-stranded overhang (e.g., EcoRI sticky-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 attached to any nucleic acid molecule containing a sequence complementary to the restriction site single-stranded overhang. A ligase (such as T4 DNA ligase) can be used to ligate the two fragments.

[0159] In some cases, the method also includes the use of oligonucleotide-labeled cell component binding reagents, for example, when it is necessary to quantify one or more cell components, such as surface proteins. The oligonucleotide-labeled cell component binding reagents used in the embodiments include cell component binding reagents, such as antibodies or binding fragments thereof, conjugated to cell component binding reagent-specific oligonucleotides, which contain an identifier sequence for the cell component binding reagent that binds to the cell component binding reagent-specific oligonucleotide. In this case, the capturing magnetic beads may include nucleic acids configured to capture, for example, specifically binding cell component binding reagent-specific oligonucleotide domains. In this way, protein expression can be analyzed in conjunction with gene expression analysis, for example, when multi-omics analysis, such as transcriptomic and proteomic combined analysis, is required. In this case, the method may include preparing a capture sample using oligonucleotide-labeled cell component binding reagents and then providing capture of cell component binding reagent-specific oligonucleotides released from the captured, segmented cells. Further details regarding the use of oligonucleotide-labeled cell component binding agents can be found in U.S. Patent Application Publications Nos. US20180267036 and US20200248263, the contents of which are incorporated herein by reference.

[0160] In some cases, the method also involves separating the captured nucleic acids from other components of the segmented capture particles. Recovery of attached target barcode assemblies based on solid supports can be performed by using magnetic beads and an externally applied magnetic field.

[0161] If desired, a given workflow may include a pooling step, in which a product composition, for example, consisting of captured nucleic acids, synthetic first-strand cDNA, or synthetic double-stranded cDNA, is bound or pooled with a product composition obtained from one or more additional samples, such as particles, like cells, and / or vesicles. In some cases, the pooling step is performed after a step of hybridization between barcode nucleic acids and target nucleic acids, for example, as described above. The number of different product compositions produced from different samples, such as cells, that are bound or pooled in these embodiments may vary, wherein in some cases, the number ranges from 2 to 1,000,000, for example 3 to 200,000, including 4 to 100,000, for example 5 to 50,000, and in some cases, the number ranges from 100 to 10,000, for example 1,000 to 5,000. The product composition may be amplified before or after mixing by methods such as polymerase chain reaction (PCR), as detailed below.

[0162] Once the target barcode molecules are aggregated, all other processing can be performed in a single reaction vessel. Other processing may include, for example, reverse transcription, amplification, lysis, dissociation, and / or nucleic acid extension. These additional processing reactions can be carried out in microwells, meaning that it is not necessary to first mix the labeled target nucleic acid molecules from multiple cells.

[0163] This disclosure provides methods for generating target-barcode conjugates using any convenient scheme, such as reverse transcription or nucleotide extension (e.g., sample-indexed oligonucleotides or cell component = reagent-specific oligonucleotides). Target-barcode conjugates may contain a barcode and a complementary sequence of all or part of the target nucleic acid (i.e., a barcode-encoded cDNA molecule, such as a randomly barcode-encoded cDNA molecule). Reverse transcription of the associated RNA molecule can be achieved by adding a 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 about 12 to 18 nucleotides, and binds to an endogenous poly(A) tail at the 3' end of mammalian mRNA. Random hexanucleotide primers can bind to mRNA at various complementary sites. Target-specific oligonucleotide primers typically selectively priming the mRNA of interest.

[0164] In some implementations, labeled RNA molecules can be generated by reverse transcription of mRNA molecules through the addition of reverse transcription primers. In some implementations, the reverse transcription primers are oligo(dT) primers, random hexanucleotide primers, or target-specific oligonucleotide primers. 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 typically selectively prepare the mRNA of interest.

[0165] In some implementations, the target is a cDNA molecule. For example, a reverse transcriptase can be used to reverse transcribe an mRNA molecule, such as the Moroni mouse leukemia virus (MMLV) reverse transcriptase, to produce a cDNA molecule with a poly(dC) tail. The barcode may include a target-binding region with a poly(dG) tail. 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 to copy to the 5' end of the barcode. By doing so, a cDNA molecule containing a barcode (e.g., a molecular marker) sequence at the 3' end of the cDNA molecule is obtained.

[0166] Reverse transcription can occur repeatedly to produce multiple labeled cDNA molecules. The methods disclosed herein may involve 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. Methods may also involve performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 reverse transcription reactions.

[0167] One or more nucleic acid amplification reactions can be performed to produce multiple copies of a labeled target nucleic acid molecule. Amplification can be performed in a multiplexed manner, where multiple target nucleic acid sequences are amplified simultaneously. The amplification reaction can be used to add a sequencing adaptor to the nucleic acid molecule. If present, the amplification reaction may include at least a portion of an amplified sample label. The amplification reaction may include at least a portion of an amplified cellular label and / or barcode sequence (e.g., a molecular marker). The amplification reaction may include at least a portion of an amplified sample tag, cellular label, spatial label, barcode sequence (e.g., a molecular marker), target nucleic acid, or a combination thereof. The amplification reaction comprises amplifying multiple nucleic acids at values ​​or ranges 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 any two of these values. The method may also include performing one or more cDNA synthesis reactions to produce one or more cDNA copies of a target-barcode molecule containing sample markers, cell markers, spatial markers and / or barcode sequences (e.g., molecular markers).

[0168] In some implementations, amplification can be performed using polymerase chain reaction (PCR). As described herein, PCR can refer to a reaction that amplifies a specific DNA sequence in vitro by simultaneously extending complementary DNA strands with primers. As described herein, PCR can include derivative forms of reactions, including but not limited to RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, digital PCR, and assembly PCR.

[0169] Amplification of labeled nucleic acids can include non-PCR-based methods. Examples of non-PCR-based methods include, but are not limited to, multiple substitution amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand substitution 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 transcriptional amplification or RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets, ligase chain reaction (LCR) and Qβ replicase (Qβ) methods, using palindromic probes, strand substitution amplification, oligonucleotide-driven amplification using restriction endonucleases to hybridize primers to nucleic acid sequences, amplification methods that generate double-strand cleavage before extension and amplification, strand substitution amplification using nucleic acid polymerases lacking 5' exonuclease activity, rolling circle amplification, and network branching amplification (RAM). In some embodiments, amplification does not produce circular transcripts.

[0170] In some embodiments, the methods disclosed herein further include performing a polymerase chain reaction on labeled nucleic acids (e.g., labeled-RNA, labeled-DNA, labeled-cDNA) to generate labeled amplicones (e.g., randomly labeled amplicones). The labeled amplicon may be a double-stranded molecule. A double-stranded molecule may include a double-stranded RNA molecule, a double-stranded DNA molecule, or an RNA molecule that hybridizes to a DNA molecule. One or both strands of the double-stranded molecule may contain a sample marker, a spatial marker, a cellular marker, and / or a barcode sequence (e.g., a molecular marker). The labeled amplicon may be a single-stranded molecule. A single-stranded molecule may include DNA, RNA, or a combination thereof. The nucleic acids of this disclosure may include synthetic or modified nucleic acids. Therefore, the method may include generating an amplicon composition from a first-stranded cDNA domain containing the captured nucleic acid.

[0171] Amplification may involve the use of one or more non-natural nucleotides. Non-natural nucleotides may include light-labile or triggerable nucleotides. Examples of non-natural nucleotides may include, but are not limited to, peptide nucleic acids (PNAs), morpholinonucleotides and locked nucleic acids (LNAs), as well as glycol nucleic acids (GNAs) and threononucleotides (TNAs). Non-natural nucleotides may be added to one or more cycles of the amplification reaction. The addition of non-natural nucleotides can be used to identify products as specific cycles or time points in the amplification reaction.

[0172] Performing one or more amplification reactions may include using one or more primers. One or more primers may contain, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. One or more primers may contain 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 nucleotides. One or more primers may contain fewer than 12 to 15 nucleotides. One or more primers may anneal to at least a portion of multiple labeled targets (e.g., randomly labeled targets). One or more primers can anneal to the 3' or 5' ends of multiple labeled targets. One or more primers can anneal to the internal regions of multiple labeled targets. The internal regions can be at least approximately 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, or 410 units away from the 3' ends of the multiple labeled targets. The distance between 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. One or more primers may include a fixed set of primers. One or more primers may include at least one or more custom primers. One or more primers may include at least one or more control primers. One or more primers may include at least one or more gene-specific primers.

[0173] One or more primers may include universal primers. Universal primers may be annealed to universal primer binding sites. One or more custom primers may be annealed to a first sample marker, a second sample marker, a spatial marker, a cellular marker, a barcode sequence (e.g., a molecular marker), a target, or any combination thereof. One or more primers may include universal primers and custom primers. Custom primers may be designed to amplify one or more targets. Targets may comprise a subset of all nucleic acids in one or more samples. Targets may comprise a subset of all labeled targets in one or more samples. One or more primers may contain at least 96 or more custom primers. One or more primers may contain at least 960 or more custom primers. One or more primers may contain at least 9600 or more custom primers. One or more custom primers may be annealed to two or more different labeled nucleic acids. Two or more different marker nucleic acids can correspond to one or more genes.

[0174] Any amplification scheme can be used in the methods disclosed herein. For example, in one scheme, the first round of PCR can amplify molecules attached to beads using gene-specific primers and primers targeting the universal Illumina sequencing primer 1 sequence. The second round of PCR can amplify the first PCR product using nested gene-specific primers, with Illumina sequencing primer 2 sequence inserted on both sides, and primers corresponding to the universal Illumina sequencing primer 1 sequence. The third round of PCR adds P5 and P7 and a sample index, converting the PCR product into an Illumina sequencing library. Sequencing at 150 bp x 2 can reveal cellular markers and barcode sequences (e.g., molecular markers) on read 1, genes on read 2, and the sample index on index 1 read.

[0175] In some embodiments, chemical cleavage can be used to remove nucleic acids from the substrate. For example, chemical groups or modified bases present in the nucleic acid can be used to facilitate its removal from the solid support. For example, enzymes can be used to remove nucleic acids from the substrate. For example, nucleic acids can be removed from the substrate by digestion with restriction endonucleases. For example, nucleic acids containing dUTPs or ddUTPs can be treated with uracil-d-glycosylation enzymes (UDG) to remove nucleic acids from the substrate. For example, enzymes that perform nucleotide excision, such as base excision repair enzymes, such as depurine pyrimidine endonucleases, can be used to remove nucleic acids from the substrate. In some embodiments, photolytic cleavage and light can be used to remove nucleic acids from the substrate. In some embodiments, cleavable adapters can be used to remove nucleic acids from the substrate. For example, cleavable adapters may contain at least one of biotin / avidin, biotin / streptavidin, biotin / neutrophil, Ig-protein A, a photostable adapter, an acid- or base-unstable adapter group, or an aptamer.

[0176] When the probe is a gene-specific probe, the molecule can hybridize with the probe and be reverse transcribed and / or amplified. In some implementations, it can be amplified after the nucleic acid has been synthesized (e.g., reverse transcribed). 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.

[0177] In some implementations, amplification can be performed on a substrate, for example, using bridging amplification. Homopolymer tailing of the cDNA can be performed to generate compatible ends for bridging amplification using oligomeric (dT) probes on the substrate. In bridging amplification, the primer complementary to the 3' end of the template nucleic acid can be the first primer in each pair covalently attached to the solid particle. When the sample containing the template nucleic acid is contacted with the particle and subjected to a single thermal cycle, the template molecule can anneal to the first primer and extend the first primer forward by adding nucleotides, forming a double-stranded molecule consisting of the template molecule and a newly formed DNA strand complementary to the template. In the heating step of the next cycle, the double-stranded molecule can denature, releasing the template molecule from the particle and leaving the complementary DNA strand attached to the particle by the first primer. In the annealing phase of the subsequent annealing and extension steps, the complementary strand can hybridize with a second primer that is complementary to the fragment of the complementary strand at the site where it was removed from the first primer. This hybridization allows the complementary strand to form a bridge between the first and second primers, being fixed to the first primer by covalent bonding and to the second primer by hybridization. In the extension phase, the second primer can be used to extend in reverse by adding nucleotides to the same reaction mixture, thus converting the bridge into a double-stranded bridge. Then, at the start of the next cycle, the double-stranded bridge can denature to produce two single-stranded nucleic acid molecules, each with one end attached to the particle surface via the first and second primers respectively, and the other end of each unattached. In the annealing and extension steps of the second cycle, each strand can hybridize with another complementary primer that was not previously used on the same particle to form a new single-stranded bridge. The two previously unused primers are then elongated after hybridization, converting the two new bridges into double-stranded bridges.

[0178] The amplification reaction may include 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 multiple nucleic acids.

[0179] Amplification of labeled nucleic acids can include PCR-based or non-PCR-based methods. Amplification of labeled nucleic acids can include exponential amplification of labeled nucleic acids. Amplification of labeled nucleic acids can include amplification of the straight strand of labeled nucleic acids. Amplification can be performed using polymerase chain reaction (PCR). PCR can refer to a reaction that amplifies a specific DNA sequence in vitro by simultaneously extending complementary DNA strands with primers. PCR can include various derivative forms of reactions, 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.

[0180] In some implementations, the amplification of labeled nucleic acids includes 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 loop-to-loop amplification. Other non-PCR-based amplification methods include multiple cycles of DNA-dependent RNA polymerase-driven RNA transcriptional amplification or RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets, ligase chain reaction (LCR) and Qβ replicase (Qβ) methods, using palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using restriction endonucleases, amplification methods that hybridize primers to nucleic acid sequences and generate double-strand cleavages before extension and amplification, strand displacement amplification using nucleic acid polymerases lacking 5' exonuclease activity, rolling circle amplification, and network branching amplification (RAM).

[0181] In some embodiments, the methods disclosed herein further include performing a nested polymerase chain reaction on the amplified amplicons (e.g., targets). The amplicons can be double-stranded molecules. 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 molecule may contain a sample tag or molecular identifier. Alternatively, the amplicons can be single-stranded molecules. Single-stranded molecules can comprise DNA, RNA, or combinations thereof. The nucleic acids of the present invention can comprise synthetic or modified nucleic acids.

[0182] In some embodiments, the method includes repeating the amplified labeled nucleic acid to generate multiple amplicones. The methods disclosed herein include 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 includes performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amplification reactions.

[0183] Amplification may also involve adding one or more control nucleic acids to one or more samples containing multiple nucleic acids. The control nucleic acids may contain control markers.

[0184] Amplification may include the use of one or more non-natural nucleotides. Non-natural nucleotides may include light-labile and / or triggerable nucleotides. Examples of non-natural nucleotides include, but are not limited to, peptide nucleic acids (PNAs), morpholinonucleotides and locked nucleic acids (LNAs), as well as glycol nucleic acids (GNAs) and threononucleotides (TNAs). Non-natural nucleotides may be added to one or more cycles of the amplification reaction. The addition of non-natural nucleotides can be used to identify products as occurring at a specific cycle or time point in the amplification reaction.

[0185] Performing one or more amplification reactions may include using one or more primers. One or more primers may contain at least one or more oligonucleotides. One or more oligonucleotides may contain at least 7 to 9 nucleotides. One or more oligonucleotides may contain fewer than 12 to 15 nucleotides. One or more primers may anneal to at least a portion of a plurality of labeled nucleotides. One or more primers may anneal to the 3' or 5' end of a plurality of labeled nucleic acids. One or more primers may anneal to the internal regions of a plurality of labeled nucleic acids. The internal region can be at least approximately 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, or 410 from the 3' ends of multiple labeled nucleic acids. The distance of 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. One or more primers may include a fixed set of primers. One or more primers may include at least one or more custom primers. One or more primers may include at least one or more control primers. One or more primers may include at least one or more housekeeping gene primers. One or more primers may contain universal primers. Universal primers may be annealed to universal primer binding sites. One or more custom primers may be annealed to a first sample tag, a second sample tag, a molecular identifier tag, a nucleic acid, or a product thereof. One or more primers may comprise universal primers and custom primers. Custom primers may be designed to amplify one or more target nucleic acids. Target nucleic acids may comprise a subset of all nucleic acids in one or more samples. In some embodiments, the primers are probes attached to an array of the present disclosure.

[0186] 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.

[0187] 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.

[0188] 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, sequencing platforms provided by Illumina® (e.g., HiSeq™, MiSeq™, and / or NextSeq™ sequencing systems); Ion Torrent... TM (For example, Ion PGM)TM and / or Ion Proton TM Sequencing systems); 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. The NGS protocol will vary depending on the other NGS sequencing system used. Detailed protocols for sequencing may also include, for example, amplification (e.g., solid-phase amplification), sequencing amplicon, and analysis of sequencing data can be obtained from the manufacturer of the NGS sequencing system used.

[0189] Devices and systems

[0190] This document discloses apparatuses and systems that can be used to practice embodiments of the invention. In some embodiments, the apparatus includes: a) a substrate comprising: i) at least 100 microwells, and ii) a plurality of capture particles and / or barcode-coded beads, wherein each of the plurality of at least 100 microwells contains a single capture particle optionally having a single barcode-coded bead (e.g., when the capture magnetic beads do not include barcode-coded nucleic acids), and b) a flow cell in fluid communication with the substrate. In some embodiments, at least one inlet port and at least one outlet port are also present, wherein the at least one inlet port and at least one outlet port are in fluid communication with the flow cell via fluid channels, wherein the at least one inlet port and at least one outlet port are capable of directing fluid flow through the flow cell, thereby contacting the microwells with the fluid. In some embodiments, the apparatus further includes a valve that prevents fluid from flowing within the apparatus unless a pipette tip is inserted into a pipette tip interface with a tapered feature.

[0191] This document also discloses a system comprising: a) an apparatus comprising: i) a substrate having at least 100 micropores; 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 fluid flow through the flow cell, thereby bringing the micropores into contact with the fluid; and b) a flow controller configured to control the delivery of fluid.

[0192] In some embodiments, the system further includes a fluid comprising a particulate (e.g., cell and / or vesicle) sample, a bead suspension, an analytical 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 directly dispensed or injected into the device by the user. In some embodiments, beads and analytical reagents, in addition to the cell sample, are pre-installed in the device. In some embodiments, a flow controller is configured to disperse fluid injection and air injection into the flow cell. In some embodiments, the system further includes a distribution mechanism for enhancing the uniform distribution of cells and beads in at least 100 microwells, wherein the distribution mechanism is selected from oscillation, shaking, vortexing, circulating flow, low-frequency stirring, and high-frequency stirring, or any combination thereof. In some embodiments, the system further includes a cell lysis mechanism, for example, sonication of cells using a high-frequency piezoelectric transducer. In some embodiments, the system further includes a temperature controller for maintaining a user-specific temperature or for raising the temperature between two or more specific temperatures over two or more specific time intervals. In some embodiments, the system further includes a magnetic field controller for generating a magnetic field gradient for eluting beads from at least 100 microwells or for transporting beads through a device. In some embodiments, the system further includes an imaging system configured to capture and process images of all or part of the at least 100 microwells, wherein the imaging system further includes an illumination subsystem, an imaging subsystem, and a processor. In some embodiments, the imaging system is configured to perform bright-field, dark-field, fluorescence, 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 a distribution mechanism to enhance the uniform distribution of cells or beads in the at least 100 microwells, thereby achieving a predetermined cell or bead distribution. In some embodiments, the predetermined cell and bead distribution refers to at least 10% of the microwells simultaneously containing a single cell and a single bead. In some embodiments, the predetermined cell and bead distribution refers to at least 25% of the microwells simultaneously containing a single cell and a single bead. In some embodiments, the system further includes a selection mechanism in which information obtained from the processed image is used to identify a subset of cells representing one or more specific features, and the selection mechanism is configured to include or exclude the subset of cells from subsequent data analysis. In some embodiments, the selection mechanism includes physically removing beads co-located with cells of the identified subset of cells from at least 100 microwells. In some embodiments, the selection mechanism includes physically loading beads co-located with cells of the identified subset of cells from at least 100 microwells.In some embodiments, the selection mechanism includes using dual-encoded beads, where each individual bead is optically and encoded by an attached oligonucleotide cell marker, sequencing the cell marker attached to the bead, and co-localizing the bead with cells of an identified subset of cells to generate 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 to at least 100 microwells at a first time and a cell lysis reagent to at least 100 microwells at a second time. In some embodiments, the first and second times are the same. In some embodiments, one or more specific features are selected from 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 present specific cell surface markers, and the expression of one or more specific genetic markers.

[0193] Software stored in a computer-readable medium may also be present, programmed to perform one or more of the following sequence data analysis steps: a) decoding or decomposition of sample barcodes, cell barcodes, molecular barcodes, and target sequence data; b) automated clustering of cell markers to compensate for amplification or sequencing errors, wherein sequence data is collected as a randomly labeled target oligonucleotide molecule library; c) alignment of sequence data with known reference sequences; d) determination of the number of reads for each gene in each cell, and the number of unique transcripts for each gene in each cell; e) statistical analysis to predict confidence intervals to determine the number of transcripts for each gene in each cell; and f) statistical analysis of cell clustering or identification of rare cell subpopulations based on gene expression data.

[0194] 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 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 the apparatus configured to distribute cells and beads through the plurality of microwells.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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 is commercially available as the Rhapsody™ Single Cell Analysis System (Becton, Dickinson and Company). 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 6The sample is then sequenced, for example, using a next-generation sequencing platform as described above.

[0199] Flow cytometer

[0200] In some embodiments, the method includes isolating, obtaining, and / or enriching cells of interest. The isolation, obtaining, and / or enrichment of cells of interest has been described in U.S. Patent Application No. 2016 / 0244828, the entire contents of which are incorporated herein by reference. For example, a flow cytometry method comprising isolating, obtaining, and / or enriching cells of interest can be used. In some embodiments, the flow cytometry utilizes fluorescence-activated cell sorting.

[0201] Flow cytometry is a valuable method for cell analysis and isolation. Therefore, it has a wide range of diagnostic and therapeutic applications. Flow cytometry utilizes a fluid flow to isolate cells in a straight line, allowing them to pass through the detection device in a single row. Individual cells can be distinguished based on their position in the fluid flow and the presence of detectable markers or components (e.g., fluorophores on oligonucleotides bound to cellular components). The cells flow through a focusing interrogation point, where at least one laser directs the laser beam to a focal point within the channel. The sample fluid containing cells is hydrodynamically focused onto a very small core diameter by flowing a sheath around the sample flow at a very high volumetric rate. This small core diameter can be less than 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, or any two of these values ​​or a range thereof. The volumetric velocity of the sheath flow is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the sample volumetric velocity, or any two of these values ​​or a range thereof. This results in a very high velocity of the focused cell's linear chain, approximately meters per second. Therefore, the residence time of each cell at the excitation point is very limited, for example, 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, or 100 microseconds, or any two of these values ​​or a range thereof. Once a cell passes the query point, it cannot be redirected back to the query point because the flow rate of the linear chain cannot be reversed.

[0202] Flow cytometry is a tool for characterizing cells based on optical parameters such as light scattering and fluorescence. In flow cytometry, cells in a fluid suspension pass through a detection zone, where they are exposed to excitation light, typically from one or more lasers, and their light scattering and fluorescence properties are measured. Cells or components thereof are often labeled with fluorescent dyes to facilitate detection. By labeling different cells or components with fluorescent dyes of different spectra, multiple different cells or components can be detected simultaneously. In some implementations, the analyzer includes multiple photodetectors, one for each scattering parameter to be measured and one for each different dye to be detected. The acquired data includes signals for each light scattering parameter and fluorescence emission measurement.

[0203] Biological cells are separated by adding sorting or collection capabilities to a flow cytometer. Cells detected in the separation stream that possess one or more desired characteristics are removed individually from the sample stream by mechanical or electrical means. This flow cytometry sorting method has been used to sort different types of cells, separate sperm for breeding with X and Y chromosomes, sort chromosomes for genetic analysis, and isolate specific organisms from complex biological populations.

[0204] Common flow cytometry techniques utilize droplet sorting, where a linearly separated fluid flow of cells breaks down into droplets. These droplets, containing cells of interest, become charged and are deflected into a collection tube by an electric field. Droplet sorting systems can form droplets at rates of approximately 100, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7500, 10000, 20000, 30000, 40000, 50000, 60000, 75000, 100000, 200000, 300000, and 40000. 0 drops / second, 500,000 drops / second, 600,000 drops / second, 750,000 drops / second, 1,000,000 drops / second, or any two of these values ​​or a range thereof, for fluid flow through nozzles with diameters less than 1000 micrometers, 750 micrometers, 600 micrometers, 500 micrometers, 400 micrometers, 300 micrometers, 200 micrometers, 100 micrometers, 75 micrometers, 60 micrometers, 50 micrometers, 40 micrometers, 30 micrometers, 20 micrometers, 1 micrometer, or any two of these values ​​or a range thereof. Droplet sorting requires droplets to break off from the flow at a fixed distance from the nozzle tip. This distance is typically on the order of a few millimeters from the nozzle tip and can be maintained by vibrating the nozzle tip at a predefined frequency to keep the fluid flow undisturbed.

[0205] As they pass through an observation point located below the nozzle tip, linearly separated cells in the flow can be characterized. A cell is identified as one that meets approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 75, 100, 200, 300, 400, 500, 600, or 750 criteria, or values ​​or ranges between any two of these, or values ​​greater than 1000 criteria. The time it takes for the cell to reach its landing point and for it to break off from the flow is predictable. A brief charge can be applied to the fluid flow before the droplet containing the selected cells breaks off from the flow, and then grounded immediately after the droplet breaks off. The sorted droplet retains its charge when it breaks off from the fluid flow, while all other droplets are uncharged. Charged droplets deviate from the downward trajectory of other droplets under the influence of the electric field and are collected in the sample tube. Uncharged droplets fall directly into the draining device.

[0206] Flow cytometers may also include devices for recording and analyzing measurement and analysis data. For example, a computer connected to the detection electronics can be used for data storage and analysis. Data may be stored in tabular form, with each row corresponding to the data for one cell and columns corresponding to each measurement parameter. Data from the flow cytometer is stored using standard file formats, such as the “FCS” file format, to facilitate data analysis using separate procedures and / or machines. Using current analytical methods, data is typically displayed as two-dimensional (“2D”) graphs for ease of visualization, but other methods can also be used to visualize multidimensional data.

[0207] Parameters measured using flow cytometry typically include excitation light scattered by the cell in the predominantly forward direction, called forward scattering (“FSC”); excitation light scattered by the cell in the predominantly sideways direction, called side scattering (“SSC”); light emitted by fluorescent molecules in one or more channels (frequency ranges) of the spectrum, called FL1, FL2, etc., or fluorescent dyes primarily measured in that channel. Different cell types can be identified by scattering parameters and the fluorescence emission produced by labeling various cellular proteins with dyes.

[0208] Fluorescence-activated cell sorting (FACS) is a special type of flow cytometer. It provides a method for sorting a heterogeneous mixture of cells into two or more containers or wells of a microtiter plate, one cell at a time, based on the specific light scattering and fluorescence characteristics of each cell. It records the fluorescence signal from individual cells and physically separates cells of particular interest. The acronym FACS is a trademark owned by Becton Dickinson.

[0209] The cell suspension is placed near the center of a narrow, rapidly flowing liquid stream. The stream is formulated to create a large separation between cells relative to their diameter on average (Poisson distribution). Vibrational mechanisms cause the cell stream to break into individual droplets. The system is tuned so that the probability of more than one cell in a droplet is very low. Before breaking into droplets, the stream passes through one or more laser intersections, where the fluorescence characteristics of each cell of interest are measured. To collect the cells, a charge is applied to the flow cell for a period of time, during which one or more droplets form and break off from the stream. These charged droplets then fall into the target container via an electrostatic deflection system, based on the charge on the droplets.

[0210] Non-limiting examples of suitable cell sorting devices include BD FACSJazz TM Cell sorter, BDFACSseq TM Cell sorting instrument, or any other cell sorting instrument, including Bio-Rad Laboratories, Inc. (Hercules, CA) S3e™ Cell Sorter, Sony Biotechnology Inc. (San Jose, CA) SH800 Cell Sorter, Beckman Coulter Inc. (Brea, CA) MoFlo™ XDP Cell Sorter.

[0211] In some implementations, the method utilizes cell surface marker tagging and staining for cell sorting. In some implementations, the method utilizes the magnetic tagging of cell surface markers to consume uninterested cells, interfering cells, and debris. In some implementations, the method includes determining one or more of a patient's genotypes based on obtained sequence information; determining the patient's phenotype based on obtained sequence information; determining one or more gene mutations in the patient based on sequence information; and predicting the patient's susceptibility to one or more diseases. At least one of the one or more diseases is cancer or a hereditary disease.

[0212] FACS can use antibodies targeting detectable cell markers to tag and stain cell surface markers, thereby classifying cells. Antibodies include monoclonal and polyclonal antibodies conjugated to fluorophores. Detectable cell markers include cell surface markers of interest. In some embodiments, magnetic depletion can be based on tagging cell surface markers with magnetic beads containing antibodies, including monoclonal and polyclonal antibodies, conjugating them to the surface of non-target cells, interfering with cells and / or debris. Non-limiting examples of cell surface markers include CD surface markers, growth factors / cytokines, 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, CD25BB515, CD161 PE, CD45RA PerCP-Cy™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 and chemokine receptors include ACVR 1B, ALK4, ACVR2A, ACVR2B, BMPR1A, BMPR2, CSF1R, MCSFR, CSF2RB, EGFR, EPHA2, EPHA4, EPHB2, EPHB4, and ERBB2. Examples of nuclear receptors include androgen receptors, CAR, ER α, ERβ, ESRRA, ESRRB, ESRRG, FXR, glucocorticoid receptors, LXR-a, LXR-b, PPARA, PPARD, PPARG, PXR, SXR, estrogen receptor β, progesterone receptors, RARA, RARB, RARG, RORA, RXRA, RXRB, THRA, THRB, and vitamin D3 receptors. Other examples of 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.

[0213] Compositions and kits

[0214] The invention also includes kits and compositions that can be used to practice the various methods of the invention. The compositions include, for example, magnetic beads for trapping as described above. In some cases, the magnetic beads for trapping may include, for example, barcoded nucleic acids as described above.

[0215] The kit of the present invention may include, for example, capture magnetic beads as described above. Where the capture magnetic beads do not include barcoded nucleic acids, the kit may also include, for example, barcoded beads as described above. The kit may also include one or more additive components for practicing embodiments of the method. For example, the kit may include one or more of the following: primers, polymerases (e.g., thermostable polymerases with hot-start properties or similar properties, reverse transcriptases, etc.), dsDNAases, exonucleases, dNTPs, metal cofactors, 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 stabilizing components (e.g., DTT), stimulus-responsive polymers, or any other desired kit components, such as devices, for example, as described above, solid supports, containers, boxes, such as tubes, beads, plates, microfluidic chips, etc. The components of the kit may be in separate containers, or multiple components may be contained in one container.

[0216] In addition to the components described above, the main kit may also include (in some embodiments) instructions for practicing the main method. These instructions may be present in the main kit in various forms, one or more of which may be present in the kit. One form of these instructions is that the information is printed on a suitable medium or substrate, such as one or more sheets of paper with the information printed on them, on the kit packaging, on a packaging insert, and so on. Another form of these instructions is a computer-readable medium containing the information, such as a floppy disk, optical disc (CD), portable flash drive, etc. Yet another form of these instructions may be a website address, allowing the information to be accessed via the Internet from a remote location.

[0217] Notwithstanding the appended claims, this disclosure is also defined by the following terms:

[0218] 1. A method for barcoding nucleic acids in particles, the method comprising:

[0219] a) Combine the sample with trapping magnetic beads containing a portion for trapping sample particles to produce a trapping sample;

[0220] b) The capture particles of the captured sample are fragmented using a magnetic field-mediated fragmentation scheme to produce fragmented capture particles, wherein the fragmented capture particles are spatially approximated to bead-bound barcode nucleic acids containing target-binding regions; and

[0221] 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.

[0222] 2. The method according to Clause 1, wherein the bead-bound barcode nucleic acid is tethered to a capture magnetic bead.

[0223] 3. The method according to Clause 1, wherein the bead-bound barcode nucleic acid is tethered to a barcode-bearing bead, which is different from the capturing magnetic bead.

[0224] 4. The method according to any one of the preceding clauses, wherein the divided particles are divided into micropores.

[0225] 5. The method according to any one of the preceding clauses, wherein the capturing portion contains a specific binding member.

[0226] 6. The method according to Clause 5, wherein the specific binding member comprises an antibody or a binding fragment thereof.

[0227] 7. The method according to any one of the preceding clauses, wherein the bead-bound barcode nucleic acid further comprises a cell marker domain.

[0228] 8. The method according to any one of the preceding clauses, wherein the bead-bound barcode nucleic acid further comprises a unique molecular index field.

[0229] 9. The method according to any one of the preceding clauses, wherein the bead-bound barcode nucleic acid further comprises a universal primer-binding domain.

[0230] 10. The method according to any one of Clauses 1 to 6, wherein the bead-bound barcode nucleic acid comprises the following structure: bead-5'-universal primer-binding domain-cell marker domain-unique molecular index domain-target-binding region-3'.

[0231] 11. The method according to any one of the preceding clauses, wherein the target binding region comprises an oligodT domain, a gene-specific domain, or a random sequence domain.

[0232] 12. The method according to any one of the preceding clauses, wherein the particles include subcellular particles.

[0233] 13. The method according to Clause 12, wherein the subcellular-sized particles include vesicles.

[0234] 14. The method according to any one of the preceding clauses, wherein the particles comprise cells.

[0235] 15. The method according to any one of the preceding clauses, wherein the method further comprises separating the captured nucleic acid from other components of the segmented captured particle, for example separating the nucleic acid from an oligonucleotide-labeled cell component binding reagent.

[0236] 16. The method according to Clause 15, wherein separation includes using an applied magnetic field.

[0237] 17. The method according to any one of the preceding clauses, wherein the method further comprises collecting the captured nucleic acids.

[0238] 18. The method according to any one of the preceding clauses, wherein the method further comprises placing the captured nucleic acid under cDNA synthesis reaction conditions to generate a first-strand cDNA domain comprising the captured nucleic acid.

[0239] 19. The method of claim 18, wherein the method further comprises generating an amplicon composition from a first-strand cDNA domain comprising the captured nucleic acid.

[0240] 20. The method according to Clause 19, wherein the amplicon composition is generated from a first-strand cDNA domain containing the captured nucleic acid using one or more rounds of amplification.

[0241] 21. The method according to Clause 19 or Clause 20, wherein the amplicon composition comprises a next-generation sequencing (NGS) library.

[0242] 22. The method according to Clause 21, wherein the amplicon composition comprises an NGS adaptor containing nucleic acid.

[0243] 23. The method according to Clause 21 or Clause 22, wherein the method further comprises a next-generation sequencing (NGS) library.

[0244] 24. A method for sequencing nucleic acids of particles, the method comprising:

[0245] a) Combining a sample containing particles with trapping magnetic beads, the trapping magnetic beads comprising:

[0246] i) Barcode nucleic acids containing target-binding regions, and

[0247] ii) The capture portion that specifically binds to particles;

[0248] To generate captured samples;

[0249] b) Use a magnetic field-mediated partitioning scheme to partition the captured particles of the captured sample into the micropores to produce partitioned captured particles;

[0250] c) The fragmented capture particles are cleaved so that the released nucleic acids bind to the target binding domain of the barcode nucleic acid to produce captured nucleic acids;

[0251] d) The captured nucleic acid is placed under the conditions of a cDNA synthesis reaction to produce a first-strand cDNA domain containing the captured nucleic acid;

[0252] e) Constructing an NGS library from the first-strand cDNA domain containing the captured nucleic acids; and

[0253] f) Sequencing the NGS library to sequence the nucleic acids of the target particles.

[0254] 25. The method according to Clause 24, wherein the particles include subcellular particles.

[0255] 26. The method according to Clause 25, wherein the subcellular-sized particles include vesicles.

[0256] 27. The method according to any one of the preceding clauses, wherein the particles comprise cells.

[0257] 28. The method according to Clause 24, wherein the method further comprises separating the captured nucleic acid from other components of the segmented captured particle.

[0258] 29. The method according to Clause 28, wherein separation includes using an applied magnetic field.

[0259] 30. The method according to any one of Clauses 24 to 29, wherein the method further comprises mixing the captured nucleic acid before placing the captured nucleic acid under the conditions of a cDNA synthesis reaction.

[0260] 31. The method according to any one of Clauses 24 to 30, wherein the NGS library is generated from a first-strand cDNA domain containing the captured nucleic acid using one or more rounds of amplification.

[0261] 32. The method according to Clause 31, wherein the amplicon composition comprises an NGS adaptor containing nucleic acid.

[0262] 33. The method according to Clauses 24 to 32, wherein the capture portion comprises an antibody or a binding fragment thereof.

[0263] 34. The method according to any one of Clauses 24 to 33, wherein the barcode nucleic acid further comprises a cell marker domain.

[0264] 35. The method according to any one of Clauses 24 to 34, wherein the barcode nucleic acid further comprises a unique molecular index field.

[0265] 36. The method according to any one of Clauses 24 to 35, wherein the barcode nucleic acid further comprises a universal primer binding domain.

[0266] 37. The method according to any one of Clauses 24 to 33, wherein the barcode nucleic acid comprises the following structure: bead-5'-universal primer binding domain-cell marker domain-unique molecular index domain-target binding region-3'.

[0267] 38. The method according to any one of Clauses 24 to 37, wherein the target binding region comprises an oligodT domain, a gene-specific domain, or a random sequence domain.

[0268] 39. The method according to any one of Clauses 24 to 38, wherein the partitioning includes the use of a flow pool.

[0269] 40. The method according to Clause 39, wherein the flow cell comprises a substrate.

[0270] 41. Capture magnetic beads containing barcode nucleic acids and a capture portion that specifically binds to particles.

[0271] 42. The capturing magnetic beads according to Clause 41, wherein the capturing portion comprises an antibody or a binding fragment thereof.

[0272] 43. The method according to any one of Clauses 41 to 42, wherein the barcode nucleic acid further comprises a cell marker domain.

[0273] 44. The capture magnetic bead according to any one of Clauses 41 to 43, wherein the barcode nucleic acid further comprises a unique molecular index field.

[0274] 45. The method according to any one of Clauses 41 to 44, wherein the barcode nucleic acid further comprises a universal primer binding domain.

[0275] 46. ​​The capture magnetic bead according to any one of Clauses 41 to 42, wherein the barcode nucleic acid comprises the following structure: bead-5'-universal primer binding domain-cell marker domain-unique molecular index domain-target binding region-3'.

[0276] 47. The capturing magnetic bead according to any one of Clauses 41 to 46, wherein the target binding region comprises an oligodT domain, a gene-specific domain, or a random sequence domain.

[0277] 48. An apparatus comprising:

[0278] a) A substrate comprising 100 or more micropores, wherein the micropores contain trapping magnetic beads, the trapping magnetic beads comprising:

[0279] i) Barcode nucleic acids containing target-binding regions, and

[0280] ii) the capture portion that specifically binds to particles; and

[0281] b) A flow cell in fluid communication with the substrate.

[0282] 49. The device according to clause 48, wherein the capturing portion comprises an antibody or a binding fragment thereof.

[0283] 50. The apparatus according to any one of Clauses 48 to 49, wherein the barcode nucleic acid further comprises a cell marker domain.

[0284] 51. The apparatus according to any one of Clauses 48 to 50, wherein the barcode nucleic acid further comprises a unique molecular index field.

[0285] 52. The method according to any one of Clauses 48 to 51, wherein the barcode nucleic acid further comprises a universal primer binding domain.

[0286] 53. The device according to any one of Clauses 48 to 49, wherein the barcode nucleic acid comprises the following structure: bead-5'-universal primer binding domain-cell marker domain-unique molecular index domain-target binding region-3'.

[0287] 54. The device according to any one of Clauses 48 to 53, wherein the target binding region comprises an oligodT domain, a gene-specific domain, or a random sequence domain.

[0288] 55. A system that comprises:

[0289] a) A substrate comprising 100 or more micropores, wherein the micropores contain trapping magnetic beads, the trapping magnetic beads comprising:

[0290] i) Barcode nucleic acids containing target-binding regions, and

[0291] ii) The capture portion that specifically binds to particles;

[0292] b) A flow cell in fluid communication with the substrate; and

[0293] c) Flow controller; wherein the flow controller is configured to control the delivery of fluid to the flow tank.

[0294] 56. The system according to Clause 55, wherein the capture portion comprises an antibody or a binding fragment thereof.

[0295] 57. The system according to any one of Clauses 55 to 56, wherein the barcode nucleic acid further comprises a cell marker domain.

[0296] 58. The system according to any one of Clauses 55 to 57, wherein the barcode nucleic acid further comprises a unique molecular index field.

[0297] 59. The method according to any one of Clauses 55 to 58, wherein the barcode nucleic acid further comprises a universal primer binding domain.

[0298] 60. The system according to any one of Clauses 55 to 56, wherein the barcode nucleic acid comprises the following structure: bead-5'-universal primer-binding domain-cell marker domain-unique molecular index domain-target-binding region-3'.

[0299] 61. The system according to any one of Clauses 55 to 60, wherein the target binding region comprises an oligodT domain, a gene-specific domain, or a random sequence domain.

[0300] 62. A reagent kit comprising

[0301] (a) A trapping magnetic bead comprising a trapping portion that specifically binds to target particles; and

[0302] (b) Barcode nucleic acid.

[0303] 63. The kit according to Clause 62, wherein the capture magnetic beads contain barcoded nucleic acids.

[0304] 64. The kit according to Clause 62, wherein barcode nucleic acid is tethered to a barcode-bearing bead separated from the capture magnetic bead.

[0305] 65. The kit according to any one of Clauses 62 to 64, wherein the kit further comprises:

[0306] The apparatus includes:

[0307] (i) a substrate containing 100 or more micropores; and

[0308] (ii) A flow cell in fluid communication with the substrate.

[0309] 66. The kit according to Clauses 62 to 65, wherein the capture portion contains an antibody or a binding fragment thereof.

[0310] 67. The kit according to any one of Clauses 62 to 66, wherein the barcode nucleic acid further comprises a cell marker domain.

[0311] 68. The kit according to any one of Clauses 62 to 67, wherein the barcode nucleic acid further comprises a unique molecular index field.

[0312] 69. The kit according to any one of Clauses 62 to 68, wherein the barcode nucleic acid further comprises a universal primer binding domain.

[0313] 70. The kit according to any one of Clauses 62 to 63, wherein the barcode nucleic acid comprises the following structure: bead-5'-universal primer binding domain-cell marker domain-unique molecular index domain-target binding region-3'.

[0314] 71. The kit according to any one of Clauses 62 to 70, wherein the target binding region comprises an oligodT domain, a gene-specific domain, or a random sequence domain.

[0315] In at least some of the previously described embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, unless such substitution is technically impractical. Those skilled in the art will understand that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter as defined in the appended claims.

[0316] Regarding the use of substantially any plural and / or singular terms herein, those skilled in the art may translate from plural to singular and / or from singular to plural depending on the context and / or application. For clarity, various singular / plural arrangements may be explicitly stated herein. It should be noted that, unless the context explicitly specifies otherwise, the singular forms used in this description and appended claims include plural references. Unless otherwise stated, any inclusion of “or” herein is intended to cover “and / or”.

[0317] Those skilled in the art will understand that, generally speaking, the terms used herein, and especially in the appended claims (e.g., the body of the appended claims), are generally intended to be “open” terms (e.g., the term “comprising” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” etc.). Those skilled in the art will also understand that if an intention is to introduce a particular number of claims, this intention will be explicitly stated in the claims, and without such a statement, this intention does not exist. For example, to aid understanding, the appended claims below may contain the use of the introductory phrases “at least one” and “one or more” to introduce the claim statement. However, the use of such phrases should not be construed as implying that a claim statement introduced by “one” limits any particular claim to only one embodiment of such a statement, even if the same claim includes the introductory phrases “one or more” or “at least one” (e.g., the absence of a quantifier before an element should be interpreted as “at least one” or “one or more”). Furthermore, even when a specific number of claims is explicitly listed, those skilled in the art will recognize that such a statement should be interpreted as indicating at least the listed number (e.g., a simple statement of "two statements" without other modifiers means at least two statements, or two or more statements). Moreover, in the use of conventions such as "at least one of A, B, and C, etc.", such a structure is generally intended to be understood by those skilled in the art in a certain sense (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems with: 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.). In the use of conventions such as "at least one of A, B, or C, etc.", such a structure is generally intended to be understood by those skilled in the art in a certain sense (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, systems with: 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 also understand that virtually any separating word or phrase representing two or more optional clauses, whether in the description, claims, or drawings, should be understood to preclude the possibility of including one, one, or both of the clauses. For example, the phrase “A or B” would be understood to include the possibility of “A” or “B” or “A and B”.

[0318] Furthermore, given that the features or aspects of this disclosure are described according to the Markush Group, those skilled in the art will recognize that this disclosure is also described according to any individual member or subgroup member of the Markush Group.

[0319] 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.

[0320] 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.

[0321] 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.

[0322] Therefore, the scope of the invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied in the appended claims. In the claims, 35 USC § 112(f) or 35 USC § 112(6) is expressly defined as being invoked only if the exact phrase “method is” or the exact phrase “step is” is used at the beginning of the limitation period of the claim; if such an exact phrase is not used in the limiting clause of the claim, then 35 USC § 112(f) or 35 USC § 112(6) is not invoked.

Claims

1. A reagent kit comprising: (a) Capturing magnetic beads, which contain a capturing portion that specifically binds to target particles; as well as (b) Barcode nucleic acid.

2. The kit according to claim 1, wherein the capturing magnetic beads comprise barcoded nucleic acids.

3. The kit according to claim 1, wherein the barcode nucleic acid is tethered to a barcode-bearing bead separated from the capture magnetic bead.

4. The kit according to any one of claims 1 to 3, wherein the kit further comprises: The apparatus includes: (i) A substrate containing 100 or more micropores; as well as (ii) A flow cell in fluid communication with the substrate.

5. The kit according to any one of claims 1 to 4, wherein the capture portion comprises an antibody or a binding fragment thereof.

6. The kit according to any one of claims 1 to 5, wherein the barcode nucleic acid further comprises a cell marker domain.

7. The kit according to any one of claims 1 to 6, wherein the barcode nucleic acid further comprises a unique molecular index field.

8. The kit according to any one of claims 1 to 7, wherein the barcode nucleic acid further comprises a universal primer binding domain.

9. The kit according to any one of claims 1 to 2, wherein the barcode nucleic acid comprises the following structure: bead-5'-universal primer binding domain-cell marker domain-unique molecular index domain-target binding region-3'.

10. The kit according to any one of claims 1 to 9, wherein the target binding region comprises an oligodT domain, a gene-specific domain, or a random sequence domain.

Images