WO2024006734A1

WO2024006734A1 - Methods for preparing and using mhc multimer reagents compositions

Info

Publication number: WO2024006734A1
Application number: PCT/US2023/069134
Authority: WO
Inventors: Payam Shahi; Bruce Alexander ADAMS; Wyatt James MCDONNELL
Original assignee: 10X Genomics, Inc.
Priority date: 2022-06-27
Filing date: 2023-06-27
Publication date: 2024-01-04

Abstract

The present disclosure relates generally to the field of immunology, and particularly relates to methods useful for preparing major histocompatibility complex (MHC) multimer reagents and methods for using the same for characterization of antigen-binding molecules (ABMs) produced by immune cells, e.g., B cells and T cells.

Description

METHODS FOR PREPARING AND USING MHC MULTIMER REAGENTS COMPOSITIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application Serial No. 63/355,952, filed on June 27, 2022. The disclosure of the abovereferenced application is herein expressly incorporated by reference it its entirety, including any drawings.

FIELD

[0002] The present disclosure relates generally to the field of immunology, and particularly relates to methods useful for preparing major histocompatibility complex (MHC) multimer reagents and methods for using the same for characterization of antigen-binding molecules (ABMs) produced by immune cells, e.g., B cells and T cells.

INCORPORATION OF THE SEQUENCE LISTING

[0003] This application contains a Sequence Listing, which is hereby incorporated herein by reference in its entirety. The accompanying Sequence Listing, named “057862-615001WO _SequenceListing_ST26.xml,” was created on June 26, 2023 and is 2,383 bytes.

BACKGROUND

[0004] Antigen-binding molecules (ABMs) that bind to antigens of interest can be developed as new immunotherapeutic agents. In particular, many ABMs developed in the past decades as therapeutic agents are antibodies (Abs), or antigen-binding fragments thereof, which bind to extracellular antigens or cell-surface antigens. Other therapeutic ABMs, such as B cell receptors, T cell receptors (TCRs), TCR-like antibodies and antigen-binding fragments thereof, that can recognize intracellular antigens, such as tumor antigens and certain virus-associated antigens, have also been developed.

[0005] The recognition of antigenic structures by the cellular immune system is mediated by surface molecules of the “major histocompatibility complex” (MHC). Reagents such as peptide-MHC (pMHC) multimers may be used the detection and isolation of antigen- specific T- cells. This is because antigen-expressing cells process antigens into short peptides which after binding are presented in a specific peptide binding fold of the MHC molecule and thus can be recognized by the T cells.

[0006] Currently, the preparation of MHC multimer reagents typically involves a number of complex biochemical reactions wherein proteins expressed in a recombinant manner generally must be folded correctly in vitro, biotinylated and afterwards caused to form MHC multimers in the correct molar ratio. At present, most MHC multimer reagents are often prepared using a very complex and cumbersome process. First, MHC components are expressed in the form of recombinant proteins and purified from host cells. Following urea denaturation, the MHC portions are folded in the presence of high peptide/epitope concentrations, and are subsequently isolated or purified as pMHC monomers, hr a further step, the recombinant MHC monomers are biotinylated and, after a second purification, are multimerized via a solid support such as streptavidin.

[0007] Existing techniques for the preparation of MHC multimer reagents are often timeconsuming, vulnerable, e.g., with respect to the efficiency of the biotinylation reaction, and costly. A simplification of the process of preparation would significantly promote the broader use of this methodology in basic research and also in the clinical diagnostic field.

SUMMARY

[0008] The present disclosure provides, inter alia, methods for preparing major histocompatibility complex (MHC) multimer reagents and methods for using the same for characterization of antigen-binding molecules (ABMs) such as antibodies, B cell receptors, T cell receptors (TCRs), and TCR-like antibodies (Abs) obtained from biological samples.

[0009] In one aspect, provided herein are methods for preparing a major histocompatibility complex (MHC) multimer reagent, the method comprising: (a) providing a mixture including: (i) a plurality of empty MHC monomers linked to first binding moieties, (ii) a second binding moiety including a plurality of binding sites capable of binding to the first binding moieties, and (iii) a plurality of antigens; (b) in the mixture, (i) loading the plurality of antigens to at least a subset of the plurality of MHC monomers linked to the first binding moieties, and (ii) binding the first binding moieties to at least a subset of the plurality of binding sites of the second binding moiety, thereby generating a MHC multimer reagent including the second binding moiety bound to the plurality of antigen -loaded MHC monomers.

[0010] Non-limiting exemplary embodiments of the methods of the disclosure can include one or more of the following features. In some embodiments, the loading in (b)(i) and the binding in (b)(ii) are performed simultaneously. In some embodiments, the second binding moiety includes a core support attached to a detectable label capable of emitting a detectable signal. In some embodiments, the detectable label is or includes a fluorophore, a magnetic particle, or a mass tag. In some embodiments, the fluorophore molecule is or includes phycoerythrin (PE), allophycocyanin (APC), Alexa Fluor 405, Pacific Blue, Alexa Fluor 488, Alexa Fluor 647, Alexa Fluor 700, DyLight 405, DyLight 550, DyLight 650, fluorescein isothiocyanate (FITC), peridinin chlorophyll protein (PerCP), StarBright Violet 440, StarBright Violet 515, StarBright 610, StarBright Violet 670, or StarBright Blue 700. In some embodiments, the second binding moiety further includes a reporter oligonucleotide. In some embodiments, the reporter oligonucleotide includes a reporter barcode sequence. In some embodiments, the reporter oligonucleotide is attached to the core support and/or to the detectable label.

[0011] In some embodiments of the disclosure, the methods further include including quenching the MHC multimer reagent generated in (b) with first binding moieties that are not linked to MHC monomers. In some embodiments, the plurality binding sites of the second binding moiety of the generated MHC multimer reagent are each either bound to an antigen- loaded MHC monomer or bound a first binding moiety.

[0012] In some embodiments of the methods disclosed herein, the first binding moieties are biotinylated or include a biotin moiety. In some embodiments, the core support of the second binding moiety includes one or more biotin-binding sites. In some embodiments, the core support is or includes a biotin-binding protein selected from streptavidin, avidin, deglycosylated avidin (e.g., Neu tr Avidin™), traptavidin, tamavidin, xenavidin, bradavidin, AVR2 (avidin related protein 2), AVR4 (avidin related protein 4), and variants, mutants, derivatives, and homologs of any thereof. In some embodiments, the MHC multimer reagent generated in (b) includes a core support including four biotin-binding sites, and wherein at least one, two, three, or four of the biotin-binding sites are each bound to an antigen-loaded MHC monomer (or biotinylated MHC monomer). In some embodiments, biotin-binding sites of the core support not bound to the antigen- loaded MHC monomer (or biotinylated MHC monomer) are bound to biotin.

[0013] In some embodiments, the MHC monomers are selected from the group consisting of MHC class I monomers and MHC class II monomers. In some embodiments, the MHC monomers are of the same MHC class. In some embodiments, the MHC monomers are of different MHC classes.

[0014] In some embodiments, the plurality of antigens is selected from oligopeptides, proteins, polysaccharides, lipids, liposomes, and infectious agents. Tn some embodiments, the plurality of antigen-loaded MHC monomers include different antigens. In some embodiments, the plurality of antigen-loaded MHC monomers include the same antigen.

[0015] In some embodiments, the ratio of the second binding moiety to empty MHC monomers in the mixture ranges from about 5: 1 to about 1:5 (w/w). In some embodiments, the ratio of the second binding moiety to empty MHC monomers in the mixture is about 1:2 (w/w).

[0016] In some embodiments, step (b) of the methods disclosed herein is carried out at a temperature ranging from about 0°C to about 37°C, from about 4°C to about 25°C, or from 4°C to about 22°C. In some embodiments, step (b) is carried out at a temperature of about 4°C. In some embodiments, step (b) is carried out for a duration of less than about 120 minutes, less than about 100 minutes, less than about 90 minutes, less than about 60 minutes, less than about 45 minutes, less than about 30 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes. In some embodiments, the step (b) is carried out at room temperature (e.g., 21°C - 24°C) and for a duration of about 5 minutes. In some embodiments, the MHC multimer reagent generated in (b) is a barcoded MHC multimer reagent including the second binding moiety that is attached to a plurality of antigen-loaded MHC monomers and further attached to the reporter oligonucleotide.

[0017] In another aspect of the disclosure, provided herein are barcoded MHC multimer reagents prepared according to a method described herein.

[0018] In yet another aspect, provided herein are methods for characterizing an antigenbinding molecule (ABM), the methods include: (a) providing a reaction mixture including: (i) a plurality of immune cells and/or a plurality of cell beads comprising immune cells, and (ii) a plurality of barcoded MHC multimer reagents as described herein; (b) partitioning the mixture, or a portion thereof, into a plurality of partitions, wherein the partitioning provides a first partition including: (i) a first immune cell and/or a first cell bead comprising a first immune cell, (ii) a first barcoded MHC multimer reagent, and (iii) a plurality of nucleic acid barcode molecules including a partition-specific barcoded sequence; and (c) generating barcoded nucleic acid molecules, wherein the barcoded nucleic acid molecules include a first barcoded nucleic acid molecule including (i) a first nucleic acid sequence encoding at least a portion of an antigenbinding molecule (ABM) expressed by the first immune cell and/or a first cell bead comprising a first immune cell, or a reverse complement thereof and (ii) the partition- specific barcode sequence or a reverse complement thereof, and a second barcoded nucleic acid molecule including (i) the first reporter barcode sequence or a reverse complement thereof and (ii) the partition- specific barcode sequence or a reverse complement thereof.

[0019] Non-limiting exemplary embodiments of the methods for characterizing an ABM disclosed herein can include one or more of the following features. In some embodiments, the methods further included generating a third barcoded nucleic acid molecule including (i) the partition barcode sequence or a reverse complement thereof and (ii) a second nucleic acid sequence or a reverse complement thereof, the second nucleic acid sequence encoding a different portion of the ABM expressed by the first immune cell and/or a first cell bead comprising a first immune cell. In some embodiments, the methods further include generating a fourth barcoded nucleic acid molecule including (i) the partition- specific barcode sequence or a reverse complement thereof and a third nucleic acid sequence, wherein the third nucleic acid sequence is a sequence of an mRNA analyte of the first immune cell and/or a first cell bead comprising a first immune cell, or a reverse complement thereof, or a cDNA sequence of the mRNA analyte of the first immune cell and/or a first cell bead comprising a first immune cell, or a reverse complement thereof.

[0020] In some embodiments, the methods disclosed herein further include determining a sequence of the first barcoded nucleic acid molecule or an amplicon thereof, and determining a sequence of the second barcoded nucleic molecule or an amplicon thereof.

[0021] In some embodiments, the plurality of immune cells and/or cell beads comprising immune cells includes B cells. In some embodiments, the first immune cell is a B cell bound to the antigen(s) of the first barcoded MHC multimer reagent. In some embodiments, the ABM produced by the B cell is a B cell receptor (BCR), an antibody (Ab) or an antigen-binding fragment thereof. In some embodiments, the plurality of immune cells and/or cell beads comprising immune cells includes T cells. In some embodiments, the first immune cell is a T cell bound to the antigen(s) of the first barcoded MHC multimer reagent. In some embodiments, the ABM produced by the T cell is a TCR.

[0022] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows an exemplary microfluidic channel structure for partitioning individual biological particles in accordance with some embodiments of the disclosure.

[0024] FIG. 2 shows an exemplary microfluidic channel structure for the controlled partitioning of beads into discrete droplets.

[0025] FIG. 3 shows an exemplary barcode carrying bead.

[0026] FIG. 4 illustrates another example of a barcode carrying bead.

[0027] FIG. 5 schematically illustrates an example microwell array.

[0028] FIG. 6 schematically illustrates an example workflow for processing nucleic acid molecules.

[0029] FIGS. 7A-7C schematically illustrate examples of labelling agents.

[0030] FIG. 8 shows an exemplary microfluidic channel structure for delivering barcode carrying beads to droplets.

[0031] FIG. 9 depicts an example of a barcode carrying bead.

[0032] FIGS. 10A, 10B and 10C schematically depict an example workflow for processing nucleic acid molecules.

[0033] FIG. 11 pictorially summarizes the results of antigen-mapping experiments using MHC multimer reagents prepared by an exemplary one- step preparation method described herein.

[0034] FIG. 12 pictorially summarizes the results of additional antigen-mapping experiments using MHC multimer reagents prepared by one-step preparation methods described herein.

DETAILED DESCRIPTION

[0035] Provided herein are methods useful for preparing major histocompatibility complex (MHC) multimer reagents. Also provided are, inter alia, are MHC multimer reagents prepared by the methods disclosed herein. Further provided are methods for characterizing antigenbinding molecules (AMBs) produced by immune cells using one or more MHC multimer reagents as described herein.

[0036] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols generally identify similar components, unless context dictates otherwise. The illustrative alternatives described in the detailed description, drawings, and claims are not meant to be limiting. Other alternatives may be used and other changes may be made without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this application.

DEFINITIONS

[0037] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

[0038] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and/or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”.

[0039] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0040] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0041] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0042] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. In some embodiments, the term “about” indicates the designated value ± up to 10%, up to + 5%, or up to ± 1%.

[0043] Headings, e.g., (a), (b), (i) etc., are presented merely for ease of reading the specification and claims. The use of headings in the specification or claims does not require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

[0044] Use of ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.

[0045] The term “barcode,” as used herein, generally refers to a label, or identifier, that conveys or is capable of conveying information about an analyte. A barcode can be part of an analyte. A barcode can be independent of an analyte. A barcode can be a tag attached to an analyte (e.g., nucleic acid molecule) or a combination of the tag in addition to an endogenous characteristic of the analyte (e.g., size of the analyte or end sequence(s)). A barcode may be unique. Barcodes can have a variety of different formats. For example, barcodes can include: polynucleotide barcodes, random nucleic acid and/or amino acid sequences, and synthetic nucleic acid and/or amino acid sequences. A barcode can be attached to an analyte in a reversible or irreversible manner. A barcode can be added to, for example, a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before, during, and/or after sequencing of the sample. Barcodes can allow for identification and/or quantification of individual sequencing -reads.

[0046] As used herein, the term “barcoded nucleic acid molecule” generally refers to a nucleic acid molecule that results from, for example, the processing of a nucleic acid barcode molecule with a nucleic acid sequence (e.g., nucleic acid sequence complementary to a nucleic acid primer sequence encompassed by the nucleic acid barcode molecule). The nucleic acid sequence may be a targeted sequence or a non-targeted sequence. The nucleic acid barcode molecule may be coupled to or attached to the nucleic acid molecule comprising the nucleic acid sequence. For example, a nucleic acid barcode molecule described herein may be hybridized to an analyte (e.g., a messenger RNA (mRNA) molecule) of a cell. Reverse transcription can generate a barcoded nucleic acid molecule that has a sequence corresponding to the nucleic acid sequence of the mRNA and the barcode sequence (or a reverse complement thereof). The processing of the nucleic acid molecule comprising the nucleic acid sequence, the nucleic acid barcode molecule, or both, can include a nucleic acid reaction, such as, in non-limiting examples, reverse transcription, nucleic acid extension, ligation, etc. The nucleic acid reaction may be performed prior to, during, or following barcoding of the nucleic acid sequence to generate the barcoded nucleic acid molecule. For example, the nucleic acid molecule comprising the nucleic acid sequence may be subjected to reverse transcription and then be attached to the nucleic acid barcode molecule to generate the barcoded nucleic acid molecule, or the nucleic acid molecule comprising the nucleic acid sequence may be attached to the nucleic acid barcode molecule and subjected to a nucleic acid reaction (e.g., extension, ligation) to generate the barcoded nucleic acid molecule. A barcoded nucleic acid molecule may serve as a template, such as a template polynucleotide, that can be further processed (e.g., amplified) and sequenced to obtain the target nucleic acid sequence. For example, in the methods and systems described herein, a barcoded nucleic acid molecule may be further processed (e.g., amplified) and sequenced to obtain the nucleic acid sequence of the nucleic acid molecule (e.g., mRNA).

[0047] The term “partition,” as used herein, generally, refers to a space or volume that may be suitable to contain one or more species or conduct one or more reactions. A partition can be a physical container, compartment, or vessel, such as a droplet, a flowcell, a reaction chamber, a reaction compartment, a tube, a well, or a microwell. The partition may isolate space or volume from another space or volume. The droplet may be a first phase (e.g., aqueous phase) in a second phase (e.g., oil) immiscible with the first phase. The droplet may be a first phase in a second phase that does not phase separate from the first phase, such as, for example, a capsule or liposome in an aqueous phase. A partition may comprise one or more other (inner) partitions. In some cases, a partition may be a virtual compartment that can be defined and identified by an index (e.g., indexed libraries) across multiple and/or remote physical compartments. For example, a physical compartment may comprise a plurality of virtual compartments.

[0048] The term “percent identity,” as used herein in the context of two or more proteins, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acids that are the same, e.g., about 70%, 72%, 74%, 75%, 76%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9%9, 99.5%, 99.9%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. See, e.g., the NCBI web site at ncbi.nlm.nih.gov/BLAST. In some embodiments, this definition includes sequence comparison performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences. In some embodiments, this definition also includes sequences that have modifications such as deletions and/or additions (e.g., insertions), as well as those that have substitutions. Such modifications can occur naturally or synthetically. In some embodiments, sequence identity can be calculated over a region that is at least about 20 amino acids in length, or over a region that is 10-100 amino acids in length, or over the entire length of a given sequence. Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res (1984) 12:387), BLASTP, BLASTN, FASTA (Atschul et al., J Mol Biol (1990) 215:403). In some embodiments, sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof. Additional methodologies that can suitably be utilized to determine structural similarity or identity amino acid sequences include those relying on position-specific structure-scoring matrix (P3SM) that incorporates structure-prediction scores from Rosetta, as well as those based on a length-normalized edit distance as described previously in, e.g., Setcliff et al., Cell Host & Microbe 23(6), May 2018.

[0049] The term “subject,” as used herein, generally refers to an animal, such as a mammal (e.g., human) or avian (e.g., bird), or other organism, such as a plant. For example, the subject can be a vertebrate, a mammal, a rodent (e.g., a mouse), a primate, a simian or a human. Animals may include, but are not limited to, farm animals, sport animals, and pets. A subject can be a healthy or asymptomatic individual, an individual that has or is suspected of having a disease (e.g., cancer) or a pre-disposition to the disease, and/or an individual that is in need of therapy or suspected of needing therapy. A subject can be a patient. A subject can be a microorganism or microbe (e.g., bacteria, fungi, archaea, viruses). The term “non-human animals” includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, non-human primates, and other mammals, such as e.g., sheep, dogs, cows, chickens, and non-mammals, such as amphibians, reptiles, etc.

[0050] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub- combination was individually and explicitly disclosed herein.

METHODS FOR PREPARING MHC MULTIMER REAGENTS

[0051] As described in more detail below, one aspect of the disclosure relates to new approaches and methods for preparation of MHC multimer reagent, the method include: (a) providing a mixture including: (i) a plurality of empty MHC monomers linked to first binding moieties, (ii) a second binding moiety including a plurality of binding sites capable of binding to the first binding moieties, and (iii) a plurality of antigens; (b) in the mixture, (i) loading the plurality of antigens to at least a subset of the plurality of MHC monomers linked to the first binding moieties, and (ii) binding the first binding moieties to at least a subset of the plurality of binding sites of the second binding moiety, thereby generating a MHC multimer reagent including the second binding moiety bound to the plurality of antigen-loaded MHC monomers. In some embodiments, the MHC monomers are operably linked to first binding moieties. The term “operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.

[0052] Non-limiting exemplary embodiments of the methods of the disclosure can include one or more of the following features. In some embodiments, the loading in (b)(i) and the binding in (b)(ii) are carried out in a single reaction volume. In some embodiments, the loading in (b)(i) and the binding in (b)(ii) are carried out simultaneously in a single reaction volume.

[0053] In some embodiments, the second binding moiety includes a core support attached to a detectable label, e.g., a fluorophore capable of emitting a detectable signal. In some embodiments, the detectable label is or includes a fluorophore, a magnetic particle, or a mass tag. In some embodiments, the detectable label includes a fluorophore molecule. In some embodiments, the detectable label is or includes a fluorophore, a magnetic particle, or a mass tag. In some embodiments, the fluorophore molecule is or includes phycoerythrin (PE), allophycocyanin (APC), Alexa Fluor 405, Pacific Blue, Alexa Fluor 488, Alexa Fluor 647, Alexa Fluor 700, DyLight 405, DyLight 550, DyLight 650, fluorescein isothiocyanate (FITC), peridinin chlorophyll protein (PerCP), StarBright Violet 440, StarBright Violet 515, StarBright 610, StarBright Violet 670, or StarBright Blue 700.

[0054] In some embodiments, the second binding moiety further includes a reporter oligonucleotide. In some embodiments, the reporter oligonucleotide further includes one or more functional sequences useful in the processing of the reporter oligonucleotide and/or barcoded nucleic acid molecules comprising a sequence of the reporter oligonucleotide. Suitable functional sequences include, but are not limited to, adapter sequences, primer sequences, primer binding sequences, unique molecular identifiers (UMIs), and hybridization or probing sequences, e.g., for identification of presence of the sequences or for pulling down reporter oligonucleotide and barcoded nucleic acids, or any of a number of other potential functional sequences. In some embodiments, the reporter oligonucleotide includes a reporter barcode sequence. In some embodiments, the reporter barcode sequence identifies a target antigen. In some embodiments, the target antigen is biotinylated. In some embodiments, the target antigen is an oligopeptide, a protein, a polysaccharide, a lipid, a liposome, an infectious agent, or a target MHC molecule complex.

[0055] The reporter oligonucleotide can be attached e.g., coupled) to the core support and/or to the detectable label. Accordingly, in some embodiments, the reporter oligonucleotide can be attached (e.g., coupled) to the core support. In some embodiments, the reporter oligonucleotide can be attached (e.g., coupled) to the detectable label. Attachment (coupling) of the reporter oligonucleotide can be attached (e.g., coupled) to the core support and/or to the detectable label can be achieved through any of a variety of direct or indirect, covalent or non- covalent associations or attachments. For example, oligonucleotides and/or core supports can be covalently attached to the detectable label, e.g., via a linker, using chemical conjugation techniques (e.g., Lightning-Link® antibody labelling kits available from Innova Biosciences), as well as other non-covalent attachment mechanisms. Antibody and oligonucleotide biotinylation techniques are available. See, e.g., Fang, et al., “Fluoride-Cleavable Biotinylation Phosphoramidite for 5'-end-Labelling and Affinity Purification of Synthetic Oligonucleotides,” Nucleic Acids Res. Jan. 15, 2003; 3 l(2):708-715. Likewise, protein and peptide biotinylation techniques have been developed and are readily available. See, e.g., U.S. Pat. No. 6,265,552. Furthermore, click reaction chemistry such as 5’ Azide oligos and Alkyne-NHS for click chemistry, 4’-Amino oligos for HyNic-4B chemistry, a Methyltetrazine-PEG5-NHS Ester reaction, a TCO-PEG4-NHS Ester reaction, strain-promoted alkync-azidc cycloaddition (SPAAC), or the like, can be used to couple reporter oligonucleotide to the core support and/or the detectable label.

[0056] In some embodiments of the disclosure, the methods further include including quenching the MHC multimer reagent generated in (b) with first binding moieties that are not linked to MHC monomers. In some embodiments, the plurality of binding sites of the second binding moiety of the generated MHC multimer reagent are each either bound to an antigen- loaded MHC monomer or bound a first binding moiety. In some embodiments, the plurality of binding sites of the second binding moiety are capable of reversibly (e.g., releasably) binding to the first binding moieties.

[0057] In some embodiments of the methods disclosed herein, the first binding moieties are biotinylated or include a biotin moiety.

[0058] In some embodiments, the core support of the second binding moiety includes one or more biotin-binding sites. In some embodiments, the core support is or includes a biotinbinding protein Suitable biotin-binding proteins include, but are not limited to streptavidin, avidin, deglycosylated avidin (e.g., NeutrAvidin™), traptavidin, tamavidin, xenavidin, bradavidin, AVR2 (avidin related protein 2), AVR4 (avidin related protein 4), and variants, mutants, derivatives, and homologs of any thereof. In some embodiments, the biotin-binding protein is selected from streptavidin, avidin, deglycosylated avidin (e.g., Neutr Avidin™), traptavidin, tamavidin, xenavidin, bradavidin, AVR2 (avidin related protein 2), and AVR4 (avidin related protein 4).

[0059] In some embodiments, the MHC multimer reagent generated in (b) includes a core support including four biotin-binding sites, and wherein at least one, two, three, or four of the biotin-binding sites are each bound to an antigen-loaded MHC monomer (or biotinylated MHC monomer). In some embodiments, biotin-binding sites of the core support not bound to the antigen-loaded MHC monomer (or biotinylated MHC monomer) are bound to biotin.

[0060] MHC monomers suitable for the methods and compositions disclosed herein may be MHC class I molecules and MHC class II molecules. In some embodiments, the MHC monomers are of the same MHC class. In some embodiments, the MHC monomers are of different MHC classes. In instances in which the MHC monomers are of MHC class I, the MHC class I monomers may be human MHC class I molecules. In instances in which the MHC monomers are human MHC class I molecules, the human MHC class I molecules may be human leukocyte antigen (HLA)-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G molecules. In instances in which the MHC monomers are HLA-A molecules, the HLA-A molecules may be of allele A*01:01, A*02:01, A*02:03, A*02:06, A*02:07, A*03:01, A* 11:01, A*23:01, A*24:02, A*25:01, A*26:01, A*29:02, A*30:01, A*31:01, A*32:01, A*33:O3, A*34:02, A*68:01, A*68:02, or A*74:01. In instances in which the MHC monomers are HLA-B molecules, the HLA-B molecules may be of allele B*07:02, B*08:01, B* 14:02, B* 15:01, B* 15:02, B*15:03, B*18:01, B*35:01, B*38:02, B*40:01, B*40:02, B*42:01, B*44:02, B*44:03, B*45:01, B*46:01, B*49:01, B*51:01, B*52:01, B*53:01, B*54:01, B*55:02, B*57:01 or B*58:01. In instances in which the MHC monomers are HLA-C molecules, the HLA-C molecules may be of allele C*01:02, C*02:02, C*03:02, C*O3:O3, C*03:04, C*04:01, C*05:01, C*06:02, C*07:01, C*07:02, C*08:01, C*08:02, C*12:03, C*14:02, C*16:01, C*17:01 or C*18:01.

[0061] In instances in which the MHC monomers are MHC class II molecules, the MHC class II molecules may be human MHC class II molecules. In instances in which the MHC monomers are human MHC class II molecules, the human MHC class II molecules may be a HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ or HLA-DR molecules. In instances in which the MHC monomers are HLA-DR molecules, the HLA-DR molecules may be of allele DRB1*O1O1, DRB1*O3O1, DRBl*0401, DRB1*O7O1, DRB1*O8O1, DRB1*11O1, DRB1*13O1, DRB1*15O1, DRB3*0101, DRB3*0202, DRB4*0101 or DRB5*0101. In instances in which the MHC monomers are HLA-DP molecules, the HLA-DP molecules may be of allele DPAl*0103, DPAl*0202, DPABl*0401 or DPABl*0402. In instances in which the MHC monomers are HLA-DQ molecules, the HLA-DQ molecules may be of allele DQAl*0101, DQBl*0301 or DQB 1*0402.

[0062] In any of methods disclosed herein, the MHC monomers may be of the same allele or of different alleles. For example, the MHC monomers may all be MHC class I molecules. In instances in which all MHC monomers are MHC class I molecules, they may be MHC class I molecules of the same or different alleles. In some embodiments, the MHC monomers may all be MHC class II molecules. In instances in which all MHC monomers are MHC class IT molecules, they may be of the same or different alleles. Tn some embodiments, a subset of the MHC monomers may be MHC class II molecules and the remaining MHC monomers may be MHC class I molecules.

[0063] In a particular embodiment, the MHC monomers may include a MHC class I molecule described in the European Patent Publication No. EP 385722 Al. In some embodiments, the MHC monomers may include a MHC class 1 molecule comprising a heavy chain comprising an alpha- 1 domain and an alpha-2 domain connected by a disulfide bridge, wherein the heavy chain includes the amino acid sequence of SEQ ID NO: 1 described in EP 385722 Al. In some embodiments, the heavy chain includes an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1 described in EP 385722 Al. In some embodiments, the MHC monomers may include a MHC class I molecule comprising a heavy chain comprising an alpha- 1 domain and an alpha-2 domain connected by a disulfide bridge, wherein the heavy chain comprising an amino acid sequence selected from (a) SEQ ID NO: 1 described in EP385722A1, or (b) an amino acid sequence having at least 80% identity to (a) wherein a mutant cysteine residue is positioned in the alpha- 1 domain at amino acid residue 84 or 85 and a mutant cysteine residue is positioned in the alpha-2 domain at amino acid residue 139.

[0064] The plurality of antigens can generally include any antigen types. Non-limiting examples of antigens suitable for the methods and compositions of the disclosure include oligopeptides, proteins, polysaccharides, lipids, liposomes, and infectious agents. For example, an antigen may be a peptide or a peptide fragment of a target antigen associated with an infectious agent, such as a viral, bacterial, parasitic, protozoal or prion agent. In some instances, the antigen, a peptide or peptide fragment of which may be a target antigenic peptide, may be an antigen associated a viral agent. In these instances, the viral agent may be an influenza virus, a coronavirus, a retrovirus, a rhinovirus, or a sarcoma virus. In other instances, the viral agent may be severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), a SARS-CoV-2, a Middle East respiratory syndrome coronavirus (MERS-CoV), or human immunodeficiency virus (HIV), influenza, respiratory syncytial virus, or Ebola virus. Examples of viral antigens include, but are not limited to, corona virus spike (S) protein, an influenza hemagglutinin protein, an HIV envelope protein or any other a viral glycoprotein. In addition or alternatively, the antigen may be an antigen associated with a tumor or a cancer. Antigens associated with a tumor or cancer, include any of epidermal growth factor receptor (EGFR), CD38, platelet-derived growth factor receptor (PDGFR) alpha, insulin growth factor receptor (IGFR), CD20, CD 19, CD47, ERBB2IP, TP53, KRAS, MAGEA1, LC3A2, KIAA0368, CADPS2, CTSB or human epidermal growth factor receptor 2 (HER2). Further, the antigen may be an checkpoint molecule associated with tumors or cancers (e.g., CD38, PD-1, CTLA-4, T1G1T, LAG-3, VISTA, TIM-3), or it may be a cytokine, a GPCR, a cell-based co-stimulatory molecule, a cell-based co-inhibitory molecule, an ion channel, or a growth factor. Further still, the antigen may be associated with a degenerative condition or disease.

[0065] In some embodiments, the plurality of antigen-loaded MHC monomers of the MHC multimer reagent disclosed herein can include different antigens. In some embodiments, the plurality of antigen-loaded MHC monomers of the MHC multimer reagents can include the same antigen. For example, in some embodiments, the MHC multimer reagents of the disclosure can include a core support comprising four biotin-binding sites, and wherein at least one, two, three, or four of the biotin-binding sites arc each bound to an MHC monomer (or biotinylated MHC monomer) loaded with different antigen.

[0066] As discussed above, while any amounts of the second binding moiety and empty MHC monomers can be used in the mixture at step (a) of the disclosed methods, care should be taken to ensure that the antigen-loaded MHC multimer reagents are formed in correct molar ratio(s). In some embodiments, the ratio of the second binding moiety to empty MHC monomers in the mixture ranges from about 5: 1 to about 1:5 (w/w), for example from about 5: 1 to about 1:5 (w/w), from about 5: 1 to about 1:1 (w/w), from about 4: 1 to about 1:2 (w/w), from about 3:1 to about 1:3 (w/w), from about 2: 1 to about 1:5 (w/w), from about 5:1 to about 2:1 (w/w), from about 1: 1 to about 1:2 (w/w), from about 1:2 to about 1:4 (w/w), from about 1:3 to about 1:5 (w/w), from about 5: 1 to about 1:5 (w/w), or from about 5:1 to about 2:5 (w/w). In some embodiments, the ratio of the second binding moiety to empty MHC monomers in the mixture ranges from about 1:1 to about 1:2 (w/w), from about 1: 1.5 to about 1:2 (w/w), from about 1:1.5 to about 1: 1.5 (w/w), from about 1.5:1 to about 1:2 (w/w), or from about 1.5:1 to about 1:2 (w/w). In some embodiments, the ratio of the second binding moiety to empty MHC monomers in the mixture is about 3.1 (w/w), about 2.1 (w/w), about 1.1 (w/w), about 1:2 (w/w), or about 1 :3 (w/w). In some embodiments, the ratio of the second binding moiety to empty MHC monomers in the mixture is about 1:2 (w/w).

[0067] In some embodiments, step (b) of the methods disclosed herein is carried out at suitable temperatures to achieve a desired assembly efficacy, for example those ranging from about 0°C to about 37°C, e.g., from about 5°C to about 15°C, from about 10°C to about 20°C, from about 10°C to about 30°C, from about 15°C to about 25°C, from about 20°C to about 30°C, from about 25°C to about 35°C, from about 20°C to about 25°C, from about 15°C to about 30°C, from about 10°C to about 37°C, from about 15°C to about 20°C, from about 20°C to about 25°C, from about 21°C to about 24°C, or from about 20°C to about 35°C. In some embodiments, step

(b) of the methods disclosed herein is carried out at a temperature ranging from about 0°C to about 37°C, from about 4°C to about 25°C, or from 4°C to about 22°C. In some embodiments, step (b) is carried out at a temperature of about 4°C.

[0068] In some embodiments, step (b) of the methods disclosed herein is carried out for suitable durations to achieve a desired assembly efficacy. In some embodiments, step (b) is carried out for a duration of less than about 120 minutes, for example, less than about 110 minutes, less than about 100 minutes, less than about 90 minutes, less than about 60 minutes, less than about 45 minutes, less than about 30 minutes, less than about 20 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes. In some embodiments, step (b) is carried out for a duration of from about 5 minutes to about 60 minutes, for example, about 5 minutes to about 50 minutes, about 5 minutes to about 40 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 20 minutes, about 10 minutes to about 40 minutes, about 10 minutes to about 50 minutes, about 10 minutes to about 60 minutes, about 15 minutes to about 30 minutes, about 15 minutes to about 40 minutes, or about 15 minutes to about 60 minutes. In some embodiments, step (b) of the methods disclosed herein is carried out for a duration of about 15 minutes to about 20 minutes. In some embodiments, step (b) of the methods disclosed herein is carried out for a duration of about 15 minutes to about 30 minutes. In some embodiments, step (b) of the methods disclosed herein is carried out for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, or about 30 minutes.

[0069] In some embodiments, step (b) is carried out on ice, e.g., at a temperature ranging from about 0°C to about 6°C. In some embodiments, the step (b) is carried out on ice for a duration of about 5 minutes, about 15 minutes, about 30 minutes, or about 45 minutes. In some embodiments, the step (b) is carried out on ice (e.g., about 0°C to about 6°C) for a duration of about 30 minutes.

[0070] In some embodiments, step (b) is carried out at room temperature e.g., 21°C - 24°C) and for a duration of about 5 minutes. In some embodiments, the MHC multimer reagent generated in (b) is a barcoded MHC multimer reagent including the second binding moiety that is attached to a plurality of antigen-loaded MHC monomers and further attached to the reporter oligonucleotide.

[0071] In some embodiments, the methods disclosed herein further include the isolation and/or purification of the MHC multimer reagents, e.g., barcoded MHC multimer reagents prepared by the disclosed methods. Strategics, methods, and reagents suitable for the isolation and/or purification of MHC multimer reagents are known in the art. Accordingly, MHC multimer reagents, e.g., barcoded MHC multimer reagents, prepared by the disclosed methods are also within the scope of this application.

METHODS FOR CHARACTERIZATION OF ANTIGEN-BINDING MOLECULES (AMBS)

[0072] As described in more detail below, one aspect of the disclosure relates to new approaches and methods for characterization of antigen-binding molecules, e.g., antibodies, B cell receptors, T cell receptors (TCRs), and TCR-like antibodies (Abs). [0073] In some embodiments, the disclosed methods include: (a) providing a reaction mixture including: (i) a plurality of immune cells and/or a plurality of cell beads comprising immune cells, and (ii) a plurality of barcoded MHC multimer reagents as described herein; (b) partitioning the mixture, or a portion thereof, into a plurality of partitions, wherein the partitioning provides a first partition including: (i) a first immune cell and/or a first cell bead comprising a first immune cell, (ii) a first barcoded MHC multimer reagent, and (iii) a plurality of nucleic acid barcode molecules including a partition- specific barcoded sequence; and (c) generating barcoded nucleic acid molecules, wherein the barcoded nucleic acid molecules include a first barcoded nucleic acid molecule including (i) a first nucleic acid sequence encoding at least a portion of an antigen-binding molecule (ABM) expressed by the first immune cell and/or a first cell bead comprising a first immune cell, or a reverse complement thereof and (ii) the partition-specific barcode sequence or a reverse complement thereof, and a second barcoded nucleic acid molecule including (i) the first reporter barcode sequence or a reverse complement thereof and (ii) the partition-specific barcode sequence or a reverse complement thereof.

[0074] Non-limiting exemplary embodiments of the methods for characterizing an ABM disclosed herein can include one or more of the following features. In some embodiments, the methods further included generating a third barcoded nucleic acid molecule including (i) the partition barcode sequence or a reverse complement thereof and (ii) a second nucleic acid sequence or a reverse complement thereof, the second nucleic acid sequence encoding a different portion of the ABM expressed by the first immune cell and/or a first cell bead comprising a first immune cell. Suitable methods, compositions, systems, and kits for single cell analysis of antigen-binding molecules produced by immune cells and/or antigen binding activity are disclosed in US20180105808A1, US20180179590A1, US20190338353A1, and US20190367969A1, each of which are incorporated by reference herein in their entirety.

[0075] In some embodiments, the methods further include generating a fourth barcoded nucleic acid molecule including (i) the partition -specific barcode sequence or a reverse complement thereof and a third nucleic acid sequence, wherein the third nucleic acid sequence is a sequence of an mRNA analyte of the first immune cell and/or a first cell bead comprising a first immune cell, or a reverse complement thereof, or a cDNA sequence of the mRNA analyte of the first immune cell and/or a first cell bead comprising a first immune cell, or a reverse complement thereof.

[0076] In some embodiments, the methods disclosed herein further include determining a sequence of the first barcoded nucleic acid molecule or an amplicon thereof, and determining a sequence of the second barcoded nucleic molecule or an amplicon thereof.

[0077] In some embodiments, the methods described herein further include (i) identifying the ABM as expressed by the first immune cell and/or a first cell bead comprising a first immune cell based on the determined sequence of the first barcoded nucleic acid molecule or amplicon thereof and (ii) identifying the first immune cell and/or a first cell bead comprising a first immune cell having bound the target antigen based on the determined sequence of the second barcoded nucleic acid molecule or amplicon thereof. In some embodiments, the methods described herein further include determining a sequence of the third barcoded nucleic acid molecule or an amplicon thereof. In some embodiments, the methods further include determining a sequence of the fourth barcoded nucleic acid molecule or an amplicon thereof.

[0078] In some embodiments, the plurality of immune cells and/or cell beads comprising immune cells includes B cells. In some embodiments, the B cells include a plasmablast, a plasma cell, a memory B cell, a regulatory B cell, and/or a lymphoplasmacytoid cell. In some embodiments, the first immune cell is a B cell bound to the antigen(s) of the first barcoded MHC multimer reagent. In some embodiments, the ABM produced by the B cell is a B cell receptor (BCR), an antibody (Ab) or an antigen-binding fragment thereof. In some embodiments, the plurality of immune cells and/or cell beads comprising immune cells includes T cells. In some embodiments, the T cells include a CD8+ T cytotoxic lymphocyte cell and/or a CD4+ T helper lymphocyte cell. In some embodiments, the CD8+ T cytotoxic lymphocyte cell is selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, effector CD8+ T cells, CD8+ stem memory T cells, bulk CD8+ T cells. In some embodiments, the CD4+ T helper lymphocyte cell is selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, effector CD4+ T cells, CD4+ stem memory T cells, and bulk CD4+ T cells. In some embodiments, the T cell is an exhausted T cell or a non-exhausted T cell. In some embodiments, the first immune cell is a T cell bound to the antigen(s) of the first barcoded MHC multimer reagent. In some embodiments, the ABM produced by the T cell is a TCR.

Systems and methods for partitioning

[0079] In some aspects, such as those that have been described above, the methods provided herein include a step of partitioning, or include a step of generating barcoded nucleic acid molecules, or may include an additional processing step(s). This description sets forth examples, embodiments and characteristics of steps of the methods and of reagents useful in the methods.

[0080] In an aspect, the systems and methods described herein provide for the compartmentalization, depositing, or partitioning of one or more particles (e.g., biological particles, macromolecular constituents of biological particles, beads, reagents, etc. ) into discrete compartments or partitions (referred to interchangeably herein as partitions), where each partition maintains separation of its own contents from the contents of other partitions.

[0081] In some embodiments disclosed herein, the partitioned biological particle is a labelled cell, e.g. a labelled immune cell (e.g., B cell or T cell). In some embodiments, the labelled immune cell is a B cell which expresses an antigen-binding molecule (e.g., an immune receptor, an antibody or a functional fragment thereof) on its surface. In other examples, the partitioned particle can be a labelled cell engineered to express antigen-binding molecules (e.g., an immune receptors, antibodies or functional fragments thereof). In some embodiments, the labelled immune cell is a B cell. In some embodiments, the labelled immune cell is a T cell.

[0082] The term “partition,” as used herein, generally, refers to a space or volume that can be suitable to contain one or more cells, one or more species of features or compounds, or conduct one or more reactions. A partition can be a physical container, compartment, or vessel, such as a droplet, a flow cell, a reaction chamber, a reaction compartment, a tube, a well, or a microwell. A partition can be a volume or sub-volume wherein diffusion of contents beyond the volume or sub-volume is inhibited. For example, the partitions can include a porous matrix that is capable of entraining and/or retaining materials within its matrix.

[0083] In some embodiments, the compartments or partitions include partitions that are flowable within fluid streams. These partitions can include, for example, micro-vesicles that have an outer barrier surrounding an inner fluid center or core, or, in some cases, the partitions can include a porous matrix that is capable of entraining and/or retaining materials within its matrix. In some aspects, partitions comprise droplets of aqueous fluid within a non-aqueous continuous phase (e.g., oil phase). A variety of different vessels are described in, for example, U.S. Patent Application Publication No. 2014/0155295. Emulsion systems for creating stable droplets in non-aqueous or oil continuous phases are described in detail in, e.g., U.S. Patent Application Publication No. 2010/010511.

[0084] In some embodiments, a partition herein includes a space or volume that can be suitable to contain one or more species or conduct one or more reactions. A partition can be a physical compartment, such as a droplet or well. The partition can be an isolated space or volume from another space or volume. The droplet can be a first phase (e.g., aqueous phase) in a second phase (e.g., oil) immiscible with the first phase. The droplet can be a first phase in a second phase that does not phase separate from the first phase, such as, for example, a capsule or liposome in an aqueous phase. A partition can include one or more other (inner) partitions. In some cases, a partition can be a virtual compartment that can be defined and identified by an index (e.g., indexed libraries) across multiple and/or remote physical compartments. For example, a physical compartment can include a plurality of virtual compartments.

[0085] A partition can include one or more particles. A partition can include one or more types of particles. For example, a partition of the present disclosure can include one or more biological particles, e.g., labelled immune cells, and/or macromolecular constituents thereof. A partition can include one or more gel beads. A partition can include one or more cell beads. A partition can include a single gel bead, a single cell bead, or both a single cell bead and single gel bead. A partition can include one or more reagents. Alternatively, a partition can be unoccupied. For example, a partition cannot comprise a bead. Unique identifiers, such as barcodes, can be injected into the droplets previous to, subsequent to, or concurrently with droplet generation, such as via a bead, as described elsewhere herein. Microfluidic channel networks (e.g., on a chip) can be utilized to generate partitions as described herein. Alternative mechanisms can also be employed in the partitioning of individual biological particles, including porous membranes through which aqueous mixtures of cells are extruded into non-aqueous fluids.

[0086] The partitions can be flowable within fluid streams. The partitions can include, for example, micro-vesicles that have an outer barrier surrounding an inner fluid center or core. In some cases, the partitions can include a porous matrix that is capable of entraining and/or retaining materials (e.g., expressed antibodies or antigen-binding fragments thereof) within its matrix (e.g., via a capture agent configured to couple to both the matrix and the expressed antibody or antigen-binding fragment thereof). The partitions can be droplets of a first phase within a second phase, wherein the first and second phases are immiscible. For example, the partitions can be droplets of aqueous fluid within a non-aqueous continuous phase e.g., oil phase). In another example, the partitions can be droplets of a non-aqueous fluid within an aqueous phase. In some examples, the partitions can be provided in a water-in-oil emulsion or oil-in-water emulsion. A variety of different vessels is described in, for example, U.S. Patent Application Publication No. 2014/0155295. Emulsion systems for creating stable droplets in non-aqueous or oil continuous phases are described in, for example, U.S. Patent Application Publication No. 2010/0105112.

[0087] In the case of droplets in an emulsion, allocating individual particles (e.g., labelled immune cells) to discrete partitions can, in one non-limiting example, be accomplished by introducing a flowing stream of particles in an aqueous fluid into a flowing stream of a nonaqueous fluid, such that droplets are generated at the junction of the two streams. Fluid properties (e.g., fluid flow rates, fluid viscosities, etc.), particle properties (e.g., volume fraction, particle size, particle concentration, etc.), microfluidic architectures (e.g., channel geometry, etc.), and other parameters can be adjusted to control the occupancy of the resulting partitions (e.g., number of biological particles per partition, number of beads per partition, etc.). For example, partition occupancy can be controlled by providing the aqueous stream at a certain concentration and/or flow rate of particles. To generate single biological particle partitions, the relative flow rates of the immiscible fluids can be selected such that, on average, the partitions can contain less than one biological particle per partition in order to ensure that those partitions that are occupied are primarily singly occupied. In some cases, partitions among a plurality of partitions can contain at most one biological particle (e.g., bead, DNA, cell, such as a labelled B cell or T cell, or cellular material). In some embodiments, the various parameters (e.g., fluid properties, particle properties, microfluidic architectures, etc.) can be selected or adjusted such that a majority of partitions are occupied, for example, allowing for only a small percentage of unoccupied partitions. The flows and channel architectures can be controlled as to ensure a given number of singly occupied partitions, less than a certain level of unoccupied partitions and/or less than a certain level of multiply occupied partitions.

[0088] In the case of droplets in emulsion, allocating individual cells to discrete partitions can generally be accomplished by introducing a flowing stream of cells in an aqueous fluid into a flowing stream of a non-aqueous fluid, such that droplets are generated at the junction of the two streams. By providing the aqueous cell-containing stream at a certain concentration of cells, the occupancy of the resulting partitions (e.g., number of cells per partition) can be controlled. For example, where single cell partitions are desired, the relative flow rates of the fluids can be selected such that, on average, the partitions contain less than one cell per partition, in order to ensure that those partitions that are occupied, are primarily singly occupied. In some embodiments, the relative flow rates of the fluids can be selected such that a majority of partitions are occupied, e.g., allowing for only a small percentage of unoccupied partitions. In some embodiments, the flows and channel architectures are controlled as to ensure a desired number of singly occupied partitions, less than a certain level of unoccupied partitions and less than a certain level of multiply occupied partitions.

[0089] In some embodiments, the methods described herein can be performed such that a majority of occupied partitions include no more than one cell per occupied partition. In some embodiments, the partitioning process is performed such that fewer than 25%, fewer than 20%, fewer than 15%, fewer than 10%, fewer than 5%, fewer than 2%, or fewer than 1% the occupied partitions contain more than one cell. In some embodiments, fewer than 20% of the occupied partitions include more than one cell. In some embodiments, fewer than 10% of the occupied partitions include more than one cell per partition. In some embodiments, fewer than 5% of the occupied partitions include more than one cell per partition. In some embodiments, it is desirable to avoid the creation of excessive numbers of empty partitions. For example, from a cost perspective and/or efficiency perspective, it may be desirable to minimize the number of empty partitions. While this can be accomplished by providing sufficient numbers of cells into the partitioning zone, the Poissonian distribution can optionally be used to increase the number of partitions that include multiple cells. As such, in some embodiments described herein, the flow of one or more of the cells, or other fluids directed into the partitioning zone are performed such that no more than 50% of the generated partitions, no more than 25% of the generated partitions, or no more than 10% of the generated partitions are unoccupied. Further, in some aspects, these flows are controlled so as to present non-Poissonian distribution of single occupied partitions while providing lower levels of unoccupied partitions. Restated, in some aspects, the above noted ranges of unoccupied partitions can be achieved while still providing any of the single occupancy rates described above. For example, in some embodiments, the use of the systems and methods described herein creates resulting partitions that have multiple occupancy rates of less than 25%, less than 20%, less than 15%), less than 10%, and in some embodiments, less than 5%, while having unoccupied partitions of less than 50%), less than 40%, less than 30%, less than 20%, less than 10%, and in some embodiments, less than 5%.

[0090] Although described in terms of providing substantially singly occupied partitions, above, in some embodiments, the methods as described herein include providing multiply occupied partitions, e.g., containing two, three, four or more cells and/or beads comprising nucleic acid barcode molecules within a single partition.

[0091] In some embodiments, the reporter oligonucleotides contained within a partition are distinguishable from the reporter oligonucleotides contained within other partitions of the plurality of partitions. This can be accomplished by incorporating one or more partition-specific barcode sequences into the reporter barcode sequence of the reporter oligonucleotides contained within the partition.

[0092] In some embodiments, it may be desirable to incorporate multiple different barcode sequences within a given partition, either attached to a single bead or multiple beads within the partition. For example, in some cases, a mixed, but known barcode sequences set can provide greater assurance of identification in the subsequent processing, e.g., by providing a stronger address or attribution of the barcodes to a given partition, as a duplicate or independent confirmation of the output from a given partition.

Micro fluidic channel structures

[0093] Microfluidic channel networks (e.g., on a chip) can be utilized to generate partitions as described herein. Alternative mechanisms can also be employed in the partitioning of individual biological particles, including porous membranes through which aqueous mixtures of cells are extruded into non-aqueous fluids.

[0094] FIG. 1 shows an example of a microfluidic channel structure 100 for partitioning individual biological particles. The channel structure 100 can include channel segments 102, 104, 106 and 108 communicating at a channel junction 110. In operation, a first aqueous fluid 112 that includes suspended biological particles (e.g., cells, for example, labelled immune cells, B cells, or T cells) 114 can be transported along channel segment 102 into junction 110, while a second fluid 116 that is immiscible with the aqueous fluid 112 is delivered to the junction 110 from each of channel segments 104 and 106 to create discrete droplets 118, 120 of the first aqueous fluid 112 flowing into channel segment 108, and flowing away from junction 110. The channel segment 108 can be fluidically coupled to an outlet reservoir where the discrete droplets can be stored and/or harvested. A discrete droplet generated can include an individual biological particle 114 (such as droplets 118). A discrete droplet generated can include more than one individual biological particle e.g., labelled immune cell, e.g., B cell, or T cell) 114 (not shown in FIG. 1). A discrete droplet can contain no biological particle 114 (such as droplet 120). Each discrete partition can maintain separation of its own contents (e.g., individual biological particle 114) from the contents of other partitions.

[0095] The second fluid 116 can comprise an oil, such as a fluorinated oil, that includes a fluoro surfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets 118, 120. Examples of particularly useful partitioning fluids and fluorosurfactants are described, for example, in U.S. Patent Application Publication No. 2010/0105112.

[0096] As will be appreciated, the channel segments described herein can be coupled to any of a variety of different fluid sources or receiving components, including reservoirs, tubing, manifolds, or fluidic components of other systems. As will be appreciated, the microfluidic channel structure 100 can have other geometries. For example, a microfluidic channel structure can have more than one channel junction. For example, a microfluidic channel structure can have 2, 3, 4, or 5 channel segments each carrying particles (e.g., biological particles, cell beads, and/or gel beads) that meet at a channel junction. Fluid can be directed to flow along one or more channels or reservoirs via one or more fluid flow units. A fluid flow unit can comprise compressors (e.g., providing positive pressure), pumps (e.g., providing negative pressure), actuators, and the like to control flow of the fluid. Fluid can also or otherwise be controlled via applied pressure differentials, centrifugal force, electrokinetic pumping, vacuum, capillary or gravity flow, or the like.

[0097] The generated droplets can include two subsets of droplets: (1) occupied droplets 118, containing one or more biological particles 114, e.g., labelled engineered cells, labelled immune cells, B cells, or T cells, and (2) unoccupied droplets 120, not containing any biological particles 114. Occupied droplets 118 can include singly occupied droplets (having one biological particle, such as one labelled immune cell, B cells, or T cell) and multiply occupied droplets (having more than one biological particle, such as multiple engineered cells, labelled immune cells, B cells, or T cells). As described elsewhere herein, in some cases, the majority of occupied partitions can include no more than one biological particle, e.g., labelled immune cell, e.g., B cell, or T cell, per occupied partition and some of the generated partitions can be unoccupied (of any biological particle, or labelled engineered cell, labelled immune cell, B cell, or T cell). In some cases, though, some of the occupied partitions can include more than one biological particle, e.g., labelled engineered cell, labelled immune cell, B cell, or T cell. In some cases, the partitioning process can be controlled such that fewer than about 25% of the occupied partitions contain more than one biological particle, and in many cases, fewer than about 20% of the occupied partitions have more than one biological particle, while in some cases, fewer than about 10% or even fewer than about 5% of the occupied partitions include more than one biological particle per partition.

[0098] In some cases, it can be desirable to minimize the creation of excessive numbers of empty partitions, such as to reduce costs and/or increase efficiency. While this minimization can be achieved by providing a sufficient number of biological particles (e.g., biological particles, such as labelled engineered cell, immune B cells, B cells, or T cells 114) at the partitioning junction 110, such as to ensure that at least one biological particle is encapsulated in a partition, the Poissonian distribution can expectedly increase the number of partitions that include multiple biological particles. As such, where singly occupied partitions are to be obtained, at most about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less of the generated partitions can be unoccupied.

[0099] In some cases, the flow of one or more of the biological particles, such as B cells, e.g., memory B cells, or plasma cells, (e.g., in channel segment 102), or other fluids directed into the partitioning junction (e.g., in channel segments 104, 106) can be controlled such that, in many cases, no more than about 50% of the generated partitions, no more than about 25% of the generated partitions, or no more than about 10% of the generated partitions are unoccupied. These flows can be controlled so as to present a non-Poissonian distribution of single-occupied partitions while providing lower levels of unoccupied partitions. The above noted ranges of unoccupied partitions can be achieved while still providing any of the single occupancy rates described above. For example, in many cases, the use of the systems and methods described herein can create resulting partitions that have multiple occupancy rates of less than about 25%, less than about 20%, less than about 15%, less than about 10%, and in many cases, less than about 5%, while having unoccupied partitions of less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less.

[0100] As will be appreciated, the above-described occupancy rates are also applicable to partitions that include both biological particles (e.g., labelled B cells or T cells) and additional reagents, including, but not limited to, beads (e.g., gel beads) carrying barcoded nucleic acid molecules (e.g., nucleic acid barcode molecules or barcoded oligonucleotides) (described in relation to FIGS. 1 and 2). The occupied partitions (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the occupied partitions) can include both a bead comprising barcoded nucleic acid nucleic acid molecules (e.g., nucleic acid barcode molecules) and a biological particle.

[0101] FIG. 8 shows an example of a microfluidic channel structure 800 for delivering barcode carrying beads to droplets. The channel structure 800 can include channel segments 801, 802, 804, 806 and 808 communicating at a channel junction 810. In operation, the channel segment 801 may transport an aqueous fluid 812 that includes a plurality of beads 814 (e.g., with nucleic acid molecules, e.g., nucleic acid barcode molecules or barcoded oligonucleotides, molecular tags) along the channel segment 801 into junction 810. The plurality of beads 814 may be sourced from a suspension of beads. For example, the channel segment 801 may be connected to a reservoir comprising an aqueous suspension of beads 814. The channel segment 802 may transport the aqueous fluid 812 that includes a plurality of biological particles 816 along the channel segment 802 into junction 810. The plurality of biological particles 816 may be sourced from a suspension of biological particles. For example, the channel segment 802 may be connected to a reservoir comprising an aqueous suspension of biological particles 816. In some instances, the aqueous fluid 812 in either the first channel segment 801 or the second channel segment 802, or in both segments, can include one or more reagents, as further described below. A second fluid 818 that is immiscible with the aqueous fluid 812 (e.g., oil) can be delivered to the junction 810 from each of channel segments 804 and 806. Upon meeting of the aqueous fluid 812 from each of channel segments 801 and 802 and the second fluid 818 from each of channel segments 804 and 806 at the channel junction 810, the aqueous fluid 812 can be partitioned as discrete droplets 1420 in the second fluid 818 and flow away from the junction 810 along channel segment 808. The channel segment 808 may deliver the discrete droplets to an outlet reservoir fluidly coupled to the channel segment 808, where they may be harvested. As an alternative, the channel segments 801 and 802 may meet at another junction upstream of the junction 810. At such junction, beads and biological particles may form a mixture that is directed along another channel to the junction 810 to yield droplets 820. The mixture may provide the beads and biological particles in an alternating fashion, such that, for example, a droplet comprises a single bead and a single biological particle.

[0102] In another aspect, in addition to or as an alternative to droplet-based partitioning, biological particles (e.g., cells) may be comprised within (e.g., encapsulated within) a particulate material to form a “cell bead.”

[0103] A cell bead can contain a biological particle (e.g., a cell) or macromolecular constituents (e.g., RNA, DNA, proteins, etc.) of a biological particle. A cell bead may include a single cell or multiple cells, or a derivative of the single cell or multiple cells. For example, after lysing and washing the cells, inhibitory components from cell lysates can be washed away and the macromolecular constituents can be bound as cell beads. Systems and methods disclosed herein can be applicable to both cell beads (and/or droplets or other partitions) containing biological particles and cell beads (and/or droplets or other partitions) containing macromolecular constituents of biological particles. Cell beads may be or include a cell, cell derivative, cellular material and/or material derived from the cell in, within, or encased in a matrix, such as a polymeric matrix. In some cases, a cell bead may comprise a live cell. Tn some instances, the live cell may be capable of being cultured when enclosed in a gel or polymer matrix, or of being cultured when comprising a gel or polymer matrix. In some instances, the polymer or gel may be diffusively permeable to certain components and diffusively impermeable to other components (e.g., macromolecular constituents).

[0104] Cell beads can provide certain potential advantages of being more storable and more portable than droplet-based partitioned biological particles. Furthermore, in some cases, it may be desirable to allow biological particles to incubate for a select period of time before analysis, such as in order to characterize changes in such biological particles over time, either in the presence or absence of different stimuli (or reagents).

[0105] Suitable polymers or gels may include one or more of disulfide cross-linked polyacrylamide, agarose, alginate, polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate, PEG-thiol, PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronic acid, collagen, fibrin, gelatin, or elastin. The polymer or gel may comprise any other polymer or gel.

[0106] Encapsulation of biological particles may be performed by a variety of processes. Such processes may combine an aqueous fluid containing the biological particles with a polymeric precursor material that may be capable of being formed into a gel or other solid or semi-solid matrix upon application of a particular stimulus to the polymer precursor. The conditions sufficient to polymerize or gel the precursors may comprise any conditions sufficient to polymerize or gel the precursors. Such stimuli can include, for example, thermal stimuli e.g., either heating or cooling), photo-stimuli (e.g., through photo-curing), chemical stimuli (e.g., through crosslinking, polymerization initiation of the precursor (e.g., through added initiators)), electromagnetic radiation, mechanical stimuli, or any combination thereof.

[0107] In some cases, air knife droplet or aerosol generators may be used to dispense droplets of precursor fluids into gelling solutions in order to form cell beads that include individual biological particles or small groups of biological particles. Likewise, membranebased encapsulation systems may be used to generate cell beads comprising encapsulated biological particles as described herein. Microfluidic systems of the present disclosure, such as that shown in FIG. 1, may be readily used in encapsulating biological particles (e.g., cells) as described herein. Exemplary methods for encapsulating biological particles (e.g., cells) are also further described in U.S. Patent Application Pub. No. US 2015/0376609 and PCT/US2018/016019, which are hereby incorporated by reference in their entirety. In particular, and with reference to FIG. 1, the aqueous fluid 112 comprising (i) the biological particles 114 and (ii) the polymer precursor material (not shown) is flowed into channel junction 110, where it is partitioned into droplets 118, 120 through the flow of non-aqueous fluid 116. In the case of encapsulation methods, non-aqueous fluid 116 may also include an initiator (not shown) to cause polymerization and/or crosslinking of the polymer precursor to form the bead that includes the entrained biological particles. Examples of polymer precursor/initiator pairs include those described in U.S. Patent Application Publication No. 2014/0378345, which is entirely incorporated herein by reference for all purposes.

[0108] In some cases, encapsulated biological particles can be selectively releasable from the cell bead, such as through passage of time or upon application of a particular stimulus, that degrades the bead sufficiently to allow the biological particles (e.g., cell), or its other contents to be released from the bead, such as into a partition e.g., droplet). Exemplary stimuli suitable for degradation of the bead are described in U.S. Patent Application Publication No. 2014/0378345, which is entirely incorporated herein by reference for all purposes.

[0109] The polymer or gel may be diffusively permeable to chemical or biochemical reagents. The polymer or gel may be diffusively impermeable to macromolecular constituents of the biological particle. In this manner, the polymer or gel may act to allow the biological particle to be subjected to chemical or biochemical operations while spatially confining the macromolecular constituents to a region of the droplet defined by the polymer or gel.

[0110] The polymer or gel may be functionalized to bind to targeted analytes, such as nucleic acids, proteins, carbohydrates, lipids or other analytes. The polymer or gel may be polymerized or gelled via a passive mechanism. The polymer or gel may be stable in alkaline conditions or at elevated temperature. The polymer or gel may have mechanical properties similar to the mechanical properties of the bead. For instance, the polymer or gel may be of a similar size to the bead. The polymer or gel may have a mechanical strength (e.g. tensile strength) similar to that of the bead. The polymer or gel may be of a lower density than an oil. The polymer or gel may be of a density that is roughly similar to that of a buffer. The polymer or gel may have a tunable pore size. The pore size may be chosen to, for instance, retain denatured nucleic acids. The pore size may be chosen to maintain diffusive permeability to exogenous chemicals such as sodium hydroxide (NaOH) and/or endogenous chemicals such as inhibitors. The polymer or gel may be biocompatible. The polymer or gel may maintain or enhance cell viability. The polymer or gel may be biochemically compatible. The polymer or gel may be polymerized and/or depolymerized thermally, chemically, enzymatically, and/or optically.

[0111] The encapsulation of biological particles may constitute the partitioning of the biological particles into which other reagents are co-partitioned. Alternatively or in addition, encapsulated biological particles may be readily deposited into other partitions (e.g., droplets) as described above.

Wells

[0112] As described herein, one or more processes can be performed in a partition, which can be a well. The well can be a well of a plurality of wells of a substrate, such as a microwell of a microwell array or plate, or the well can be a microwell or microchamber of a device (e.g., microfluidic device) comprising a substrate. The well can be a well of a well array or plate, or the well can be a well or chamber of a device (e.g., fluidic device). In some embodiments, a well of a fluidic device is fluidically connected to another well of the fluidic device. Accordingly, the wells or microwells can assume an “open” configuration, in which the wells or microwells are exposed to the environment (e.g., contain an open surface) and are accessible on one planar face of the substrate, or the wells or microwells can assume a “closed” or “sealed” configuration, in which the microwells are not accessible on a planar face of the substrate. In some instances, the wells or microwells can be configured to toggle between “open” and “closed” configurations. For instance, an “open” microwell or set of microwells can be “closed” or “sealed” using a membrane (e.g., semi-permeable membrane), an oil (e.g., fluorinated oil to cover an aqueous solution), or a lid, as described elsewhere herein. The wells or microwells can be initially provided in a “closed” or “sealed” configuration, wherein they arc not accessible on a planar surface of the substrate without an external force. For instance, the “closed” or “sealed” configuration can include a substrate such as a sealing film or foil that is puncturable or pierceable by pipette tip(s). Suitable materials for the substrate include, without limitation, polyester, polypropylene, polyethylene, vinyl, and aluminum foil.

[0113] In some embodiments, the well can have a volume of less than 1 milliliter (mL). For example, the well can be configured to hold a volume of at most 1000 microliters (pL), at most 100 pL, at most 10 pL, at most 1 pL, at most 100 nanoliters (nL), at most 10 nL, at most 1 nL, at most 100 picoliters (pL), at most 10 (pL), or less. The well can be configured to hold a volume of about 1000 pL, about 100 |aL, about 10 pL, about 1 pL, about 100 nL, about 10 nL, about 1 nL, about 100 pL, about 10 pL, etc. The well can be configured to hold a volume of at least 10 pL, at least 100 pL, at least 1 nL, at least 10 nL, at least 100 nL, at least 1 pL, at least 10 pL, at least 100 pL, at least 1000 pL, or more. The well can be configured to hold a volume in a range of volumes listed herein, for example, from about 5 nL to about 20 nL, from about 1 nL to about 100 nL, from about 500 pL to about 100 pL, etc. The well can be of a plurality of wells that have varying volumes and can be configured to hold a volume appropriate to accommodate any of the partition volumes described herein.

[0114] In some instances, a microwell array or plate includes a single variety of micro wells. In some instances, a micro well array or plate includes a variety of micro wells. For instance, the microwell array or plate can include one or more types of microwells within a single microwell array or plate. The types of microwells can have different dimensions (e.g., length, width, diameter, depth, cross-sectional area, etc.), shapes (e.g., circular, triangular, square, rectangular, pentagonal, hexagonal, heptagonal, octagonal, nonagonal, decagonal, etc.), aspect ratios, or other physical characteristics. The microwell array or plate can include any number of different types of microwells. For example, the microwell array or plate can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more different types of microwells. A well can have any dimension (e.g., length, width, diameter, depth, cross-sectional area, volume, etc.), shape (e.g., circular, triangular, square, rectangular, pentagonal, hexagonal, heptagonal, octagonal, nonagonal, decagonal, other polygonal, etc.), aspect ratios, or other physical characteristics described herein with respect to any well.

[0115] In certain instances, the microwell array or plate includes different types of micro wells that are located adjacent to one another within the array or plate. For example, a micro well with one set of dimensions can be located adjacent to and in contact with another microwell with a different set of dimensions. Similarly, microwells of different geometries can be placed adjacent to or in contact with one another. The adjacent microwells can be configured to hold different articles; for example, one microwell can be used to contain a cell, cell bead, or other sample (e.g., cellular components, nucleic acid molecules, nucleic acid barcode molecules, etc.) while the adjacent microwell can be used to contain a droplet, bead, or other reagent. In some cases, the adjacent micro wells can be configured to merge the contents held within, e.g., upon application of a stimulus, or spontaneously, upon contact of the articles in each microwell.

[0116] As is described elsewhere herein, a plurality of partitions can be used in the systems, compositions, and methods described herein. For example, any suitable number of partitions (e.g., wells or droplets) can be generated or otherwise provided. For example, in the case when wells are used, at least about 1,000 wells, at least about 5,000 wells, at least about 10,000 wells, at least about 50,000 wells, at least about 100,000 wells, at least about 500,000 wells, at least about 1,000,000 wells, at least about 5,000,000 wells at least about 10,000,000 wells, at least about 50,000,000 wells, at least about 100,000,000 wells, at least about 500,000,000 wells, at least about 1,000,000,000 wells, or more wells can be generated or otherwise provided. Moreover, the plurality of wells can include both unoccupied wells (e.g., empty wells) and occupied wells.

[0117] A well can include any of the reagents described herein, or combinations thereof. These reagents can include, for example, barcode molecules, enzymes, adapters, and combinations thereof. The reagents can be physically separated from a sample (for example, a cell, cell bead, or cellular components, e.g., proteins, nucleic acid molecules, etc.) that is placed in the well. This physical separation can be accomplished by containing the reagents within, or coupling to, a bead that is placed within a well. The physical separation can also be accomplished by dispensing the reagents in the well and overlaying the reagents with a layer that is, for example, dissolvable, meltable, or permeable prior to introducing the polynucleotide sample into the well. This layer can be, for example, an oil, wax, membrane (e.g., semi- permeable membrane), or the like. The well can be sealed at any point, for example, after addition of the bead, after addition of the reagents, or after addition of either of these components. The sealing of the well can be useful for a variety of purposes, including preventing escape of beads or loaded reagents from the well, permitting select delivery of certain reagents (e.g., via the use of a semi-permeable membrane), for storage of the well prior to or following further processing, etc.

[0118] Once sealed, the well may be subjected to conditions for further processing of a cell (or cells) in the well. For instance, reagents in the well may allow further processing of the cell, e.g., cell lysis, as further described herein. Alternatively, the well (or wells such as those of a well-based array) comprising the cell (or cells) may be subjected to freeze-thaw cycling to process the cell (or cells), e.g., cell lysis. The well containing the cell may be subjected to freezing temperatures (e.g., 0°C, below 0°C, -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -35°C, - 40°C, -45°C, -50°C, -55°C, -60°C, -65°C, -70°C, -80°C, or -85°C). Freezing may be performed in a suitable manner, e.g. , sub-zero freezer or a dry ice/ethanol bath. Following an initial freezing, the well (or wells) comprising the cell (or cells) may be subjected to freeze-thaw cycles to lyse the cell (or cells). In one embodiment, the initially frozen well (or wells) are thawed to a temperature above freezing (e.g., 4°C or above, 8°C or above, 12°C or above, 16°C or above, 20°C or above, room temperature, or 25°C or above). In another embodiment, the freezing is performed for less than 10 minutes (e.g., 5 minutes or 7 minutes) followed by thawing at room temperature for less than 10 minutes (e.g., 5 minutes or 7 minutes). This freeze-thaw cycle may be repeated a number of times, e.g., 2, 3, 4 or more times, to obtain lysis of the cell (or cells) in the well (or wells). Tn one embodiment, the freezing, thawing and/or freeze/thaw cycling is performed in the absence of a lysis buffer. Additional disclosure related to freeze-thaw cycling is provided in WO2019165181A1, which is incorporated herein by reference in its entirety.

[0119] A well can include free reagents and/or reagents encapsulated in, or otherwise coupled to or associated with beads or droplets. In some embodiments, any of the reagents described in this disclosure can be encapsulated in, or otherwise coupled to a droplet or bead with any chemicals, particles, and elements suitable for sample processing reactions involving biomolecules, such as, but not limited to, nucleic acid molecules and proteins. For example, a bead or droplet used in a sample preparation reaction for DNA sequencing can include one or more of the following reagents: enzymes, restriction enzymes (e.g., multiple cutters), ligase, polymerase, fluorophores, oligonucleotide barcodes, adapters, buffers, nucleotides (e.g., dNTPs, ddNTPs) and the like.

[0120] Additional examples of reagents include, but are not limited to: buffers, acidic solution, basic solution, temperature-sensitive enzymes, pH-sensitive enzymes, light-sensitive enzymes, metals, metal ions, magnesium chloride, sodium chloride, manganese, aqueous buffer, mild buffer, ionic buffer, inhibitor, enzyme, protein, polynucleotide, antibodies, saccharides, lipid, oil, salt, ion, detergents, ionic detergents, non-ionic detergents, oligonucleotides, nucleotides, deoxyribonucleotide triphosphates (dNTPs), dideoxyribonucleotide triphosphates (ddNTPs), DNA, RNA, peptide polynucleotides, complementary DNA (cDNA), double stranded DNA (dsDNA), single stranded DNA (ssDNA), plasmid DNA, cosmid DNA, chromosomal DNA, genomic DNA, viral DNA, bacterial DNA, mtDNA (mitochondrial DNA), mRNA, rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, ribozyme, riboswitch and viral RNA, polymerase, ligase, restriction enzymes, proteases, nucleases, protease inhibitors, nuclease inhibitors, chelating agents, reducing agents, oxidizing agents, fluorophores, probes, chromophores, dyes, organics, emulsifiers, surfactants, stabilizers, polymers, water, small molecules, pharmaceuticals, radioactive molecules, preservatives, antibiotics, aptamers, and pharmaceutical drug compounds. As described herein, one or more reagents in the well can be used to perform one or more reactions, including but not limited to: cell lysis, cell fixation, permeabilization, nucleic acid reactions, e.g., nucleic acid extension reactions, amplification, reverse transcription, transposase reactions (e.g., tagmentation), etc.

[0121] The wells disclosed herein can be provided as a part of a kit. For example, a kit can include instructions for use, a microwell array or device, and reagents (e.g., beads). The kit can include any useful reagents for performing the processes described herein, e.g., nucleic acid reactions, barcoding of nucleic acid molecules, sample processing (e.g., for cell lysis, fixation, and/or permeabilization).

[0122] In some cases, a well includes a bead or droplet that includes a set of reagents that has a similar attribute, for example, a set of enzymes, a set of minerals, a set of oligonucleotides, a mixture of different barcode molecules, a mixture of identical barcode molecules. In other cases, a bead or droplet includes a heterogeneous mixture of reagents. In some cases, the heterogeneous mixture of reagents can include all components necessary to perform a reaction. In some cases, such mixture can include all components necessary to perform a reaction, except for 1, 2, 3, 4, 5, or more components necessary to perform a reaction. In some cases, such additional components are contained within, or otherwise coupled to, a different droplet or bead, or within a solution within a partition (e.g., microwell) of the system.

[0123] A non-limiting example of a microwell array in accordance with some embodiments of the disclosure is schematically presented in FIG. 5. In this example, the array can be contained within a substrate 500. The substrate 500 includes a plurality of wells 502. The wells 502 can be of any size or shape, and the spacing between the wells, the number of wells per substrate, as well as the density of the wells on the substrate 500 can be modified, depending on the particular application. In one such example application, a sample molecule 506, which can include a cell or cellular components (e.g., nucleic acid molecules) is co-partitioned with a bead 504, which can include a nucleic acid barcode molecule coupled thereto. The wells 502 can be loaded using gravity or other loading technique (e.g., centrifugation, liquid handler, acoustic loading, optoelectronic, etc.). In some instances, at least one of the wells 502 contains a single sample molecule 506 (e.g., cell) and a single bead 504.

[0124] Reagents can be loaded into a well either sequentially or concurrently. In some cases, reagents are introduced to the device either before or after a particular operation. In some cases, reagents (which can be provided, in certain instances, in droplets or beads) are introduced sequentially such that different reactions or operations occur at different steps. The reagents (or droplets or beads) can also be loaded at operations interspersed with a reaction or operation step. For example, droplets or beads including reagents for fragmenting polynucleotides (e.g., restriction enzymes) and/or other enzymes (e.g., transposases, ligases, polymerases, etc.) can be loaded into the well or plurality of wells, followed by loading of droplets or beads including reagents for attaching nucleic acid barcode molecules to a sample nucleic acid molecule. Reagents can be provided concurrently or sequentially with a sample, e.g., a cell or cellular components (e.g., organelles, proteins, nucleic acid molecules, carbohydrates, lipids, etc.). Accordingly, use of wells can be useful in performing multi-step operations or reactions.

[0125] As described elsewhere herein, the nucleic acid barcode molecules and other reagents can be contained within a bead or droplet. These beads or droplets can be loaded into a partition (e.g., a microwell) before, after, or concurrently with the loading of a cell, such that each cell is contacted with a different bead or droplet. This technique can be used to attach a unique nucleic acid barcode molecule to nucleic acid molecules obtained from each cell. Alternatively or in addition, the sample nucleic acid molecules can be attached to a support. For example, the partition (e.g., microwell) can include a bead which has coupled thereto a plurality of nucleic acid barcode molecules. The sample nucleic acid molecules, or derivatives thereof, can couple or attach to the nucleic acid barcode molecules attached on the support. The resulting barcoded nucleic acid molecules can then be removed from the partition, and in some instances, pooled and sequenced. In such cases, the nucleic acid barcode sequences can be used to trace the origin of the sample nucleic acid molecule. For example, polynucleotides with identical barcodes can be determined to originate from the same cell or partition, while polynucleotides with different barcodes can be determined to originate from different cells or partitions.

[0126] The samples or reagents can be loaded in the wells or microwells using a variety of approaches. For example, the samples (e.g., a cell, cell bead, or cellular component) or reagents (as described herein) can be loaded into the well or microwell using an external force, e.g., gravitational force, electrical force, magnetic force, or using mechanisms to drive the sample or reagents into the well, for example, via pressure-driven flow, centrifugation, optoelectronics, acoustic loading, electrokinetic pumping, vacuum, capillary flow, etc. In certain cases, a fluid handling system can be used to load the samples or reagents into the well. The loading of the samples or reagents can follow a Poissonian distribution or a non-Poissonian distribution, e.g., super Poisson or sub-Poisson. The geometry, spacing between wells, density, and size of the microwells can be modified to accommodate a useful sample or reagent distribution; for example, the size and spacing of the microwells can be adjusted such that the sample or reagents can be distributed in a super-Poissonian fashion.

[0127] In one non-limiting example, the microwell array or plate includes pairs of microwells, in which each pair of microwells is configured to hold a droplet e.g., including a single cell) and a single bead (such as those described herein, which can, in some instances, also be encapsulated in a droplet). The droplet and the bead (or droplet containing the bead) can be loaded simultaneously or sequentially, and the droplet and the bead can be merged, e.g., upon contact of the droplet and the bead, or upon application of a stimulus (e.g., external force, agitation, heat, light, magnetic or electric force, etc.). In some cases, the loading of the droplet and the bead is super-Poissonian. In other examples of pairs of microwells, the wells are configured to hold two droplets including different reagents and/or samples, which are merged upon contact or upon application of a stimulus. In such instances, the droplet of one microwell of the pair can include reagents that can react with an agent in the droplet of the other microwell of the pair. For example, one droplet can include reagents that are configured to release the nucleic acid barcode molecules of a bead contained in another droplet, located in the adjacent micro well. Upon merging of the droplets, the nucleic acid barcode molecules can be released from the bead into the partition (e.g., the microwell or microwell pair that are in contact), and further processing can be performed (e.g., barcoding, nucleic acid reactions, etc.). In cases where intact or live cells are loaded in the microwells, one of the droplets can include lysis reagents for lysing the cell upon droplet merging.

[0128] In some embodiments, a droplet can be partitioned into a well. The droplets can be selected or subjected to pre-processing prior to loading into a well. For instance, the droplets can include cells, and only certain droplets, such as those containing a single cell (or at least one cell), can be selected for use in loading of the wells. Such a pre-selection process can be useful in efficient loading of single cells, such as to obtain a non-Poissonian distribution, or to pre-filter cells for a selected characteristic prior to further partitioning in the wells. Additionally, the technique can be useful in obtaining or preventing cell doublet or multiplet formation prior to or during loading of the microwell.

[0129] In some embodiments, the wells can include nucleic acid barcode molecules attached thereto. The nucleic acid barcode molecules can be attached to a surface of the well (e.g., a wall of the well). The nucleic acid barcode molecules may be attached to a droplet or bead that has been partitioned into the well. The nucleic acid barcode molecule e.g., a partition barcode sequence) of one well can differ from the nucleic acid barcode molecule of another well, which can permit identification of the contents contained with a single partition or well. In some embodiments, the nucleic acid barcode molecule can include a spatial barcode sequence that can identify a spatial coordinate of a well, such as within the well array or well plate. In some embodiments, the nucleic acid barcode molecule can include a unique molecular identifier for individual molecule identification. In some instances, the nucleic acid barcode molecules can be configured to attach to or capture a nucleic acid molecule from or within a sample or cell distributed in the well. For example, the nucleic acid barcode molecules can include a capture sequence that can be used to capture or hybridize to a nucleic acid molecule e.g., RNA, DNA) from or within the sample. In some embodiments, the nucleic acid barcode molecules can be releasable from the microwell. In some instances, the nucleic acid barcode molecules may be releasable from the bead or droplet. For example, the nucleic acid barcode molecules can include a chemical cross-linker which can be cleaved upon application of a stimulus (e.g., photo-, magnetic, chemical, biological, stimulus). The released nucleic acid barcode molecules, which can be hybridized or configured to hybridize to a sample nucleic acid molecule, can be collected and pooled for further processing, which can include nucleic acid processing (e.g., amplification, extension, reverse transcription, etc.) and/or characterization (e.g., sequencing). In some instances nucleic acid barcode molecules attached to a bead in a well may be hybridized to sample nucleic acid molecules, and the bead with the sample nucleic acid molecules hybridized thereto may be collected and pooled for further processing, which can include nucleic acid processing (e.g., amplification, extension, reverse transcription, etc.) and/or characterization (e.g., sequencing). In such cases, the unique partition barcode sequences can be used to identify the cell or partition from which a nucleic acid molecule originated.

[0130] Characterization of samples within a well can be performed. Such characterization can include, in non-limiting examples, imaging of the sample (e.g., cell, cell bead, or cellular components) or derivatives thereof. Characterization techniques such as microscopy or imaging can be useful in measuring sample profiles in fixed spatial locations. For example, when cells are partitioned, optionally with beads, imaging of each micro well and the contents contained therein can provide useful information on cell doublet formation (e.g., frequency, spatial locations, etc.), cell-bead pair efficiency, cell viability, cell size, cell morphology, expression level of a biomarker (e.g., a surface marker, a fluorescently labeled molecule therein, etc.), cell or bead loading rate, number of cell-bead pairs, etc. In some instances, imaging can be used to characterize live cells in the wells, including, but not limited to: dynamic live-cell tracking, cellcell interactions (when two or more cells are co-partitioned), cell proliferation, etc. Alternatively or in addition to, imaging can be used to characterize a quantity of amplification products in the well.

[0131] In operation, a well can be loaded with a sample and reagents, simultaneously or sequentially. When cells or cell beads are loaded, the well can be subjected to washing, e.g., to remove excess cells from the well, micro well array, or plate. Similarly, washing can be performed to remove excess beads or other reagents from the well, microwell array, or plate. In the instances where live cells are used, the cells can be lysed in the individual partitions to release the intracellular components or cellular analytes. Alternatively, the cells can be fixed or permeabilized in the individual partitions. The intracellular components or cellular analytes can couple to a support, e.g., on a surface of the microwell, on a solid support (e.g., bead), or they can be collected for further downstream processing. For example, after cell lysis, the intracellular components or cellular analytes can be transferred to individual droplets or other partitions for barcoding. Alternatively, or in addition, the intracellular components or cellular analytes (e.g., nucleic acid molecules) can couple to a bead including a nucleic acid barcode molecule; subsequently, the bead can be collected and further processed, e.g., subjected to nucleic acid reaction such as reverse transcription, amplification, or extension, and the nucleic acid molecules thereon can be further characterized, e.g., via sequencing. Alternatively, or in addition, the intracellular components or cellular analytes can be barcoded in the well (e.g., using a bead including nucleic acid barcode molecules that are releasable or on a surface of the microwell including nucleic acid barcode molecules). The barcoded nucleic acid molecules or analytes can be further processed in the well, or the barcoded nucleic acid molecules or analytes can be collected from the individual partitions and subjected to further processing outside the partition. Further processing can include nucleic acid processing (e.g., performing an amplification, extension) or characterization (e.g., fluorescence monitoring of amplified molecules, sequencing). At any convenient, suitable, and/or useful step, the well (or microwell array or plate) can be sealed (e.g., using an oil, membrane, wax, etc.), which enables storage of the assay or selective introduction of additional reagents.

Reagents

[0132] In accordance with certain aspects, biological particles can be partitioned along with lysis reagents in order to release the contents of the biological particles within the partition. See, e.g., U.S. Pat. Pub. 2018/0216162 (now U.S. Pat. 10,428,326), U.S. Pat. Pub. 2019/0100632 (now U.S. Pat. 10,590,244), and U.S. Pat. Pub. 2019/0233878. Biological particles (e.g., cells, cell beads, cell nuclei, organelles, and the like) can be partitioned together with nucleic acid barcode molecules and the nucleic acid molecules of or derived from the biological particle (e.g., mRNA, cDNA, gDNA, etc.,) can be barcoded as described elsewhere herein. In some embodiments, biological particles are co-partitioned with barcode carrying beads (e.g., gel beads) and the nucleic acid molecules of or derived from the biological particle are barcoded as described elsewhere herein. Tn such cases, the lysis agents can be contacted with the biological particle suspension concurrently with, or immediately prior to, the introduction of the biological particles into the partitioning junction/droplet generation zone (e.g., junction 210), such as through an additional channel or channels upstream of the channel junction. In accordance with other aspects, additionally or alternatively, biological particles can be partitioned along with other reagents, as will be described further below.

[0133] Beneficially, when lysis reagents and biological particles are co-partitioned, the lysis reagents can facilitate the release of the contents of the biological particles within the partition. The contents released in a partition can remain discrete from the contents of other partitions.

[0134] As will be appreciated, the channel segments described herein can be coupled to any of a variety of different fluid sources or receiving components, including reservoirs, tubing, manifolds, or fluidic components of other systems. As will be appreciated, the microfluidic channel structures can have other geometries and/or configurations. For example, a microfluidic channel structure can have more than two channel junctions. For example, a microfluidic channel structure can have 2, 3, 4, 5 channel segments or more each carrying the same or different types of beads, reagents, and/or biological particles that meet at a channel junction. Fluid flow in each channel segment can be controlled to control the partitioning of the different elements into droplets. Fluid can be directed flow along one or more channels or reservoirs via one or more fluid flow units. A fluid flow unit can include compressors (e.g., providing positive pressure), pumps (e.g., providing negative pressure), actuators, and the like to control flow of the fluid. Fluid can also or otherwise be controlled via applied pressure differentials, centrifugal force, electrokinetic pumping, vacuum, capillary or gravity flow, or the like.

[0135] Examples of lysis agents include bioactive reagents, such as lysis enzymes that are used for lysis of different cell types, e.g., gram positive or negative bacteria, plants, yeast, mammalian, etc., such as lysozymes, achromopcptidasc, lysostaphin, labiasc, kitalasc, lyticasc, and a variety of other lysis enzymes available from, e.g., Sigma-Aldrich, Inc. (St Louis, MO), as well as other commercially available lysis enzymes. Other lysis agents can additionally or alternatively be co-partitioned with the biological particles to cause the release of the biological particle’s contents into the partitions. For example, in some cases, surfactant-based lysis solutions can be used to lyse cells e.g., labelled immune cells, B cells, or T cells), although these can be less desirable for emulsion based systems where the surfactants can interfere with stable emulsions. In some cases, lysis solutions can include non-ionic surfactants such as, for example, TritonX-100 and Tween 20. In some cases, lysis solutions can include ionic surfactants such as, for example, sarcosyl and sodium dodecyl sulfate (SDS). Electroporation, thermal, acoustic or mechanical cellular disruption can also be used in certain cases, e.g., non-emulsion based partitioning such as encapsulation of biological particles that can be in addition to or in place of droplet partitioning, where any pore size of the encapsulate is sufficiently small to retain nucleic acid fragments of a given size, following cellular disruption.

[0136] Alternatively or in addition to the lysis agents co-partitioned with the biological particles (e.g., labelled immune cells, B cells, or T cells) described above, other reagents can also be co-partitioned with the biological particles, including, for example, DNase and RNase inactivating agents or inhibitors, such as proteinase K, chelating agents, such as EDTA, and other reagents employed in removing or otherwise reducing negative activity or impact of different cell lysate components on subsequent processing of nucleic acids. In addition, in the case of encapsulated biological particles e.g., labelled immune cells, B cells, or T cells), the biological particles can be exposed to an appropriate stimulus to release the biological particles or their contents from a co-partitioned bead. For example, in some cases, a chemical stimulus can be copartitioned along with an encapsulated biological particle to allow for the degradation of the bead and release of the cell or its contents into the larger partition. In some cases, this stimulus can be the same as the stimulus described elsewhere herein for release of nucleic acid molecules (e.g., oligonucleotides) from their respective bead. In alternative aspects, this can be a different and non-overlapping stimulus, in order to allow an encapsulated biological particle to be released into a partition at a different time from the release of nucleic acid molecules into the same partition.

[0137] Additional reagents can also be co-partitioned with the biological particles (e.g., labelled immune cells, B cells, or T cells), such as endonucleases to fragment a biological particle’s DNA, DNA polymerase enzymes and dNTPs used to amplify the biological particle’s nucleic acid fragments and to attach the barcode molecular tags to the amplified fragments. Other enzymes can be co-partitioned, including without limitation, polymerase, transposase, ligase, proteinase K, DNAse, etc. Additional reagents can also include reverse transcriptase enzymes, including enzymes with terminal transferase activity, primers and oligonucleotides, and switch oligonucleotides (also referred to herein as “switch oligos” or “template switching oligonucleotides”) which can be used for template switching. In some cases, template switching can be used to increase the length of a cDNA. In some cases, template switching can be used to append a predefined nucleic acid sequence to the cDNA. In an example of template switching, cDNA can be generated from reverse transcription of a template, e.g., cellular mRNA, where a reverse transcriptase with terminal transferase activity can add additional nucleotides, e.g., polyC, to the cDNA in a template independent manner. Switch oligos can include sequences complementary to the additional nucleotides, e.g., polyG. The additional nucleotides (e.g., polyC) on the cDNA can hybridize to the additional nucleotides (e.g., polyG) on the switch oligo, whereby the switch oligo can be used by the reverse transcriptase as template to further extend the cDNA. Template switching oligonucleotides can include a hybridization region and a template region. Template switching oligonucleotides are further described in PCT Pub. No. WO2018119447, which is hereby incorporated by reference in its entirety.

[0138] Once the contents of the cells (e.g., labelled immune cells, B cells, or T cells) are released into their respective partitions, the macromolecular components (e.g., macromolecular constituents of biological particles, such as RNA, DNA, proteins, or secreted antibodies or antigen-binding fragments thereof) contained therein can be further processed within the partitions. In accordance with the methods and systems described herein, the macromolecular component contents of individual biological particles (e.g., labelled immune cells, B cells, or T cells) can be provided with unique identifiers such that, upon characterization of those macromolecular components they can be attributed as having been derived from the same biological particle or particles. The ability to attribute characteristics to individual biological particles or groups of biological particles is provided by the assignment of unique identifiers specifically to an individual biological particle or groups of biological particles. Unique identifiers, e.g., in the form of nucleic acid barcodes can be assigned or associated with individual biological particles or populations of biological particles, in order to tag or label the biological particle’s macromolecular components (and as a result, its characteristics) with the unique identifiers. These unique identifiers can then be used to attribute the biological particle’s components and characteristics to an individual biological particle or group of biological particles.

[0139] In some aspects, this is performed by co-partitioning the individual biological particle (e.g., labelled immune cell, B cell, or T cell) or groups of biological particles (e.g., labelled immune cells, B cells, or T cells) with the unique identifiers, such as described above (with reference to FIGS. 5 and 6). In some aspects, the unique identifiers are provided in the form of nucleic acid molecules (e.g., oligonucleotides) that include nucleic acid barcode sequences that can be attached to or otherwise associated with the nucleic acid contents of individual biological particle, or to other components of the biological particle, and particularly to fragments of those nucleic acids. The nucleic acid molecules are partitioned such that as between nucleic acid molecules in a given partition, the nucleic acid barcode sequences contained therein are the same, but as between different partitions, the nucleic acid molecule can, and do have differing barcode sequences, or at least represent a large number of different barcode sequences across all of the partitions in a given analysis. In some aspects, only one nucleic acid barcode sequence can be associated with a given partition, although in some cases, two or more different barcode sequences can be present.

[0140] The nucleic acid barcode sequences can include from about 6 to about 20 or more nucleotides within the sequence of the nucleic acid molecules (e.g., oligonucleotides). The nucleic acid barcode sequences can include from about 6 to about 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides. In some cases, the length of a barcode sequence can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of a barcode sequence can be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of a barcode sequence can be at most about 6, 7,

8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter. These nucleotides can be completely contiguous, i.e., in a single stretch of adjacent nucleotides, or they can be separated into two or more separate subsequences that are separated by one or more nucleotides. In some cases, separated barcode subsequences can be from about 4 to about 16 nucleotides in length. In some cases, the barcode subsequence can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequence can be at least about 4, 5, 6, 7, 8,

9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequence can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[0141] The co-partitioned nucleic acid molecules can also include other functional sequences useful in the processing of the nucleic acids from the co-partitioned biological particles e.g., labelled immune cells, B cells, or T cells). These sequences include, e.g., targeted or random/universal amplification primer sequences for amplifying the genomic DNA from the individual biological particles within the partitions while attaching the associated barcode sequences, sequencing primers or primer recognition sites, hybridization or probing sequences, e.g., for identification of presence of the sequences or for pulling down barcoded nucleic acids, or any of a number of other potential functional sequences. Other mechanisms of co-partitioning oligonucleotides can also be employed, including, e.g., coalescence of two or more droplets, where one droplet contains oligonucleotides, or microdispensing of oligonucleotides into partitions, e.g., droplets within microfluidic systems.

[0142] In an example, beads are provided that each include large numbers of the above described barcoded nucleic acid molecules (e.g., barcoded oligonucleotides) releasably attached to the beads, where all of the nucleic acid molecules attached to a particular bead will include the same nucleic acid barcode sequence, but where a large number of diverse barcode sequences are represented across the population of beads used. In some embodiments, hydrogel beads, e.g., including polyacrylamide polymer matrices, are used as a solid support and delivery vehicle for the nucleic acid molecules into the partitions, as they are capable of carrying large numbers of nucleic acid molecules, and can be configured to release those nucleic acid molecules upon exposure to a particular stimulus, as described elsewhere herein. In some cases, the population of beads provides a diverse barcode sequence library that includes at least about 1 ,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000,000 different barcode sequences, at least about 5,000,000 different barcode sequences, or at least about 10,000,000 different barcode sequences, or more. Additionally, each bead can be provided with large numbers of nucleic acid (e.g., oligonucleotide) molecules attached. In particular, the number of molecules of nucleic acid molecules including the barcode sequence on an individual bead can be at least about 1,000 nucleic acid molecules, at least about 5,000 nucleic acid molecules, at least about 10,000 nucleic acid molecules, at least about 50,000 nucleic acid molecules, at least about 100,000 nucleic acid molecules, at least about 500,000 nucleic acids, at least about 1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acid molecules, at least about 10,000,000 nucleic acid molecules, at least about 50,000,000 nucleic acid molecules, at least about 100,000,000 nucleic acid molecules, at least about 250,000,000 nucleic acid molecules and in some cases at least about 1 billion nucleic acid molecules, or more. Nucleic acid molecules of a given bead can include identical (or common) barcode sequences, different barcode sequences, or a combination of both. Nucleic acid molecules of a given bead can include multiple sets of nucleic acid molecules. Nucleic acid molecules of a given set can include identical barcode sequences. The identical barcode sequences can be different from barcode sequences of nucleic acid molecules of another set.

[0143] Moreover, when the population of beads is partitioned, the resulting population of partitions can also include a diverse barcode library that includes at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000,000 different barcode sequences, at least about 5,000,000 different barcode sequences, or at least about 10,000,000 different barcode sequences. Additionally, each partition of the population can include at least about 1,000 nucleic acid molecules, at least about 5,000 nucleic acid molecules, at least about 10,000 nucleic acid molecules, at least about 50,000 nucleic acid molecules, at least about 100,000 nucleic acid molecules, at least about 500,000 nucleic acids, at least about 1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acid molecules, at least about 10,000,000 nucleic acid molecules, at least about 50,000,000 nucleic acid molecules, at least about 100,000,000 nucleic acid molecules, at least about 250,000,000 nucleic acid molecules and in some cases at least about 1 billion nucleic acid molecules.

[0144] In some cases, it may be desirable to incorporate multiple different barcodes within a given partition, either attached to a single or multiple beads within the par tition. For example, in some cases, a mixed, but known set of barcode sequences can provide greater assurance of identification in the subsequent processing, e.g., by providing a stronger address or attribution of the barcodes to a given partition, as a duplicate or independent confirmation of the output from a given partition.

[0145] The nucleic acid molecules (e.g., oligonucleotides) are releasable from the beads upon the application of a particular stimulus to the beads. In some cases, the stimulus can be a photo-stimulus, e.g., through cleavage of a photo-labile linkage that releases the nucleic acid molecules. In other cases, a thermal stimulus can be used, where elevation of the temperature of the beads environment will result in cleavage of a linkage or other release of the nucleic acid molecules from the beads. In still other cases, a chemical stimulus can be used that cleaves a linkage of the nucleic acid molecules to the beads, or otherwise results in release of the nucleic acid molecules from the beads. In one case, such compositions include the polyacrylamide matrices described above for encapsulation of biological particles, and can be degraded for release of the attached nucleic acid molecules through exposure to a reducing agent, such as DTT.

Systems and methods for controlled partitioning

[0146] In some aspects, provided herein are systems and methods for controlled partitioning. Droplet size can be controlled by adjusting certain geometric features in channel architecture (e.g., microfluidics channel architecture). For example, an expansion angle, width, and/or length of a channel can be adjusted to control droplet size.

[0147] FIG. 2 shows an example of a microfluidic channel structure for the controlled partitioning of beads into discrete droplets. A channel structure 200 can include a channel segment 202 communicating at a channel junction 206 (or intersection) with a reservoir 204. The reservoir 204 can be a chamber. Any reference to “reservoir,” as used herein, can also refer to a “chamber.” In operation, an aqueous fluid 208 that includes suspended beads 212 can be transported along the channel segment 202 into the junction 206 to meet a second fluid 210 that is immiscible with the aqueous fluid 208 in the reservoir 204 to create droplets 216, 218 of the aqueous fluid 208 flowing into the reservoir 204. At the junction 206 where the aqueous fluid 208 and the second fluid 210 meet, droplets can form based on factors such as the hydrodynamic forces at the junction 206, flow rates of the two fluids 208, 210, fluid properties, and certain geometric parameters e.g., w, ho, a, etc.) of the channel structure 200. A plurality of droplets can be collected in the reservoir 204 by continuously injecting the aqueous fluid 208 from the channel segment 202 through the junction 206.

[0148] A discrete droplet generated can include a bead (e.g., as in occupied droplets 216). Alternatively, a discrete droplet generated can include more than one bead. Alternatively, a discrete droplet generated cannot include any beads (e.g., as in unoccupied droplet 218). In some instances, a discrete droplet generated can contain one or more biological particles, as described elsewhere herein. In some instances, a discrete droplet generated can include one or more reagents, as described elsewhere herein.

[0149] In some instances, the aqueous fluid 208 can have a substantially uniform concentration or frequency of beads 212. The beads 212 can be introduced into the channel segment 202 from a separate channel (not shown in FIG. 2). The frequency of beads 212 in the channel segment 202 can be controlled by controlling the frequency in which the beads 212 are introduced into the channel segment 202 and/or the relative flow rates of the fluids in the channel segment 202 and the separate channel. In some instances, the beads can be introduced into the channel segment 202 from a plurality of different channels, and the frequency controlled accordingly.

[0150] In some instances, the aqueous fluid 208 in the channel segment 202 can include biological particles (e.g., described with reference to FIG. 1). In some instances, the aqueous fluid 208 can have a substantially uniform concentration or frequency of biological particles. As with the beads, the biological particles (e.g., labelled immune cells, B cells, or T cells) can be introduced into the channel segment 202 from a separate channel. The frequency or concentration of the biological particles in the aqueous fluid 208 in the channel segment 202 can be controlled by controlling the frequency in which the biological particles are introduced into the channel segment 202 and/or the relative flow rates of the fluids in the channel segment 202 and the separate channel. In some instances, the biological particles can be introduced into the channel segment 202 from a plurality of different channels, and the frequency controlled accordingly. In some instances, a first separate channel can introduce beads and a second separate channel can introduce biological particles into the channel segment 202. The first separate channel introducing the beads can be upstream or downstream of the second separate channel introducing the biological particles.

[0151] The second fluid 210 can include an oil, such as a fluorinated oil, that includes a fluoro surfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets.

[0152] In some instances, the second fluid 210 cannot be subjected to and/or directed to any flow in or out of the reservoir 204. For example, the second fluid 210 can be substantially stationary in the reservoir 204. In some instances, the second fluid 210 can be subjected to flow within the reservoir 204, but not in or out of the reservoir 204, such as via application of pressure to the reservoir 204 and/or as affected by the incoming flow of the aqueous fluid 208 at the junction 206. Alternatively, the second fluid 210 can be subjected and/or directed to flow in or out of the reservoir 204. For example, the reservoir 204 can be a channel directing the second fluid 210 from upstream to downstream, transporting the generated droplets.

[0153] The channel structure 200 at or near the junction 206 can have certain geometric features that at least partly determine the sizes of the droplets formed by the channel structure 200. The channel segment 202 can have a height, ho and width, w, at or near the junction 206. By way of example, the channel segment 202 can include a rectangular cross- section that leads to a reservoir 204 having a wider cross-section (such as in width or diameter). Alternatively, the cross-section of the channel segment 202 can be other shapes, such as a circular shape, trapezoidal shape, polygonal shape, or any other shapes. The top and bottom walls of the reservoir 204 at or near the junction 206 can be inclined at an expansion angle, a. The expansion angle, a, allows the tongue (portion of the aqueous fluid 208 leaving channel segment 202 at junction 206 and entering the reservoir 204 before droplet formation) to increase in depth and facilitate decrease in curvature of the intermediately formed droplet. Droplet size can decrease with increasing expansion angle. The resulting droplet radius, Rd, can be predicted by the following equation for the aforementioned geometric parameters of ho, w, and a

( , - ^w \ /io

R_d « 0.441 1 -I- 2.2 tan a — j — =

\ ₀ / tan a

[0154] Systems and methods for controlled partitioning are described further in W02019040637, which is hereby incorporated by reference in its entirety.

[0155] The methods and systems described herein can be used to greatly increase the efficiency of single cell applications and/or other applications receiving droplet-based input. For example, following the sorting of occupied cells and/or appropriately-sized cells, subsequent operations that can be performed can include generation of amplification products, purification (e.g., via solid phase reversible immobilization (SPRI)), further processing (e.g., shearing, ligation of functional sequences, and subsequent amplification (e.g., via PCR)). These operations can occur in bulk (e.g., outside the partition). In the case where a partition is a droplet in an emulsion, the emulsion can be broken and the contents of the droplet pooled for additional operations. Additional reagents that can be co-partitioned along with the barcode bearing bead can include oligonucleotides to block ribosomal RNA (rRNA) and nucleases to digest genomic DNA from cells. Alternatively, rRNA removal agents can be applied during additional processing operations. The configuration of the constructs generated by such a method can help minimize (or avoid) sequencing of the poly-T sequence during sequencing and/or sequence the 5’ end of a polynucleotide sequence. The amplification products, for example, first amplification products and/or second amplification products, can be subject to sequencing for sequence analysis. In some cases, amplification can be performed using the Partial Hairpin Amplification for Sequencing (PHASE) method.

[0156] A variety of applications require the evaluation of the presence and quantification of different biological particle or organism types within a population of biological particles, including, for example, microbiome analysis and characterization, environmental testing, food safety testing, epidemiological analysis, e.g., in tracing contamination or the like.

[0157] Partitions including a barcode bead (e.g., a gel bead) associated with barcode molecules and a bead encapsulating cellular constituents (e.g., a cell bead) such as cellular nucleic acids can be useful in constituent analysis as is described in U.S. Patent Publication No. 2018/0216162.

Sample and cell processing

[0158] A sample can be derived from any useful source including any subject, such as a human subject. A sample can include material (e.g., one or more cells) from one or more different sources, such as one or more different subjects. Multiple samples, such as multiple samples from a single subject (e.g., multiple samples obtained in the same or different manners from the same or different bodily locations, and/or obtained at the same or different times (e.g., seconds, minutes, hours, days, weeks, months, or years apparat)), or multiple samples from different subjects, can be obtained for analysis as described herein. For example, a first sample can be obtained from a subject at a first time and a second sample can be obtained from the subject at a second time later than the first time. The first time can be before a subject undergoes a treatment regimen or procedure (e.g., to address a disease or condition), and the second time can be during or after the subject undergoes the treatment regimen or procedure. In another example, a first sample can be obtained from a first bodily location or system of a subject (e.g., using a first collection technique) and a second sample can be obtained from a second bodily location or system of the subject (e.g., using a second collection technique), which second bodily location or system can be different than the first bodily location or system. In another example, multiple samples can be obtained from a subject at a same time from the same or different bodily locations. Different samples, such as different samples collected from different bodily locations of a same subject, at different times, from multiple different subjects, and/or using different collection techniques, can undergo the same or different processing (e.g., as described herein). For example, a first sample can undergo a first processing protocol and a second sample can undergo a second processing protocol.

[0159] A sample can be a biological sample, such as a cell sample (e.g., as described herein). A sample can include one or more analyte carriers, such as one or more cells and/or cellular constituents, such as one or more cell nuclei. For example, a sample can include a plurality of cells and/or cellular constituents. Components (e.g., cells or cellular constituents, such as cell nuclei) of a sample can be of a single type or a plurality of different types. For example, cells of a sample can include one or more different types of blood cells.

[0160] A biological sample can include a plurality of cells having different dimensions and features. In some cases, processing of the biological sample, such as cell separation and sorting (e.g., as described herein), can affect the distribution of dimensions and cellular features included in the sample by depleting cells having certain features and dimensions and/or isolating cells having certain features and dimensions.

[0161] A sample may undergo one or more processes in preparation for analysis (e.g., as described herein), including, but not limited to, filtration, selective precipitation, purification, centrifugation, pcrmcabilization, isolation, agitation, heating, and/or other processes. For example, a sample may be filtered to remove a contaminant or other materials. In an example, a filtration process can include the use of microfluidics (e.g., to separate analyte carriers of different sizes, types, charges, or other features).

[0162] In an example, a sample including one or more cells can be processed to separate the one or more cells from other materials in the sample (e.g., using centrifugation and/or another process). In some cases, cells and/or cellular constituents of a sample can be processed to separate and/or sort groups of cells and/or cellular constituents, such as to separate and/or sort cells and/or cellular constituents of different types. Examples of cell separation include, but are not limited to, separation of white blood cells or immune cells from other blood cells and components, separation of circulating tumor cells from blood, and separation of bacteria from bodily cells and/or environmental materials. A separation process can include a positive selection process (e.g., targeting of a cell type of interest for retention for subsequent downstream analysis, such as by use of a monoclonal antibody that targets a surface marker of the cell type of interest), a negative selection process (e.g., removal of one or more cell types and retention of one or more other cell types of interest), and/or a depletion process (e.g., removal of a single cell type from a sample, such as removal of red blood cells from peripheral blood mononuclear cells).

[0163] Separation of one or more different types of cells can include, for example, centrifugation, filtration, microfluidic-based sorting, flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), buoyancy-activated cell sorting (BACS), or any other useful method. For example, a flow cytometry method can be used to detect cells and/or cellular constituents based on a parameter such as a size, morphology, or protein expression. Flow cytometry-based cell sorting can include injecting a sample into a sheath fluid that conveys the cells and/or cellular constituents of the sample into a measurement region one at a time. In the measurement region, a light source such as a laser can interrogate the cells and/or cellular constituents and scattered light and/or fluorescence can be detected and converted into digital signals. A nozzle system (e.g., a vibrating nozzle system) can be used to generate droplets (e.g., aqueous droplets) including individual cells and/or cellular constituents. Droplets including cells and/or cellular constituents of interest (e.g., as determined via optical detection) can be labeled with an electric charge (e.g., using an electrical charging ring), which charge can be used to separate such droplets from droplets including other cells and/or cellular constituents. For example, FACS can include labeling cells and/or cellular constituents with fluorescent markers (e.g., using internal and/or external biomarkers). Cells and/or cellular constituents can then be measured and identified one by one and sorted based on the emitted fluorescence of the marker or absence thereof. MACS can use micro- or nano-scalc magnetic particles to bind to cells and/or cellular constituents (e.g., via an antibody interaction with cell surface markers) to facilitate magnetic isolation of cells and/or cellular constituents of interest from other components of a sample (e.g., using a column-based analysis). BACS can use microbubbles (e.g., glass microbubbles) labeled with antibodies to target cells of interest. Cells and/or cellular components coupled to microbubbles can float to a surface of a solution, thereby separating target cells and/or cellular components from other components of a sample. Cell separation techniques can be used to enrich for populations of cells of interest (e.g., prior to partitioning, as described herein). For example, a sample including a plurality of cells including a plurality of cells of a given type can be subjected to a positive separation process. The plurality of cells of the given type can be labeled with a fluorescent marker (e.g., based on an expressed cell surface marker or another marker) and subjected to a FACS process to separate these cells from other cells of the plurality of cells. The selected cells can then be subjected to subsequent partition-based analysis (e.g., as described herein) or other downstream analysis. The fluorescent marker can be removed prior to such analysis or can be retained. The fluorescent marker can include an identifying feature, such as a nucleic acid barcode sequence and/or unique molecular identifier.

[0164] In another example, a first sample including a first plurality of cells including a first plurality of cells of a given type (e.g., immune cells expressing a particular marker or combination of markers) and a second sample including a second plurality of cells including a second plurality of cells of the given type can be subjected to a positive separation process. The first and second samples can be collected from the same or different subjects, at the same or different types, from the same or different bodily locations or systems, using the same or different collection techniques. For example, the first sample can be from a first subject and the second sample can be from a second subject different than the first subject. The first plurality of cells of the first sample can be provided a first plurality of fluorescent markers configured to label the first plurality of cells of the given type. The second plurality of cells of the second sample can be provided a second plurality of fluorescent markers configured to label the second plurality of cells of the given type. The first plurality of fluorescent markers can include a first identifying feature, such as a first barcode, while the second plurality of fluorescent markers can include a second identifying feature, such as a second barcode, that is different than the first identifying feature. The first plurality of fluorescent markers and the second plurality of fluorescent markers can fluoresce at the same intensities and over the same range of wavelengths upon excitation with a same excitation source (e.g., light source, such as a laser). The first and second samples can then be combined and subjected to a FACS process to separate cells of the given type from other cells based on the first plurality of fluorescent markers labeling the first plurality of cells of the given type and the second plurality of fluorescent markers labeling the second plurality of cells of the given type. Alternatively, the first and second samples can undergo separate FACS processes and the positively selected cells of the given type from the first sample and the positively selected cells of the given type from the second sample can then be combined for subsequent analysis. The encoded identifying features of the different fluorescent markers can be used to identify cells originating from the first sample and cells originating from the second sample. For example, the first and second identifying features can be configured to interact (e.g., in partitions, as described herein) with nucleic acid barcode molecules (e.g., as described herein) to generate barcoded nucleic acid products detectable using, e.g., nucleic acid sequencing.

[0165] FIG. 6 schematically shows an example workflow for processing nucleic acid molecules within a sample. A substrate 600 including a plurality of microwells 602 can be provided. A sample 606 which can include a cell, cell bead, cellular components or analytes e.g., proteins and/or nucleic acid molecules) can be co-partitioned, in a plurality of microwells 602, with a plurality of beads 604 including nucleic acid barcode molecules. During a partitioning process, the sample 606 can be processed within the partition. For instance, in the case of live cells, the cell can be subjected to conditions sufficient to lyse the cells and release the analytes contained therein. In process 620, the bead 604 can be further processed. By way of example, processes 620a and 620b schematically illustrate different workflows, depending on the properties of the bead 604.

[0166] In 620a, the bead includes nucleic acid barcode molecules that are attached thereto, and sample nucleic acid molecules (e.g., RNA, DNA) can attach, e.g., via hybridization of ligation, to the nucleic acid barcode molecules. Such attachment can occur on the bead. In process 630, the beads 604 from multiple wells 602 can be collected and pooled. Further processing can be performed in process 640. For example, one or more nucleic acid reactions can be performed, such as reverse transcription, nucleic acid extension, amplification, ligation, transposition, etc. In some instances, adapter sequences are ligated to the nucleic acid molecules, or derivatives thereof, as described elsewhere herein. For instance, sequencing primer sequences can be appended to each end of the nucleic acid molecule. In process 650, further characterization, such as sequencing can be performed to generate sequencing reads. The sequencing reads can yield information on individual cells or populations of cells, which can be represented visually or graphically, e.g., in a plot.

[0167] In 620b, the bead includes nucleic acid barcode molecules that are releasably attached thereto, as described below. The bead can degrade or otherwise release the nucleic acid barcode molecules into the well 602; the nucleic acid barcode molecules can then be used to barcode nucleic acid molecules within the well 602. Further processing can be performed either inside the partition or outside the partition. For example, one or more nucleic acid reactions can be performed, such as reverse transcription, nucleic acid extension, amplification, ligation, transposition, etc. In some instances, adapter sequences are ligated to the nucleic acid molecules, or derivatives thereof, as described elsewhere herein. For instance, sequencing primer sequences can be appended to each end of the nucleic acid molecule. In process 650, further characterization, such as sequencing can be performed to generate sequencing reads. The sequencing reads can yield information on individual cells or populations of cells, which can be represented visually or graphically, e.g., in a plot.

Multiplexing methods

[0168] In some embodiments of the disclosure, steps (b) and (c) of the methods described herein are performed in multiplex format. For example, in some embodiments, step (a) of the methods disclosed herein can include partitioning additional immune cells of the plurality of immune cells in partitions of the first plurality of partitions, and step (c) can further include determining all or a part of the nucleic acid sequences encoding ABMs produced by the additional immune cells.

[0169] Accordingly, in some embodiments, the present disclosure provides methods and systems for multiplexing, and otherwise increasing throughput of samples for analysis. For example, a single or integrated process workflow may permit the processing, identification, and/or analysis of more or multiple analytes, more or multiple types of analytes, and/or more or multiple types of analyte characterizations. For example, in the methods and systems described herein, one or more labelling agents capable of binding to or otherwise coupling to one or more cells or cell features can be used to characterize cells and/or cell features. In some instances, cell features include cell surface features. Cell surface features can include, but are not limited to, a receptor, an antigen, a surface protein, a transmembrane protein, a cluster of differentiation protein, a protein channel, a protein pump, a carrier protein, a phospholipid, a glycoprotein, a glycolipid, a cell-cell interaction protein complex, an antigen-presenting complex, a major histocompatibility complex, an engineered T-cell receptor, a T-cell receptor, a B-cell receptor, a chimeric antigen receptor, a gap junction, an adherens junction, or any combination thereof. In some instances, cell features can include intracellular analytes, such as proteins, protein modifications (e.g., phosphorylation status or other post- translational modifications), nuclear proteins, nuclear membrane proteins, or any combination thereof. A labelling agent can include, but is not limited to, a protein, a peptide, an antibody (or an epitope binding fragment thereof), a lipophilic moiety (such as cholesterol), a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bi-specific antibody, a bi-specific T-cell engager, a T-cell receptor engager, a B-cell receptor engager, a pro-body, an aptamer, a monobody, an affimer, a darpin, and a protein scaffold, or any combination thereof. A labelling agent can be or include a labelling composition (e.g., a barcoded MHC multimer reagent) described herein. The labelling agents (e.g., any of the labelling compositions and/or barcoded MHC multimer reagents described herein) can include (e.g., are attached to) a reporter oligonucleotide that is indicative of the cell surface feature to which the binding group binds. For example, the reporter oligonucleotide can include a barcode sequence that permits identification of the labelling agent. For example, a labelling agent that is specific to one type of cell feature (e.g., a first cell surface feature) can have a first reporter oligonucleotide coupled thereto, while a labelling agent that is specific to a different cell feature (e.g., a second cell surface feature) can have a different reporter oligonucleotide coupled thereto. For a description of exemplary labelling agents, reporter oligonucleotides, and methods of use, see, e.g., U.S. Pat. 10,550,429; U.S. Pat. Pub.

20190177800; and U.S. Pat. Pub. 20190367969.

[0170] In a particular example, a library of potential cell feature labelling agents can be provided, where the respective cell feature labelling agents arc associated with nucleic acid reporter molecules, such that a different reporter oligonucleotide sequence is associated with each labelling agent capable of binding to a specific cell feature. In other aspects, different members of the library can be characterized by the presence of a different oligonucleotide sequence label. For example, an antibody capable of binding to a first protein can have associated with it a first reporter oligonucleotide sequence, while an antibody capable of binding to a second protein can have a different reporter oligonucleotide sequence associated with it. The presence of the particular oligonucleotide sequence can be indicative of the presence of a particular antibody or cell feature which can be recognized or bound by the particular antibody.

[0171] Labelling agents capable of binding to or otherwise coupling to one or more cells can be used to characterize a cell as belonging to a particular set of cells. For example, labeling agents can be used to label a sample of cells or a group of cells. In this way, a group of cells can be labeled as different from another group of cells. In an example, a first group of cells can originate from a first sample and a second group of cells can originate from a second sample. Labelling agents can allow the first group and second group to have a different labeling agent (or reporter oligonucleotide associated with the labeling agent). This can, for example, facilitate multiplexing, where cells of the first group and cells of the second group can be labeled separately and then pooled together for downstream analysis. The downstream detection of a label can indicate analytes as belonging to a particular group.

[0172] In some embodiments, the reporter oligonucleotides of the additional labeling agents include a sample barcode sequence (e.g., sample index) that allows associating the antibodies with their source biological sample. In some embodiments, the reporter oligonucleotides can further include a barcode sequence that permits identification of a pretreatment condition to which the biological sample (or subject from whom the biological sample is obtained) is subjected prior to step (a) obtaining the plurality of immune cells from the biological sample. In some embodiments, the pretreatment is performed prior to the step of contacting the immune cells with the antigens.

[0173] For example, a reporter oligonucleotide can be linked to an antibody or an epitope binding fragment thereof, and labeling a cell can include subjecting the antibody-linked barcode molecule or the epitope binding fragment-linked barcode molecule to conditions suitable for binding the antibody to a molecule present on a surface of the cell. The binding affinity between the antibody or the epitope-binding fragment thereof and the molecule present on the surface can be within a desired range to ensure that the antibody or the epitope binding fragment thereof remains bound to the molecule. For example, the binding affinity can be within a desired range to ensure that the antibody or the epitope binding fragment thereof remains bound to the molecule during various sample processing steps, such as partitioning and/or nucleic acid amplification or extension. A dissociation constant (Kd) between the antibody or an epitope binding fragment thereof and the molecule to which it binds can be less than about 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, or 1 pM. For example, the dissociation constant can be less than about 10 pM. In some embodiments, the antibody or antigen-binding fragment thereof has a desired dissociation rate constant (koff), such that the antibody or antigen-binding fragment thereof remains bound to the target antigen or antigen fragment during various sample processing steps.

[0174] In another example, a reporter oligonucleotide can be coupled to a cell-penetrating peptide (CPP), and labeling cells can include delivering the CPP coupled reporter oligonucleotide into an analyte carrier. Labeling analyte carriers can include delivering the CPP conjugated oligonucleotide into a cell and/or cell bead by the cell-penetrating peptide. A CPP that can be used in the methods provided herein can include at least one non-functional cysteine residue, which can be either free or derivatized to form a disulfide link with an oligonucleotide that has been modified for such linkage. Non-limiting examples of CPPs that can be used in embodiments herein include penetratin, transportan, plsl, TAT(48-60), pVEC, MTS, and MAP. Cell-penetrating peptides useful in the methods provided herein can have the capability of inducing cell penetration for at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of cells of a cell population. The CPP can be an arginine-rich peptide transporter. The CPP can be Penetratin or the Tat peptide. In another example, a reporter oligonucleotide can be coupled to a fluorophorc or dye, and labeling cells can include subjecting the fluorophore-linked barcode molecule to conditions suitable for binding the fluorophore to the surface of the cell. In some instances, fluorophores can interact strongly with lipid bilayers and labeling cells can include subjecting the fluorophore-linked barcode molecule to conditions such that the fluorophore binds to or is inserted into a membrane of the cell. In some cases, the fluorophore is a water-soluble, organic fluorophore. In some instances, the fluorophore is Alexa 532 maleimide, tetramethylrhodamine-5-maleimide (TMR maleimide), BODIPY-TMR maleimide, Sulfo-Cy3 maleimide, Alexa 546 carboxylic acid/succinimidyl ester, Atto 550 maleimide, Cy3 carboxylic acid/succinimidyl ester, Cy3B carboxylic acid/succinimidyl ester, Atto 565 biotin, Sulforhodamine B, Alexa 594 maleimide, Texas Red maleimide, Alexa 633 maleimide, Abberior STAR 635P azide, Atto 647N maleimide, Atto 647 SE, or Sulfo-Cy5 maleimide. See, e.g., Hughes L D, et al. PLoS One. 2014 Feb. 4; 9(2):e87649, for a description of organic fluorophores.

[0175] A reporter oligonucleotide can be coupled to a lipophilic molecule, and labeling cells can include delivering the nucleic acid barcode molecule to a membrane of a cell or a nuclear membrane by the lipophilic molecule. Lipophilic molecules can associate with and/or insert into lipid membranes such as cell membranes and nuclear membranes. In some cases, the insertion can be reversible. In some cases, the association between the lipophilic molecule and the cell or nuclear membrane can be such that the membrane retains the lipophilic molecule (e.g., and associated components, such as nucleic acid barcode molecules, thereof) during subsequent processing (e.g., partitioning, cell permeabilization, amplification, pooling, etc.). The reporter nucleotide can enter into the intracellular space and/or a cell nucleus. In some embodiments, a reporter oligonucleotide coupled to a lipophilic molecule will remain associated with and/or inserted into lipid membrane (as described herein) via the lipophilic molecule until lysis of the cell occurs, e.g., inside a partition. Exemplary embodiments of lipophilic molecules coupled to reporter oligonucleotides are described in PCT/US2018/064600.

[0176] A reporter oligonucleotide can be part of a nucleic acid molecule including any number of functional sequences, as described elsewhere herein, such as a target capture sequence, a random primer sequence, and the like, and coupled to another nucleic acid molecule that is, or is derived from, the analyte.

[0177] Prior to partitioning, the cells can be incubated with the library of labelling agents, that can be labelling agents to a broad panel of different cell features, e.g., receptors, proteins, etc., and which include their associated reporter oligonucleotides. Unbound labelling agents can be washed from the cells, and the cells can then be co-partitioned (e.g., into droplets or wells) along with partition-specific barcode oligonucleotides (e.g., attached to a support, such as a bead or gel bead) as described elsewhere herein. As a result, the partitions can include the cell or cells, as well as the bound labelling agents and their known, associated reporter oligonucleotides.

[0178] In other instances, e.g., to facilitate sample multiplexing, a labelling agent that is specific to a particular cell feature can have a first plurality of the labelling agent (e.g., an antibody or lipophilic moiety) coupled to a first reporter oligonucleotide and a second plurality of the labelling agent coupled to a second reporter oligonucleotide. For example, the first plurality of the labeling agent and second plurality of the labeling agent can interact with different cells, cell populations or samples, allowing a particular report oligonucleotide to indicate a particular cell population (or cell or sample) and cell feature. In this way, different samples or groups can be independently processed and subsequently combined together for pooled analysis (e.g., partition-based barcoding as described elsewhere herein). See, e.g., U.S. Pat. Pub. 20190323088.

[0179] In some embodiments, to facilitate sample multiplexing, individual samples can be stained with lipid tags, such as cholesterol-modified oligonucleotides (CMOs, see, e.g., FIG. 7A), anti-calcium channel antibodies, or anti-ACTB antibodies. Non-limiting examples of anticalcium channel antibodies include anti-KCNN4 antibodies, anti-BK channel beta 3 antibodies, anti-alB calcium channel antibodies, and anti-CACNAlA antibodies. Examples of anti-ACTB antibodies suitable for the methods of the disclosure include, but are not limited to, mAbGEa, ACTN05, AC-15, 15G5A11/E2, BA3R, and HHF35.

[0180] As described elsewhere herein, libraries of labelling agents can be associated with a particular cell feature as well as be used to identify analytes as originating from a particular cell population, or sample. Cell populations can be incubated with a plurality of libraries such that a cell or cells include multiple labelling agents. For example, a cell can include coupled thereto a lipophilic labeling agent and an antibody. The lipophilic labeling agent can indicate that the cell is a member of a particular cell sample, whereas the antibody can indicate that the cell includes a particular analyte. In this manner, the reporter oligonucleotides and labelling agents can allow multi-analyte, multiplexed analyses to be performed.

[0181] In some instances, these reporter oligonucleotides can include nucleic acid barcode sequences that permit identification of the labelling agent which the reporter oligonucleotide is coupled to. The use of oligonucleotides as the reporter can provide advantages of being able to generate significant diversity in terms of sequence, while also being readily attachable to most biomolecules, e.g., antibodies, etc., as well as being readily detected, e.g., using sequencing or array technologies.

[0182] Attachment (coupling) of the reporter oligonucleotides to the labelling agents can be achieved through any of a variety of direct or indirect, covalent or non-covalent associations or attachments. For example, oligonucleotides can be covalently attached to a portion of a labelling agent (such a protein, e.g., an antibody or antibody fragment), e.g., via a linker, using chemical conjugation techniques (e.g., Lightning-Link® antibody labelling kits available from Innova Biosciences), as well as other non-covalent attachment mechanisms, e.g., using biotinylated antibodies and oligonucleotides (or beads that include one or more biotinylated linker, coupled to oligonucleotides) with an avidin or streptavidin linker. Antibody and oligonucleotide biotinylation techniques are available. See, e.g., Fang, et al., “Fluoride-Cleavable Biotinylation Phosphoramidite for 5 '-end-Labelling and Affinity Purification of Synthetic Oligonucleotides,” Nucleic Acids Res. Jan. 15, 2003; 3 l(2):708-715. Likewise, protein and peptide biotinylation techniques have been developed and are readily available. See, e.g., U.S. Pat. No. 6,265,552. Furthermore, click reaction chemistry such as 5’ Azide oligos and Alkyne - NHS for click chemistry, 4’-Amino oligos for HyNic-4B chemistry, a Methyltetrazine-PEG5- NHS Ester reaction, a TCO-PEG4-NHS Ester reaction, strain-promoted alkyne-azide cycloaddition (SPAAC), or the like, can be used to couple reporter oligonucleotides to labelling agents. Commercially available kits, such as those from Thunderlink and Abeam, and techniques common in the art can be used to couple reporter oligonucleotides to labelling agents as appropriate. In another example, a labelling agent is indirectly (e.g., via hybridization) coupled to a reporter oligonucleotide including a barcode sequence that identifies the label agent. For instance, the labelling agent can be directly coupled (e.g., covalently bound) to a hybridization oligonucleotide that includes a sequence that hybridizes with a sequence of the reporter oligonucleotide. Hybridization of the hybridization oligonucleotide to the reporter oligonucleotide couples the labelling agent to the reporter oligonucleotide. In some embodiments, the reporter oligonucleotides are releasable from the labelling agent, such as upon application of a stimulus. For example, the reporter oligonucleotide can be attached to the labeling agent through a labile bond (e.g., chemically labile, photolabile, thermally labile, etc.) as generally described for releasing molecules from supports elsewhere herein. In some instances, the reporter oligonucleotides described herein can include one or more functional sequences that can be used in subsequent processing, such as an adapter sequence, a unique molecular identifier (UMI) sequence, a sequencer specific flow cell attachment sequence (such as an P5, P7, or partial P5 or P7 sequence), a primer or primer binding sequence, a sequencing primer or primer biding sequence (such as an Rl, R2, or partial R1 or R2 sequence).

[0183] In some cases, the labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a monomer. In some cases, the labelling agent is presented as a multimer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a dimer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a trimer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a tetramer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a pentamer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a hexamer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a heptamer. In some cases, a labelling agent (e.g. , an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as an octamer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a nonamer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a decamer. In some cases, a labelling agent (e.g., an antigen, an antigen fragment, an antibody, an antibody fragment) is presented as a 10+-mer.

[0184] In some cases, the labelling agent can include a reporter oligonucleotide and a label (e.g., detectable label). A label (e.g., detectable label) can be a fluorophore, a radioisotope, a molecule capable of a colorimetric reaction, a magnetic particle, or any other suitable molecule or compound capable of detection. The label can be conjugated to a labelling agent (or reporter oligonucleotide) either directly or indirectly (e.g., the label can be conjugated to a molecule that can bind to the labelling agent or reporter oligonucleotide). In some cases, a label is conjugated to an oligonucleotide that is complementary to a sequence of the reporter oligonucleotide, and the oligonucleotide can be allowed to hybridize to the reporter oligonucleotide.

[0185] FIG. 7A describes exemplary labelling agents (710, 720, 730) including reporter oligonucleotides (740) attached thereto. Labelling agent 710 (e.g., any of the labelling agents described herein) is attached (either directly, e.g., covalently attached, or indirectly) to reporter oligonucleotide 740. Reporter oligonucleotide 740 can include barcode sequence 742 that identifies labelling agent 710. Reporter oligonucleotide 740 can also include one or more functional sequences 743 that can be used in subsequent processing, such as an adapter sequence, a unique molecular identifier (UMI) sequence, a sequencer specific flow cell attachment sequence (such as an P5, P7, or partial P5 or P7 sequence), a primer or primer binding sequence, or a sequencing primer or primer biding sequence (such as an Rl, R2, or partial R1 or R2 sequence).

[0186] Referring to FIG. 7A, in some instances, reporter oligonucleotide 740 conjugated to a labelling agent (e.g., 710, 720, 730) includes a functional sequence 741, a reporter barcode sequence 742 that identifies the labelling agent (e.g., 710, 720, 730), and reporter capture handle 743. Reporter capture handle sequence 743 can be configured to hybridize to a complementary sequence, such as a complementary sequence present on a nucleic acid barcode molecule (not shown), such as those described elsewhere herein. In some instances, nucleic acid barcode molecule is attached to a support (e.g., a bead, such as a gel bead), such as those described elsewhere herein (e.g., FIGS. 3, 4, 8 and 9A-9C). For example, nucleic acid barcode molecule can be attached to the support via a releasable linkage (e.g., including a labile bond), such as those described elsewhere herein (e.g., FIGS. 3, 4, 8 and 9A-9C). In some instances, reporter oligonucleotide 740 includes one or more additional functional sequences, such as those described above.

[0187] In some instances, the labelling agent 710 is a protein or polypeptide (e.g., an antigen or prospective antigen) including reporter oligonucleotide 740. Reporter oligonucleotide 740 includes reporter barcode sequence 742 that identifies polypeptide 710 and can be used to infer the presence of an analyte, e.g., a binding partner of polypeptide 710 (i.e., a molecule or compound to which polypeptide 710 can bind). In some instances, the labelling agent 710 is a lipophilic moiety (e.g., cholesterol) including reporter oligonucleotide 740, where the lipophilic moiety is selected such that labelling agent 710 integrates into a membrane of a cell or nucleus. Reporter oligonucleotide 740 includes reporter barcode sequence 742 that identifies lipophilic moiety 710 which in some instances is used to tag cells (e.g., groups of cells, cell samples, etc.) and can be used for multiplex analyses as described elsewhere herein. In some instances, the labelling agent is an antibody 720 (or an epitope binding fragment thereof) including reporter oligonucleotide 740. Reporter oligonucleotide 740 includes reporter barcode sequence 742 that identifies antibody 720 and can be used to infer the presence of, e.g., a target of antibody 720 (z.e., a molecule or compound to which antibody 720 binds). In other embodiments, labelling agent 730 includes an MHC molecule 731 including peptide 732 and reporter oligonucleotide 740 that identifies peptide 732. In some instances, the MHC molecule is coupled to a support 733. In some instances, support 733 can be or comprise a polypeptide, such as avidin, neutravidin, streptavidin, or a polysaccharide, such as dextran. In some embodiments, support 733 further comprises a detectable label, e.g., a detectable label described herein, e.g., a fluorescent label. In some instances, reporter oligonucleotide 740 can be directly or indirectly coupled to MHC labelling agent 730 in any suitable manner. For example, reporter oligonucleotide 740 can be coupled to MHC molecule 731, support 733, or peptide 732. In some embodiments, labelling agent 730 includes a plurality of MHC molecules, (e.g. is an MHC multimer, which can be coupled to a support (e.g., 733)). In some embodiments, reporter oligonucleotide 740 and MHC molecule 731 are attached to the polypeptide or polysaccharide of support 733. In some embodiments, reporter oligonucleotide 740 and MHC molecule 731 are attached to the detectable label of support 733. In some embodiments, reporter oligonucleotide 740 and an antigen (e.g., protein, polypeptide) are attached to polypeptide or polysaccharide of support 733. In some embodiments, reporter oligonucleotide 740 and an antigen (e.g., protein, polypeptide) are attached to the detectable label of support 733. There are many possible configurations of Class I and/or Class II MHC multimers that can be utilized with the methods and systems disclosed herein, e.g., MHC tetramers, MHC pentamers (MHC assembled via a coiled-coil domain, e.g., Pro5® MHC Class I Pentamers, (Prolmmune, Ltd.), MHC octamers, MHC dodecamers, MHC decorated dextran molecules (e.g., MHC Dextramer® (Immudex)), etc. For a description of exemplary labelling agents, including antibody and MHC-based labelling agents, reporter oligonucleotides, and methods of use, see, e.g., U.S. Pat. 10,550,429 and U.S. Pat. Pub. 20190367969. [0188] Referring to FIG. 7B, in some instances, reporter oligonucleotide 740 is conjugated to a support 750 that can be used to complex with or bind to an antigen (e.g., an antigen of interest or a non-target antigen). Reporter oligonucleotide 740 includes a functional sequence 741, a reporter barcode sequence 742 that identifies the antigen of interest, and reporter capture handle 743. Reporter capture handle sequence 743 can be configured to hybridize to a complementary sequence, such as a complementary sequence present on a nucleic acid barcode molecule (not shown), such as those described elsewhere herein (e.g., FIGS. 3, 4, 8 and 9A-9C). In some instances, nucleic acid barcode molecule is attached to a support (e.g., a bead, such as a gel bead), such as those described elsewhere herein (e.g., FIGS. 3, 4, 8 and 9A-9C). For example, nucleic acid barcode molecule can be attached to the support via a releasable linkage (e.g., including a labile bond), such as those described elsewhere herein. In some instances, reporter oligonucleotide 740 includes one or more additional functional sequences, such as those described above. In one other embodiment, support 750 comprises an anchor sequence 745 that is complementary to functional sequence 741. The reporter oligonucleotide 740 may be attached to support 750 via hybridization to anchor sequence 745. The anchor sequence 745 may further comprise (or may be) a functional sequence (similar to or equivalent to functional sequence 741) as described herein. In some embodiments, the anchor sequence 745 does not comprise a functional sequence. In some embodiments, reporter oligonucleotide 740 includes a functional sequence (not shown). A support 750 may comprise a binding region that can be used to complex with (or bind to) an antigen of interest. In one embodiment, the antigen of interest comprises a ligand that can be bound by the binding region of support 750.

[0189] Referring to FIG. 7B again, labeling agents for antigen receptor analysis are provided. In one embodiment, labelling agent 760 comprises a support 750 that includes an antigen of interest 753 and reporter oligonucleotide 740 that identifies the antigen 753 (e.g., via reporter barcode sequence 742). In some embodiments, the support 750 is coupled to, complexed with, or bound to a ligand 751. In some embodiments, support 750 can be a polypeptide. In some embodiments, the polypeptide can be streptavidin. In some embodiments, the polypeptide can be avidin. In some embodiments, support 750 can be a polysaccharide. In some embodiments, the polysaccharide can be dextran. In some embodiments, the polysaccharide can be a dextran. The ligand 751 can be a molecule with affinity for the binding region of the support 750. For example, the ligand 751 may be biotin and the support 750 may be a streptavidin support. In other embodiments, the ligand 751 is coupled to or conjugated to antigen 753 via a linker 752. Accordingly, in some embodiments of the disclosure, the partitioned cells are contacted with one or more biotinylated antigens. In some embodiments, the antigens can include Avitag biotinylation site and/or a His tag. Protein biotinylation techniques are available. See, e.g., Fang, et al., “Fluoride-Cleavable Biotinylation Phosphoramidite for 5 '-end- Labelling and Affinity Purification of Synthetic Oligonucleotides,” Nucleic Acids Res. Jan. 15, 2003; 31(2):708-715, and U.S. Pat. No. 6,265,552. In some embodiments, the partitioned cells are contacted with full- length coronavirus spike proteins comprising a trimerization domain. In some embodiments, reporter oligonucleotide 740 can be directly or indirectly coupled to labelling agent 760 in any suitable manner. For example, reporter oligonucleotide 740 can be coupled to the antigen 753, support 750, anchor sequence 745, or ligand 751.

[0190] Referring to FIG. 7C, a labelled cell 755 comprising an antigen receptor of interest 754 is depicted. The labelling agent 760 can be contacted with a plurality of cells comprising antigen receptors of interest. In one example, an antigen receptor of interest 754 is bound by or labeled with the labelling agent 760 via an interaction between the antigen receptor of interest 754 and the antigen 753. Further processing of the labelled cell 755 can be performed in a partition-based methods and system as further described herein.

[0191] Exemplary barcode molecules attached to a support (e.g., a bead) is shown in FIG. 9. In some embodiments, analysis of multiple analytes (e.g., RNA and one or more analytes using labelling agents described herein) can include nucleic acid barcode molecules as generally depicted in FIG. 9. In some embodiments, nucleic acid barcode molecules 910 and 920 are attached to support 930 via a releasable linkage 940 (e.g., including a labile bond) as described elsewhere herein. Nucleic acid barcode molecule 910 can include functional sequence 911, barcode sequence 912 and capture sequence 913. Nucleic acid barcode molecule 920 can include adapter sequence 921, barcode sequence 912, and adapter sequence 923, wherein adapter sequence 923 includes a different sequence than adapter sequence 913. Tn some instances, adapter 911 and adapter 921 include the same sequence. In some instances, adapter 911 and adapter 921 include different sequences. Although support 930 is shown including nucleic acid barcode molecules 910 and 920, any suitable number of barcode molecules including common barcode sequence 912 are contemplated herein. For example, in some embodiments, support 930 further includes nucleic acid barcode molecule 950. Nucleic acid barcode molecule 950 can include adapter sequence 951, barcode sequence 912 and adapter sequence 953, wherein adapter sequence 953 includes a different sequence than adapter sequence 913 and 923. In some instances, nucleic acid barcode molecules (e.g., 910, 920, 950) include one or more additional functional sequences, such as a UMI or other sequences described herein. The nucleic acid barcode molecules 910, 920 or 950 can interact with analytes as described elsewhere herein, for example, as depicted in FIGS. 10A-10C.

[0192] Referring to FIG. 10A, in an instance where cells are labelled with labeling agents, capture sequence 1023 can be complementary to an adapter sequence of a reporter oligonucleotide. Cells can be contacted with one or more reporter oligonucleotide 1020 conjugated labelling agents 1010 {e.g., polypeptide such as an antigen, antibody, or others described elsewhere herein). In some cases, the cells can be further processed prior to barcoding. For example, such processing steps can include one or more washing and/or cell sorting steps. In some instances, a cell that is bound to labelling agent 1010 which is conjugated to reporter oligonucleotide 1020, and a support 1030 {e.g., a bead, such as a gel bead) including nucleic acid barcode molecule 1090 is partitioned into a partition amongst a plurality of partitions e.g., a droplet of a droplet emulsion or a well of a microwell array). In some instances, the partition includes at most a single cell bound to labelling agent 1010. In some instances, reporter oligonucleotide 1020 conjugated to labelling agent 1010 {e.g., polypeptide such as an antigen, an antibody, pMHC molecule such as an MHC multimer, etc. ) includes a first adapter sequence 1011 {e.g., a primer sequence), a barcode sequence 1012 that identifies the labelling agent 1010 {e.g., the polypeptide such as an antigen, antibody, or peptide of a pMHC molecule or complex), and a capture handle sequence 1013. Capture handle sequence 1013 can be configured to hybridize to a complementary sequence, such as capture sequence 1023 present on a nucleic acid barcode molecule 1090 {e.g., partition-specific barcode molecule). In some instances, reporter oligonucleotide 1020 includes one or more additional functional sequences, such as those described elsewhere herein.

[0193] Barcoded nucleic acid molecules can be generated {e.g., via a nucleic acid reaction, such as nucleic acid extension, reverse transcription, or ligation) from the constructs described in FIGS. 10A-10C. For example, capture handle sequence 1013 can then be hybridized to complementary capture sequence 1023 to generate (e.g., via a nucleic acid reaction, such as nucleic acid extension or ligation) a barcoded nucleic acid molecule including cell barcode (for example, common barcode, e.g., partition-specific barcode) sequence 1022 (or a reverse complement thereof) and reporter barcode sequence 1012 (or a reverse complement thereof). In some embodiments, the nucleic acid barcode molecule 1090 (e.g., partition-specific barcode molecule) further includes a UMI. Barcoded nucleic acid molecules can then be optionally processed as described elsewhere herein, e.g., to amplify the molecules and/or append sequencing platform specific sequences to the fragments. See, e.g., U.S. Pat. Pub. 2018/0105808. Barcoded nucleic acid molecules, or derivatives generated therefrom, can then be sequenced on a suitable sequencing platform.

[0194] In some instances, analysis of multiple analytes (e.g., nucleic acids and one or more analytes using labelling agents described herein) can be performed. For example, the workflow can include a workflow as generally depicted in any of FIGS. 10A-10C, or a combination of workflows for an individual analyte, as described elsewhere herein. For example, by using a combination of the workflows as generally depicted in FIGS. 10A-10C, multiple analytes can be analyzed.

[0195] In some instances, analysis of an analyte (e.g. a nucleic acid, a polypeptide, a carbohydrate, a lipid, etc.) includes a workflow as generally depicted in FIG. 10A. A nucleic acid barcode molecule 1090 can be co-partitioned with the one or more analytes. In some instances, nucleic acid barcode molecule 1090 is attached to a support 1030 (e.g., a bead, such as a gel bead), such as those described elsewhere herein. For example, nucleic acid barcode molecule 1090 can be attached to support 1030 via a releasable linkage 1040 (e.g., including a labile bond), such as those described elsewhere herein. Nucleic acid barcode molecule 1090 can include a barcode sequence 1021 and optionally include other additional sequences, for example, a barcode sequence 1022 (e.g., common barcode, partition- specific barcode, UMI, or other functional sequences described elsewhere herein). Nucleic acid barcode molecule 1090 can include a functional sequence 1021. In some embodiments, the nucleic acid barcode molecule 1090 can include other additional sequences, for example, a barcode sequence 1022 (e.g., common barcode, partition-specific barcode, or other functional sequences described elsewhere herein), and/or a UMI sequence. The nucleic acid barcode molecule 1090 can include a capture sequence 1023 that can be complementary to another nucleic acid sequence, such that it can hybridize to a particular sequence.

[0196] For example, capture sequence 1023 can include a poly-T sequence and can be used to hybridize to mRNA. Referring to FIG. 10C, in some embodiments, nucleic acid barcode molecule 1090 includes capture sequence 1023 complementary to a sequence of RNA molecule 1060 from a cell. In some instances, capture sequence 1023 includes a sequence specific for an RNA molecule. Capture sequence 1023 can include a known or targeted sequence or a random sequence. In some instances, a nucleic acid extension reaction can be performed, thereby generating a barcoded nucleic acid product including capture sequence 1023, the functional sequence 1021, UMI and/or barcode sequence 1022, any other functional sequence, and a sequence corresponding to the RNA molecule 1060.

[0197] In another example, capture sequence 1023 can be complementary to an overhang sequence or an adapter sequence that has been appended to an analyte. In one embodiment, capture sequence 1023 is complementary to a sequence that has been appended to a nucleic acid molecule derived from an analyte of interest. In another embodiment, the nucleic acid molecule is a cDNA molecule generated in a reverse transcription reaction using an RNA analyte (e.g., an mRNA analyte) of interest. In an additional embodiment, capture sequence 1023 is complementary to a sequence that has been appended to the cDNA molecule generated from the mRNA analyte of interest. For example, referring to FIG. 10B, in some embodiments, primer 1050 includes a sequence complementary to a sequence of nucleic acid molecule 1060 (such as an RNA encoding for a BCR sequence) from a biological particle. In some instances, primer 1050 includes one or more sequences 1051 that are not complementary to RNA molecule 1060. Sequence 1051 can be a functional sequence as described elsewhere herein, for example, an adapter sequence, a sequencing primer sequence, or a sequence the facilitates coupling to a flow cell of a sequencer. In some instances, primer 1050 includes a poly-T sequence. In some instances, primer 1050 includes a sequence complementary to a target sequence in an RNA molecule. In some instances, primer 1050 includes a sequence complementary to a region of an immune molecule, such as the constant region of a BCR sequence. Primer 1050 is hybridized to nucleic acid molecule 1060 and complementary molecule 1070 is generated. For example, complementary molecule 1070 can be cDNA generated in a reverse transcription reaction. In some instances, an additional sequence can be appended to complementary molecule 1070. For example, the reverse transcriptase enzyme can be selected such that several non-templated bases 1080 (e.g., a poly-C sequence) are appended to the cDNA. In another example, a terminal transferase can also be used to append the additional sequence. Nucleic acid barcode molecule 1090 includes a sequence 1024 complementary to the non-templated bases, and the reverse transcriptase performs a template switching reaction onto nucleic acid barcode molecule 1090 to generate a barcoded nucleic acid molecule including cell (e.g., partition specific) barcode sequence 1022 (or a reverse complement thereof) and a sequence of complementary molecule 1070 (or a portion thereof). In some instances, capture sequence 1023 includes a sequence complementary to a region of an immune molecule, such as the constant region of a BCR sequence. Capture sequence 1023 is hybridized to nucleic acid molecule 1060 and a complementary molecule 1070 is generated. For example, complementary molecule 1070 can be generated in a reverse transcription reaction generating a barcoded nucleic acid molecule including cell barcode (e.g., common barcode or partition- specific barcode) sequence 1022 (or a reverse complement thereof) and a sequence of complementary molecule 1070 (or a portion thereof). Additional methods and compositions suitable for barcoding cDNA generated from mRNA transcripts including those encoding V(D)J regions of an immune cell receptor and/or barcoding methods and composition including a template switch oligonucleotide are described in International Patent Application WO2018/075693, U.S. Patent Publication No. 2018/0105808, U.S. Patent Publication No. 2015/0376609, and U.S. Patent Publication No. 2019/0367969.

[0198] In some embodiments, biological particles (e.g., cells, nuclei) from a plurality of samples (e.g., a plurality of subjects) can be pooled, sequenced, and demultiplexed by identifying mutational profiles associated with individual samples and mapping sequence data from single biological particles to their source based on their mutational profile. See, e.g., Xu J. et al., Genome Biology Vol. 20, 290 (2019); Huang Y. et al., Genome Biology Vol. 20, 273 (2019); and Heaton et al., Nature Methods volume 17, pages 615-620(2020).

[0199] Gene expression data can reflect the underlying genome and mutations and structural variants therein. As a result, the variation inherent in the captured and sequenced RNA molecules can be used to identify genotypes de novo or used to assign molecules to genotypes that were known a priori. In some embodiments, allelic variation that is present due to haplotypic states (including linkage disequilibrium of the human leucocyte antigen loci (HLA), immune receptor loci (BCR), and other highly polymorphic regions of the genome), can also be used for demultiplexing. Expressed ABM (e.g., antibodies or B cell receptors) can be used to infer germline alleles from unrelated individuals, which information may be used for demultiplexing.

[0200] In some instances, barcoding of a nucleic acid molecule may be done using a combinatorial approach. In such instances, one or more nucleic acid molecules (which may be comprised in a biological particle, e.g., a cell, e.g., a fixed cell, organelle, nucleus, or cell bead) may be partitioned (e.g., in a first set of partitions, e.g., wells or droplets) with one or more first nucleic acid barcode molecules (optionally coupled to a bead). The first nucleic acid barcode molecules or derivative thereof (e.g., complement, reverse complement) may then be attached to the one or more nucleic acid molecules, thereby generating barcoded nucleic acid molecules, e.g., using the processes described herein. The first nucleic acid barcode molecules may be partitioned to the first set of partitions such that a nucleic acid barcode molecule, of the first nucleic acid barcode molecules, that is in a partition comprises a barcode sequence that is unique to the partition among the first set of partitions. Each partition may comprise a unique barcode sequence. For example, a set of first nucleic acid barcode molecules partitioned to a first partition in the first set of partitions may each comprise a common barcode sequence that is unique to the first partition among the first set of partitions, and a second set of first nucleic acid barcode molecules partitioned to a second partition in the first set of partitions may each comprise another common barcode sequence that is unique to the second partition among the first set of partitions. Such barcode sequence (unique to the partition) may be useful in determining the cell or partition from which the one or more nucleic acid molecules (or derivatives thereof) originated.

[0201] The barcoded nucleic acid molecules from multiple partitions of the first set of partitions may be pooled and re-partitioned (e.g., in a second set of partitions, e.g., one or more wells or droplets) with one or more second nucleic acid barcode molecules. The second nucleic acid barcode molecules or derivative thereof may then be attached to the barcoded nucleic acid molecules. As with the first nucleic acid barcode molecules during the first round of partitioning, the second nucleic acid barcode molecules may be partitioned to the second set of partitions such that a nucleic acid barcode molecule, of the second nucleic acid barcode molecules, that is in a partition comprises a barcode sequence that is unique to the partition among the second set of partitions. Such barcode sequence may also be useful in determining the cell or partition from which the one or more nucleic acid molecules or first barcoded nucleic acid molecules originated. The barcoded nucleic acid molecules may thus comprise two barcode sequences (e.g., from the first nucleic acid barcode molecules and the second nucleic acid barcode molecules).

[0202] Additional barcode sequences may be attached to the barcoded nucleic acid molecules by repeating the processes any number of times (e.g., in a split-and-pool approach), thereby combinatorically synthesizing unique barcode sequences to barcode the one or more nucleic acid molecules. For example, combinatorial barcoding may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more operations of splitting (e.g., partitioning) and/or pooling (e.g., from the partitions). Additional examples of combinatorial barcoding may also be found in International Patent Publication Nos. WO2019/165318, each of which is herein entirely incorporated by reference for all purposes.

[0203] Beneficially, the combinatorial barcode approach may be useful for generating greater barcode diversity, and synthesizing unique barcode sequences on nucleic acid molecules derived from a cell or partition. For example, combinatorial barcoding comprising three operations, each with 100 partitions, may yield up to 106 unique barcode combinations. In some instances, the combinatorial barcode approach may be helpful in determining whether a partition contained only one cell or more than one cell. For instance, the sequences of the first nucleic acid barcode molecule and the second nucleic acid barcode molecule may be used to determine whether a partition comprised more than one cell. For instance, if two nucleic acid molecules comprise different first barcode sequences but the same second barcode sequences, it may be inferred that the second set of partitions comprised two or more cells.

[0204] In some instances, combinatorial barcoding may be achieved in the same compartment. For instance, a unique nucleic acid molecule comprising one or more nucleic acid bases may be attached to a nucleic acid molecule ( e.g.. a sample or target nucleic acid molecule) in successive operations within a partition ( e.g.. droplet or well) to generate a barcoded nucleic acid molecule. A second unique nucleic acid molecule comprising one or more nucleic acid bases may be attached to the barcoded nucleic acid molecule. In some instances, all the reagents for barcoding and generating combinatorially barcoded molecules may be provided in a single reaction mixture, or the reagents may be provided sequentially.

[0205] In some instances, cell beads comprising nucleic acid molecules may be barcoded. Methods and systems for barcoding cell beads are further described in PCT/US2018/067356 and U.S. Pat. Pub. No. 2019/0330694, which are hereby incorporated by reference in its entirety.

[0206] In some instances wherein a partition is a volume wherein diffusion of contents beyond the volume is inhibited, the partition contains a diffusion resistant material. Such partition may also be referred to herein as a diffusion resistant partition. The diffusion resistant material may have an increased viscosity. The diffusion resistant material may be or comprise a matrix, e.g., a polymeric matrix, or a gel. Suitable polymers or gels are disclosed herein. The matrix can be a porous matrix capable of entraining and/or retaining materials within its matrix. In some embodiments, a diffusion resistant partition comprises a single biological particle and a single bead, the single bead comprising a plurality of nucleic acid barcode molecules comprising a partition specific barcode sequence. In some embodiments, the partition specific barcode sequence is unique to the diffusion resistant partition. In some embodiments, partitioning comprises contacting a plurality of biological particles with a plurality of beads in a diffusion resistant material to provide a diffusion resistant partition comprising a single biological particle and a single bead. In some embodiments, partitioning comprises contacting a plurality of biological particles with a plurality of beads in a liquid comprising a polymeric precursor material that may be capable of being formed into a gel or other solid or semi-solid matrix, and subjecting the liquid to conditions sufficient to polymerize or gel the precursors, e.g., as described herein. In some embodiments, the biological particle may be lysed or permeabilized in the diffusion resistant partition. In some embodiments, a nucleic acid analyte of the biological particle (which may include a reporter oligonucleotide associated with a labelling agent disclosed herein) may be coupled with a nucleic acid barcode molecule in the diffusion resistant partition. In some cases, further processing, e.g. , generation of barcoded nucleic acid molecules, may be performed in the diffusion resistant partition or in bulk. For example, nucleic acid analytes, once coupled to nucleic acid barcode molecules in partitions, may be pooled and then subjected to further processing in bulk e.g., extension, reverse transcription, or other processing) to generate barcoded nucleic acid molecules. For other example, nucleic acid analytes, one coupled to nucleic acid barcode molecules in diffusion resistant partitions, may be subjected to further processing in the diffusion resistant partitions to generate barcoded nucleic acid molecules.

[0207] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0208] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and the Applicant reserves the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of information sources, including scientific journal articles, patent documents, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0209] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure, and are to be included within the spirit and purview of this application.

[0210] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.

EXAMPLES

[0211] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press;

Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference.

[0212] Additional embodiments are disclosed in further detail in the following examples, which arc provided by way of illustration and arc not in any way intended to limit the scope of this disclosure or the claims.

EXAMPLE 1

[0213] This Example describes the results of additional experiments performed to evaluate performance of some MHC multimer reagents of the disclosure in an exemplary antigen mapping workflow.

[0214] In previously reported studies, standard preparation of MHC multimer reagents relied on (i) tetramerization of MHC monomers and (ii) peptide loading of the tetrameric MHC molecules with streptavidin scaffolds in a laborious two-step process that lasted 48 hours or more (Sec, e.g., Lcisncr C. et al. One-Pot, Mix-and-Read Peptide-MHC Tetramers. 2008 Feb 27;3(2):el678. doi: 10.1371/joumal.pone.0001678). In contrast to these previous studies, significantly more efficient methods for preparing MHC multimer reagents was developed, e.g. , where tetramerization and peptide loading were performed simultaneously.

[0215] In these experiments, anti-CMV expanded T cells and MHC HLA-A02:01 monomers were used.

Two-step protocol

[0216] Immediately prior to performing staining, barcoded and fluorescently-labeled streptavidin was mixed in a tube and centrifuge at 2500xg for 5 minutes at 4°C. [0217] Tetramerization of MHC monomers:

[0218] #1. Added 125 pL storage buffer.

[0219] #2. Added 5 |aL TotalSeq Streptavidin (0.5 mg/mL of BioLegend TsC-C0952-PE or BioLegend TsC-C0956-APC).

[0220] #3. MHC HLA-A02:01 monomers were diluted in PBS to provide a final concentration of 40 ng/pl of monomers.

[0221] #4. Incubated 30 minutes on ice in the dark.

[0222] 5# Peptide Loading Step

[0223] Below is a general procedure for loading peptide- antigen into empty loadable MHC tetramers:

[0224] (a) Prepared 1/10 volume peptide stock (“NLVPMVATV”) 200 pM in PBS.

[0225] (b) Prepared a 200 pM working stock of peptide antigen by diluting 10 mM DMSO stock solution in PBS, pH 7.4 (Example: Add 1 pl of 10 mM stock solution to 49 pl PBS).

[0226] (c) 2 rxn: 10 pl empty MHC tetramer (from #1-4 above) add 1 pl peptide antigen (from #5 above).

[0227] 1. Incubation: 30 minutes on ice at 4°C.

[0228] 2. Storage: -20°C.

One-step tetramerization and peptide-loading protocol

[0229] One-Step tetramerization and peptide loading reaction contained the following components:

[0230] 31.25 pl storage buffer

[0231] 1.25 pl STA TotalSeq Streptavidin (0.625 pg total of BioLegend TsC-C0952-PE or BioLegend TsC-C0956-APC))

[0232] 28.5 pl PBS

[0233] 1.5 pl HLA-A02:01 monomers (1.7 pg/mL)

[0234] After the above components were mixed well, 10 pl of mix was immediately taken out and added 1 pl of peptide (“NLVPMVATV”).

[0235] Incubated for 30 minutes, 15 minutes, or 5 minutes, and added to cells immediately.

Cell Preparation: Anti CMV T cells and HLA-A*0201

[0236] Anti-CMV expanded T cells were prepared according to previously published protocol for fresh frozen human peripheral blood mononuclear cells for single cell RNA sequencing (CG00039).

[0237] Cell count and viability: 1.6 x 10⁶ total cells, 84% Viability. A total of 5 samples (3.2x 10’⁵ cells/sample) were prepared as follows.

[0238] Experimental samples:

[0239] 1) No Stain

[0240] 2) Two-Step Peptide loading.

[0241] 3) One-Step Peptide loading (30 min).

[0242] 4) One-Step Peptide loading (15 min).

[0243] 5) One-Step Peptide loading (5 min).

Cell Staining Step

[0244] Cell staining was performed as follows:

[0245] 1) 5 pl MHC tetramers prepared as described above were added to the cells, mixed well and incubated 15 minutes on ice in the dark. 5 pl of tetramerized STA-PE was used for staining (i.e., 1.36 x 10 ¹¹ PE molecules, 3.2 x 10’⁵ cells/group to provide 4.25 x 10’⁵ molecules/cell).

[0246] 2) Added 3 pl of anti-CD8 AF488 to samples, incubated 30 minutes on ice in the dark.

[0247] 3) Added 1 pl of 7AAD arid incubated 10 minutes in the dark.

[0248] 4) Washed 3x 1 ml of PBS + 2% FBS.

[0249] 5) Resuspended in 300 pl of PBS + 2% FBS.

[0250] 6) Analyzed using flow cytometry.

[0251] As illustrated in FIG. 11, it was observed that tetramerization and peptide-loading processes could be performed in one single reaction with the same efficiency as existing two-step methods. In particular, it was observed that the newly developed one-step method could generate peptide loaded MHC tetramers with the same staining results as the two-step method in as short as 5 minutes. In these experiments, 15-min to 30-min assembly times were found to provide the best staining quality.

EXAMPLE 2

[0252] This Example describes the results of additional experiments performed to evaluate performance of the newly developed one-step peptide loading and tetramer assembly method described in Example 1.

[0253] In these experiments, one-step peptide loading and tetramer assembly was performed either at room temperature or on ice at four different time points (5 min, 15 min, 30 min, and 45 minutes). For each condition, CMV, Flu, and HIV tetramers were assembled. For the cell staining, CMV and Flu expanded T cells were mixed in equal ratios and stained with the assembled tetramers as described (e.g., in Example 1), then analyzed by flow cytometry.

[0254] As illustrated in FIG. 12, it was observed that one-step method could generate peptide loaded MHC tetramers with the same staining results as the two-step method under a variety of time and temperature conditions, including as short as five minutes.

[0255] While particular alternatives of the present disclosure have been disclosed, it is to be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. There is no intention, therefore, of limitations to the exact abstract and disclosure herein presented.

Claims

CLAIMS WHAT IS CLAIMED IS

1. A method for preparing a major histocompatibility complex (MHC) multimer reagent, the method comprising:

(a) providing a mixture comprising:

(i) a plurality of empty MHC monomers linked to first binding moieties,

(ii) a second binding moiety comprising a plurality of binding sites capable of binding to the first binding moieties, and

(iii) a plurality of antigens;

(b) in the mixture,

(i) loading the plurality of antigens to at least a subset of the plurality of MHC monomers linked to the first binding moieties, and

(ii) binding the first binding moieties to at least a subset of the plurality of binding sites of the second binding moiety, thereby generating a MHC multimer reagent comprising the second binding moiety bound to the plurality of antigen-loaded MHC monomers.

2. The method of claim 1, wherein the loading in (b)(i) and the binding in (b)(ii) are performed simultaneously.

3. The method of any one of claims 1 to 2, wherein the second binding moiety comprises a core support attached to a detectable label capable of emitting a detectable signal.

4. The method of any one of claims 1 to 3, wherein the detectable label is or comprises a fluorophore, a magnetic particle, or a mass tag.

5. The method of claim 4, wherein the fluorophore molecule is or comprises phycoerythrin (PE), allophycocyanin (APC), Alexa Fluor 405, Pacific Blue, Alexa Fluor 488, Alexa Fluor 647, Alexa Fluor 700, DyLight 405, DyLight 550, DyLight 650, fluorescein isothiocyanate (FITC), peridinin chlorophyll protein (PerCP), StarBright Violet 440, StarBright Violet 515, StarBright 610, StarBright Violet 670, or StarBright Blue 700.

6. The method of any one of claims 1 to 5, wherein the second binding moiety further comprises a reporter oligonucleotide.

7. The method of claim 6, wherein the reporter oligonucleotide comprises a reporter barcode sequence.

8. The method of any one of claims 6 to 7, the reporter oligonucleotide is attached to the core support and/or to the detectable label.

9. The method of any one of claims 1 to 8, further comprising quenching the MHC multimer reagent generated in (b) with first binding moieties that are not linked to MHC monomers.

10. The method of any one of claims 1 to 9, wherein the plurality binding sites of the second binding moiety of the generated MHC multimer reagent are each either bound to an antigen- loaded MHC monomer or bound a first binding moiety.

11. The method of any one of claims 1 to 10, wherein the first binding moieties are biotinylated or comprise a biotin moiety.

12. The method of claim 11, wherein the core support of the second binding moiety comprises one or more biotin-binding sites.

13. The method of any one of claims 11 to 12, wherein the core support is or comprises a biotin-binding protein selected from streptavidin, avidin, deglycosylated avidin (e.g.,

Neutr Avidin™), traptavidin, tamavidin, xenavidin, bradavidin, AVR2 (avidin related protein 2), AVR4 (avidin related protein 4), and variants, mutants, derivatives, and homologs of any thereof.

14. The method of any one of claims 10 to 12, wherein the MHC multimer reagent generated in (b) comprises a core support comprising four biotin-binding sites, and wherein at least one, two, three, or four of the biotin-binding sites are each bound to an antigen-loaded MHC monomer (or biotinylated MHC monomer).

15. The method of claim 13, wherein biotin-binding sites of the core support not bound to the antigen-loaded MHC monomer (or biotinylated MHC monomer) are bound to biotin.

16. The method of any one of claims 1 to 14, wherein the MHC monomers are selected from the group consisting of MHC class I monomers and MHC class II monomers.

17. The method of claim 15, wherein the MHC monomers are of the same MHC class.

18. The method of claim 15, wherein the MHC monomers are of different MHC classes.

19. The method of any one of claims 1 to 17, wherein the plurality of antigens is selected from oligopeptides, proteins, polysaccharides, lipids, liposomes, and infectious agents.

20. The method of any one of claims 1 to 18, wherein the plurality of antigen-loaded MHC monomers comprise different antigens.

21. The method of any one of claims 1 to 18, wherein the plurality of antigen-loaded MHC monomers comprise the same antigen.

22. The method of any one of claims 1 to 20, wherein the ratio of the second binding moiety to empty MHC monomers in the mixture ranges from about 5:1 to about 1:5 (w/w).

23. The method of claim 21, wherein the ratio of the second binding moiety to empty MHC monomers in the mixture is about 1:2 (w/w).

24. The method of any one of claims 1 to 22, wherein step (b) is carried out at a temperature ranging from about 0°C to about 37°C, from about 4°C to about 25°C, or from 4°C to about 22°C.

25. The method of claim 23, wherein step (b) is carried out at a temperature of about 4°C.

26. The method of any one of claims 1 to 24, wherein step (b) is carried out for a duration of less than about 120 minutes, less than about 100 minutes, less than about 90 minutes, less than about 60 minutes, less than about 45 minutes, less than about 30 minutes, less than about 15 minutes, less than about 10 minutes, or less than about 5 minutes.

27. The method of any one of claims 1 to 25, wherein step (b) is carried out at room temperature and for a duration of about 5 minutes.

28. The method of any one of claims 6 to 26, wherein the MHC multimer reagent generated in (b) is a barcoded MHC multimer reagent comprising the second binding moiety that is attached to a plurality of antigen-loaded MHC monomers and further attached to the reporter oligonucleotide.

29. A barcoded MHC multimer reagent prepared by the method of claim 27.

30. A method for characterizing an antigen-binding molecule (ABM), the method comprising: a) providing a reaction mixture comprising: (i) a plurality of immune cells and/or a plurality of cell beads comprising immune cells, and (ii) a plurality of barcoded MHC multimer reagents according to claim 28; b) partitioning the mixture, or a portion thereof, into a plurality of partitions, wherein the partitioning provides a first partition comprising:

(i) a first immune cell and/or a first cell bead comprising a first immune cell,

(ii) a first barcoded MHC multimer reagent, and

(iii) a plurality of nucleic acid barcode molecules comprising a partition-specific barcoded sequence; and c) generating barcoded nucleic acid molecules, wherein the barcoded nucleic acid molecules comprise a first barcoded nucleic acid molecule comprising (i) a first nucleic acid sequence encoding at least a portion of an antigen-binding molecule (ABM) expressed by the first immune cell and/or a first cell bead comprising a first immune cell, or a reverse complement thereof and (ii) the partition- specific barcode sequence or a reverse complement thereof, and a second barcoded nucleic acid molecule comprising (i) the first reporter barcode sequence or a reverse complement thereof and (ii) the partition- specific barcode sequence or a reverse complement thereof.

31. The method of claim 29, further comprising generating a third barcoded nucleic acid molecule comprising (i) the partition barcode sequence or a reverse complement thereof and (ii) a second nucleic acid sequence or a reverse complement thereof, the second nucleic acid sequence encoding a different portion of the ABM expressed by the first immune cell and/or a first cell bead comprising a first immune cell.

32. The method of claim 29 or 30, further comprising generating a fourth barcoded nucleic acid molecule comprising (i) the partition-specific barcode sequence or a reverse complement thereof and a third nucleic acid sequence, wherein the third nucleic acid sequence is a sequence of an mRNA analyte of the first immune cell and/or a first cell bead comprising a first immune cell, or a reverse complement thereof, or a cDNA sequence of the mRNA analyte of the first immune cell and/or a first cell bead comprising a first immune cell or a reverse complement thereof.

33. The method of any one of claims 29 to 31, wherein the mRNA analyte docs not encode an ABM or portion thereof.

34. The method of any one of claims 29 to 33, further comprising determining a sequence of the first barcoded nucleic acid molecule or an amplicon thereof, and determining a sequence of the second barcoded nucleic molecule or an amplicon thereof.

35. The method of claim 34, further comprising (i) identifying the ABM as expressed by the first immune cell and/or a first cell bead comprising a first immune cell based on the determined sequence of the first barcoded nucleic acid molecule or amplicon thereof and (ii) identifying the first immune cell and/or a first cell bead comprising a first immune cell having bound the target antigen based on the determined sequence of the second barcoded nucleic acid molecule or amplicon thereof.

36. The method of any one of claims 31 to 35, further comprising determining a sequence of the third barcoded nucleic acid molecule or an amplicon thereof.

37. The method of any one of claims 32 to 36, further comprising determining a sequence of the fourth barcoded nucleic acid molecule or an amplicon thereof.

38. The method of claim 29 or 37, wherein the plurality of immune cells and/or cell beads comprising immune cells comprises B cells.

39. The method of claim 38, wherein the first immune cell is a B cell bound to the antigen(s) of the first barcoded MHC multimer reagent.

40. The method of claim 39, wherein the ABM produced by the B cell is a B cell receptor (BCR), an antibody (Ab) or an antigen-binding fragment thereof.

41. The method of any one of claims 29 to 31, wherein the plurality of immune cells and/or cell beads comprising immune cells comprises T cells.

42. The method of claim 41, wherein the first immune cell is a T cell bound to the antigen(s) of the first barcoded MHC multimer reagent.

43. The method of claim 42, wherein the ABM produced by the T cell is a TCR.