Methods and compositions for obtaining associated image and sequence data of single cells

Abstract

Aspects of the invention include methods of obtaining associated images and sequence data of a single cell, such as a single cell of a cell sample. Embodiments of the method include combinatorily barcoding cells with a specific binding member / oligonucleotide barcode to produce combinatorily barcoded cells, e.g., obtained from an initial cell sample. The resulting combined barcoded cells are then partitioned to produce combined barcoded single cells each having a partition of a combined barcode. Image data and sequence data of the partitioned combined barcoded single cells are then obtained, and then the image data and sequence data sharing the common combined barcode are correlated to obtain correlated image and sequence data of the single cells of the cell sample. Compositions for practicing the methods of the invention are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 USC § 119(e) to the filing date of U.S. Provisional Patent Application Serial No. 63 / 332,087, filed on April 18, 2022; the disclosure of which is incorporated herein by reference in its entirety. Background Art

[0002] Current technology allows the measurement of gene expression of single cells in a massively parallel manner (e.g., >10,000 cells) by attaching cell-specific oligonucleotide barcodes to poly(A)mRNA molecules from individual cells, as each of the cells is co-localized in a compartment with a barcoding reagent bead. One platform that allows the measurement of gene expression of single cells in a massively parallel manner is the BD Rhapsody™ Single Cell Analysis System. The BD Rhapsody™ Single Cell Analysis System is a platform that allows high-throughput capture of nucleic acids from single cells using a simple cartridge workflow and a multi-layer barcoding system. The resulting captured information can be used to generate various types of next-generation sequencing (NGS) libraries, including libraries suitable for whole transcriptome analysis, e.g., for discovery biology and targeted RNA analysis for high-sensitivity transcript detection. Shum et al., “Quantitation of mRNA Transcripts and Proteins Using the BD Rhapsody™ Single-Cell Analysis System,” Adv Exp Med Biol, 2019;1129:63-79.

[0003] Gene expression may affect protein expression. Protein-protein interactions may affect both gene expression and protein expression. Therefore, systems and methods that can quantitatively analyze protein expression in cells and simultaneously measure protein expression and gene expression in cells have recently been developed. The BD Abseq platform is one such platform. AbSeq is a method for profiling proteins in single cells. In Abseq, the usual fluorophore-labeled antibodies are replaced by nucleic acid sequence tags that can be read out at the single cell level, for example, via barcoding and NGS sequencing. "The goal of Abseq is to enable sensitive, accurate, and comprehensive characterization of proteins in large numbers of single cells. Cells are bound to antibodies against different target epitopes, just like traditional immunostaining, except that the antibodies are labeled with unique sequence tags. When the antibody binds to its target, the DNA tag is carried along, allowing the presence of the target to be inferred based on the presence of the tag. Counting the tags in this way provides an estimate of the different epitopes present in the cells detected via antibody binding." Shahi et al., "Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding", Sci Rep 7, 44447 (2017). Summary of the invention

[0004] The inventors have realized that in single cell analysis (including single cell multi-omics applications), it is highly desirable to associate image data with massively parallel NGS data. The inventors are not aware of any current schemes for associating single cell imaging data with single cell multi-omics data from the same cell. Although single cell sorted (FACS) cells can be placed into large well plates (96 wells or other) first, and then a plate-based single cell multi-omics workflow is performed on the sorted cells, this does not provide image data associated with the NGS data of the cells. Plate-based workflows cannot provide the same throughput or efficiency as massively parallel single cell multi-omics workflows. In addition, the index data is not microscope-based, and the common flow cytometer data currently used lacks 2-dimensional (spatial) information. Embodiments of the present invention meet the needs of the art for methods and compositions for easily obtaining associated image and sequencing data of single cells.

[0005] Aspects of the invention include methods for obtaining associated images and sequence data of single cells, such as single cells of a cell sample. Embodiments of the method include: combinatorially barcoding cells with specific binding members / oligonucleotide sub-barcodes to produce combinatorially barcoded cells, such as cells obtained from an initial cell sample. Next, the resulting combinatorially barcoded cells are partitioned to produce combinatorially barcoded single cells of partitions each having a combinatorial barcode. Image data and sequence data of the partitioned combinatorially barcoded single cells are then obtained, and then the image data and sequence data sharing a common combinatorial barcode are associated to obtain associated images and sequence data of single cells of a cell sample. Compositions for practicing the methods of the invention are also provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The present invention can be best understood by reading the following detailed description in conjunction with the accompanying drawings, which include the following figures:

[0007] Figure 1 Schematically illustrates a segmented merged sample indexed to individual cells according to an embodiment of the present invention.

[0008] Figure 2 Schematically illustrates the segmentation / merging of ab-oligo markers on cells to generate single cell indices that can be read by imaging and downstream single cell multi-omics according to an embodiment of the present invention.

[0009] Figure 3 An example of decoding ab-oligo identity of single cells using cyclic immunofluorescence according to an embodiment of the present invention is provided.

[0010] definition

[0011] Unless otherwise defined, technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the present disclosure belongs. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd edition, J. Wiley & Sons (New York, NY, 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Spring Harbor, NY, 1989). For the purposes of this disclosure, the following terms are defined as follows.

[0012] As used herein, an antibody can be a full-length (e.g., naturally occurring or formed by normal immunoglobulin gene fragment recombination processes) immunoglobulin molecule (e.g., IgG antibody) or an immunologically active (i.e., specifically binding) portion of an immunoglobulin molecule, such as an antibody fragment. In some embodiments, the antibody is a functional antibody fragment. For example, an antibody fragment can be a part 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. Antibody fragments can include separated fragments consisting of antibody variable regions, such as "Fv" fragments consisting of heavy and light chain variable regions, and recombinant single-chain polypeptide molecules ("scFv proteins") in which the light and heavy chain variable regions are connected 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.

[0013] As used herein, the term "association" or "associated with..." can mean that two or more substances can be identified as being in the same place at a certain point in time. "Association" can mean that two or more substances are now or have been in a similar container. The association can be an informatics association. For example, digital information about two or more substances can be stored and can be used to determine that one or more of the substances are in the same place at a certain point in time. The association can also be a physical association. In some embodiments, two or more associated substances are "tethered", "attached" or "fixed" to each other or a common solid or semi-solid surface. Association can refer to a covalent or non-covalent means for attaching a label to a solid or semi-solid support (such as a bead). The association can be a covalent bond between a target and a label. The association can include hybridization between two molecules (such as a target molecule and a label).

[0014] As used herein, the term "complementarity" can refer to the ability of accurate pairing between two nucleotides. For example, if the nucleotide at a specific position of a nucleic acid can hydrogen bond with the nucleotide of another nucleic acid, the two nucleic acids are considered to be complementary to each other at that position. The complementarity between two single-stranded nucleic acid molecules may be "partial", in which only some of the nucleotides bind, or when there is full complementarity between single-stranded molecules, the complementarity may be complete. If the first nucleotide sequence is complementary to the second nucleotide sequence, the first nucleotide sequence can be said to be the "complement" of the second sequence. If the first nucleotide sequence is complementary to the reverse sequence (i.e., the nucleotide order is reversed) of the second sequence, the first nucleotide sequence can be said to be the "reverse complement" of the second sequence. As used herein, the terms "complement", "complementarity" and "reverse complement" can be used interchangeably. It can be understood from the present disclosure that if a molecule can hybridize with another molecule, it may be the complement of the molecule being hybridized.

[0015] As used herein, the term "nucleic acid" refers to a polynucleotide sequence or a fragment thereof. Nucleic acids may include nucleotides. For cells, nucleic acids may be exogenous or endogenous. Nucleic acids may be present 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 include one or more analogs (e.g., altered backbones, sugars, or nucleobases). Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acids, iso-nucleic acids, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-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, braided glycosides, and wyosine. "Nucleic acid," "polynucleotide," "target polynucleotide," and "target nucleic acid" may be used interchangeably.

[0016] Nucleic acids may include one or more modifications (e.g., base modifications, backbone modifications) to provide new or enhanced properties (e.g., improved stability) to the nucleic acid. Nucleic acids may include nucleic acid affinity tags. Nucleosides may be base-sugar combinations. The base portion of a nucleoside may be a heterocyclic base. The two most common types of such heterocyclic bases are purine and pyrimidine. Nucleotides may be nucleosides that further comprise a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides comprising pentafuranoses, the phosphate group may be linked to the 2', 3', or 5' hydroxyl portion of the sugar. When forming nucleic acids, the phosphate group may covalently link adjacent nucleosides to each other to form a linear polymeric compound. The corresponding ends of such linear polymeric compounds may further be linked to form cyclic compounds; however, linear compounds are generally suitable. In addition, linear compounds may have internal nucleotide base complementarity and may therefore be folded in a manner that produces a fully or partially double-stranded compound. In nucleic acids, phosphate groups are generally referred to as the inner skeleton of the nucleoside that forms the nucleic acid. This connection or skeleton may be a 3' to 5' phosphodiester connection.

[0017] Nucleic acid can include modified backbone and / or modified intranucleoside connection.The modified backbone can include the backbone that retains the phosphorus atom in the backbone and the backbone that does not have the phosphorus atom in the backbone.For example, the nucleic acid backbone of suitable modification containing phosphorus atom can include thiophosphate; chiral thiophosphate; phosphorodithioate; phosphotriester; aminoalkylphosphotriester; methylphosphonate and other alkylphosphonates, such as 3'-alkylenephosphonate, 5'-alkylenephosphonate; chiral phosphonate; phosphite; phosphoramidate, including 3'-aminophosphoramidate and aminoalkylphosphoramidate; diaminophosphorate; thiocarbonylphosphoramidate; thiocarbonylalkylphosphonate; thiocarbonylalkylphosphotriester; selenophosphate; and boranophosphate with normal 3'-5' connection; 2'-5' connected analogs; and those with reverse polarity, wherein one or more internucleotide connections are 3' to 3', 5' to 5' or 2' to 2' connection.

[0018] Nucleic acids can include polynucleotide backbones formed by short-chain alkyl or cycloalkyl nucleoside interlinking, mixed heteroatoms and alkyl or cycloalkyl nucleoside interlinking, or one or more short-chain heteroatomic or heterocyclic nucleoside interlinking. These can include those with the following structures: morpholino linkage (partially formed from the sugar moiety of nucleosides); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formyl acetyl and thioformyl acetyl backbones; methylene formyl acetyl and thioformyl acetyl backbones; ribose acetyl backbones; olefin-containing backbones; aminosulfonate backbones; methylene imino and methylene hydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones with mixed N, O, S and CH2 component parts.

[0019] Nucleic acids can include nucleic acid mimics. The term "mimetic" can be intended to include polynucleotides in which only the furanose ring or both the furanose ring and the internucleotide connection are replaced by non-furanose groups, and only the replacement of the furanose ring can also be referred to as a sugar substitute. The heterocyclic base portion or the modified heterocyclic base portion can be retained in order to hybridize with an appropriate target nucleic acid. One such nucleic acid can be a peptide nucleic acid (PNA). In PNA, the sugar backbone of the polynucleotide can be replaced by an amide-containing backbone (particularly an aminoethylglycine backbone). Nucleotides can be retained and directly or indirectly combined with the aza nitrogen atoms of the amide portion of the backbone. The backbone in the PNA compound can include two or more connected aminoethylglycine units, which give the PNA an amide-containing backbone. The heterocyclic base portion can be directly or indirectly combined with the aza nitrogen atoms of the amide portion of the backbone.

[0020] Nucleic acid can include morpholino backbone structure.For example, nucleic acid can include 6 yuan of morpholino rings replacing ribose ring.In some of these embodiments, phosphorodiamidate or other non-phosphodiester internucleoside connections can replace phosphodiester connections.

[0021] Nucleic acids can include morpholino units (e.g., morpholino nucleic acids) having a connection to a heterocyclic base attached to a morpholino ring. A linking group can connect morpholino monomer units in morpholino nucleic acids. Nonionic morpholino-based oligomeric compounds can have fewer undesirable interactions with cellular proteins. Morpholino-based polynucleotides can be nonionic mimics of nucleic acids. Various compounds in the morpholino category can be connected using different linking groups. Another class of polynucleotide mimics can be called cyclohexenyl nucleic acids (CeNA). The furanose rings that are usually present in nucleic acid molecules can be replaced by cyclohexenyl rings. CeNA DMT-protected phosphoramidite monomers can be prepared and used for oligomeric compound synthesis using phosphoramidite chemistry. Incorporating CeNA monomers into nucleic acid chains can increase the stability of DNA / RNA hybrids. CeNA oligoadenylates can form complexes with nucleic acid complements having stability similar to that of natural complexes. Additional modifications may include locked nucleic acids (LNA), in which the 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring, thereby forming a 2'-C,4'-C-oxymethylene linkage, thereby forming a bicyclic sugar moiety. The linkage may be a methylene (-CH2) group bridging the 2' oxygen atom and the 4' carbon atom, where n is 1 or 2. LNA and LNA analogs may exhibit very high duplex thermal stability (Tm = +3°C to +10°C) with complementary nucleic acids, stability to 3'-exonucleolytic degradation, and good solubility properties.

[0022] Nucleic acids can also include modifications or substitutions of nucleobases (often referred to simply as "bases"). As used herein, "unmodified" or "natural" nucleobases can include purine bases (e.g., adenine (A) and guanine (G)) and pyrimidine bases (e.g., thymine (T), cytosine (C), and uracil (U)). Modified nucleobases can include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C=C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil. uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. The modified nucleobase may comprise a tricyclic pyrimidine such as phenoxazine cytidine (1H-pyrimido (5,4-b) (1,4) benzoxazin-2 (3H) -one), phenothiazine cytidine (1H-pyrimido (5,4-b) (1,4) benzothiazin-2 (3H) -one), a G-clamp such as a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido (5,4-(b) (1,4) benzoxazin-2 (3H) -one), a phenothiazine cytidine (1 H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamp such as substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3',2':4,5)pyrrolo[2,3-d]pyrimidin-2-one).

[0023] As used herein, the term "sample" may refer to a composition including a target. Suitable samples for analysis by the disclosed methods, devices, and systems include cells, tissues, organs, or organisms. A cell sample is a composition composed of a plurality of cells, such as a composition comprising a plurality of different cells, such as an aqueous composition of a single cell, wherein the number of cells may vary.

[0024] As used herein, the term "sampling device" or "device" may refer to a device that can take a portion of a sample and / or place the portion on a substrate. The sampling device may refer to, for example, a fluorescence activated cell sorting (FACS) machine, a cell sorter, a biopsy needle, a biopsy device, a tissue sectioning device, a microfluidic device, a cascade, and / or an ultramicrotome.

[0025] As used herein, the term "solid support" may refer to a discrete solid or semi-solid surface to which nucleic acids can be attached. A solid support may encompass any type of solid, porous or hollow sphere, ball, socket, cylinder or other similar configuration, composed of plastic, ceramic, metal or polymeric material (e.g., hydrogel), on which nucleic acids can be fixed (e.g., covalently or non-covalently). A solid support may include discrete particles that may be spherical (e.g., microspheres) or have a non-spherical or irregular shape, such as a cube, rectangle, cone, cylinder, cone, ellipse or disc, etc. The shape of the bead may be non-spherical. A plurality of solid supports spaced apart in an array may not include a substrate. A solid support may be used interchangeably with the term "bead".

[0026] As used herein, the term "target" may refer to a composition that can be analyzed according to embodiments of the invention. Exemplary suitable targets for analysis by the disclosed methods, devices, and systems include oligonucleotides, DNA, RNA, mRNA, microRNA, tRNA, etc. The target may be single-stranded or double-stranded. In some embodiments, the target may be a protein, peptide, or polypeptide. In some embodiments, the target is a lipid. As used herein, "target" may be used interchangeably with "substance."

[0027] As used herein, the term "reverse transcriptase" may refer to a group of enzymes having reverse transcriptase activity (i.e., catalyzing the synthesis of DNA from an RNA template). Typically, such enzymes include, but are not limited to, retroviral reverse transcriptases, retrotransposon reverse transcriptases, reverse transcriptases of plasmid reverse transcriptases, reverse transcriptases of bacteria, reverse transcriptases derived from type II introns, and their mutants, variants, or derivatives. Non-retroviral reverse transcriptases include non-LTR retrotransposon reverse transcriptases, reverse transcriptases of plasmid reverse transcriptases, reverse transcriptases of type II introns, and reverse transcriptases of type II introns. Examples of type II intron reverse transcriptases include Lactococcus lactis LI.LtrB intron reverse transcriptases, Thermosynechococcus elongatus TeI4c intron reverse transcriptases, or Geobacillus stearothermophilus GsI-IIC intron reverse transcriptases. Other types of reverse transcriptases may include many types of non-retroviral reverse transcriptases (i.e., retroviral, group II intron, and diversity-generating retroelements, etc.). DETAILED DESCRIPTION

[0028] Aspects of the invention include methods for obtaining associated images and sequence data of single cells, such as single cells of a cell sample. Embodiments of the method include: combinatorially barcoding cells with specific binding members / oligonucleotide sub-barcodes to produce combinatorially barcoded cells, such as cells obtained from an initial cell sample. Next, the resulting combinatorially barcoded cells are partitioned to produce combinatorially barcoded single cells of partitions each having a combinatorial barcode. Image data and sequence data of the partitioned combinatorially barcoded single cells are then obtained, and then the image data and sequence data sharing a common combinatorial barcode are associated to obtain associated images and sequence data of single cells of a cell sample. Compositions for practicing the methods of the invention are also provided.

[0029] Before describing the present invention in more detail below, it should be understood that the present invention is not limited to the specific embodiments described, as such may of course vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0030] Where a numerical range is provided, it is understood that each intervening value (based on one-tenth of the unit of the lower limit unless the context clearly dictates otherwise) between the upper and lower limits of that range and any other stated or intervening value in that range is included in the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included in the invention, subject to any specifically excluded limits in the stated range. Where the stated range includes one or both limits, ranges excluding one or both of those included limits are also included in the invention.

[0031] Certain ranges appearing herein are presented by preceding the numerical value with the term "about". The term "about" is used herein to provide literal support for the exact number that follows it, as well as a number that is close to or approximately the number that follows the term. In determining whether a number is close to or approximately a specifically cited number, the close or approximate unquoted number may be a number that provides a substantial equivalent of the specifically cited number in the context in which it appears.

[0032] 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 the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0033] All publications and patents cited in this specification are incorporated herein by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and discloses and describes the methods and / or materials related thereto. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0034] It is noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is also noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for use of exclusive terminology such as "only," "only" and the like in connection with the recitation of claim elements or use of a "negative" limitation.

[0035] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method may be performed in the order of events recited or in any other order that is logically possible.

[0036] Although the system and method have been or will be described for the sake of grammatical fluency and functional interpretation, it should be clearly understood that unless expressly recited pursuant to 35 USC §112, the claims should not be construed as necessarily limited in any way to the "means" or "step" limitations, but should be given the full scope of meaning and equivalents provided by the claims pursuant to the judicial doctrine of equivalents, and to the extent a claim is expressly recited pursuant to 35 USC §112, it will be given full legal equivalents pursuant to 35 USC §112.

[0037] method

[0038] As summarized above, a method for obtaining associated image and sequence data of a single cell is provided, the single cell being, for example, a single cell of an initial cell sample. Associated image and sequence data means a combined data set comprising both image data and nucleic acid sequence data, which data set can be attributed to the same cell so that it can be considered to be derived from the same cell. In other words, associated image and sequence data are data sets comprising both image data and nucleic acid sequence data obtained from the same cell. Image data is data obtained from a cell using an imaging technique. The term "image" is used in its conventional sense to refer to a representation of an object (e.g., a cell) produced by radiation (e.g., via illumination). Image data is data that together constitutes the representation, and can be data obtained using any convenient scheme. In some embodiments, the image data obtained in the method of the present invention is microscopic image data. Microscopic image data refers to image data obtained when an object and an object region (e.g., a cell) that cannot be seen by the naked eye are observed using a microscope. Nucleic acid sequence data refers to data obtained using nucleic acid sequencing technology, which identifies the sequence of nucleotides in a nucleic acid molecule. Nucleic acid sequencing data from a cell includes the sequence of one or more nucleic acid sequences (e.g., RNA molecules) present in the cell. Such data can be obtained using a variety of sequencing protocols, including next-generation sequencing (NGS).

[0039] As summarized above, aspects of the method include: combinatorially barcoding cells of a cell sample with specific binding member / oligonucleotide sub-barcodes to produce combinatorially barcoded cells; partitioning the combinatorially barcoded cells to produce partitioned combinatorially barcoded single cells each having a combinatorial barcode; obtaining image data and sequence data of the partitioned combinatorially barcoded single cells; and associating image data and sequence data sharing a common combinatorial barcode; to obtain associated image and sequence data of single cells of the cell sample. Embodiments of each of these steps will now be described in more detail.

[0040] Combinatorial barcoding of cells from a cell sample using specific binding members / oligonucleotide sub-barcodes

[0041] Embodiments of the method include barcoding the cell combination of a cell sample with specific binding members / oligonucleotide sub-barcodes. Combination barcoded cells mean cells that modify the initial cell sample so that they are stably associated with a unique sub-barcode combination (provided by a combination of specific binding members / oligonucleotide sub-barcodes), which together constitute a unique combination barcode of the cell. Stable association with it means that the specific binding members / oligonucleotide sub-barcodes of a given combination barcode of a cell constituting the combination barcode are attached to the surface of the cell, and the attachment method makes the specific binding members / oligonucleotide sub-barcodes not dissociate from the cell during the conditions experienced by the cell under the method of the present invention, for example, as described in more detail below. In some cases, stable association is provided by specific binding interactions, for example, as described in more detail below. In the combinatorially barcoded cells of the embodiments of the present invention, a combinatorial scheme that associates a unique combination of specific binding members / oligonucleotide sub-barcodes with a given cell is used to stably associate a unique combination of sub-barcodes with the cell, wherein the unique combination is obtained from an initial set of specific binding members / oligonucleotide sub-barcodes. The combinatorial scheme employed in the embodiments of the present invention includes a split / pool scheme, for example, as described in more detail below.

[0042] The specific binding member / oligonucleotide sub-barcode provides a sub-barcode for the cell, which together provides a unique combination barcode. The specific binding member / oligonucleotide sub-barcode comprises a specific binding member component and an oligonucleotide sub-barcode component, wherein the specific binding member component and the oligonucleotide sub-barcode component are stably associated with each other, for example, by an appropriate bond or linking group (e.g., a covalent bond). Therefore, the specific binding member / oligonucleotide sub-barcode can be regarded as having a specific binding member conjugated to the oligonucleotide sub-barcode component. Embodiments of each of these components will now be described in more detail.

[0043] The specific binding member components of the specific binding members / oligonucleotide sub-barcodes used in the embodiments of the present invention can vary. The term "specific binding" refers to the direct association between two molecules due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bond interactions (including interactions such as salt bridges and water bridges). Specific binding members describe members of a pair of molecules that have binding specificity for each other. The members of a specific binding pair can be naturally derived, or produced in whole or in part synthetically. One member of a molecular pair has an area or cavity on its surface that specifically binds to and is complementary to a specific spatial and polar organization of another member of the molecular pair. Therefore, the members of the pair have the property of specific binding to each other. Examples of specific binding member pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. The specific binding members of the binding pair exhibit high affinity and binding specificity for binding to each other. Typically, the affinity between paired specific binding members is expressed by K. d (dissociation constant), which is 10 -6 M or lower, such as 10 -7 M or less, including 10 -8 M or lower, e.g. 10 -9 M or lower, 10 -10 M or lower, 10 -11 M or lower, 10 -12 M or lower, 10 -13 M or lower, 10 -14 M or less, including 10 -15M or lower. "Affinity" refers to the strength of binding, and increased binding affinity is associated with a lower KD. In an embodiment, affinity is determined by surface plasmon resonance (SPR), for example, as used by a Biacore system. The affinity of one molecule to another molecule is determined by, for example, measuring the binding kinetics of the interaction at 25 ° C. "Affinity" refers to the strength of binding, and increased binding affinity is associated with a lower KD. In an embodiment, affinity is determined by surface plasmon resonance (SPR), for example, as used by a Biacore system. The affinity of one molecule to another molecule is determined by, for example, measuring the binding kinetics of the interaction at 25 ° C. Specific binding members can vary, wherein examples of specific binding members include but are not limited to polypeptides, nucleic acids, carbohydrates, lipids, peptoids, etc. In some cases, specific binding members are proteinaceous. As used herein, the term "proteinaceous" refers to a portion consisting of amino acid residues. A portion of a protein can be a polypeptide. In some cases, a specific binding member of a protein is an antibody. In certain embodiments, a specific binding member of a protein is an antibody fragment, for example, a binding fragment of an antibody that specifically binds to a polymerized dye. As used herein, the terms "antibody" and "antibody molecule" are used interchangeably and refer to a protein consisting of one or more polypeptides that are substantially encoded by all or part of the recognized immunoglobulin genes. The recognized immunoglobulin genes, for example in humans, include kappa (k), lambda (l) and heavy chain loci, which together include numerous variable region genes and constant region genes μ (u), δ (d), γ (g), σ (e) and α (a), which encode IgM, IgD, IgG, IgE and IgA isotypes, respectively. The immunoglobulin light or heavy chain variable region consists of a "framework" region (FR) interrupted by three hypervariable regions (also called "complementarity determining regions" or "CDRs"). The extent of the framework region and CDR has been precisely defined (see "Sequences of Proteins of Immunological Interest", E. Kabat et al., U.S. Department of Health and Human Services, (1991)). The numbering of all antibody amino acid sequences discussed herein conforms to the Kabat system. The framework region sequences of different light or heavy chains are relatively conserved within the substance. The framework region of an antibody, i.e., the combined framework region of the light and heavy chains that constitute it, is used to position and arrange the CDR. The CDR is primarily responsible for binding to the epitope of the antigen. The term antibody is intended to include full-length antibodies and may refer to natural antibodies from any organism, engineered antibodies, or antibodies recombinantly generated for experiments, treatments, or other purposes further defined below.Target antibody fragments include but are not limited to Fab, Fab', F(ab')2, Fv, scFv or other antigen binding subsequences of antibodies, which are either produced by modifying complete antibodies or synthesized de novo using recombinant DNA technology. Antibodies can be monoclonal or polyclonal, and can have other specific activities (e.g., antagonists, agonists, neutralizing, inhibitory or stimulating antibodies) to cells. It should be understood that antibodies can have additional conservative amino acid substitutions, which have substantially no effect on antigen binding or other antibody functions. In certain embodiments, specific binding members are Fab fragments, F(ab')2 fragments, scFv, double antibodies or three antibodies. In certain embodiments, specific binding members are antibodies. In some cases, specific binding members are mouse antibodies or their binding fragments. In some cases, specific binding members are recombinant antibodies or their binding fragments.

[0044] The specific binding member / oligonucleotide sub-barcode can specifically bind to any convenient cell marker. In some cases, the specific binding member / oligonucleotide sub-barcode binds to a cell surface marker, wherein the target cell surface marker includes but is not limited to a ubiquitous cell surface marker, i.e., a cell surface marker that is at least predicted to be on all cells of a given cell sample to be processed in a given workflow according to the present invention. Tests of ubiquitous cell surface markers that can specifically bind to specific binding members / oligonucleotide sub-barcodes include but are not limited to: CD44, CD45, beta-2 microglobulin, etc.

[0045] In addition to the specific binding member component, the specific binding member / oligonucleotide sub-barcode also includes an oligonucleotide sub-barcode component. The length of the oligonucleotide sub-barcode component can vary, ranging from 10 nt to 500 nt in some cases, such as 15 nt to 100 nt. In some cases, as required, the oligonucleotide sub-barcode component can be composed of ribonucleic acid or deoxyribonucleic acid. The oligonucleotide sub-barcode of an embodiment of the present invention can include an image marking area, as well as other domains that can be used for embodiments of the present invention, wherein such domains can include a unique identifier of a specific binding member, a capture sequence, a primer binding site, etc.

[0046] The image marker region of the oligonucleotide sub-barcode component is a domain or subsequence, i.e., a segment, of the oligonucleotide sub-barcode component that serves as a specific binding site for a labeled oligonucleotide employed in the imaging step of an embodiment of the present invention, for example, as described in more detail below. The sequence of the image marker region can be used as an identifier for a label (such as a fluorescent label) of a labeled oligonucleotide hybridized to the image marker region. Thus, the sequence of the image marker region corresponds to the label of the labeled oligonucleotide bound to the image marker region. The image marker region can have any convenient sequence and vary in length, in some cases ranging from 5 nt to 100 nt, such as 10 nt to 50 nt. A given oligonucleotide sub-barcode component can contain a single image marker region, or two or more image marker regions, such as three or more image marker regions, wherein in some cases, the number of image marker regions ranges from one to five, such as two to three.

[0047] In addition to the image marking area, the oligonucleotide sub-barcode component can also include one or more of a unique identifier of a specific binding member, a capture sequence, a primer binding site, etc. The unique identifier of a specific binding member can be used, for example, for identifying a domain or region of a specific binding member by its sequence. The unique identifier can be, for example, a nucleotide sequence with any suitable length, such as about 4 nucleotides to about 200 nucleotides. In certain embodiments, the unique identifier is a nucleotide sequence with a length of 25 nucleotides to about 45 nucleotides. In certain embodiments, the length that the unique identifier can have is below, about below, less than, greater than: 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 200 nucleotides, or in the range between any two of the above values.

[0048] The oligonucleotide component can include a capture sequence, for example, a domain or region that acts as a binding site for a target binding region, for example, a target binding region of a bead-bound barcode nucleic acid, such as described above. As desired, the target capture sequence can vary and can be specific or random or semi-random. In some cases, the capture sequence hybridizes with the target binding region of the bead-bound nucleic acid, for example, as described in more detail below. In some cases, the capture sequence is a poly (A) sequence that is configured to hybridize with the oligodT target binding region, such as described in more detail below. In such cases, the length of the poly (A) capture sequence can vary, ranging from 3 nt to 50 nt in some cases, such as 5 nt to 25 nt. The capture sequence can be positioned at the 5' end of the oligonucleotide component when present.

[0049] The oligonucleotide component can include a primer binding site. The primer binding site can be configured to bind to a primer, such as one used in preparing a sequenceable nucleic acid, when present. For example, the oligonucleotide component can include a universal primer. A universal primer can refer to a universal or common nucleotide sequence in all specific binding members / oligonucleotide sub-barcodes used in a given workflow. In some cases, the length of the primer binding site can be as follows or about as follows: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or any two of these nucleotides or a range. The length of the primer binding site can vary, and can be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The length of the universal primer can vary, and in some cases can range from 5 to 30 nucleotides in length. The primer binding site can be positioned at the 5' end of the oligonucleotide sub-barcode component.

[0050] As described above, in specific binding members / oligonucleotide sub-barcodes, specific binding members are conjugated to oligonucleotide sub-barcode components. Oligonucleotide components can be conjugated to specific binding members components by various mechanisms. In certain embodiments, oligonucleotide components can be covalently conjugated to specific binding members components. In certain embodiments, oligonucleotide components can be non-covalently conjugated to specific binding members components. In certain embodiments, oligonucleotide components are conjugated to specific binding members component reagents through joints. Joints can be, for example, cuttable or separable with specific binding members and / or oligonucleotide components. In certain embodiments, joints can include chemical groups that reversibly attach oligonucleotides to specific binding members. Chemical groups can be conjugated to joints, for example, by amine groups. In certain embodiments, joints can include chemical groups that form stable bonds with another chemical group conjugated to specific binding members components. For example, chemical groups can be UV light cleavable groups, disulfide bonds, streptavidin, biotin, amines, etc. In certain embodiments, chemical groups can be conjugated to specific binding members components by primary amines or N-termini on amino acids such as lysine. Commercially available conjugation kits, such as Protein-Oligo conjugation kits (Solulink, Inc., San Diego, California), Thunder-Link® oligo conjugation systems (Innova Biosciences, Cambridge, United Kingdom), etc., can be used to conjugate oligonucleotide components to specific binding member components. Oligonucleotide components can be conjugated to any suitable site of a specific binding member component (e.g., a protein binding reagent), as long as it does not interfere with the specific binding between the specific binding member component and its cellular component target. The method of conjugating an oligonucleotide to a specific binding member (e.g., an antibody) has previously been disclosed in, for example, U.S. Patent No. 6,531,283, the contents of which are incorporated herein by reference. The stoichiometry of the oligonucleotide for a specific binding member can vary.

[0051] Further details regarding specific binding members / oligonucleotide sub-barcode reagents and components thereof that can be used in embodiments of the present invention are provided in U.S. Patent Application Publication No. US2018 / 0088112; U.S. Patent Application Publication No. 2018 / 0200710; U.S. Patent Application Publication No. US2018 / 0346970; U.S. Patent Application Publication No. 2019 / 0056415; U.S. Patent Application Publication No. US 2020 / 0248263; U.S. Patent Application Publication No. 2020 / 0299672; and U.S. Patent Application Publication No. 2021 / 0171940, the disclosures of which are incorporated herein by reference.

[0052] A given combinatorial barcoded cell may comprise one or more specific binding members / oligonucleotide sub-barcodes stably associated therewith. In some cases, a given combinatorial barcoded cell comprises a plurality of, i.e., two or more different specific binding members / oligonucleotide sub-barcodes stably associated therewith, wherein the different specific binding members / oligonucleotide sub-barcodes differ from one another at least with respect to the cell markers (e.g., cell surface proteins) to which they specifically bind. In some cases, the number of different specific binding members / oligonucleotide sub-barcodes stably associated with a combinatorially labeled cell ranges from two to ten, such as from two to five, for example, from three to four.

[0053] In embodiments of the methods of the present invention, the cells of a cell sample may be combinatorially barcoded using any convenient scheme. In some cases, combinatorial barcoding includes one or more split / merge iterations that sequentially contact the cells of the cell sample with different specific binding members / oligonucleotide sub-barcodes. In some cases, each split / merge iteration includes: allocating the cells of the cell sample into different compartments; introducing different (i.e., distinct) specific binding members / oligonucleotide sub-barcodes that differ from each other by oligonucleotide sub-barcode components into different compartments to generate sub-barcoded cells; and merging sub-barcoded cells of different compartments.

[0054] In a given segmentation / merging iteration, the cells of a cell sample are distributed in different compartments so that they are partitioned from each other. The number of cells distributed in different compartments therein can vary, and in some cases the range is 5 to 1,000, such as 5 to 500, including 5 to 100, for example 25 to 100. In some cases, the compartment is present in substrate, such as the hole of a orifice plate, such as a large orifice plate. The example of the orifice plate to which the cell sample can be distributed comprises 36 orifice plates, 96 orifice plates and 384 orifice plates, wherein in some embodiments, the orifice plate is 36 or 96 orifice plates. In order to distribute the cells of a cell sample in different compartments, any convenient scheme can be adopted, for example, the aliquots of a cell sample are distributed such as being pipetted in the compartment, so that the sample is flowed on the surface of the orifice plate, etc.

[0055] After the distribution of cells, different specific binding members / oligonucleotide sub-barcodes that differ from each other due to oligonucleotide sub-barcode components are introduced into different compartments to produce sub-barcoded cells. Different specific binding members / oligonucleotide sub-barcodes can be introduced into each compartment so that the cells of different compartments are stably associated with the specific binding members / oligonucleotide sub-barcodes introduced into those compartments. In this way, the cells of different compartments are stably associated with different specific binding members / oligonucleotide sub-barcodes. In this step, the number of different specific binding members / oligonucleotide sub-barcodes introduced into different compartments can vary, and in some cases the range is 5 to 1,000, such as 5 to 500, wherein the number is similar to the number of compartments in some cases. The compartmentalized cells stably associated with specific binding members / oligonucleotide sub-barcodes can be referred to as sub-barcoded cells.

[0056] After the sub-barcoded cells are generated, the sub-barcoded cells of the different compartments can be combined or merged, for example to generate a combined composition of the sub-barcoded cells. The sub-barcoded cells can be combined or merged using any convenient scheme. For example, the liquid compositions of the different compartments are recovered from the compartments and combined, for example, into a suitable tube of sufficient volume.

[0057] Each split / sub-barcode / merge sequence in a given combinatorial labeling workflow can be referred to as an iteration. A given combinatorial labeling workflow can have any desired number of iterations, where more iterations provide more complex barcodes and there are greater numbers of cells that can be processed in a given assay. In some cases, the number of split / merge iterations ranges from two to ten, such as two to five.

[0058] Combinatorially barcoded cells are partitioned to generate combinatorially barcoded single cells each having a combinatorial barcode in the partitions

[0059] After generating the combinatorial barcoded cells, for example as described, embodiments of the method include partitioning the combinatorial barcoded cells to generate partitioned combinatorial barcoded single cells each having a combinatorial barcode. In some cases, partitioning includes allocating the combinatorial barcoded cells into partitions or compartments such that the compartments contain a single combinatorial barcoded cell. Partitioning means placing the combinatorial barcoded cells into small reaction chambers, which can be fluid-divided structures defined by solid materials, such as microwells configured to accommodate combinatorial barcoded cells. In some embodiments of the disclosed methods, devices, and systems, a plurality of microwells randomly distributed across a substrate are used. In some embodiments, the plurality of microwells are distributed on a substrate in an ordered pattern, such as an ordered array. In some embodiments, the plurality of microwells are distributed on a substrate in a random pattern, such as a random array. The microwells can be manufactured in a variety of shapes and sizes. Suitable hole geometry includes but is not limited to cylindrical, elliptical, cubic, conical, hemispherical, rectangular or polyhedral, for example, a three-dimensional geometry consisting of several planes, for example, a rectangular cuboid, a hexagonal column, an octagonal column, an inverted triangular pyramid, an inverted tetrahedral pyramid, an inverted pentagonal pyramid, an inverted hexagonal pyramid or an inverted truncated pyramid. In certain embodiments, non-cylindrical micropores, such as holes with an elliptical or square footprint, can provide advantages in terms of being able to accommodate larger cells. In certain embodiments, the upper edge and / or lower edge of the pore wall can be rounded to avoid sharp corners, and thereby reduce the electrostatic force that may rise at sharp edges or points due to the concentration of the electrostatic field. Therefore, the use of rounded corners can improve the ability to recover beads from micropores. Micropore size can be characterized in terms of absolute size. In some cases, the average diameter range of the micropore 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 still 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 methods of the present invention can vary, in some cases ranging from about 200 μm. 3 to about 800,000 μm 3 In some embodiments, the micropore volume is at least 200 μm 3 , at least 500 μm 3, at least 1,000 μm 3 , at least 10,000 μm 3 , at least 25,000 μm 3 , at least 50,000 μm 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 700,000 μm 3 , up to 600,000 μm 3 , 500,000 μm 3 , up to 400,000 μm 3 , up to 300,000 μm 3 , up to 200,000 μm 3 , up to 100,000 μm 3 , up to 50,000 μm 3 , up to 25,000 μm 3 , up to 10,000 μm 3 , up to 1,000 μm 3 , up to 500 μm 3 or up to 200 μm 3. The number of micropores in a given device employed in embodiments of the present invention may vary, wherein in some cases, the number is 100 or more, such as 250 or more, such as 500 or more, including 1000 or more, such as 5,000 or more, such as 10,000 or more, wherein in some cases, the number is 15,000 or less, such as 12,500 or less. Micropores suitable for embodiments of the present invention are further described in PCT application serial number PCT / US2016 / 014612 disclosed as WO / 2016 / 118915, the disclosure of which is incorporated herein by reference. As used herein, a substrate may refer to a solid support type. A substrate may, for example, include a plurality of micropores. For example, a substrate may be a well array including two or more micropores. In some embodiments, a micropore may include a small reaction chamber of a defined volume. In some embodiments, a micropore may entrap one or more cells. In some embodiments, a micropore may entrap only one cell. In some embodiments, a micropore may entrap one or more solid supports. In some embodiments, a micropore may entrap only one solid support. In some embodiments, a microwell captures a single cell and a single solid support (e.g., a bead). Although the number of wells, e.g., microwells, in a well plate, e.g., a microwell array, can vary in a given dispensing step, in some cases, the number ranges from 5 to 500, such as 5 to 100.

[0060] When the combinatorial barcoded cells are partitioned, any convenient scheme can be used to position the combinatorial barcoded cells in compartments (e.g., microwells of a microwell array). The present disclosure provides methods for compartmentalizing the combinatorial barcoded cells into compartments so as to partition the combinatorial barcoded cells. For example, a collection of combinatorial barcoded cells can be introduced into a structure (e.g., a microwell) to partition the combinatorial barcoded cells. The combinatorial barcoded cells can be contacted, for example, by gravity flow, wherein the combinatorial barcoded cells can settle into the partition structure. In some cases, an aqueous composition of the combinatorial barcoded cells is contacted, for example, by flowing it through a microwell array, so that the combinatorial barcoded cells are deposited into the microwells. The aqueous composition containing the combinatorial barcoded cells can flow through a flow cell that is fluidly connected to the microwell. Suitable protocols and systems for partitioning capture particles into microwells are described in microwells suitable for use in embodiments of the present invention, and further described in PCT application serial number PCT / US2016 / 014612, published as WO / 2016 / 118915, the disclosure of which is incorporated herein by reference. To partition cells of a cell sample, any convenient protocol may be employed, for example, dispensing aliquots of the cell sample such as pipetting into compartments, flowing the sample over the surface of a well plate, etc.

[0061] In some embodiments, partitioning the plurality of combinatorially barcoded cells further comprises providing particles (e.g., beads) comprising particle (e.g., bead)-bound nucleic acids to the partitions comprising single cells, wherein the bound nucleic acids are used to prepare a nucleic acid sequence-ready composition, such as a sequence-ready library, from the combinatorially barcoded cells. In some cases, the particle (e.g., bead)-bound nucleic acids comprise a target binding region that, for example, binds to a complementary sequence in a target nucleic acid material in the combinatorial cells and a sequence of a capture oligonucleotide sub-barcode component. For example, where the target nucleic acid material is cellular mRNA and the oligonucleotide sub-barcode component comprises a poly(A) capture sequence, the bead-bound nucleic acid may comprise a poly(T) domain as a target binding region. In addition to the target binding region, many of the bound nucleic acids further comprise one or more additional domains, such as, but not limited to, a cell marker domain, a barcode domain, a molecular indexing domain (e.g., a unique molecular identifier (UMI) domain), a universal primer binding domain, and the like. Further details about particles with bound nucleic acids that can be provided in compartments can be found in U.S. Patent Application Publication No. US2018 / 0088112; U.S. Patent Application Publication No. 2018 / 0200710; U.S. Patent Application Publication No. US2018 / 0346970; U.S. Patent Application Publication No. 2019 / 0056415; U.S. Patent Application Publication No. US 2020 / 0248263; U.S. Patent Application Publication No. 2020 / 0299672; and U.S. Patent Application Publication No. 2021 / 0171940, the disclosures of which are incorporated herein by reference. Beads with bound nucleic acids can be provided in compartments using any convenient scheme, including but not limited to those described above for partitioning cells, and further described in PCT Application Serial No. PCT / US2016 / 014612, published as WO / 2016 / 118915, the disclosures of which are incorporated herein by reference. As desired, particles (eg, beads) can be partitioned into cells before or after, or in some cases in combination with, combining the barcoded cells.

[0062] Obtaining combined image and sequence data of partitioned barcoded single cells

[0063] As summarized above, after generating the partitioned combinatorial barcoded single cells each having a combinatorial barcode, image data and sequence data of the partitioned combinatorial barcoded single cells are obtained. In an embodiment, image data of the partitioned barcoded single cells are obtained before obtaining sequence data of the partitioned combinatorial barcoded single cells.

[0064] Image data acquisition

[0065] The combined barcoded single cells of the partitions can be imaged using any convenient scheme to obtain image data of the single cells of the partitions. The image data obtained can vary. Image data of any combined barcoded cells of the target can be obtained, and obtained from the partition containing the combined barcoded cells of the target. The type of image data obtained can vary, and can include live cell image data. Any convenient scheme can be used to obtain image data of the combined barcoded cells in the partitions, wherein examples of imaging schemes that can be used include but are not limited to microscope imaging schemes, such as phase contrast microscopy, fluorescence microscopy, quantitative phase contrast microscopy, holographic tomography, BD Rhapsody system, etc. For example, images can be generated by fluorescence imaging. Imaging can include microscopy, such as bright field imaging, oblique illumination, dark field imaging, dispersion coloring, phase contrast, differential interference contrast, interference reflection microscopy, fluorescence, confocal and single plane illumination, or any combination thereof. Imaging can include imaging a portion of the sample (e.g., a slide / array). Imaging can include imaging at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the cells of the partition. In some cases, imaging can be completed in discrete steps (e.g., images may not need to be continuous). Imaging can include taking at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more different images. Imaging can include taking at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more different images. In the case of expectation, the image data can include images taken from two or more different imaging iterations, wherein each imaging iteration includes a labeling step and an imaging step thereafter. In such cases, obtaining image data from the cells of the partition can be considered as a cyclic imaging step.

[0066] In an embodiment of the present invention, obtaining image data of a partitioned combinatorially barcoded single cell includes obtaining a partition-specific fluorescent barcode of a target partition and thereby obtaining a partition-specific fluorescent barcode of the combinatorially barcoded cells present therein. Therefore, the method may include, for each target combinatorially barcoded cell, obtaining a partition-specific fluorescent barcode of the partition containing the cell and thereby obtaining a partition-specific fluorescent barcode of the cell. A partition-specific fluorescent barcode is a collection of fluorescent signals obtained in a given partition that corresponds to an image-marked region of the combinatorial barcode of the cells in the partition. In some cases, the collection of fluorescent signals that makes up a given barcode has a specific sequence, such as a time series that corresponds to the time at which the time series was obtained in a given workflow and / or in the position of an image-marked region on an oligonucleotide sub-barcode component corresponding to a given fluorescent signal of a barcode.

[0067] In some cases, the partition-specific fluorescent barcode is obtained by two or more imaging iterations, such as two to twenty iterations, including two to ten iterations, wherein each imaging iteration comprises: contacting the partitioned combinatorial barcoded single cells with one or more labeled oligonucleotides bound to the image tag region of the oligonucleotide sub-barcode component of the specific binding member / oligonucleotide sub-barcode to produce a labeled partitioned combinatorial barcoded single cell; and capturing an image of the labeled partitioned combinatorial barcoded single cell to obtain a fluorescent signal from the label of the labeled oligonucleotide hybridized to the image tag region. In such embodiments, the labeled oligonucleotide is contacted with a labeled oligonucleotide bound to the image tag region of the oligonucleotide sub-barcode component of the combinatorial barcoded cell. The labeled oligonucleotide is an oligonucleotide that hybridizes to the image tag region and contains a detectable label. In the labeled oligonucleotides used in embodiments of the present invention, the detectable label can be a portion of a labeled nucleic acid that hybridizes to the image tag region of the oligonucleotide sub-barcode unit. In such cases, the length of the labeled nucleic acid can vary, in some cases ranging from 5 nt to 100 nt in length, and contains one or more detectable portions bound thereto. In some embodiments, the detectable portion comprises an optical portion, a luminescent portion, an electrochemically active portion, a nanoparticle, or a combination thereof. In some embodiments, the luminescent portion comprises a chemiluminescent portion, an electroluminescent portion, a photoluminescent portion, or a combination thereof. In some embodiments, the photoluminescent portion comprises a fluorescent portion, a phosphorescent portion, or a combination thereof. In some embodiments, the fluorescent portion comprises a fluorescent dye. In some embodiments, the nanoparticle comprises a quantum dot. In some embodiments, the method comprises reacting to convert a detectable portion precursor into a detectable portion. Detectable portions that can be used in embodiments of the present invention include those described in U.S. Patent Application Publication No. US2018 / 0088112; U.S. Patent Application Publication No. 2018 / 0200710; U.S. Patent Application Publication No. US2018 / 0346970; U.S. Patent Application Publication No. 2019 / 0056415; U.S. Patent Application Publication No. US 2020 / 0248263; U.S. Patent Application Publication No. 2020 / 0299672; and U.S. Patent Application Publication No. 2021 / 0171940, the disclosures of which are incorporated herein by reference.

[0068] For example, as described above, the combined barcoded single cells of the partitions are contacted with one or more labeled oligonucleotides that are bound to the image tag region of the oligonucleotide sub-barcode component of the specific binding member / oligonucleotide sub-barcode to produce the combined barcoded single cells of the partitions of the label. In the combined barcoded single cell set of the labeled partitions, the single cells in the compartments contain the image tag region of the sub-barcode component hybridized with the labeled oligonucleotide. In order to facilitate imaging, the same labeled oligonucleotides with the same label, such as fluorescent dyes, can be contacted with all the combined labeled cells in all the partitions. Those combined labeled cells with image tag regions complementary to the labeled oligonucleotides will hybridize with the labeled oligonucleotides and be detectable in subsequent imaging steps.

[0069] After generating the combined barcoded single cells of the labeled partitions, the detectable labels of the combined barcoded single cells of the labeled partitions can be detected to obtain a fluorescent signal from the label. The detection of the fluorescent signal can be performed using any convenient scheme, wherein such scheme can include exciting the cells with light of a suitable wavelength and detecting the light from the label associated with the cells. As summarized above, the signals of the combined barcoded cells from the partitions can be obtained in continuous iterations, wherein each detection iteration includes a labeling step and a subsequent detection step. In such cases, obtaining image data from the cells of the partitions can be regarded as a cyclic imaging step, so that the cyclic imaging step is used to obtain partition-specific fluorescent barcodes. In such embodiments, for example, the combined barcoded single cell set of the partitions can be contacted with a first labeled oligonucleotide, which is specifically bound to the first image domain of the sub-barcode component that can be associated with different cells of the partitioned cells. A first subset of image data can be obtained from the cells. The differentiated cells can then be contacted with a second labeled oligonucleotide, which is specifically bound to a second image domain of a sub-barcode component that can be associated with different cells of the partitioned cells, and then a second subset of image data can be obtained from the cell. This process can be repeated for any desired number of iterations. In some cases, the range of imaging iterations for obtaining image data is two to twenty times, such as two to ten times. Between each iteration, the previous set of labeled oligonucleotides can be removed from the cells of the partition, for example, by washing the cells. Alternatively, the labeling used in the previous iteration can be inactivated so that it is not detectable in the subsequence imaging iteration. In some other embodiments, the labeling set selected for use in a given imaging iteration scheme can be selected so that the labeling is distinguishable in terms of excitation and / or emission maximum values. In such cases, a single labeling step can be used, wherein two or more different labeled oligonucleotides are introduced in the partition under hybridization conditions. After removing any unbound labeled oligonucleotides, the labeled partitioned cells can then be circulated through two or more imaging steps, wherein the difference between each imaging step is the excitation and / or detection of the cell. Thus, a partition-specific fluorescent barcode for a given partition can be obtained by first contacting the given partition with a plurality of different labeled oligonucleotides, wherein a subset of the plurality of different labeled oligonucleotides binds to corresponding image-labeled regions of specific binding members / oligonucleotide sub-barcodes associated with cells present in the partition. After removing unbound labeled oligonucleotides, the remaining bound labeled oligonucleotides can be detected to obtain a fluorescent barcode for the partition, wherein the detection scheme can be iterative, such as a cyclic imaging scheme.

[0070] Sequence data acquisition

[0071] For example, as described above, the partitioning of combinatorial barcoded cells results in combinatorial barcoded cells that are spatially proximate to the partitions of a particle (e.g., a bead) having bound cell marker domain nucleic acids that include a target binding region, e.g., as described above. When the cell marker domain nucleic acid is in close proximity to a target of the combinatorial barcoded single cell, the target can hybridize with the cell marker domain nucleic acid. If desired, the cell marker domains comprising nucleic acids can be contacted at a non-exhaustive ratio such that each different target can associate with a different cell marker domain comprising nucleic acid having its own unique UMI.

[0072] After the combinatorial barcoded cells are partitioned, as described above, the combinatorial barcoded cells can be lysed to release the target molecules, so that the released target molecules (e.g., nucleic acids) can bind to the target binding region of the cell marker domain nucleic acid to produce captured nucleic acids. Cell lysis can be accomplished by any of a variety of means, for example, by chemical or biochemical means, by osmotic shock, or by means of thermal lysis, mechanical lysis, or optical lysis. The particles can be lysed by adding a cell lysis buffer, which includes a detergent (e.g., SDS, lithium dodecyl sulfate, TritonX-100, Tween-20, or NP-40), an organic solvent (e.g., methanol or acetone), or a digestive enzyme (e.g., proteinase K, pepsin, or trypsin) or any combination thereof. In order to increase the association of the target and the barcode, the diffusion rate of the target molecule can be changed by, for example, lowering the temperature of the lysate and / or increasing the viscosity of the lysate. In some embodiments, the sample can be lysed using filter paper. The filter paper can be soaked with a lysis buffer on top of the filter paper. The filter paper can be applied to the sample under pressure, which can promote the cleavage of the sample and the hybridization of the target of the sample with the substrate. In some embodiments, the cleavage can be performed by mechanical cleavage, thermal cleavage, optical cleavage and / or chemical cleavage. Chemical cleavage can include the use of digestive enzymes such as proteinase K, pepsin and trypsin. The cleavage can be performed by adding a lysis buffer to the substrate. The lysis buffer can include Tris HCl. The lysis buffer can include at least about 0.01 M, 0.05 M, 0.1 M, 0.5 M or 1 M or more Tris HCl. The lysis buffer can include up to about 0.01 M, 0.05 M, 0.1 M, 0.5 M or 1 M or more TrisHCL. The lysis buffer can include about 0.1 M Tris HCl. The pH of the lysis buffer can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or higher. The pH of the lysis buffer can be up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. In some embodiments, the pH of the lysis buffer is about 7.5. The lysis buffer may include a salt (e.g., LiCl). The concentration of the salt in the lysis buffer may be at least about 0.1 M, 0.5 M, or 1 M or more. The concentration of the salt in the lysis buffer may be up to about 0.1 M, 0.5 M, or 1 M or more. In some embodiments, the concentration of the salt in the lysis buffer is about 0.5 M. The lysis buffer may include a detergent (e.g., SDS, lithium dodecyl sulfate, triton X, tween, NP-40). The concentration of the detergent 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 more.The concentration of the detergent in the lysis buffer may be at most about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6% or 7% or more. In some embodiments, the concentration of the detergent in the lysis buffer is about 1% lithium dodecyl sulfate. The time used in the method for lysis may depend on the amount of detergent used. In some embodiments, the more detergent is used, the less time is required for lysis. The lysis buffer may include a chelating agent (e.g., EDTA, EGTA). The concentration of the chelating agent in the lysis buffer may be at least about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM or 30 mM or more. The concentration of the chelating agent in the lysis buffer may be at most about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM or 30 mM or more. In some embodiments, the concentration of the chelating agent in the lysis buffer is about 10 mM. The lysis buffer may include a reducing agent (e.g., β-mercaptoethanol, DTT). The concentration of the reducing agent in the lysis buffer may be at least about 1 mM, 5 mM, 10 mM, 15 mM, or 20 mM or more. The concentration of the reducing agent in the lysis buffer may be at most about 1 mM, 5 mM, 10 mM, 15 mM, or 20 mM or more. In some embodiments, the concentration of the reducing agent in the lysis buffer is about 5 mM. In some embodiments, the lysis buffer may include about 0.1 MTris HCl (about pH 7.5), about 0.5 M LiCl, about 1% lithium dodecyl sulfate, about 10 mM EDTA, and about 5 mM DTT. Lysis may be performed at a temperature of about 4°C, 10°C, 15°C, 20°C, 25°C, or 30°C. Lysis may be performed for about 1 minute, 5 minutes, 10 minutes, 15 minutes, or 20 minutes or more. Lysed cells can include at least about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, or 700,000 or more target nucleic acid molecules. Lysed cells can include up to about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, or 700,000 or more target nucleic acid molecules.

[0073] After the cells of the lysed combined barcoded cells are released therefrom, the nucleic acid molecules can be randomly associated with the cell marker domain nucleic acids of the co-located solid support (e.g., beads). The association can include hybridization of the target recognition region of the cell marker domain nucleic acid with the complementary portion of the target nucleic acid molecule (e.g., the oligo (dT) of the barcode can interact with the poly (A) tail of the target). The assay conditions (e.g., buffer pH, ionic strength, temperature, etc.) for hybridization can be selected to promote the formation of specific, stable hybrids. In some embodiments, the nucleic acid molecules released from the lysed cells can be associated with multiple probes on the substrate (e.g., hybridized with the probes on the substrate). When the probe includes oligo (dT), the mRNA molecule can be hybridized with the probe and reverse transcribed. The oligo (dT) portion of the oligonucleotide can serve as a primer for the first strand synthesis of the cDNA molecule, for example, when subjected to DNA synthesis reaction conditions to produce a first strand cDNA domain comprising a capture nucleic acid. The cell marker domain nucleic acid can also hybridize with a complementary capture sequence of an oligonucleotide sub-barcode component (e.g., a poly(A) sequence) of a combination barcoded cell-associated specific binding member / oligonucleotide sub-barcode. In this way, the cell marker domain nucleic acid can serve as a primer for reverse transcription using the oligonucleotide sub-barcode as a template, e.g., as described in more detail below.

[0074] Where desired, a given workflow may include a merging step, wherein a product composition consisting of, for example, captured nucleic acid, a first-strand cDNA synthesized, or a double-stranded cDNA synthesized is combined or merged with a product composition obtained from one or more additional samples, such as a cell that is combined with barcoded. In some cases, the merging step is performed just after the hybridization step between the cell marker domain nucleic acid and the target nucleic acid, for example, as reviewed above. The number of different product compositions produced from different samples (e.g., cells) combined or merged in such embodiments may vary, wherein in some cases, the number ranges from 2 to 1,000,000, such as 3 to 200,000, including 4 to 100,000, such as 5 to 50,000, wherein in some cases, the number ranges from 100 to 10,000, such as 1,000 to 5,000. Before or after merging, the product composition may be, for example, amplified by polymerase chain reaction (PCR), as described in more detail below. Once the target-cell domain marker molecules have been merged, all further processing may be performed in a single reaction vessel. Further processing may comprise, for example, reverse transcription reactions, amplification reactions, cleavage reactions, dissociation reactions and / or nucleic acid extension reactions. Further processing reactions may be performed within microwells, ie without first combining labeled target nucleic acid molecules from multiple cells.

[0075] The present disclosure provides methods for creating target-cell marker domain conjugates using any convenient scheme such as reverse transcription or nucleotide extension. Target-cell marker domain conjugates can include complementary sequences of all or part of the cell marker domain and target nucleic acid. Reverse transcription of the associated RNA molecule can occur by adding a reverse transcription primer together with a reverse transcriptase. The reverse transcription primer can be an oligo (dT) primer, a random hexanucleotide primer or a target-specific oligonucleotide primer. Oligo (dT) primer length can be or can be about 12 to 18 nucleotides, and binds to endogenous poly (A) tails at the 3' end of mammalian mRNA. Random hexanucleotide primers can bind to mRNA at a variety of complementary sites. Target-specific oligonucleotide primers typically selectively initiate target mRNA. Reverse transcription can occur repeatedly to produce multiple cDNA molecules. The methods disclosed herein can 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 reverse transcription reactions. The methods can include performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 reverse transcription reactions.

[0076] One or more nucleic acid amplification reactions can be performed to create multiple copies of target nucleic acid molecules. Amplification can be performed in a multiplex manner, wherein multiple target nucleic acid sequences are amplified simultaneously. Amplification reactions can be used to add sequencing adapters to nucleic acid molecules. Amplification reactions can include at least a portion of amplified sample labels (if present). Amplification reactions can include at least a portion of amplified cell labels and / or barcode sequences (e.g., molecular markers). Amplification reactions can include at least a portion of amplified sample labels, cell labels, spatial markers, barcode sequences (e.g., molecular markers), target nucleic acids, or a combination thereof. Amplification reactions can include amplifying 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% of multiple nucleic acids or a range or number between any two of these values. The method can further include performing one or more cDNA synthesis reactions to generate cDNA copies of one or more target-barcode molecules, the target barcode molecules comprising a sample marker, a cell marker, a spatial marker, and / or a barcode sequence (eg, a molecular marker).

[0077] In some embodiments, amplification can be performed using polymerase chain reaction (PCR). As used herein, PCR can refer to a reaction for amplifying a specific DNA sequence in vitro by simultaneous primer extension of complementary strands of DNA. As used herein, PCR can encompass derivative forms of the reaction, including but not limited to RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, digital PCR, and assembled PCR.

[0078] Amplification of nucleic acids may include 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 RNA transcription amplification driven by a DNA-dependent RNA polymerase or RNA-guided DNA synthesis and transcription for amplification of DNA or RNA targets; ligase chain reaction (LCR); and Qβ replicase (Qβ) method; use of palindromic probes; strand displacement amplification; oligonucleotide-driven amplification using restriction endonucleases; amplification methods in which primers hybridize to nucleic acid sequences and the resulting duplexes are cleaved prior to extension reactions and amplification; strand displacement amplification using a nucleic acid polymerase lacking 5' exonuclease activity; rolling circle amplification; and branch extension amplification (RAM). In some embodiments, amplification does not produce circularized transcripts.

[0079] In some embodiments, the methods disclosed herein further include performing a polymerase chain reaction on a nucleic acid (e.g., RNA, DNA, cDNA) to produce a labeled amplicon (e.g., a randomly labeled amplicon). The labeled amplicon can be a double-stranded molecule. The double-stranded molecule can include a double-stranded RNA molecule, a double-stranded DNA molecule, or an RNA molecule hybridized with a DNA molecule. One or both chains of the double-stranded molecule can include a sample marker, a spatial marker, a cell marker, and / or a barcode sequence (e.g., a molecular marker). The labeled amplicon can be a single-stranded molecule. The single-stranded molecule can include DNA, RNA, or a combination thereof. The nucleic acid disclosed herein can include a synthetic or altered nucleic acid. Therefore, the method can include generating an amplicon composition from a first-strand cDNA domain comprising a capture nucleic acid.

[0080] Amplification can include the use of one or more non-natural nucleotides. Non-natural nucleotides can include light unstable or triggerable nucleotides. Examples of non-natural nucleotides can include but are not limited to peptide nucleic acids (PNA), morpholinos and locked nucleic acids (LNA), and glycol nucleic acids (GNA) and threose nucleic acids (TNA). Non-natural nucleotides can be added to one or more cycles of the amplification reaction. The addition of non-natural nucleotides can be used to identify the product as a specific cycle or time point in the amplification reaction.

[0081] Carrying out one or more amplification reactions can include using one or more primers. One or more primers can include, 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 can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or more nucleotides. One or more primers can include less than 12 to 15 nucleotides. One or more primers can anneal to at least a portion of a target (e.g., a randomly labeled target) of a plurality of labels. One or more primers can anneal to 3' ends or 5' ends of a target of a plurality of labels. One or more primers can anneal to an internal region of a target of a plurality of labels. The inner region can be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900 or 1000 nucleotides from the 3' end of the target of multiple labels. One or more primers can include a fixed primer set. One or more primers can include at least one or more custom primers. The one or more primers may include at least one or more control primers.The one or more primers may include at least one or more gene-specific primers.

[0082] One or more primers may include universal primers. Universal primers may anneal to universal primer binding sites. One or more custom primers may anneal to a first sample marker, a second sample marker, a spatial marker, a cell 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. The target may include a subset of the total nucleic acid in one or more samples. The target may include a subset of the total marker target in one or more samples. One or more primers may include at least 96 or more custom primers. One or more primers may include at least 960 or more custom primers. One or more primers may include at least 9600 or more custom primers. One or more custom primers may anneal to two or more different labeled nucleic acids. Two or more different labeled nucleic acids may correspond to one or more genes.

[0083] Any amplification scheme can be used in the method of the present disclosure. For example, in one scheme, the first round of PCR can use gene-specific primers and primers for universal Illumina sequencing primer 1 sequence to amplify molecules attached to beads. The second round of PCR can use nested gene-specific primers flanking Illumina sequencing primer 2 sequence and primers for universal Illumina sequencing primer 1 sequence to amplify the first PCR product. The third round of PCR adds P5 and P7 and sample indexes to turn the PCR products into Illumina sequencing libraries. Sequencing using 150 bp x 2 sequencing can reveal cell markers and barcode sequences (e.g., molecular markers) on read 1, genes on read 2, and sample indexes on index 1 reads.

[0084] In some embodiments, chemical cleavage can be used to remove nucleic acids from substrates. For example, chemical groups or modified bases present in nucleic acids can be used to facilitate their removal from solid supports. For example, enzymes can be used to remove nucleic acids from substrates. For example, nucleic acids can be removed from substrates by restriction endonuclease digestion. For example, treatment of nucleic acids containing dUTP or ddUTP with uracil-d-glycosylase (UDG) can be used to remove nucleic acids from substrates. For example, enzymes that perform nucleotide excision, such as base excision repair enzymes, such as apurinic / apyrimidinic (AP) endonucleases, can be used to remove nucleic acids from substrates. In some embodiments, photocleavable groups and light can be used to remove nucleic acids from substrates. In some embodiments, cleavable linkers can be used to remove nucleic acids from substrates. For example, cleavable linkers can include at least one of biotin / avidin, biotin / streptavidin, biotin / neutravidin, Ig-protein A, photolabile linkers, acid or base labile linker groups, or aptamers.

[0085] In some embodiments, amplification can be performed on a substrate, for example, with bridging amplification. The cDNA can be homopolymer tailed so that a compatible end for bridging amplification is generated on a substrate using an oligo (dT) probe. In bridging amplification, a primer complementary to the 3' end of the template nucleic acid can be the first primer of each pair covalently attached to a solid particle. When a sample containing template nucleic acid contacts the particle and a single thermal cycle is performed, the template molecule can anneal to the first primer and the first primer is extended in the forward direction by adding nucleotides to form a duplex molecule composed of the template molecule and the newly formed DNA chain complementary to the template. In the heating step of the next cycle, the duplex molecule can be denatured, releasing the template molecule from the particle and leaving a complementary DNA chain attached to the particle by the first primer. In the annealing phase of the subsequent annealing and extension steps, the complementary chain can be hybridized with the second primer, which is complementary to the segment of the complementary chain at the position removed from the first primer. This hybridization can cause complementary strands to form a bridge between the first and second primers, which is fixed to the first primer by a covalent bond and to the second primer by hybridization. In the extension phase, the second primer can be extended in the reverse direction by adding nucleotides in the same reaction mixture, thereby converting the bridge into a double-stranded bridge. Then start the next cycle, and the double-stranded bridge can be denatured to produce two single-stranded nucleic acid molecules, one end of each single-stranded nucleic acid molecule is attached to the particle surface via the first and second primers respectively, and the other end of each single-stranded nucleic acid molecule is not attached. In the annealing and extension steps of this second cycle, each chain can be hybridized with other complementary primers that were not previously used on the same particle, to form a new single-stranded bridge. Two previously unused primers that are now hybridized are extended to convert two new bridges into double-stranded bridges. The amplification reaction can 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 a plurality of nucleic acids.

[0086] The amplification of the labeled nucleic acid may include a PCR-based method or a non-PCR-based method. The amplification of the labeled nucleic acid may include an exponential amplification of the labeled nucleic acid. The amplification of the labeled nucleic acid may include a linear amplification of the labeled nucleic acid. The amplification may be performed by polymerase chain reaction (PCR). PCR may refer to a reaction for in vitro amplification of a specific DNA sequence by simultaneous primer extension of complementary strands of DNA. PCR may encompass derivative forms of the reaction, including but not limited to RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, digital PCR, inhibition PCR, semi-inhibition PCR, and assembly PCR.

[0087] In some embodiments, amplification of the labeled nucleic acid includes a non-PCR-based method. Examples of non-PCR-based methods include, but are not limited to, multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, rolling circle amplification, or loop-to-loop amplification. Other non-PCR-based amplification methods include: multiple cycles of RNA transcription amplification driven by a DNA-dependent RNA polymerase or RNA-guided DNA synthesis and transcription for amplification of DNA or RNA targets; ligase chain reaction (LCR); Qβ replicase (Qβ); use of palindromic probes; strand displacement amplification; oligonucleotide-driven amplification using restriction endonucleases; amplification methods in which primers hybridize to nucleic acid sequences and the resulting duplexes are cleaved prior to extension reactions and amplification; strand displacement amplification using a nucleic acid polymerase lacking 5' exonuclease activity; rolling circle amplification; and / or branch extension amplification (RAM).

[0088] In some embodiments, the methods disclosed herein further include performing a nested polymerase chain reaction on the amplified amplicon (e.g., target). The amplicon can be a double-stranded molecule. The double-stranded molecule can include a double-stranded RNA molecule, a double-stranded DNA molecule, or an RNA molecule hybridized with a DNA molecule. One or both strands of the double-stranded molecule can include a sample tag or a molecular identifier label. Alternatively, the amplicon can be a single-stranded molecule. The single-stranded molecule can include DNA, RNA, or a combination thereof. The nucleic acid of the present invention can include a synthetic or altered nucleic acid.

[0089] In some embodiments, the method includes repeating the amplification of the labeled nucleic acid to produce a plurality of amplicons. The method disclosed herein may 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.

[0090] Amplification can further include adding one or more control nucleic acids to one or more samples including multiple nucleic acids. Amplification can further include adding one or more control nucleic acids to multiple nucleic acids. The control nucleic acid can include a control marker.

[0091] Amplification can include the use of one or more non-natural nucleotides. Non-natural nucleotides can include light unstable and / or triggerable nucleotides. Examples of non-natural nucleotides include but are not limited to peptide nucleic acids (PNA), morpholinos and locked nucleic acids (LNA), and glycol nucleic acids (GNA) and threose nucleic acids (TNA). Non-natural nucleotides can be added to one or more cycles of the amplification reaction. The addition of non-natural nucleotides can be used to identify the product as a specific cycle or time point in the amplification reaction.

[0092] Carrying out one or more amplification reactions can include using one or more primers. One or more primers can include one or more oligonucleotides. One or more oligonucleotides can include at least about 7 to 9 nucleotides. One or more oligonucleotides can include less than 12 to 15 nucleotides. One or more primers can anneal to at least a portion of a plurality of labeled nucleic acids. One or more primers can anneal to the 3' end and / or 5' end of a plurality of labeled nucleic acids. One or more primers can anneal to an internal region of a plurality of labeled nucleic acids. The inner region can be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900 or 1000 nucleotides from the 3' end of the nucleic acid of multiple labels. One or more primers can include a fixed primer set. One or more primers can 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 include universal primers. Universal primers may anneal to universal primer binding sites. One or more custom primers may anneal to the first sample tag, the second sample tag, a molecular identifier tag, a nucleic acid or its product. One or more primers may include universal primers and custom primers. Custom primers may be designed to amplify one or more target nucleic acids. Target nucleic acids may include a subset of the total nucleic acids in one or more samples. In certain embodiments, primers are probes attached to arrays of the present disclosure.

[0093] In some embodiments, barcoding (e.g., randomly barcoding) multiple targets in a sample further comprises 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 comprises generating multiple index polynucleotides from multiple targets in a sample. For example, for an index library of barcoded targets comprising a first index target and a second index target, the marker region of the polynucleotide of the first index can differ from the marker region of the polynucleotide of the second index by less than, about less than, at least less than, or at most less than: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 nucleotides, or a number or range between any two of these values. In some embodiments, generating an index library of barcoded targets comprises contacting a plurality of targets (e.g., mRNA molecules) with a plurality of oligonucleotides comprising a poly(T) region and a tag region; and performing first strand synthesis using a reverse transcriptase to generate single-stranded labeled cDNA molecules each comprising a cDNA region and a tag region, wherein the plurality of targets comprises at least two mRNA molecules of different sequences, and the plurality of oligonucleotides comprises at least two oligonucleotides of different sequences. Generating an index library of barcoded targets may further comprise amplifying the single-stranded labeled cDNA molecules to generate double-stranded labeled cDNA molecules; and performing nested PCR on the double-stranded labeled cDNA molecules to generate labeled amplicons. In some embodiments, the method may comprise generating adapter-tagged amplicons.

[0094] Barcoding (e.g., random barcoding) can include labeling a single nucleic acid (e.g., DNA or RNA) molecule using a nucleic acid barcode or tag. In some embodiments, it involves adding a DNA barcode or tag to a cDNA molecule when the cDNA molecule is generated from mRNA. Nested PCR can be performed to minimize PCR amplification bias. Adapters can be added for sequencing using, for example, next generation sequencing (NGS). The sequencing results can be used to determine the cell markers, molecular markers, and sequences of nucleotide fragments of one or more copies of the target.

[0095] In certain embodiments, the provided methods further comprise subjecting the prepared expression library (e.g., the amplicon composition produced as described above) to a sequencing protocol, such as an NGS protocol. The protocol can be performed on any suitable NGS sequencing platform. The target NGS sequencing platform includes, but is not limited to, sequencing platforms provided by Illumina® (e.g., HiSeq™, MiSeq™, and / or NextSeq™ sequencing systems); Ion Torrent™ (e.g., Ion PGM™ and / or Ion Proton™ sequencing systems); Pacific Biosciences (e.g., PACBIO RS II Sequel sequencing systems); Life Technologies™ (e.g., SOLiD sequencing systems); Oxford Nanopore (e.g., Minion), Roche (e.g., 454 GS FLX+ and / or GS Junior sequencing systems); or any other target sequencing platform. The NGS protocol will vary depending on the specific NGS sequencing system employed. The detailed protocol for sequencing (e.g., which may include further amplification (e.g., solid phase amplification), sequencing the amplicon, and analyzing the sequencing data) can be obtained from the manufacturer of the NGS sequencing system employed.

[0096] In some cases, the method further comprises a cell component binding reagent using an oligonucleotide label, such as in an application where one or more cell components (e.g., surface proteins) are expected to be detected (e.g., quantitatively). The cell component binding reagent of the oligonucleotide label used in such embodiments comprises a cell component binding reagent coupled to a cell component binding reagent specific oligonucleotide, such as an antibody or its binding fragment, and the cell component binding reagent specific oligonucleotide includes the identifier sequence of the cell component binding reagent associated with the cell component binding reagent specific oligonucleotide. In such cases, the magnetic capture bead can include a nucleic acid configured to capture the domain of the cell component binding reagent specific oligonucleotide, such as specifically binding to the domain. In this way, protein expression can be determined together with gene expression, such as, in the case of expecting multi-omics analysis, such as, a combined analysis of transcriptome and proteome. In such cases, the method can include preparing a sample captured with a cell component binding reagent labeled with an oligonucleotide, and then providing the capture of the cell component binding reagent specific oligonucleotide released from the captured partition. Further details regarding the use of oligonucleotide-labeled cellular component binding reagents can be found in U.S. Published Patent Application Nos. US20180267036 and US20200248263; the disclosures of which are incorporated herein by reference.

[0097] Further details regarding methods for obtaining sequence data from single cells, for example as described above, are provided in U.S. Patent Application Publication No. US2018 / 0088112; U.S. Patent Application Publication No. 2018 / 0200710; U.S. Patent Application Publication No. US2018 / 0346970; U.S. Patent Application Publication No. 2019 / 0056415; U.S. Patent Application Publication No. US 2020 / 0248263; U.S. Patent Application Publication No. 2020 / 0299672; and U.S. Patent Application Publication No. 2021 / 0171940, the disclosures of which are incorporated herein by reference.

[0098] The sequencing protocol generates sequence data for the combinatorial barcoded cells. The sequence data can then be easily associated with the image data of the combinatorial barcoded cells, so that image data and sequence data obtained from the same combinatorial barcoded cells can be paired. In other words, a given image data set and a given sequence data set can be associated as being obtained from the same combinatorial barcoded cells, for example, as described in more detail below.

[0099] Associating image data and sequence data that share a common composite barcode

[0100] After obtaining image data and sequence data (e.g., as described above), the resulting image data and sequence data obtained from a given partition, and thus the cells in the partition, are associated. Association means pairing the image and sequence data as originating from the same partition, and therefore from the combinatorially barcoded cells present in the partition when the image data of the partition was obtained. Thus, image data and sequence data obtained from the same combinatorially barcoded cells can be paired. In other words, a given image data set and a given sequence data set can be identified as being obtained from the same combinatorially barcoded cells, and then paired or otherwise associated with each other. In this way, associated image and sequence data of single cells of a cell sample can be obtained.

[0101] The image data and sequence data are associated by using the combinatorial barcode of the combinatorially barcoded cell from which the image and sequence data were obtained. In the sequence data obtained (e.g., as described above), sequence reads of both the cell target and the oligonucleotide barcode subunits of the combinatorially barcoded cell are obtained. In other words, for each combinatorially barcoded cell assayed in a given workflow, the sequence of the oligonucleotide sub-barcode associated with the cell and the sequence of the target nucleic acid from the cell (e.g., mRNA from the cell) are obtained. For each combinatorially barcoded cell, these obtained sequences are obtained using a protocol (which may be a next-generation sequencing protocol), as described above, in which a library is generated from the original sequence, wherein each member of a given library generated from the same partition shares a common cell marker. Therefore, sequence reads from the cell target nucleic acid and the oligonucleotide sub-barcode obtained from the same combinatorially barcoded cell share the same cell marker, i.e., they all have a common cell marker. When associating cell and image data, all reads with the same cell marker domain (i.e., all reads that share a common cell marker) from both the reads of the target nucleic acid and the reads of the oligonucleotide sub-barcode can be paired or associated. Such pairing or association produces a set of reads that contains reads of both the target nucleic acid and the oligonucleotide sub-barcode nucleic acid, and these reads can be identified as originating from the same combinatorially barcoded cell.

[0102] Next, the resulting sequence data of the reads containing both the target nucleic acid and the oligonucleotide sub-barcode nucleic acid can be matched with the image data, i.e., paired or associated. As reviewed above, the image data of the combined barcoded cells contains a series of fluorescent signals obtained from different labeled oligonucleotides detected from cells of a given combination label during the imaging step. This series or set of fluorescent signals obtained from the same partition can be referred to as a partition-specific fluorescent barcode. Different partitions of a given workflow will have their own unique partition-specific fluorescent barcodes. A given fluorescent signal constituting such a partition-specific fluorescent barcode can be assigned to a given portion of the sequence read, because the sequence of the labeled oligonucleotide from which the fluorescent signal is obtained is known. Therefore, each partition-specific fluorescent barcode obtained for a given combined barcoded cell present in the partition can be used to determine the sequence of different image-marked regions associated with the combined barcoded cell. Since the sequence of the image-marked region is present in the read of the oligonucleotide sub-barcode, a given partition-specific fluorescent barcode can be determined to be associated with a given sequence data set. Once a partition-specific fluorescent barcode is associated with a given sequence data set, the sequence data can be determined to be obtained from the same combined barcoded cell in the partition from which the partition-specific fluorescent barcode was obtained. In other words, a series of image marker region sequences for a given partition can be obtained from a series of fluorescent signals obtained from the partition. This series or collection of image marker region sequences can then be used to identify all sequence data obtained from the partition. This identification can be accomplished by determining that sequence reads having both of the following were obtained from combined cells: (a) a common cell barcode; and (b) a partition that identifies a collection of image marker region sequences; the combined cell is present in the partition from which the partition-specific fluorescent barcode was obtained. Once sequence data is assigned to a given partition, the sequence data can be easily associated with image data obtained from the partition. In this way, associated image and sequence data for single cells of a cell sample can be obtained.

[0103] Reagent test kit

[0104] Aspects of the present invention further include kits and compositions that can be used to practice various embodiments of the methods of the present invention. The kits of the present invention may include: a population of specific binding members / oligonucleotide sub-barcodes; a population of labeled oligonucleotides bound to an image marker region of an oligonucleotide sub-barcode component of a specific binding member / oligonucleotide sub-barcode; and beads comprising a bead-bound nucleic acid, the nucleic acid comprising a cell marker domain and a target binding region, for example, as described above. The population of specific binding members / oligonucleotide sub-barcodes may include a varying number of different specific binding members / oligonucleotide sub-barcodes that differ from one another in terms of specific binding members and / or oligonucleotide sub-barcodes, for example, differ from one another in terms of image marker regions present in the sub-barcode component. Although the number of different specific binding members / oligonucleotide sub-barcodes of a given population may vary, in some cases, the number ranges from 5 to 1,000, such as 10 to 500. The population of labeled oligonucleotides present in the kit may also vary, wherein in some cases the number of differently labeled oligonucleotides that differ from one another in terms of their oligonucleotide sequence and / or label ranges from 5 to 1,000, such as 10 to 500, for example 10 to 100.

[0105] The kit may further comprise one or more additional components that can be used to practice an embodiment of the method. For example, the kit may comprise components used to generate combinatorial barcoded cells, such as macroporous plates, liquid containers such as tubes, etc. In addition, the kit may comprise one or more components used to obtain sequence data, such as one or more of the following: primers, polymerases (e.g., thermostable polymerases, reverse transcriptases, etc., both of which have hot start properties), dsDNA enzymes, exonucleases, dNTPs, metal cofactors, one or more nuclease inhibitors (e.g., RNase inhibitors and / or DNase inhibitors), one or more molecular crowding agents (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 (e.g., as described above), solid supports, containers, boxes, such as tubes, beads, plates, microfluidic chips, etc. The components of the kit may be present in separate containers, or multiple components may be present in a single container.

[0106] In addition to the above-mentioned components, the subject test kit can further comprise (in certain embodiments) instructions for practicing the subject method. These instructions can be present in the subject test kit in various forms, one or more of which can be present in the test kit. A form in which these instructions can exist is as printed information on a suitable medium or substrate, for example, one or more sheets of paper of information printed thereon in the packaging of test kit, package inserts etc. Another form of these instructions is a computer-readable medium of recorded information thereon, for example a disk, an optical disc (CD), a portable flash drive etc. Another form in which these instructions can exist is a website address, which can be used to access the information at the removal site via the Internet.

[0107] The following is offered by way of illustration and not by way of limitation.

[0108] experiment

[0109] Figures 1 to 3 An illustration of a workflow according to an embodiment of the present invention is provided.

[0110] Notwithstanding the appended claims, the present disclosure is also defined by the following clauses:

[0111] 1. A method for obtaining associated image and sequence data of a single cell of a cell sample, the method comprising:

[0112] combinatorially barcoding the cells of the cell sample with specific binding member / oligonucleotide sub-barcodes to produce combinatorially barcoded cells;

[0113] partitioning the combinatorially barcoded cell to generate partitioned combinatorially barcoded single cells each having a combinatorial barcode;

[0114] obtaining image data and sequence data of combined barcoded single cells of the partitions; and

[0115] associating the image data and sequence data that share a common combined barcode;

[0116] To obtain correlated images and sequence data of single cells of the cell sample.

[0117] 2. The method according to clause 1, wherein combinatorial barcoding comprises one or more split / pool iterations of sequentially contacting cells of the cell sample with different specific binding members / oligonucleotide sub-barcodes.

[0118] 3. The method of clause 2, wherein each split / merge iteration comprises:

[0119] distributing cells of the cell sample into different compartments;

[0120] introducing different specific binding members / oligonucleotide sub-barcodes that differ from each other by oligonucleotide sub-barcode components into the different compartments to generate sub-barcoded cells; and

[0121] The sub-barcoded cells of the different compartments are combined.

[0122] 4. The method according to clause 3, wherein the number of said different compartments ranges from 5 to 100.

[0123] 5. A method according to any one of clauses 3 and 4, wherein the compartments are wells of an orifice plate.

[0124] 6. A method according to any one of clauses 2 to 5, wherein the number of split / merge iterations ranges from two to five.

[0125] 7. A method according to any preceding clause, wherein the specific binding member / oligonucleotide sub-barcode comprises a specific binding member conjugated to an oligonucleotide sub-barcode component.

[0126] 8. The method according to clause 7, wherein the specific binding member comprises an antibody or a binding fragment thereof.

[0127] 9. A method according to any one of clauses 7 and 8, wherein the oligonucleotide sub-barcode component comprises an image marker region.

[0128] 10. The method of clause 9, wherein the oligonucleotide sub-barcode component further comprises one or more of a unique identifier of the specific binding member, a capture sequence, and a primer binding site.

[0129] 11. The method according to any of the preceding clauses, wherein said partitioning comprises distributing said combinatorially barcoded cells into partitions comprising single combinatorially barcoded cells.

[0130] 12. The method according to clause 11, wherein the distributing comprises introducing the combinatorially barcoded cells into a flow cell having microwells on a bottom surface thereof.

[0131] 13. The method of clause 12, wherein the method further comprises providing beads comprising a bead-bound nucleic acid comprising a cell marker domain and a target binding region in the partition comprising a single combinatorially barcoded cell.

[0132] 14. The method according to clause 13, wherein the bead-bound nucleic acid further comprises one or more of a molecular indexing domain and a universal primer binding domain.

[0133] 15. A method according to any of the preceding clauses, wherein obtaining image data of the partitioned combined barcoded single cells comprises one or more imaging iterations, each imaging iteration comprising:

[0134] contacting the partitioned combinatorially barcoded single cells with one or more labeled oligonucleotides that bind to image-tagged regions of the oligonucleotide sub-barcode component of the specific binding member / oligonucleotide sub-barcode to produce labeled partitioned combinatorially barcoded single cells; and

[0135] Capture images of the labeled partitioned combined barcoded single cells.

[0136] 16. The method according to clause 15, wherein the partitioned combinatorially barcoded single cells are contacted with two to five different labeled oligonucleotides that bind to different image marker regions.

[0137] 17. A method according to any one of clauses 15 and 16, wherein the one or more labelled oligonucleotides are fluorescently labelled.

[0138] 18. A method according to any one of clauses 15 to 17, wherein the number of imaging iterations ranges from two to twenty.

[0139] 19. The method according to any of the preceding clauses, wherein obtaining sequence data of the partitioned combinatorially barcoded single cells comprises employing a next generation sequencing protocol.

[0140] 20. A method according to any preceding clause, wherein the sequencing data comprises multi-omics data.

[0141] 21. A kit for obtaining correlated image and sequence data of a single cell of a cell sample, the kit comprising:

[0142] a population of specific binding members / oligonucleotide sub-barcodes;

[0143] a population of labeled oligonucleotides that bind to image-labeled regions of the oligonucleotide sub-barcode component of the specific binding member / oligonucleotide sub-barcode; and

[0144] A bead comprising a bead-bound nucleic acid comprising a cell marker domain and a target binding region.

[0145] 22. A kit according to clause 21, wherein the specific binding member / oligonucleotide sub-barcode comprises a specific binding member conjugated to an oligonucleotide sub-barcode component.

[0146] 23. A kit according to clause 22, wherein the specific binding member comprises an antibody or a binding fragment thereof.

[0147] 24. A kit according to any one of clauses 22 and 23, wherein the oligonucleotide barcode component comprises an image marker region.

[0148] 25. The kit according to clause 24, wherein the oligonucleotide sub-barcode component further comprises one or more of a unique identifier of the specific binding member, a capture sequence, a primer binding site.

[0149] 26. A kit according to any one of clauses 21 to 25, wherein the labelled oligonucleotide is fluorescently labelled.

[0150] 27. A kit according to any one of clauses 21 to 26, wherein the bead-bound nucleic acid further comprises one or more of a molecular indexing domain and a universal primer binding domain.

[0151] 28. A kit according to any one of clauses 21 to 26, wherein the kit further comprises a multiwell plate.

[0152] 29. A kit according to clause 28, wherein the multi-well plate comprises a 36 to 96 well plate.

[0153] 30. A kit according to any one of clauses 21 to 29, wherein the kit further comprises a flow cell having microwells on its bottom surface.

[0154] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0155] Therefore, the above only illustrates the principle of the present invention.It should be understood that those skilled in the art will be able to design various arrangements, which, although not explicitly described or shown in this article, embody the principle of the present invention and are included in its spirit and scope.In addition, all examples and conditional language described in this article are mainly intended to help readers understand the principles of the present invention and the concepts contributed by the inventor to promote this field, and should be interpreted as not being limited to such specific examples and conditions.In addition, all statements describing the principles, aspects and embodiments of the present invention and their specific examples are intended to cover both their structural equivalents and functional equivalents.In addition, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, that is, any element that performs the same function developed, regardless of the structure.In addition, regardless of whether such disclosure is explicitly described in the claims, any content disclosed herein is not intended to be dedicated to the public.

[0156] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Instead, the scope and spirit of the present invention are embodied by the appended claims. In the claims, 35 USC §112(f) or 35 USC §112(6) is expressly defined as being referenced for use in a claim only if the exact phrase "means for..." or the exact phrase "step for..." is recited at the beginning of a limitation in the claim; if a limitation in a claim does not use such exact phrases, 35 USC §112(f) or 35 USC §112(6) will not be referenced.

Claims

1. A method for obtaining associated image and sequence data of a single cell of a cell sample, the method comprising: combinatorially barcoding the cells of the cell sample with specific binding member / oligonucleotide sub-barcodes to produce combinatorially barcoded cells; partitioning the combinatorially barcoded cell to generate partitioned combinatorially barcoded single cells each having a combinatorial barcode; obtaining image data and sequence data of combined barcoded single cells of the partitions; as well as associating the image data and sequence data that share a common combined barcode; To obtain correlated images and sequence data of single cells of the cell sample. 2 . The method of claim 1 , wherein combinatorial barcoding comprises one or more split / pool iterations of sequentially contacting cells of the cell sample with different specific binding members / oligonucleotide sub-barcodes.

3. The method of claim 2, wherein each split / merge iteration comprises: distributing cells of the cell sample into different compartments; introducing different specific binding members / oligonucleotide sub-barcodes that differ from each other by oligonucleotide sub-barcode components into the different compartments to generate sub-barcoded cells; and The sub-barcoded cells of the different compartments are combined. The method according to claim 3 , wherein the compartments are wells of an orifice plate.

5. The method according to any one of the preceding claims, wherein the specific binding member / oligonucleotide sub-barcode comprises a specific binding member conjugated to an oligonucleotide sub-barcode component.

6. The method of claim 5, wherein the specific binding member comprises an antibody or a binding fragment thereof.

7. The method of claim 6, wherein the oligonucleotide sub-barcode component comprises an image marker region.

8. The method of claim 7, wherein the oligonucleotide sub-barcode component further comprises one or more of a unique identifier of the specific binding member, a capture sequence, and a primer binding site.

9. The method of any of the preceding claims, wherein said partitioning comprises distributing said combinatorially barcoded cells into partitions comprising single combinatorially barcoded cells.

10. The method of claim 9, wherein the distributing comprises introducing the combinatorially barcoded cells into a flow cell having microwells on a bottom surface thereof.

11. The method of claim 10, wherein the method further comprises providing beads comprising a bead-bound nucleic acid comprising a cell marker domain and a target binding region in the partition comprising a single combinatorially barcoded cell.

12. The method of any one of the preceding claims, wherein obtaining image data of the partitioned combined barcoded single cells comprises one or more imaging iterations, each imaging iteration comprising: contacting the partitioned combinatorially barcoded single cells with one or more labeled oligonucleotides that bind to image-tagged regions of the oligonucleotide sub-barcode component of the specific binding member / oligonucleotide sub-barcode to produce labeled partitioned combinatorially barcoded single cells; as well as Capture images of the labeled partitioned combined barcoded single cells.

13. The method of any of the preceding claims, wherein obtaining sequence data of the partitioned combinatorially barcoded single cells comprises employing a next generation sequencing protocol.

14. The method of any of the preceding claims, wherein the sequencing data comprises multi-omics data.

15. A kit for obtaining correlated image and sequence data of a single cell of a cell sample, the kit comprising: a population of specific binding members / oligonucleotide sub-barcodes; a population of labeled oligonucleotides that bind to image-labeled regions of the oligonucleotide sub-barcode component of the specific binding member / oligonucleotide sub-barcode; and A bead comprising a bead-bound nucleic acid comprising a cell marker domain and a target binding region.

Images