WO2023215861A1 - Réactifs pour caractériser des molécules de liaison à l'antigène à partir de cellules immunitaires - Google Patents

Réactifs pour caractériser des molécules de liaison à l'antigène à partir de cellules immunitaires Download PDF

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Publication number
WO2023215861A1
WO2023215861A1 PCT/US2023/066653 US2023066653W WO2023215861A1 WO 2023215861 A1 WO2023215861 A1 WO 2023215861A1 US 2023066653 W US2023066653 W US 2023066653W WO 2023215861 A1 WO2023215861 A1 WO 2023215861A1
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nucleic acid
sequence
labelling
cell
detectable label
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PCT/US2023/066653
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English (en)
Inventor
Bixun WANG
Wyatt James MCDONNELL
Bruce Alexander ADAMS
Michael John Terry STUBBINGTON
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10X Genomics, Inc.
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Publication of WO2023215861A1 publication Critical patent/WO2023215861A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present disclosure relates generally to the field of immunology, and particularly relates to compositions and methods useful for characterization of antigen-binding molecules (ABMs) produced by immune cells, e.g., B cells and T cells.
  • ABSMs antigen-binding molecules
  • Antigen-binding molecules that bind to antigens of interest can be developed as new immunotherapeutic agents.
  • many ABMs developed 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.
  • High-throughput approaches and systems are particularly useful for the identification and characterization of these ABMs.
  • Such approaches and systems utilize reagents for antigen mapping, e.g., reagents where antigens are operably coupled with nucleic acid barcode sequences that identify the antigens.
  • the present disclosure provides, inter alia, compositions and methods useful 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. Characterization of ABMs having desirable properties, e.g., that recognize and bind to cells displaying tumor or viral antigens, can be useful in the development of new immunotherapies to treat cancers and/or infectious disease. Also provided in some embodiments of the disclosure are reagents, kits, and methods for the identification and/or characterization of these ABMs, including without limitation determining sequences, complete and/or partial sequences, of ABMs. In certain embodiments, the sequences comprise or essentially consist of CDR3s segments. In certain embodiments, sequences are paired VH and VL chains from BCRs. In certain embodiments, these are paired sequences from TCRs.
  • the labelling compositions include a detectable label; a core support attached to the detectable label; and a nucleic acid molecule attached to the detectable label, wherein the nucleic acid molecule includes: (a) a reporter oligonucleotide including (i) a reporter barcode sequence and (ii) a capture handle sequence, or (b) an anchor nucleic acid molecule configured to directly or indirectly attach to the reporter oligonucleotide of (a).
  • a labeling composition which is a mixture of labeling compositions each one including a detectable label; one, two, three or four core supports(s) attached to the detectable label; and a nucleic acid molecule attached to the detectable label, wherein the nucleic acid molecule includes: (a) a reporter oligonucleotide including (i) a reporter barcode sequence and (ii) a capture handle sequence, or (b) an anchor nucleic acid molecule configured to directly or indirectly attach to the reporter oligonucleotide of (a).
  • each core support comprises multiple target antigen conjugation sites.
  • the labelling composition comprises: a detectable label; a core support attached to the detectable label, wherein the core support is streptavidin or any streptavidin alternative, wherein the detectable label is PE; and a nucleic acid molecule attached to the detectable label, wherein the nucleic acid molecule comprises: (a) a reporter oligonucleotide comprising (i) a reporter barcode sequence and (ii) a capture handle sequence, or (b) an anchor nucleic acid molecule configured to directly or indirectly attach to the reporter oligonucleotide of (a).
  • Non-limiting exemplary embodiments of the labelling compositions disclosed herein can include one or more of the following features.
  • the anchor nucleic acid molecule includes a sequence configured to attach, by complementarity base pairing, to an anchoring sequence present in the reporter oligonucleotide.
  • the anchor nucleic acid is hybridized to the anchoring sequence of the report oligonucleotide.
  • the reporter oligonucleotide further includes one or more functional sequences selected from an adapter sequence, a primer sequence, a primer recognition sequence, and a unique molecular identifier (UMI).
  • UMI unique molecular identifier
  • the detectable label includes a fluorophore, a magnetic particle, or a mass tag.
  • the fluorophore is or includes one or more of the following: 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, and StarBright Blue 700.
  • PE phycoerythrin
  • APC allophycocyanin
  • Alexa Fluor 405 Pacific Blue
  • Alexa Fluor 488 Alexa Fluor 647
  • Alexa Fluor 700 Alexa Fluor 700
  • DyLight 405 DyLight 550
  • DyLight 650 fluorescein isothiocyanate
  • the core support includes a biotin-binding agent.
  • the biotin-binding agent is or includes a biotin-binding protein selected from streptavidin, or a streptavidin alternative including without limitation, avidin, deglycosylated avidin (e.g., Neu tr AvidinTM), traptavidin, tamavidin, xenavidin, bradavidin, AVR2 (avidin related protein 2), AVR4 (avidin related protein 4), and variants, mutants, derivatives, and homologs of any thereof.
  • the labelling composition of the disclosure is a first labelling composition wherein the core support comprises (e.g., coupled to) a target antigen, and wherein the reporter barcode sequence is a first reporter barcode sequence that identifies the target antigen.
  • the target antigen is biotinylated.
  • the target antigen is an oligopeptide, a protein, a polysaccharide, a lipid, a liposome, an infectious agent, or a target MHC molecule complex, e.g., a peptide-MHC (pMHC) molecule complex.
  • the labelling composition of the disclosure is a second labelling composition that is not coupled to the target antigen and wherein the reporter barcode sequence is a second reporter barcode sequence that identifies the second labelling composition.
  • the second labelling composition includes a non-target antigen.
  • the non-target antigen includes a biotin moiety.
  • the present disclosure provides a set of labelling compositions.
  • the set includes a first labelling composition as described herein and a second labelling composition as described herein.
  • the detectable label of the first labelling composition is a first detectable label and wherein the detectable label of the second labelling composition is a second detectable label.
  • the first detectable label and the second detectable label are the same. Tn some embodiments, the first detectable label and the second detectable label arc different.
  • the methods identify uncharacterized ABM(s).
  • 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, comprising or expected to comprise sequences of the ABM(s)and (ii) a plurality of first labelling compositions as described herein or a set of labelling compositions as described herein; wherein the reaction mixture is subject to conditions suitable for binding between the ABM(s) and the labelling composition comprising a target antigen; (b) partitioning the reaction 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) the first labelling composition, and (ii) a plurality of nucleic
  • the step of generating barcoded nucleic acid molecules comprises steps of subjecting the reaction to conditions suitable for: hybridizing capture sequence and capture handle sequence; performing nucleic acid amplification(s), and/or extension reaction(s), or combination thereof, so as to generate barcoded nucleic acid molecules, including partition- specific barcoded cDNA of ABMs or complement thereof, and/or a nucleic acid molecules comprising the partition-specific barcode connected to reporter barcode sequence, or complement thereof; further processing nucleic acid sequence to generate sequencing libraries; determining sequences from the sequencing libraries; and analyzing sequences determined from the sequencing libraries.
  • multiple analytes including without limitation target antigens, ABMs, expression of immune receptors sequences, including without limitation B cell receptors and T cell receptors, expression of other target sequence(s), gene expression (transcriptome), and/or other cell characteristics are characterized.
  • analyzing comprises determining a correlation between different analytes thereof, c.g. target antigens and ABMs, and/or expression of immune receptors sequences, including without limitation B cell receptors and T cell receptors, and/or expression of other target sequence(s), and/or gene expression (transcriptome), and/or other cell characteristic, and/or any combination thereof.
  • hybridizing capture sequence and capture handle sequence is conducted in the partition.
  • the subsequence steps of amplification(s), and/or extension reaction(s), or combination thereof are conducted in the partition and/or in bulk outside of the partition.
  • the plurality of nucleic acid barcode molecules including a partitionspecific barcode sequence further comprise a capture sequence as described herein.
  • the capture sequence is the same. In some embodiments where the capture sequence is the same, the capture sequence is designed so that it captures multiple analytes.
  • Analytes could be immune receptor sequences, gene expression nucleic acid sequences, labeling compositions, and/or any other cell specific features.
  • the capture sequence is any one of the capture sequences described herein, or a combination thereof.
  • Nonlimiting examples of capture handle sequence include, a poly-T capture sequence, target specific capture sequence(s) and capture sequence that is complementary to the capture handle sequence.
  • Non-limiting exemplary embodiments of the methods disclosed herein can include one or more of the following features.
  • the methods further include 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 immune cell.
  • 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 immune cell or a reverse complement thereof, or a cDNA sequence of the mRNA analyte of the immune cell or a reverse complement thereof.
  • the mRNA analyte does not encode an ABM or portion thereof.
  • the methods described 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.
  • the methods described herein further include (i) identifying the ABM as expressed by the 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 having bound the target antigen based on the determined sequence of the second barcoded nucleic acid molecule or amplicon thereof.
  • the methods described herein further include determining a sequence of the third barcoded nucleic acid molecule or an amplicon thereof.
  • the methods further include determining a sequence of the fourth barcoded nucleic acid molecule or an amplicon thereof.
  • the plurality of immune cells includes B cells.
  • the first immune cell is a B cell bound to the target antigen of the first labelling composition.
  • the ABM produced by the B cell is a B cell receptor (BCR), an antibody (Ab), or an antigen-binding fragment thereof.
  • the plurality of immune cells includes B cells and the ratio of first labelling compositions to immune cells in the reaction mixture is 200,000: 1 or higher, and/or the ratio of second labelling compositions to immune cells in the reaction mixture is 200,000:1 or higher.
  • the plurality of immune cells includes B cells and the ratio of first labelling compositions to immune cells in the reaction mixture is between about 50,000:1 and about 200,000:1, and/or the ratio of second labelling compositions to immune cells in the reaction mixture is between about 50,000: 1 and about 200,000:1.
  • the plurality of immune cells includes T cells.
  • the first immune cell is a T cell bound to the target antigen of the labelling composition.
  • the ABM produced by the T cell is a TCR.
  • the plurality of immune cells includes T cells and the ratio of first labelling compositions to immune cells in the reaction mixture is 250,000: 1 or higher, and/or the ratio of second labelling compositions to immune cells in the reaction mixture is 250,000: 1 or higher.
  • the plurality of immune cells includes T cells and the ratio of first labelling compositions to immune cells in the reaction mixture is 500,000: 1 or higher, and/or the ratio of second labelling compositions to immune cells in the reaction mixture is 500,000: 1 or higher. In some embodiments, the plurality of immune cells includes T cells and the ratio of first labelling compositions to immune cells in the reaction mixture is 1,000,000:1 or higher, and/or the ratio of second labelling compositions to immune cells in the reaction mixture is 1,000,000:1 or higher. [021] Tn some embodiments, the reaction mixture includes a set of labelling compositions as described herein and wherein the partitioning of (b) further provides a second partition including a second immune cell bound to the second labelling composition of the set of labelling compositions.
  • the methods further include, prior to the partitioning of (b), sorting the plurality of immune cells according to a flow cytometry profile based on the detectable label.
  • the reaction mixture further includes an additional target antigen composition including the target antigen operably linked to an additional detectable label.
  • the additional detectable label is different from the first detectable label, wherein the sorting includes gating according to threshold detection levels of the first detectable label and the additional detectable label.
  • the reaction mixture includes a set of labelling compositions including a first labelling composition and a second composition as describe herein, wherein the first detectable label and the second detectable label are different, and wherein the method further includes, prior to the partitioning of (b), sorting the plurality of immune cells according to a flow cytometry profile based on the first detectable label and the second detectable label.
  • the sorting includes gating according to threshold detection levels of the first detectable label and the second detectable label.
  • kits for preparing labelling compositions that involve intramolecular ligation using a splint nucleic acid molecule (e.g., a splint oligonucleotide).
  • a splint nucleic acid molecule e.g., a splint oligonucleotide
  • the methods for preparing labelling compositions disclosed herein include: (a) providing an oligonucleotide comprising a first annealing region and a second annealing region positioned adjacently to each other; (b) hybridizing the provided splint oligonucleotide with: (i) a core support attached an anchor nucleic acid molecule comprising a sequence capable of hybridizing to the first annealing region of the splint oligonucleotide; and (ii) a reporter oligonucleotide comprising (1) a reporter barcode sequence, (2) a capture handle sequence, and (3) a sequence capable of hybridizing to the second annealing region of the splint oligonucleotide; and (c) ligating the anchor nucleic acid molecule and the reporter oligonucleotide hybridized adjacently on the splint oligonucleotide to generate a continuous ligation product attached to the core support.
  • Non-limiting exemplary embodiments of the methods of preparing labelling compositions disclosed herein can include one or more of the following features.
  • the methods further include (d) coupling an antigen to the core support.
  • the core support is further coupled to a detectable label.
  • the ligating of (c) is performed by using SplintR® ligase, Taq DNA ligase, E.
  • the core support includes a biotin-binding agent.
  • the biotin-binding agent is or comprises a biotin-binding protein selected from streptavidin, avidin, deglycosylated avidin (e.g., Neu tr AvidinTM), traptavidin, tamavidin, xenavidin, bradavidin, AVR2, AVR4, and variants, mutants, derivatives, and homologs of any thereof.
  • the antigen is biotinylated.
  • the antigen is an oligopeptide, a protein, a polysaccharide, a lipid, a liposome, an infectious agent, or a target MHC molecule complex, e.g., a pMHC molecule complex.
  • kits useful for the identification and/or characterization of ABMs include (a) a labeling composition and/or a set of labelling compositions as described herein wherein the core support includes a biotin-binding agent; and (b) instructions for conjugating an antigen that is or includes a biotin moiety to the labeling composition and/or the set of labelling compositions.
  • the present disclosure provides partitions including (a) an immune cell, (b) a first labelling composition as described herein, and (c) a plurality of nucleic acid barcode molecules including a partition-specific barcode sequence.
  • the partitions further include a second labelling composition as described herein.
  • the detectable label of the first labelling composition is a first detectable label and wherein the detectable label of the second labelling composition is a second detectable label.
  • the first detectable label and the second detectable label are the same.
  • the first detectable label and the second detectable label are different.
  • the core support comprises a biotin binding molecule.
  • the core support comprises streptavidin, or any streptavidin alternative.
  • FIG. 1 shows an exemplary microfluidic channel structure for partitioning individual biological particles in accordance with some embodiments of the disclosure.
  • FIG. 2 shows an exemplary microfluidic channel structure for the controlled partitioning of beads into discrete droplets.
  • FIG. 3 shows an exemplary barcode carrying bead.
  • FIG. 4 illustrates another example of a barcode carrying bead.
  • FIG. 5 schematically illustrates an example microwell array.
  • FIG. 6 schematically illustrates an example workflow for processing nucleic acid molecules.
  • FIGS. 7A-7C schematically illustrate examples of labelling agents.
  • FIG. 8 shows an exemplary microfluidic channel structure for delivering barcode carrying beads to droplets.
  • FIG. 9 depicts an example of a barcode carrying bead.
  • FIGS. 10A, 10B, 10C, and 10D schematically depict an example workflow for processing nucleic acid molecules.
  • FIGS. 11A-11C are schematic representations of non-limiting examples of the labelling reagents in accordance with some embodiments of the disclosure.
  • FIGS. 11A-11B schematically illustrate non-limiting examples of labelling compositions that can be useful for identification and/or characterization of ABMs.
  • FIG. 11C schematically illustrates a non-limiting example of the methods for preparing labelling compositions that involve intramolecular ligation using a splint nucleic acid molecule as disclosed herein.
  • FIG. 11D schematically illustrates a nonlimiting example of the methods for preparing labelling compositions that involve 3-component conjugation.
  • one non-limiting embodiment of a labeling composition comprises one detectable label, one core attached to the detectable label and one nucleic acid molecule attached to the detectable label.
  • the labeling composition comprises one detectable label, two cores attached to the detectable label and one nucleic acid molecule attached to the detectable label.
  • the labeling composition comprises one detectable label, three cores attached to the detectable label and one nucleic acid molecule attached to the detectable label.
  • the labeling composition comprises one detectable label, four, or more cores attached to the detectable label, and one nucleic acid molecule attached to the detectable label.
  • the invention provides a mixture of labelling compositions each one comprising one detectable label, one, two, three or four core(s) attached to the detectable label, and one nucleic acid.
  • the core support comprises a target antigen.
  • each core comprises multiple conjugation sites for the attachment of a target antigen.
  • each core comprises one, two, three, four or more conjugation sites for the attachment of a target antigen, and thus the labeling composition allows the binding of multiple ABMs.
  • the core is streptavidin and the detectable label is PE.
  • the nucleic acid molecule of the labeling composition comprises a functional sequence, a barcode, and a capture handle sequence.
  • FIGS. 12A-12D pictorially summarize the results of experiments evaluating performance of some labelling reagents of the disclosure in an antigen mapping workflow.
  • FIGS. 13A-13C schematically summarize the results of experiments illustrating that increasing the number of molecules per cell results in improved separation of positive and negative populations via FACs as well as improved data from an exemplary antigen-mapping workflow performed with T cells.
  • FIG. 13A shows flow cytometry plots from the cell labeling experiments, with forward side scatter shown on the Y axis and PE signal on the X axis.
  • FIG. 13B shows a barcode rank plot of antigen UMI counts (Y axis, log scale) by cell barcode (X axis, log scale).
  • FIG. 13C shows a plot of antigen barcode UMI counts by cell density.
  • FIGS. 14A-14D schematically summarize the results of experiments illustrating that increasing the number of molecules per cell results in improved separation of positive and negative populations via FACs as well as improved data from an exemplary antigen-mapping workflow performed with B cells.
  • FIG. 14A shows flow cytometry plots from the cell labeling experiments using barcoded antigen molecules assembled at a 4:1 antigen to conjugate ratio. Forward side scatter is shown on the Y axis and PE signal on the X axis.
  • FIG. 14B shows a barcode rank plot of antigen UMI counts (Y axis, log scale) by cell barcode (X axis, log scale).
  • FIG. 14C shows a plot of antigen barcode UMI counts by cell density.
  • FIG. 14D shows median antigen barcode UMI counts per cell.
  • FIG. 15 shows flow cytometry results from experiments performed to test the impact of (1) the number of barcoded antigen reagent molcculcs/ccll ratios during cell labeling and (2) antigen/barcoded streptavidin ratios during barcoded reagent assembly, in an end to end high throughput single-cell workflow for antigen/BCR mapping, using a transgenic mouse model.
  • compositions and methods useful for characterization of antigen-binding molecules such as antibodies, B cell receptors (BCRs), T cell receptors (TCRs), and TCR-like antibodies (Abs) obtained from biological samples.
  • ABMs antigen-binding molecules
  • BCRs B cell receptors
  • TCRs T cell receptors
  • Abs TCR-like antibodies
  • Characterization of ABMs having desirable properties, e.g., that recognize and bind to cells displaying tumor or viral antigens can be useful in the development of new immunotherapies to treat cancers and/or infectious disease.
  • reagents and kits for the identification and/or characterization of these ABMs are also provided in some embodiments of the disclosure.
  • barcode 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 nucleic acid reaction e.g., extension, ligation
  • 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.
  • 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).
  • partition 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.
  • 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.
  • a physical compartment may comprise a plurality of virtual compartments.
  • one aspect of the present disclosure relates to labelling compositions that can be useful for, (see, e.g., FIG. 11A), identification and/or characterization of ABMs, e.g., antibodies, BCRs, TCRs, and TCR-like antibodies.
  • ABMs e.g., antibodies, BCRs, TCRs, and TCR-like antibodies.
  • the labelling compositions include a detectable label (e.g., a fluorophore (“FL”), a magnetic particle, or a mass tag; a core support (“core”) attached to the detectable label; and a nucleic acid molecule attached to the detectable label, wherein the nucleic acid molecule includes: (a) a reporter oligonucleotide including (i) a reporter barcode sequence and (ii) a capture handle sequence, or (b) an anchor nucleic acid molecule configured to directly or indirectly attach to the reporter oligonucleotide of (a).
  • a detectable label e.g., a fluorophore (“FL”), a magnetic particle, or a mass tag
  • core core support
  • nucleic acid molecule attached to the detectable label
  • the nucleic acid molecule includes: (a) a reporter oligonucleotide including (i) a reporter barcode sequence and (ii) a capture handle sequence, or (b) an anchor nu
  • Non-limiting exemplary embodiments of the labelling compositions disclosed herein can include one or more of the following features.
  • the core support e.g., biotin-binding agent, e.g., streptavidin
  • the reporter oligonucleotide are both directly attached to the detectable label (e.g., a fluorophore, e.g., PE).
  • the labelling compositions of the present disclosure wherein both the core support and reporter oligonucleotide are directly conjugated to the detectable label have demonstrated superior performance in nucleic acid labelling as compared to reference/control labelling compositions wherein the oligomer and detectable label are both conjugated to the core support (see, e.g., Example 1).
  • the superior performance of the labelling compositions disclosed herein is due to the core support being directly conjugated to the detectable label and not being directly conjugated to the reporter oligonucleotide, which in turn allows for greater accessibility to conjugation sites (e.g., biotinbinding sites) on the core support, and accessibility of the core support to multiple conjugation sites on the detectable molecule.
  • conjugation sites e.g., biotinbinding sites
  • FIG. 11D one non-limiting embodiment of a labeling composition is illustrated and this embodiment comprises one detectable label, one core support attached to the detectable label and one nucleic acid molecule attached to the detectable label.
  • the labeling composition comprises one detectable label, two core supports attached to the detectable label and one nucleic acid molecule attached to the detectable label. In another embodiment, the labeling composition comprises one detectable label, three core supports attached to the detectable label and one nucleic acid molecule attached to the detectable label. In another embodiment, the labeling composition comprises one detectable label, four, or more core supports attached to the detectable label, and one nucleic acid molecule attached to the detectable label.
  • the invention provides a mixture of labelling compositions each one comprising one detectable label, one, two, three or four core support (s) attached to the detectable label, and one nucleic acid.
  • the core support comprises a target antigen.
  • each core support comprises multiple conjugation sites for the attachment of a target antigen. In some embodiments, each core comprises one, two, three, four or more conjugation sites for the attachment of a target antigen, and thus the labeling composition allows the binding of multiple ABMs.
  • the core support is streptavidin and the detectable label is PE.
  • the nucleic acid molecule of the labeling composition comprises a functional sequence, a barcode, and a capture handle sequence.
  • the anchor nucleic acid molecule includes a sequence configured to attach, by complementarity base pairing, to an anchoring sequence present in the reporter oligonucleotide (see, e.g., FIG. 11B).
  • the labelling compositions of the disclosure include an anchor nucleic acid that is hybridized to the anchoring sequence of the reporter oligonucleotide.
  • 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.
  • the detectable label includes a fluorophore, a magnetic particle, or a mass tag.
  • the detectable label includes a fluorophore molecule.
  • the fluorophore is or includes one or more of the following: 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, and StarBright Blue 700.
  • PE phycoerythrin
  • APC allophycocyanin
  • Alexa Fluor 405 Pacific Blue
  • Alexa Fluor 488 Alexa Fluor 647
  • Alexa Fluor 700 Alexa Fluor 700
  • DyLight 405 DyLight 550
  • the core support includes a biotin-binding agent.
  • the biotin-binding agent is or includes a biotin-binding protein.
  • suitable biotinbinding proteins include, but are not limited to streptavidin, or any streptavidin alternative including without limitation avidin, deglycosylated avidin (e.g., NeutrAvidinTM), traptavidin, tamavidin, xenavidin, bradavidin, AVR2, AVR4, and variants, mutants, derivatives, and homologs of any thereof.
  • the biotin-binding agent is or includes a biotinbinding protein selected from streptavidin, avidin, deglycosylated avidin (e.g., NeutrAvidinTM), traptavidin, tamavidin, xenavidin, bradavidin, AVR2, and AVR4.
  • a biotinbinding protein selected from streptavidin, avidin, deglycosylated avidin (e.g., NeutrAvidinTM), traptavidin, tamavidin, xenavidin, bradavidin, AVR2, and AVR4.
  • the labelling composition can be prepared by attaching (coupling) the core support and the reporter oligonucleotide (or anchor nucleic acid molecule) to the detectable label.
  • the core support and/or the reporter oligonucleotide (or anchor nucleic acid molecule) is directly attached to the detectable label.
  • the core support and/or the reporter oligonucleotide (or anchor nucleic acid molecule) is indirectly attached to the detectable label. Attachment (coupling) can be achieved through any of a variety of direct or indirect, covalent or non-covalent associations or attachments.
  • 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.
  • 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 the core support and the reporter oligonucleotide (or anchor nucleic acid molecule) to the detectable label.
  • 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 labelling composition of the disclosure is a first labelling composition coupled to a target antigen, wherein the reporter barcode sequence is a first reporter barcode sequence that identifies the target antigen.
  • the target antigen is biotinylated.
  • the target antigen is an oligopeptide, a protein, a polysaccharide, a lipid, a liposome, an infectious agent, or a target MHC molecule complex.
  • the target MHC molecule complex is a peptide MHC molecule complex.
  • the labelling composition of the disclosure is a second labelling composition that is not coupled to the target antigen and wherein the reporter barcode sequence is a second reporter barcode sequence that identifies the second labelling composition.
  • the second labelling composition includes a non-target antigen.
  • the non-target antigen is or includes a biotin moiety.
  • the first and/or second labeling compositions may be comprised in a cell bead.
  • the present disclosure provides a set of labelling compositions.
  • the set of labelling compositions can include 2, 3, 4, 5, 6, 7, 8, 9, or 10 labelling compositions in accordance with the present disclosure.
  • the set includes a first labelling composition as described herein and a second labelling composition as described herein.
  • the detectable label of the first labelling composition is a first detectable label and wherein the detectable label of the second labelling composition is a second detectable label.
  • the first detectable label and the second detectable label arc the same.
  • the first detectable label and the second detectable label arc different.
  • the set of labeling compositions may be comprised in a cell bead.
  • the present disclosure provides partitions including (a) an immune cell, (b) a first labelling composition as described herein, and (c) a plurality of nucleic acid barcode molecules including a partition- specific barcode sequence.
  • the partitions further include a second labelling composition as described herein.
  • the detectable label of the first labelling composition is a first detectable label and wherein the detectable label of the second labelling composition is a second detectable label.
  • the first detectable label and the second detectable label are the same. In some embodiments, the first detectable label and the second detectable label are different.
  • kits useful for the practice of a method described herein are kits useful for the practice of a method described herein.
  • the kits include one or more labeling composition as described herein.
  • at least one of the labelling composition in the kits includes a core support which includes a biotin-binding agent as describe herein such as, for example, streptavidin.
  • kits can further include instructions for using the components of the kit to practice a method described herein.
  • the kit can include instructions for conjugating an antigen that is or includes a biotin moiety to the labeling composition.
  • the instructions for practicing the method are generally recorded on a suitable recording medium.
  • the instructions can be printed on a substrate, such as paper or plastic, etc.
  • the instructions can be present in the kit as a package insert, in the labeling of the container of the kit or components thereof (e.g., associated with the packaging or sub-packaging), etc.
  • the instructions can be present as an electronic storage data fde present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc.
  • the actual instructions arc not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet), can be provided.
  • An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.
  • 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).
  • antigen-binding molecules e.g., antibodies, B cell receptors, T cell receptors (TCRs), and TCR-like antibodies (Abs).
  • ABSMs antigen-binding molecules
  • 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 first labelling compositions as described herein or a set of labelling compositions as described herein; (b) partitioning the reaction 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) the first labelling composition, and (ii) a plurality of nucleic acid barcode molecules including a partition-specific barcode 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
  • Non-limiting exemplary embodiments of the methods disclosed herein can include one or more of the following features.
  • the methods further include 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 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 arc disclosed in
  • 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 immune cell or a reverse complement thereof, or a cDNA sequence of the mRNA analyte of the immune cell or a reverse complement thereof.
  • the mRNA analyte does not encode an ABM or portion thereof.
  • 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.
  • the methods described herein further include (i) identifying the ABM as expressed by the 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 having bound the target antigen based on the determined sequence of the second barcoded nucleic acid molecule or amplicon thereof.
  • the methods described herein further include determining a sequence of the third barcoded nucleic acid molecule or an amplicon thereof.
  • the methods further include determining a sequence of the fourth barcoded nucleic acid molecule or an amplicon thereof.
  • the plurality of immune cells includes B cells.
  • the B cells include a plasmablast, a plasma cell, a memory B cell, a regulatory B cell, and/or a lymphoplasmacytoid cell.
  • the first immune cell is a B cell bound to the target antigen of the first labelling composition.
  • the ABM produced by the B cell is a B cell receptor (BCR), an antibody (Ab), or an antigen-binding fragment thereof.
  • the plurality of immune cells includes B cells and the ratio of first labelling compositions to immune cells in the reaction mixture is between about 50,000:1 and about 500,000:1; for example, between about 50,000:1 and about 400,000:1; for other examples, between about between about 50,000: 1 and about 200,000: 1 ; for yet other examples, between about 100,000:1 and about 200,000:1; for yet other examples, between about 150,000: 1 and about 200,000: 1 .
  • the plurality of immune cells includes B cells and the ratio of second labelling compositions to immune cells in the reaction mixture is between about 50,000: 1 and about 500,000:1; for example, between about 50,000:1 and about 400,000:1; for other examples, between about between about 50,000:1 and about 200,000:1; for yet other examples, between about 100,000:1 and about 200,000:1; for yet other examples, between about 150,000:1 and about 200,000:1.
  • the plurality of immune cells includes B cells and the reaction mixture includes a set of labelling composition including first and second labelling compositions, wherein the ratio of first labelling compositions to immune cells in the reaction mixture is greater than 200,000:1 (e.g., greater than 200,000:1, greater than 250,000:1, greater than 300,000:1, greater than 350,000:1, greater than 400,000:1, greater than 450,000:1, or greater than 500,000:1), and/or the ratio of second labelling compositions to immune cells in the reaction mixture is 200,000:1 or higher (e.g., greater than 200,000:1, greater than 250,000:1, greater than 300,000: 1, greater than 350,000: 1, greater than 400,000: 1, greater than 450,000: 1, or greater than 500,000:1).
  • the plurality of immune cells includes B cells and the reaction mixture includes a set of labelling composition including first and second labelling compositions, wherein (i) the ratio of first labelling compositions to immune cells in the reaction mixture is between about 50,000:1 and about 500,000:1 (e.g., between about 50,000:1 and about 400,000:1; between about between about 50,000:1 and about 200,000:1; between about 100,000:1 and about 200,000: 1 ; or between about 150,000: 1 and about 200,000: 1 ), and/or the ratio of second labelling compositions to immune cells in the reaction mixture is between about 50,000:1 and about 500,000:1 (e.g., between about 50,000:1 and about 400,000:1; between about between about 50,000:1 and about 200,000:1; between about 100,000:1 and about 200,000:1; or between about 150,000:1 and about 200,000:1).
  • the ratio of first labelling compositions to immune cells in the reaction mixture is between about 50,000:1 and about 500,000:1 (e.g., between about
  • the ratio of first labelling compositions to immune cells in the reaction mixture is different from the ratio of second labelling compositions to immune cells in the reaction mixture. In some embodiments, the ratio of first labelling compositions to immune cells in the reaction mixture is the same as the ratio of second labelling compositions to immune cells in the reaction mixture.
  • the plurality of immune cells includes B cells and the reaction mixture includes a set of labelling composition including additional labelling compositions, wherein the ratio of one or more of the additional labelling compositions to immune cells in the reaction mixture is greater than 200,000:1 such as, for example, greater than 200,000:1, greater than 250,000:1, greater than 300,000:1, greater than 350,000:1, greater than 400,000:1, greater than 450,000:1, or greater than 500,000:1.
  • the ratio of one or more of the additional labelling compositions to immune cells in the reaction mixture is between about 50,000:1 and about 500,000:1, such as, for example, between about 50,000:1 and about 400,000:1; between about between about 50,000:1 and about 200,000:1; between about 100,000:1 and about 200,000:1; or between about 150,000:1 and about 200,000:1.
  • the plurality of immune cells includes T cells.
  • the T cells include a CD8+ T cytotoxic lymphocyte cell and/or a CD4+ T helper lymphocyte cell.
  • 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.
  • 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.
  • the T cell is an exhausted T cell or a non-exhausted T cell.
  • the first immune cell is a T cell bound to the target antigen of the labelling composition.
  • the ABM produced by the T cell is a TCR.
  • the plurality of immune cells includes T cells and the ratio of first labelling compositions to immune cells in the reaction mixture is greater than 200,000:1 such as, for example, greater than 200,000:1, greater than 250,000:1, greater than 300,000:1, greater than 350,000:1, greater than 400,000:1, greater than 450,000:1, or greater than 500,000:1.
  • the ratio of first labelling compositions to immune cells in the reaction mixture is between about 200,000:1 to about 3,000,000:1 such as, for example, from between about 500,000:1 to about 2,000,000:1, or for other example between about 750,000:1 to about 1.5 million: 1.
  • the plurality of immune cells includes T cells and the ratio of second labelling compositions to immune cells in the reaction mixture is greater than 200,000: 1 such as, for example, greater than 200,000:1, greater than 250,000:1, greater than 300,000:1, greater than 350,000:1, greater than 400,000:1, greater than 450,000:1, or greater than 500,000:1.
  • the ratio of second labelling compositions to immune cells in the reaction mixture is between about 200,000:1 to about 3,000,000:1 such as, for example, from between about 500,000:1 to about 2,000,000:1, or for other example between about 750,000:1 to about 1.5 million: 1.
  • the plurality of immune cells includes T cells and the reaction mixture includes a set of labelling composition including first and second labelling compositions, wherein the ratio of first labelling compositions to immune cells in the reaction mixture is greater than 200,000:1 (e.g., greater than 200,000:1, greater than 250,000:1, greater than 300,000:1, greater than 350,000:1, greater than 400,000:1, greater than 450,000:1, or greater than 500,000:1), and/or the ratio of second labelling compositions to immune cells in the reaction mixture is 200,000:1 or higher (e.g., greater than 200,000:1, greater than 250,000:1, greater than 300,000: 1, greater than 350,000: 1, greater than 400,000: 1, greater than 450,000: 1, or greater than 500,000:1).
  • the plurality of immune cells includes T cells and the reaction mixture includes a set of labelling composition including first and second labelling compositions, wherein (i) the ratio of first labelling compositions to immune cells in the reaction mixture is between about 200,000:1 to about 3,000,000:1 (e.g., between about 500,000:1 to about 2,000,000:1, or for other example between about 750,000:1 to about 1.5 million:l), and/or the ratio of second labelling compositions to immune cells in the reaction mixture is between about 200,000:1 to about 3,000,000:1 (e.g., between about 500,000:1 to about 2,000,000:1, or for other example between about 750,000:1 to about 1 .5 million:! ).
  • the ratio of first labelling compositions to immune cells in the reaction mixture is between about 200,000:1 to about 3,000,000:1 (e.g., between about 500,000:1 to about 2,000,000:1, or for other example between about 750,000:1 to about 1 .5 million:! ).
  • the ratio of first labelling compositions to immune cells in the reaction mixture is different from the ratio of second labelling compositions to immune cells in the reaction mixture. In some embodiments, the ratio of first labelling compositions to immune cells in the reaction mixture is the same as the ratio of second labelling compositions to immune cells in the reaction mixture.
  • a first solution is provided, wherein the first solution provides the first labeling compositions at a particular concentration (e.g., number of first labeling compositions per unit volume).
  • an immune cell solution is also provided, such that the immune cell solution provides immune cells at a particular concentration (e.g., number of cells per unit volume).
  • the concentration of immune cells is determined, e.g., by cell counting methods.
  • a reaction mixture is created by mixing a volume of the first solution and a volume of the immune cell solution such that the reaction mixture contains a total number of first labelling compositions and a total number of immune cells, at a first labeling composition/immune cell ratio disclosed herein.
  • a second solution is also provided, wherein the second solution provides the second labeling compositions at a particular concentration (e.g., number of second labeling compositions per unit volume).
  • creating the reaction mixture further comprises mixing a volume of the second solution with the volume of the first solution and the volume of the second solution such that the reaction mixture further contains a total number of second labeling compositions at a second labeling composition/immune cell ratio disclosed herein.
  • the ratio of first labeling compositions/immune cells is about the same or the same as the ratio of second labeling compositions/immune cells.
  • the first solution and the second solution is prepared using a kit disclosed herein, and according to kit instructions.
  • the kit instructions include instructions for using a specified number of cells and a specified volume of the first and/or second solutions to create the reaction mixture.
  • the reaction mixture includes a set of labelling compositions as described herein and wherein the partitioning of (b) further provides a second partition including a second immune cell bound to the second labelling composition of the set of labelling compositions.
  • the methods further include, prior to the partitioning of (b), sorting the plurality of immune cells according to a flow cytometry profile based on the detectable label.
  • the reaction mixture further includes an additional target antigen composition including the target antigen operably linked to an additional detectable label.
  • the additional detectable label is different from the first detectable label, wherein the sorting includes gating according to threshold detection levels of the first detectable label and the additional detectable label.
  • the additional detectable label is different from the first detectable label, wherein the sorting includes gating for the presence of both the first detectable label and the additional detectable label. In some embodiments, the sorting includes gating for the presence (e.g., above threshold) of the first detectable label and the absence (e.g., below threshold) of the additional detectable label. In some embodiments, the sorting includes gating for the absence of the first detectable label and the presence of the additional detectable label. In some embodiments, the sorting includes gating for the presence of one or more biomarkers of the partitioned immune cell e.g., B cell or T cell).
  • the reaction mixture includes a set of labelling compositions including a first labelling composition and a second composition as describe herein, wherein the first detectable label and the second detectable label are different, and wherein the method further includes, prior to the partitioning of (b), sorting the plurality of immune cells according to a flow cytometry profile based on the first detectable label and the second detectable label.
  • the sorting includes gating according to threshold detection levels of the first detectable label and the second detectable label.
  • the sorting includes gating for the presence of both the first detectable label and the second detectable label.
  • the sorting includes gating for the presence (e.g., above threshold) of the first detectable label and the absence (e.g., below threshold) of the second detectable label. In some embodiments, the sorting includes gating for the absence of the first detectable label and the presence of the second detectable label. In some embodiments, the sorting includes gating for the presence of one or more biomarkers of the partitioned immune cell (e.g., B cell or T cell).
  • the partitioned immune cell e.g., B cell or T cell
  • 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.
  • 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.
  • a partition can be a volume or sub-volume, wherein diffusion of contents beyond the volume or sub-volume is inhibited.
  • the partitions can include a porous matrix that is capable of entraining and/or retaining materials within its matrix.
  • the partitioned particle is a labelled cell, e.g. a labelled immune cell (e.g., B cell or T cell), which expresses an antibody.
  • 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.
  • the partitioned particle can be a labelled cell engineered to express antigen-binding molecules (e.g., an immune receptors, antibodies or functional fragments thereof).
  • the labelled immune cell is a B cell.
  • the labelled immune cell is a T cell.
  • partition 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.
  • 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.
  • partitions comprise droplets of aqueous fluid within a non-aqueous continuous phase (e.g., oil phase).
  • 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.
  • 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.
  • 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.
  • a physical compartment can include a plurality of virtual compartments.
  • the methods and system described herein provide for the compartmentalization, depositing or partitioning of individual cells from a sample material containing cells after at least one labelling agent or reporter agent molecule has been bound to a cell surface feature of a cell, into discrete partitions, where each partition maintains separation of its own contents from the contents of other partitions.
  • Identifiers including unique identifiers (e.g., UMI) and common or universal tags, e.g., barcodes, can be previously, subsequently or concurrently delivered to the partitions that hold the compartmentalized or partitioned cells, in order to allow for the later attribution of the characteristics of the individual cells to one or more particular compartments.
  • identifiers including unique identifiers and common or universal tags can be coupled to labelling agents and previously, subsequently or concurrently delivered to the partitions that hold the compartmentalized or partitioned cells, in order to allow for the later attribution of the characteristics of the individual cells to one or more particular compartments.
  • Identifiers including unique identifiers and common or universal tags, e.g., barcodes can be delivered, for example on an oligonucleotide, to a partition via any suitable mechanism, for example by coupling the barcoded oligonucleotides to a microcapsule, e.g., bead.
  • the barcoded oligonucleotides are reversibly (e.g., releasably) coupled to a microcapsule (e.g., bead).
  • the microcapsule (e.g., bead) suitable for the compositions and methods of the disclosure can have different surface chemistries and/or physical volumes.
  • the microcapsule (e.g., bead) includes a polymer gel.
  • the polymer gel is a polyacrylamide.
  • suitable microcapsule include microparticles, nanoparticles, and beads (e.g., microbeads).
  • the microcapsule includes a bead.
  • the partition can be a droplet in an emulsion.
  • a partition may include one or more particles.
  • a partition may include one or more types of particles.
  • a partition of the present disclosure may include one or more biological particles, e.g., labelled immune cells, and/or macromolecular constituents thereof.
  • a partition may include one or more gel beads.
  • a partition may include one or more cell beads.
  • a partition may include a single gel bead, a single cell bead, or both a single cell bead and single gel bead.
  • a partition may include one or more reagents.
  • a partition may be unoccupied.
  • a partition may not 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 microcapsule (e.g., bead), as described elsewhere herein.
  • the methods and systems of the present disclosure may comprise methods and systems for generating one or more partitions such as droplets.
  • the droplets may comprise a plurality of droplets in an emulsion.
  • the droplets may comprise droplets in a colloid.
  • the emulsion may comprise a microemulsion or a nanoemulsion.
  • the droplets may be generated with aid of a microfluidic device and/or by subjecting a mixture of immiscible phases to agitation (e.g., in a container).
  • a combination of the mentioned methods may be used for droplet and/or emulsion formation.
  • the partitions described herein may comprise small volumes, for example, less than about 10 microliters ( .L), 5pL, 1 pL, 10 nanoliters (nL), 5 nL, 1 nL, 900 picoliters (pL), 800 pL, 700 pL, 600 pL, 500 pL, 400pL, 300 pL, 200 pL, lOOpL, 50 pL, 20 pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less.
  • small volumes for example, less than about 10 microliters ( .L), 5pL, 1 pL, 10 nanoliters (nL), 5 nL, 1 nL, 900 picoliters (pL), 800 pL, 700 pL, 600 pL, 500 pL, 400pL, 300 pL, 200 pL, lOOpL, 50 pL, 20 pL, 10 pL
  • the droplets may have overall volumes that are less than about 1000 pL, 900 pL, 800 pL, 700 pL, 600 pL, 500 pL, 400pL, 300 pL, 200 pL, lOOpL, 50 pL, 20 pL, 10 pL, 1 pL, or less.
  • sample fluid volume e.g., including co-partitioned biological particles and/or beads
  • the sample fluid volume within the partitions may be less than about 90% of the above described volumes, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% of the above described volumes.
  • partitioning species may generate a population or plurality of partitions.
  • any suitable number of partitions can be generated or otherwise provided. For example, at least about 1,000 partitions, at least about 5,000 partitions, at least about 10,000 partitions, at least about 50,000 partitions, at least about 100,000 partitions, at least about 500,000 partitions, at least about 1,000,000 partitions, at least about 5,000,000 partitions at least about 10,000,000 partitions, at least about 50,000,000 partitions, at least about 100,000,000 partitions, at least about 500,000,000 partitions, at least about 1,000,000,000 partitions, or more partitions can be generated or otherwise provided.
  • the plurality of partitions may comprise both unoccupied partitions (e.g., empty partitions) and occupied partitions.
  • Droplets can be formed by creating an emulsion by mixing and/or agitating immiscible phases.
  • Mixing or agitation may comprise various agitation techniques, such as vortexing, pipetting, tube flicking, or other agitation techniques.
  • mixing or agitation may be performed without using a microfluidic device.
  • the droplets may be formed by exposing a mixture to ultrasound or sonication.
  • Microfluidic devices or platforms comprising microfluidic channel networks can be utilized to generate partitions such as droplets and/or emulsions 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.
  • Methods and systems for generating partitions such as droplets, methods of encapsulating biological particles in partitions, methods of increasing the throughput of droplet generation, and various geometries, architectures, and configurations of microfluidic devices and channels are described in U.S. Patent Publication Nos. 2019/0367997 and 2019/0064173, each of which is entirely incorporated herein by reference for all purposes.
  • individual particles can be partitioned to discrete partitions by introducing a flowing stream of particles in an aqueous fluid into a flowing stream or reservoir of a non-aqueous fluid, such that droplets may be generated at the junction of the two streams/reservoir, such as at the junction of a microfluidic device provided elsewhere herein.
  • the methods of the present disclosure may comprise generating partitions and/or encapsulating particles, such as biological particles, in some cases, individual biological particles such as single cells.
  • reagents may be encapsulated and/or partitioned (e.g., copartitioned with biological particles) in the partitions.
  • Various mechanisms may be employed in the partitioning of individual particles.
  • An example may comprise porous membranes through which aqueous mixtures of cells may be extruded into fluids (e.g., non-aqueous fluids).
  • the partitions can be flowable within fluid streams.
  • the partitions can include, for example, microvesicles that have an outer barrier surrounding an inner fluid center or core.
  • 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 antigenbinding fragment thereof).
  • the partitions can be droplets of a first phase within a second phase, wherein the first and second phases are immiscible.
  • the partitions can be droplets of aqueous fluid within a non-aqueous continuous phase (e.g., oil phase).
  • the partitions can be droplets of a non-aqueous fluid within an aqueous phase.
  • 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.
  • 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.
  • 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.).
  • partition occupancy can be controlled by providing the aqueous stream at a certain concentration and/or flow rate of particles.
  • 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.
  • 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).
  • the various parameters 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.
  • the method further includes individually partitioning one or more single tumor cells from the second tumor sample in a partition of a second plurality of partitions. In some embodiments, the method further includes individually partitioning one or more single cells from a plurality of cells (e.g., from a second sample) in a partition of a second plurality of partitions.
  • At least one of the first and second plurality of partitions includes a microwell, a flow cell, a reaction chamber, a reaction compartment, or a droplet. In some embodiments, at least one of the first and second plurality of partitions includes individual droplets in emulsion. In some embodiments, the partitions of the first plurality and/or the second plurality of partition have the same reaction volume.
  • 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.
  • the occupancy of the resulting partitions e.g., number of cells per partition
  • 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.
  • 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.
  • 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.
  • the methods described herein can be performed such that a majority of occupied partitions include no more than one cell per occupied partition.
  • 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.
  • fewer than 20% of the occupied partitions include more than one cell.
  • fewer than 10% of the occupied partitions include more than one cell per partition.
  • 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.
  • the Poissonian distribution can optionally be used to increase the number of partitions that include multiple cells.
  • 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 arc controlled so as to present 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.
  • 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%.
  • the methods as described herein include providing multiply occupied partitions, e.g., containing two, three, four or more cells and/or microcapsules (e.g., beads) comprising nucleic acid barcode molecules within a single partition.
  • 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.
  • 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.
  • 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.
  • 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.
  • suspended biological particles e.g., cells, for example, labelled immune cells, B cells, or T cells
  • 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.
  • 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.
  • an oil such as a fluorinated oil
  • fluoro surfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets 118, 120.
  • 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.
  • the microfluidic channel structure 100 can have other geometries.
  • a micro fluidic channel structure can have more than one channel junction.
  • 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.
  • 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).
  • the majority of occupied partitions can include no more than one biological particle, e.g., labelled immune cell, e.g.
  • some of the generated partitions can be unoccupied (of any biological particle, or labelled engineered cell, labelled immune cell, B cell, or T cell).
  • 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.
  • 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.
  • the creation of excessive numbers of empty partitions 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.
  • biological particles e.g., biological particles, such as labelled engineered cell, immune B cells, B cells, or T cells 11
  • flows can be controlled so as to present a non-Poissonian distribution of single-occupied partitions (e.g., no more than about 50%, about 25%, or about 10% unoccupied).
  • single-occupied partitions e.g., no more than about 50%, about 25%, or about 10% unoccupied.
  • the above noted ranges of unoccupied partitions can be achieved while still providing any of the single occupancy rates described above.
  • 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, microcapsules or beads (e.g., gel beads) carrying nucleic acid barcode molecules (e.g., oligonucleotides).
  • biological particles e.g., labelled B cells or T cells
  • additional reagents including, but not limited to, microcapsules or beads (e.g., gel beads) carrying nucleic acid barcode molecules (e.g., oligonucleotides).
  • a partition of the plurality of partitions may comprise a single biological particle (e.g., a single cell or a single nucleus of a cell).
  • a partition of the plurality of partitions may comprise multiple biological particles.
  • Such partitions may be referred to as multiply occupied partitions, and may comprise, for example, two, three, four or more cells and/or beads (e.g., beads) comprising nucleic acid barcode molecules within a single partition.
  • the flow characteristics of the biological particle and/or bead containing fluids and partitioning fluids may be controlled to provide for such multiply occupied partitions.
  • the flow parameters may be controlled to provide a given occupancy rate at greater than about 50% of the partitions, greater than about 75%, and in some cases greater than about 80%, 90%, 95%, or higher.
  • 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.
  • 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.
  • 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.
  • the channel segment 802 may be connected to a reservoir comprising an aqueous suspension of biological particles 816.
  • 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
  • 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.
  • the channel segments 801 and 802 may meet at another junction upstream of the junction 810.
  • 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.
  • biological particles in addition to or as an alternative to droplet based partitioning, biological particles (e.g., cells) can be encapsulated within a microcapsule (e.g., bead) that comprises an outer shell, layer or porous matrix in which is entrained one or more individual biological particles or small groups of biological particles.
  • biological particles in addition to or as an alternative to droplet-based partitioning, biological particles (e.g., cells) may be encapsulated within a particulate material to form a “cell bead.”
  • biological particles in addition to or as an alternative to droplet-based partitioning, biological particles (e.g., cells) may be comprised within a particulate material to form a “cell bead.”
  • the microcapsule or cell bead can include other reagents.
  • Encapsulation of biological particles e.g., labelled engineered cell, immune cells, B cells, or T cells, can be performed by a variety of processes. Such processes can combine an aqueous fluid containing the biological particles with a polymeric precursor material that can 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.
  • 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)), mechanical stimuli, or a combination thereof.
  • 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)
  • mechanical stimuli e.g., mechanical stimuli, or a combination thereof.
  • microcapsules e.g., beads (e.g., cell beads) comprising biological particles, e.g., labelled engineered cells, immune cells, B cells, or T cells
  • biological particles e.g., labelled engineered cells, immune cells, B cells, or T cells
  • air knife droplet or aerosol generators can be used to dispense droplets of precursor fluids into gelling solutions in order to form microcapsules or cell beads that include individual biological particles or small groups of biological particles (e.g., labelled B cells or T cells).
  • membrane based encapsulation systems can be used to generate microcapsules or cell beads comprising encapsulated biological particles (e.g., B cells or plasma cells) as described herein.
  • Microfluidic systems of the present disclosure can 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 Pub. No. WO2018140966A1.
  • the aqueous fluid 112 comprising (i) the biological particles (e.g., labelled B cells or T cells) 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.
  • non-aqueous fluid 116 can also include an initiator (not shown) to cause polymerization and/or crosslinking of the polymer precursor to form the microcapsule (e.g., bead) that includes the entrained biological particles.
  • an initiator not shown
  • polymer precursor/initiator pairs include those described in U.S. Patent Application Publication No. 2014/0378345.
  • the activation agent can include a cross-linking agent, or a chemical that activates a cross-linking agent within the formed droplets.
  • the activation agent can include a polymerization initiator.
  • the polymer precursor comprises a mixture of acrylamide monomer with a N,N’- bis-(acryloyl)cystamine (BAC) comonomer
  • an agent such as tetraethylmethylenediamine (TEMED) can be provided within the second fluid streams 116 in channel segments 104 and 106, which can initiate the copolymerization of the acrylamide and BAC into a cross-linked polymer network, or hydrogel.
  • TEMED tetraethylmethylenediamine
  • the TEMED can diffuse from the second fluid 116 into the aqueous fluid 112 comprising the linear polyacrylamide, which will activate the crosslinking of the polyacrylamide within the droplets 118, 120, resulting in the formation of gel (e.g., hydrogel) microcapsules or cell beads, as solid or semi-solid beads or particles entraining the cells (e.g., labelled B cells or T cells) 114.
  • gel e.g., hydrogel
  • cells e.g., labelled B cells or T cells
  • alginate droplets formed followed by exposure to divalent metal ions (e.g., Ca 2+ ions), can be used as an encapsulation process using the described processes.
  • divalent metal ions e.g., Ca 2+ ions
  • agarose droplets can also be transformed into capsules through temperature based gelling (e.g., upon cooling, etc.).
  • encapsulated biological particles can be selectively releasable from the microcapsulc or cell bead, such as through passage of time or upon application of a particular stimulus, that degrades the encapsulating material (e.g., microcapsule, cell bead) sufficiently to allow the biological particles (e.g., labelled B cells or T cells), or its other contents to be released from the encapsulating material, such as into a partition (e.g., droplet).
  • a partition e.g., droplet
  • degradation of the polymer can be accomplished through the introduction of an appropriate reducing agent, such as DTT or the like, to cleave disulfide bonds that cross-link the polymer matrix. See, for example, U.S. Patent Application Publication No. 2014/0378345.
  • the biological particle e.g., labelled immune cell, B cell, or T cell
  • the conditions sufficient to polymerize or gel the precursors can include exposure to heating, cooling, electromagnetic radiation, and/or light.
  • the conditions sufficient to polymerize or gel the precursors can include any conditions sufficient to polymerize or gel the precursors.
  • a polymer or gel can be formed around the biological particle (e.g., labelled B cell or T cell).
  • the polymer or gel can be diffusively permeable to chemical or biochemical reagents.
  • the polymer or gel can be diffusively impermeable to macromolecular constituents (e.g., secreted antibodies or antigen-binding fragments thereof) of the biological particle (e.g., labelled immune cell, B cell, or T cell).
  • the polymer or gel can act to allow the biological particle (e.g., labelled immune cell, B cell, or T cell) 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.
  • the polymer or gel can include one or more of disulfide crosslinked 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 can include any other polymer or gel.
  • the polymer or gel can be functionalized (e.g., coupled to a capture agent) to bind to targeted analytes (e.g., secreted antibodies or antigen-binding fragment thereof), such as nucleic acids, proteins, carbohydrates, lipids or other analytes.
  • targeted analytes e.g., secreted antibodies or antigen-binding fragment thereof
  • the polymer or gel can be polymerized or gelled via a passive mechanism.
  • the polymer or gel can be stable in alkaline conditions or at elevated temperature.
  • the polymer or gel can have mechanical properties similar to the mechanical properties of the bead. For instance, the polymer or gel can be of a similar size to the bead.
  • the polymer or gel can have a mechanical strength (e.g. tensile strength) similar to that of the bead.
  • the polymer or gel can be of a lower density than an oil.
  • the polymer or gel can be of a density that is roughly similar to that of a buffer.
  • the polymer or gel can have a tunable pore size.
  • the pore size can be chosen to, for instance, retain denatured nucleic acids.
  • the pore size can 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 can be biocompatible.
  • the polymer or gel can maintain or enhance cell viability.
  • the polymer or gel can be biochemically compatible.
  • the polymer or gel can be polymerized and/or depolymerized thermally, chemically, enzymatically, and/or optically.
  • the encapsulation of biological particles may constitute the partitioning of the biological particles into which other reagents are co-partitioned.
  • encapsulated biological particles may be readily deposited into other partitions (e.g., droplets) as described above.
  • Nucleic acid barcode molecules may be delivered to a partition e.g., a droplet or well) via a solid support or carrier (e.g., a bead).
  • a solid support or carrier e.g., a bead
  • nucleic acid barcode molecules are initially associated with the solid support and then released from the solid support upon application of a stimulus, which allows the nucleic acid barcode molecules to dissociate or to be released from the solid support.
  • nucleic acid barcode molecules are initially associated with the solid support (e.g., bead) and then released from the solid support upon application of a biological stimulus, a chemical stimulus, a thermal stimulus, an electrical stimulus, a magnetic stimulus, and/or a photo stimulus.
  • the solid support may be a bead.
  • a solid support e.g., a bead, may be porous, non- porous, hollow, solid, semi-solid, and/or a combination thereof. Beads may be solid, semi-solid, semi-fluidic, fluidic, and/or a combination thereof.
  • a solid support e.g., a bead
  • a solid support may be at least partially dissolvable, disruptable, and/or degradable.
  • a solid support e.g., a bead
  • the solid support e.g., a bead, may be a gel bead.
  • a gel bead may be a hydrogel bead.
  • a gel bead may be formed from molecular precursors, such as a polymeric or monomeric species.
  • a semi-solid support, e.g., a bead may be a liposomal bead.
  • Solid supports, e.g., beads may comprise metals including iron oxide, gold, and silver.
  • the solid support, e.g., the bead may be a silica bead.
  • the solid support, e.g., a bead can be rigid.
  • the solid support, e.g., a bead may be flexible and/or compressible.
  • a partition may comprise one or more unique identifiers, such as barcodes.
  • Barcodes may be previously, subsequently or concurrently delivered to the partitions that hold the compartmentalized or partitioned biological particle.
  • barcodes may be injected into droplets or deposited in microwells previous to, subsequent to, or concurrently with droplet generation or providing of reagents in the microwells, respectively.
  • the delivery of the barcodes to a particular partition allows for the later attribution of the characteristics of the individual biological particle to the particular partition.
  • Barcodes may be delivered, for example on a nucleic acid molecule (e.g., via a nucleic acid barcode molecule), to a partition via any suitable mechanism. Nucleic acid barcode molecules can be delivered to a partition via a bead. Beads are described in further detail below.
  • nucleic acid barcode molecules can be initially associated with the bead and then released from the bead. Release of the nucleic acid barcode molecules can be passive (e.g., by diffusion out of the bead). In addition or alternatively, release from the bead can be upon application of a stimulus which allows the nucleic acid barcode molecules to dissociate or to be released from the bead. Such stimulus may disrupt the bead, an interaction that couples the nucleic acid barcode molecules to or within the bead, or both.
  • Such stimulus can include, for example, a thermal stimulus, photo- stimulus, chemical stimulus (e.g., change in pH or use of a reducing agent(s)), a mechanical stimulus, a radiation stimulus; a biological stimulus (e.g., enzyme), or any combination thereof.
  • a thermal stimulus e.g., a thermal stimulus, photo- stimulus, chemical stimulus (e.g., change in pH or use of a reducing agent(s)), a mechanical stimulus, a radiation stimulus; a biological stimulus (e.g., enzyme), or any combination thereof.
  • a bead may be porous, non-porous, solid, semi-solid, semi-fluidic, fluidic, and/or a combination thereof.
  • a bead may be dissolvable, disruptable, and/or degradable.
  • Degradable beads, as well as methods for degrading beads are described in PCT/US2014/044398, which is hereby incorporated by reference in its entirety.
  • any combination of stimuli e.g., stimuli described in PCT/US2014/044398 and US Patent Application Pub. No. 2015/0376609, hereby incorporated by reference in its entirety, may trigger degradation of a bead.
  • a change in pH may enable a chemical agent (e.g., DTT) to become an effective reducing agent.
  • a bead may not be degradable.
  • the bead may be a gel bead.
  • a gel bead may be a hydrogel bead.
  • a gel bead may be formed from molecular precursors, such as a polymeric or monomeric species.
  • a semi-solid bead may be a liposomal bead.
  • Solid beads may comprise metals including iron oxide, gold, and silver.
  • the bead may be a silica bead.
  • the bead can be rigid. In other cases, the bead may be flexible and/or compressible.
  • a bead may be of any suitable shape.
  • bead shapes include, but are not limited to, spherical, non- spherical, oval, oblong, amorphous, circular, cylindrical, and variations thereof.
  • Beads may be of uniform size or heterogeneous size.
  • the diameter of a bead may be at least about 10 nanometers (nm), 100 nm, 500 nm, 1 micrometer (pm), 5pm, 10pm, 20pm, 30pm, 40pm, 50pm, 60pm, 70pm, 80pm, 90pm, 100pm, 250pm, 500pm, 1mm, or greater.
  • a bead may have a diameter of less than about 10 nm, 100 nm, 500 nm, 1pm, 5pm, 10pm, 20pm, 30pm, 40pm, 50pm, 60pm, 70pm, 80pm, 90pm, 100pm, 250pm, 500pm, 1mm, or less.
  • a bead may have a diameter in the range of about 40-75pm, 500pm.
  • beads can be provided as a population or plurality of beads having a relatively monodisperse size distribution. Where it may be desirable to provide relatively consistent amounts of reagents within partitions, maintaining relatively consistent bead characteristics, such as size, can contribute to the overall consistency.
  • the beads described herein may have size distributions that have a coefficient of variation in their cross- sectional dimensions of less than 50%, less than 40%, less than 30%, less than 20%, and in some cases less than 15%, less than 10%, less than 5%, or less.
  • a bead may comprise natural and/or synthetic materials.
  • a bead can comprise a natural polymer, a synthetic polymer or both natural and synthetic polymers. See, e.g., PCT/US2014/044398, which is hereby incorporated by reference in its entirety.
  • Beads may also be formed from materials other than polymers, including lipids, micelles, ceramics, glassceramics, material composites, metals, other inorganic materials, and others.
  • the bead may comprise covalent or ionic bonds between polymeric precursors (e.g., monomers, oligomers, linear polymers), nucleic acid barcode molecules (e.g., oligonucleotides), primers, and other entities.
  • the covalent bonds can be carboncarbon bonds, thioether bonds, or carbon-heteroatom bonds.
  • a plurality of nucleic acid barcode molecules may be attached to a bead.
  • the nucleic acid barcode molecules may be attached directly or indirectly to the bead.
  • the nucleic acid barcode molecules may be covalently linked to the bead.
  • the nucleic acid barcode molecules are covalently linked to the bead via a linker.
  • the linker is a degradable linker.
  • the linker comprises a labile bond configured to release said nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules.
  • the labile bond comprises a disulfide linkage.
  • Activation of disulfide linkages within a bead can be controlled such that only a small number of disulfide linkages are activated. Methods of controlling activation of disulfide linkages within a bead are described in PCT/US2014/044398, which is hereby incorporated by reference in its entirety.
  • a bead may comprise an acrydite moiety, which in certain aspects may be used to attach one or more nucleic acid barcode molecules (e.g., barcode sequence, nucleic acid barcode molecule, barcoded oligonucleotide, primer, or other oligonucleotide) to the bead.
  • nucleic acid barcode molecules e.g., barcode sequence, nucleic acid barcode molecule, barcoded oligonucleotide, primer, or other oligonucleotide
  • precursors e.g., monomers, cross-linkers
  • precursors that are polymerized to form a bead may comprise acrydite moieties, such that when a bead is generated, the bead also comprises acrydite moieties.
  • the acrydite moieties can be attached to a nucleic acid molecule, e.g., a nucleic acid barcode molecule described herein.
  • precursors comprising a functional group that is reactive or capable of being activated such that it becomes reactive can be polymerized with other precursors to generate gel beads comprising the activated or activatable functional group.
  • the functional group may then be used to attach additional species e.g., disulfide linkers, primers, other oligonucleotides, etc.) to the gel beads.
  • additional species e.g., disulfide linkers, primers, other oligonucleotides, etc.
  • a bond may be cleavable via other nucleic acid molecule targeting enzymes, such as restriction enzymes (e.g., restriction endonucleases), as described further below.
  • restriction enzymes e.g., restriction endonucleases
  • Species may be encapsulated in beads during bead generation (e.g., during polymerization of precursors). Such species may or may not participate in polymerization. See, e.g., PCT/US2014/044398, which is hereby incorporated by reference in its entirety.
  • Such species may include, for example, nucleic acid molecules (e.g., oligonucleotides), reagents for a nucleic acid amplification reaction (e.g., primers, polymerases, dNTPs, co-factors (e.g., ionic cofactors), buffers) including those described herein, reagents for enzymatic reactions (e.g., enzymes, co-factors, substrates, buffers), reagents for nucleic acid modification reactions such as polymerization, ligation, or digestion, and/or reagents for template preparation (e.g., tagmentation) for one or more sequencing platforms (e.g., Nextera® for Illumina®).
  • nucleic acid molecules e.g., oligonucleotides
  • reagents for a nucleic acid amplification reaction e.g., primers, polymerases, dNTPs, co-factors (e.g., ionic
  • Such species may include one or more enzymes described herein, including without limitation, polymerase, reverse transcriptase, restriction enzymes (e.g., endonuclease), transposase, ligase, proteinase K, DNAse, etc.
  • Such species may include one or more reagents described elsewhere herein (e.g., lysis agents, inhibitors, inactivating agents, chelating agents, stimulus).
  • species may be partitioned in a partition (e.g., droplet) during or subsequent to partition formation.
  • Such species may include, without limitation, the abovementioned species that may also be encapsulated in a bead.
  • beads can be non-covalently loaded with one or more reagents.
  • the beads can be non-covalently loaded by, for instance, subjecting the beads to conditions sufficient to swell the beads, allowing sufficient time for the reagents to diffuse into the interiors of the beads, and subjecting the beads to conditions sufficient to de-swell the beads.
  • the swelling of the beads may be accomplished, for instance, by placing the beads in a thermodynamically favorable solvent, subjecting the beads to a higher or lower temperature, subjecting the beads to a higher or lower ion concentration, and/or subjecting the beads to an electric field.
  • the swelling of the beads may be accomplished by various swelling methods.
  • the de-swelling of the beads may be accomplished, for instance, by transferring the beads in a thermodynamically unfavorable solvent, subjecting the beads to lower or high temperatures, subjecting the beads to a lower or higher ion concentration, and/or removing an electric field.
  • the de-swelling of the beads may be accomplished by various de-swelling methods. Transferring the beads may cause pores in the bead to shrink. The shrinking may then hinder reagents within the beads from diffusing out of the interiors of the beads. The hindrance may be due to steric interactions between the reagents and the interiors of the beads.
  • the transfer may be accomplished microfluidically. For instance, the transfer may be achieved by moving the beads from one co-flowing solvent stream to a different co-flowing solvent stream.
  • the swellability and/or pore size of the beads may be adjusted by changing the polymer composition of the bead.
  • any suitable number of molecular tag molecules can be associated with a bead such that, upon release from the bead, the molecular tag molecules (e.g. , primer, e.g. , barcoded oligonucleotide) are present in the partition at a pre-defined concentration.
  • the pre-defined concentration may be selected to facilitate certain reactions for generating a sequencing library, e.g., amplification, within the partition.
  • the predefined concentration of the primer can be limited by the process of producing oligonucleotide bearing beads.
  • a nucleic acid barcode molecule may contain one or more barcode sequences.
  • a plurality of nucleic acid barcode molecules may be coupled to a bead.
  • the one or more barcode sequences may include sequences that are the same for all nucleic acid molecules coupled to a given bead and/or sequences that are different across all nucleic acid molecules coupled to the given bead.
  • the nucleic acid molecule may be incorporated into the bead.
  • Nucleic acid barcode molecules can comprise one or more functional sequences for coupling to an analyte or analyte tag such as a reporter oligonucleotide.
  • Such functional sequences can include, e.g., a template switch oligonucleotide (TSO) sequence, a primer sequence e.g., a poly T sequence, or a nucleic acid primer sequence complementary to a target nucleic acid sequence and/or for amplifying a target nucleic acid sequence, a random primer, and/or a primer sequence for messenger RNA).
  • TSO template switch oligonucleotide
  • the nucleic acid barcode molecule can further comprise a unique molecular identifier (UMI).
  • UMI unique molecular identifier
  • the nucleic acid barcode molecule can comprise one or more functional sequences, for example, for attachment to a sequencing flow cell, such as, for example, a P5 sequence (or a portion thereof) for Illumina® sequencing.
  • the nucleic acid barcode molecule or derivative thereof e.g., oligonucleotide or polynucleotide generated from the nucleic acid molecule can comprise another functional sequence, such as, for example, a P7 sequence (or a portion thereof) for attachment to a sequencing flow cell for Illumina sequencing.
  • the nucleic acid molecule can comprise an R1 primer sequence for Illumina sequencing. In some cases, the nucleic acid molecule can comprise an R2 primer sequence for Illumina sequencing.
  • a functional sequence can comprise a partial sequence, such as a partial barcode sequence, partial anchoring sequence, partial sequencing primer sequence (e.g., partial R1 sequence, partial R2 sequence, etc.), a partial sequence configured to attach to the flow cell of a sequencer (e.g., partial P5 sequence, partial P7 sequence, etc.), or a partial sequence of any other type of sequence described elsewhere herein.
  • a partial sequence may contain a contiguous or continuous portion or segment, but not all, of a full sequence, for example.
  • a downstream procedure may extend the partial sequence, or derivative thereof, to achieve a full sequence of the partial sequence, or derivative thereof.
  • nucleic acid molecules e.g., oligonucleotides, polynucleotides, etc.
  • uses thereof as may be used with compositions, devices, methods and systems of the present disclosure, are provided in U.S. Patent Pub. Nos. 2014/0378345 and 2015/0376609, each of which is entirely incorporated herein by reference.
  • FIG. 3 illustrates an example of a barcode carrying bead.
  • a nucleic acid barcode molecule 302 can be coupled to a bead 304 by a releasable linkage 306, such as, for example, a disulfide linker.
  • the same bead 304 may be coupled (e.g., via releasable linkage) to one or more other nucleic acid barcode molecules 318, 320.
  • the nucleic acid barcode molecule 302 may be or comprise a barcode. As noted elsewhere herein, the structure of the barcode may comprise a number of sequence elements.
  • the nucleic acid barcode molecule 302 may comprise a functional sequence 308 that may be used in subsequent processing.
  • the functional sequence 308 may include one or more of a sequencer specific flow cell attachment sequence (e.g., a P5 sequence for Illumina® sequencing systems) and a sequencing primer sequence (e.g., a R1 primer for Illumina® sequencing systems), or partial sequence(s) thereof.
  • the nucleic acid barcode molecule 302 may comprise a barcode sequence 310 for use in barcoding the sample (e.g., DNA, RNA, protein, etc.).
  • the barcode sequence 310 can be bead-specific such that the barcode sequence 310 is common to all nucleic acid barcode molecules (e.g., including nucleic acid barcode molecule 302) coupled to the same bead 304.
  • the barcode sequence 310 can be partition- specific such that the barcode sequence 310 is common to all nucleic acid barcode molecules coupled to one or more beads that are partitioned into the same partition.
  • the nucleic acid barcode molecule 302 may comprise sequence 312 complementary to an analyte of interest, e.g., a priming sequence. Sequence 312 can be a poly-T sequence complementary to a poly- A tail of an mRNA analyte, a targeted priming sequence, and/or a random priming sequence.
  • the nucleic acid barcode molecule 302 may comprise an anchoring sequence 314 to ensure that the specific priming sequence 312 hybridizes at the sequence end (e.g., of the mRNA).
  • the anchoring sequence 314 can include a random short sequence of nucleotides, such as a 1-mer, 2-mer, 3-mer or longer sequence, which can ensure that a poly-T segment is more likely to hybridize at the sequence end of the poly-A tail of the mRNA.
  • the nucleic acid barcode molecule 302 may comprise a unique molecular identifying sequence 316 (e.g., unique molecular identifier (UMI)).
  • the unique molecular identifying sequence 316 may comprise from about 5 to about 8 nucleotides.
  • the unique molecular identifying sequence 316 may compress less than about 5 or more than about 8 nucleotides.
  • the unique molecular identifying sequence 316 may be a unique sequence that varies across individual nucleic acid barcode molecules (e.g., 302, 318, 320, etc.) coupled to a single bead (e.g., bead 304).
  • the unique molecular identifying sequence 316 may be a random sequence (e.g., such as a random N-mer sequence).
  • the UMI may provide a unique identifier of the starting analyte (e.g., mRNA) molecule that was captured, in order to allow quantitation of the number of original expressed RNA molecules.
  • FIG. 3 shows three nucleic acid barcode molecules 302, 318, 320 coupled to the surface of the bead 304, an individual bead may be coupled to any number of individual nucleic acid barcode molecules, for example, from one to tens to hundreds of thousands, millions, or even a billion of individual nucleic acid barcode molecules.
  • the respective barcodes for the individual nucleic acid barcode molecules can comprise both common sequence segments or relatively common sequence segments (e.g., 308, 310, 312, etc.) and variable or unique sequence segments (e.g., 316) between different individual nucleic acid barcode molecules coupled to the same bead.
  • a biological particle e.g., cell, DNA, RNA, etc.
  • the nucleic acid barcode molecules 302, 318, 320 can be released from the bead 304 in the partition.
  • the poly-T segment e.g., 312
  • one of the released nucleic acid barcode molecules e.g., 302
  • Reverse transcription may result in a cDNA transcript of the mRNA, but which transcript includes each of the sequence segments 308, 310, 316 of the nucleic acid barcode molecule 302.
  • nucleic acid barcode molecule 302 comprises an anchoring sequence 3144
  • cDNA transcripts of the individual mRNA molecules from any given partition may include a common barcode sequence segment 310.
  • the transcripts made from the different mRNA molecules within a given partition may vary at the unique molecular identifying sequence 312 segment (e.g., UMI segment).
  • UMI segment unique molecular identifying sequence
  • the number of different UMIs can be indicative of the quantity of mRNA originating from a given partition, and thus from the biological particle (e.g., cell).
  • the transcripts can be amplified, cleaned up and sequenced to identify the sequence of the cDNA transcript of the mRNA, as well as to sequence the barcode segment and the UMI segment. While a poly-T primer sequence is described, other targeted or random priming sequences may also be used in priming the reverse transcription reaction. Likewise, although described as releasing the barcoded oligonucleotides into the partition, in some cases, the nucleic acid barcode molecules bound to the bead (e.g., gel bead) may be used to hybridize and capture the mRNA on the solid phase of the bead, for example, in order to facilitate the separation of the RNA from other cell contents.
  • the nucleic acid barcode molecules bound to the bead e.g., gel bead
  • the nucleic acid barcode molecules bound to the bead may be used to hybridize and capture the mRNA on the solid phase of the bead, for example, in order to facilitate the separation of the RNA from other cell contents.
  • RNA molecules on the beads may be subjected to reverse transcription or other nucleic acid processing, additional adapter sequences may be added to the barcoded nucleic acid molecules, or other nucleic acid reactions (e.g., amplification, nucleic acid extension) may be performed.
  • the beads or products thereof e.g., barcoded nucleic acid molecules
  • the beads comprising nucleic acid barcode molecules may be introduced into a partition (e.g., well or droplet) prior to, during, or following introduction of a sample into the partition.
  • the nucleic acid molecules of a sample may be subjected to barcoding, which may occur on the bead (in cases where the nucleic acid molecules remain coupled to the bead) or following release of the nucleic acid barcode molecules into the partition.
  • captured analytes from various partitions may be collected, pooled, and subjected to further processing (e.g., reverse transcription, adapter attachment, amplification, clean up, sequencing).
  • further processing e.g., reverse transcription, adapter attachment, amplification, clean up, sequencing
  • the beads from various partitions may be collected, pooled, and subjected to further processing (e.g., reverse transcription, adapter attachment, amplification, clean up, sequencing).
  • one or more of the processing methods e.g., reverse transcription, may occur in the partition.
  • conditions sufficient for barcoding, adapter attachment, reverse transcription, or other nucleic acid processing operations may be provided in the partition and performed prior to clean up and sequencing.
  • a bead may comprise a capture sequence or binding sequence configured to bind to a corresponding capture sequence or binding sequence.
  • a bead may comprise a plurality of different capture sequences or binding sequences configured to bind to different respective corresponding capture sequences or binding sequences.
  • a bead may comprise a first subset of one or more capture sequences each configured to bind to a first corresponding capture sequence, a second subset of one or more capture sequences each configured to bind to a second corresponding capture sequence, a third subset of one or more capture sequences each configured to bind to a third corresponding capture sequence, and etc.
  • a bead may comprise any number of different capture sequences.
  • a bead may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different capture sequences or binding sequences configured to bind to different respective capture sequences or binding sequences, respectively.
  • a bead may comprise at most about 10, 9, 8, 7, 6, 5, 4, 3, or 2 different capture sequences or binding sequences configured to bind to different respective capture sequences or binding sequences.
  • the different capture sequences or binding sequences may be configured to facilitate analysis of a same type of analyte.
  • the different capture sequences or binding sequences may be configured to facilitate analysis of different types of analytes (with the same bead).
  • the capture sequence may be designed to attach to a corresponding capture sequence.
  • such corresponding capture sequence may be introduced to, or otherwise induced in, a biological particle (e.g., cell, cell bead, etc.) for performing different assays in various formats (e.g., barcoded antibodies comprising the corresponding capture sequence, barcoded MHC dextramers comprising the corresponding capture sequence, barcoded guide RNA molecules comprising the corresponding capture sequence, etc.), such that the corresponding capture sequence may later interact with the capture sequence associated with the bead.
  • a biological particle e.g., cell, cell bead, etc.
  • a biological particle e.g., cell, cell bead, etc.
  • formats e.g., barcoded antibodies comprising the corresponding capture sequence, barcoded MHC dextramers comprising the corresponding capture sequence, barcoded guide RNA molecules comprising the corresponding capture sequence, etc.
  • a capture sequence coupled to a bead may be configured to attach to a linker molecule, such as a splint molecule, wherein the linker molecule is configured to couple the bead (or other support) to other molecules through the linker molecule, such as to one or more analytes or one or more other linker molecules.
  • a linker molecule such as a splint molecule
  • FIG. 4 illustrates another example of a barcode carrying bead.
  • a nucleic acid barcode molecule 405, such as an oligonucleotide, can be coupled to a bead 404 by a releasable linkage 406, such as, for example, a disulfide linker.
  • the nucleic acid barcode molecule 405 may comprise a first capture sequence 460.
  • the same bead 404 may be coupled (e.g., via releasable linkage) to one or more other nucleic acid molecules 403, 407 comprising other capture sequences.
  • the nucleic acid barcode molecule 405 may be or comprise a barcode.
  • the structure of the barcode may comprise a number of sequence elements, such as a functional sequence 408 (e.g., flow cell attachment sequence, sequencing primer sequence, etc.), a barcode sequence 410 (e.g., bead-specific sequence common to bead, partition- specific sequence common to partition, etc.), and a unique molecular identifier 412 (e.g., unique sequence within different molecules attached to the bead), or partial sequences thereof.
  • the capture sequence 460 may be configured to attach to a corresponding capture sequence 465.
  • the corresponding capture sequence 465 may be coupled to another molecule that may be an analyte or an intermediary carrier. For example, as illustrated in FIG.
  • the corresponding capture sequence 465 is coupled to a guide RNA molecule 462 comprising a target sequence 464, wherein the target sequence 464 is configured to attach to the analyte.
  • Another oligonucleotide molecule 407 attached to the bead 404 comprises a second capture sequence 480 which is configured to attach to a second corresponding capture sequence 485.
  • the second corresponding capture sequence 485 is coupled to an antibody 482.
  • the antibody 482 may have binding specificity to an analyte (e.g., surface protein). Alternatively, the antibody 482 may not have binding specificity.
  • Another oligonucleotide molecule 403 attached to the bead 404 comprises a third capture sequence 470 which is configured to attach to a third corresponding capture sequence 475. As illustrated in FIG. 4, the third corresponding capture sequence 475 is coupled to a molecule 472.
  • the molecule 472 may or may not be configured to target an analyte.
  • the other oligonucleotide molecules 403, 407 may comprise the other sequences (e.g., functional sequence, barcode sequence, UMI, etc.) described with respect to oligonucleotide molecule 405. While a single oligonucleotide molecule comprising each capture sequence is illustrated in FIG.
  • the bead may comprise a set of one or more oligonucleotide molecules each comprising the capture sequence.
  • the bead may comprise any number of sets of one or more different capture sequences.
  • the bead 404 may comprise other capture sequences.
  • the bead 404 may comprise fewer types of capture sequences (e.g., two capture sequences).
  • the bead 404 may comprise oligonucleotide molecule(s) comprising a priming sequence, such as a specific priming sequence such as an mRNA specific priming sequence (e.g., poly-T sequence), a targeted priming sequence, and/or a random priming sequence, for example, to facilitate an assay for gene expression.
  • a priming sequence such as a specific priming sequence such as an mRNA specific priming sequence (e.g., poly-T sequence), a targeted priming sequence, and/or a random priming sequence, for example, to facilitate an assay for gene expression.
  • the barcoded oligonucleotides may be released (e.g., in a partition), as described elsewhere herein.
  • the nucleic acid molecules bound to the bead e.g., gel bead
  • analytes e.g., one or more types of analytes
  • a bead injected or otherwise introduced into a partition may comprise releasably, cleavably, or reversibly attached barcodes.
  • a bead injected or otherwise introduced into a partition may comprise activatable barcodes.
  • a bead injected or otherwise introduced into a partition may be degradable, disruptable, or dissolvable beads.
  • Barcodes can be releasably, cleavably or reversibly attached to the beads such that barcodes can be released or be releasable through cleavage of a linkage between the barcode molecule and the bead, or released through degradation of the underlying bead itself, allowing the barcodes to be accessed or be accessible by other reagents, or both.
  • cleavage may be achieved through reduction of di-sulfide bonds, use of restriction enzymes, photo-activated cleavage, or cleavage via other types of stimuli (e.g., chemical, thermal, pH, enzymatic, etc.) and/or reactions, such as described elsewhere herein.
  • Releasable barcodes may sometimes be referred to as being activatablc, in that they arc available for reaction once released.
  • an activatable barcode may be activated by releasing the barcode from a bead (or other suitable type of partition described herein).
  • Other activatable configurations are also envisioned in the context of the described methods and systems.
  • the degradation of a bead may refer to the disassociation of a bound or entrained species from a bead, both with and without structurally degrading the physical bead itself.
  • the degradation of the bead may involve cleavage of a cleavable linkage via one or more species and/or methods described elsewhere herein.
  • entrained species may be released from beads through osmotic pressure differences due to, for example, changing chemical environments. See, e.g., PCT/US2014/044398, which is hereby incorporated by reference in its entirety.
  • a degradable bead may be introduced into a partition, such as a droplet of an emulsion or a well, such that the bead degrades within the partition and any associated species (e.g., oligonucleotides) are released within the droplet when the appropriate stimulus is applied.
  • the free species e.g., oligonucleotides, nucleic acid molecules
  • barcodes that are releasably, cleavably or reversibly attached to the beads described herein include barcodes that are released or releasable through cleavage of a linkage between the barcode molecule and the bead, or that are released through degradation of the underlying bead itself, allowing the barcodes to be accessed or accessible by other reagents, or both.
  • a species e.g., oligonucleotide molecules comprising barcodes
  • a solid support e.g., a bead
  • the U-excising element may comprise a singlestranded DNA (ssDNA) sequence that contains at least one uracil.
  • the species may be attached to a solid support via the ssDNA sequence containing the at least one uracil.
  • the species may be released by a combination of uracil-DNA glycosylase (e.g., to remove the uracil) and an endonuclease (e.g., to induce an ssDNA break). If the endonuclease generates a 5’ phosphate group from the cleavage, then additional enzyme treatment may be included in downstream processing to eliminate the phosphate group, e.g., prior to ligation of additional sequencing handle elements, e.g., Illumina full P5 sequence, partial P5 sequence, full R1 sequence, and/or partial R1 sequence.
  • additional enzyme treatment may be included in downstream processing to eliminate the phosphate group, e.g., prior to ligation of additional sequencing handle elements, e.g., Illumina full P5 sequence, partial P5 sequence, full R1 sequence, and/or partial R1 sequence.
  • the barcodes that are releasable as described herein may sometimes be referred to as being activatable, in that they are available for reaction once released.
  • an activatable barcode may be activated by releasing the barcode from a bead (or other suitable type of partition described herein).
  • Other activatable configurations are also envisioned in the context of the described methods and systems.
  • the polymer can include poly(acrylamide-co-acrylic acid) crosslinked with disulfide linkages.
  • the preparation of the polymer can include a two-step reaction.
  • poly(acrylamide-co-acrylic acid) can be exposed to an acylating agent to convert carboxylic acids to esters.
  • the poly(acrylamide-co-acrylic acid) can be exposed to 4-(4,6- dimethoxy-l,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM).
  • DTMM 4-(4,6- dimethoxy-l,3,5-triazin-2-yl)-4-methylmorpholinium chloride
  • the polyacrylamide- co-acrylic acid can be exposed to other salts of 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4- methylmorpholinium.
  • the ester formed in the first step can be exposed to a disulfide crosslinking agent.
  • the ester can be exposed to cystamine (2,2’-dithiobis(ethylamine)).
  • the biological particle can be surrounded by polyacrylamide strands linked together by disulfide bridges.
  • the biological particle can be encased inside of or comprise a gel or matrix (e.g., polymer matrix) to form a “cell bead.”
  • a cell bead can contain biological particles (e.g., labelled immune cell, B cell, or T cell) or macromolecular constituents (e.g., RNA, DNA, proteins, secreted antibodies or antigenbinding fragments thereof etc.) of biological particles.
  • a cell bead can 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 (i) cell beads (and/or droplets or other partitions) containing biological particles and (ii) cell beads (and/or droplets or other partitions) containing macromolecular constituents of biological particles.
  • Encapsulated biological particles can provide certain potential advantages of being more storable and more portable than droplet-based partitioned biological particles. Furthermore, in some cases, it can be desirable to allow biological particles e.g., labelled immune cells, B cells, or T cells) 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 e.g., cytokines, antigens, etc.).
  • biological particles e.g., labelled immune cells, B cells, or T cells
  • encapsulation can allow for longer incubation than partitioning in emulsion droplets, although in some cases, droplet partitioned biological particles can also be incubated for different periods of time, e.g., at least 10 seconds, at least 30 seconds, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours, or at least 10 hours or more.
  • the encapsulation of biological particles e.g., labelled immune cells, B cells, or T cells
  • encapsulated biological particles can be readily deposited into other partitions (e.g., droplets) as described above.
  • 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).
  • 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.
  • the wells or microwells can be configured to toggle between “open” and “closed” configurations.
  • 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 are not accessible on a planar surface of the substrate without an external force.
  • 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.
  • the well can have a volume of less than 1 milliliter (mL).
  • 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 pL, 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.
  • a microwell array or plate includes a single variety of microwells.
  • a microwell array or plate includes a variety of microwells.
  • 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.
  • 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.
  • the microwell array or plate includes different types of microwells 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 microcapsulc, droplet, bead, or other reagent.
  • the adjacent microwells 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.
  • a plurality of partitions can be used in the systems, compositions, and methods described herein.
  • any suitable number of partitions e.g., wells or droplets
  • 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.
  • the plurality of wells can include both unoccupied wells (e.g., empty wells) and occupied wells.
  • 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 microcapsule or 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 microcapsule or 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.
  • the well may be subjected to conditions for further processing of a cell (or cells) in the well.
  • reagents in the well may allow further processing of the cell, e.g., cell lysis, as further described herein.
  • the well (or wells such as those of a wellbased 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.
  • 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
  • the well (or wells) comprising the cell (or cells) may be subjected to freeze-thaw cycles to lyse the cell (or cells).
  • 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).
  • 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).
  • 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.
  • a well can include free reagents and/or reagents encapsulated in, or otherwise coupled to or associated with, microcapsules, beads, or droplets.
  • any of the reagents described in this disclosure can be encapsulated in, or otherwise coupled to, a microcapsule, 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.
  • 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.
  • 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,
  • 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.
  • kits 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).
  • a well includes a microcapsule, 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.
  • a microcapsule, bead, or droplet includes a heterogeneous mixture of reagents.
  • the heterogeneous mixture of reagents can include all components necessary to perform a reaction.
  • 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.
  • such additional components are contained within, or otherwise coupled to, a different microcapsule, droplet, or bead, or within a solution within a partition (e.g., microwell) of the system.
  • FIG. 5 A non-limiting example of a microwell array in accordance with some embodiments of the disclosure is schematically presented in FIG. 5.
  • 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.
  • 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.
  • a single sample molecule 506 e.g., cell
  • Reagents can be loaded into a well either sequentially or concurrently.
  • reagents are introduced to the device either before or after a particular operation.
  • reagents (which can be provided, in certain instances, in microcapsules, droplets, or beads) are introduced sequentially such that different reactions or operations occur at different steps.
  • the reagents (or microcapsules, droplets, or beads) can also be loaded at operations interspersed with a reaction or operation step.
  • microcapsules 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 microcapsules, 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.
  • the nucleic acid barcode molecules and other reagents can be contained within a microcapsule, bead, or droplet. These microcapsules, 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 microcapsule, 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.
  • a partition e.g., a microwell
  • the partition e.g., microwell
  • the partition 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. Tn such cases, the nucleic acid barcode sequences can be used to trace the origin of the sample nucleic acid molecule.
  • 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.
  • the samples or reagents can be loaded in the wells or microwells using a variety of approaches.
  • the samples e.g., a cell, cell bead, or cellular component
  • reagents as described herein
  • 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.
  • 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.
  • 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).
  • a droplet e.g., including a single cell
  • 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.).
  • a stimulus e.g., external force, agitation, heat, light, magnetic or electric force, etc.
  • the loading of the droplet and the bead is super-Poissonian.
  • 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.
  • 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.
  • 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 microwell.
  • 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.).
  • the partition e.g., the microwell or microwell pair that are in contact
  • further processing e.g., barcoding, nucleic acid reactions, etc.
  • one of the droplets can include lysis reagents for lysing the cell upon droplet merging.
  • a droplet or microcapsule (e.g., bead) can be partitioned into a well.
  • the droplets can be selected or subjected to pre-processing prior to loading into a well.
  • 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 preselection 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.
  • the technique can be useful in obtaining or preventing cell doublet or multiplet formation prior to or during loading of the microwell.
  • 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.
  • 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.
  • the nucleic acid barcode molecule can include a unique molecular identifier for individual molecule identification.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • nucleic acid processing e.g., amplification, extension, reverse transcription, etc.
  • characterization e.g., sequencing
  • the unique partition barcode sequences can be used to identify the cell or partition from which a nucleic acid molecule originated.
  • 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.
  • imaging of each microwell 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.
  • a biomarker e.g., a surface marker, a fluorescently labeled molecule therein, etc.
  • 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.
  • imaging can be used to characterize a quantity of amplification products in the well.
  • a well can be loaded with a sample and reagents, simultaneously or sequentially.
  • the well can be subjected to washing, e.g., to remove excess cells from the well, microwell array, or plate. Similarly, washing can be performed to remove excess beads or other reagents from the well, microwell array, or plate.
  • 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.
  • the intracellular components or cellular analytes e.g., nucleic acid molecules
  • 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).
  • nucleic acid processing e.g., performing an amplification, extension
  • characterization e.g., fluorescence monitoring of amplified molecules, sequencing.
  • the well or microwell array or plate
  • the well can be sealed (e.g., using an oil, membrane, wax, etc.), which enables storage of the assay or selective introduction of additional reagents.
  • 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
  • nucleic acid barcode molecules e.g., mRNA, cDNA, gDNA, etc.,
  • 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.
  • 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.
  • the partitioning junction/droplet generation zone e.g., junction 210
  • biological particles can be partitioned along with other reagents, as will be described further below.
  • 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.
  • 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.
  • the microfluidic channel structures can have other geometries and/or configurations.
  • a microfluidic channel structure can have more than two channel junctions.
  • 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.
  • 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, achromopeptidase, lysostaphin, labiase, kitalase, lyticase, 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.
  • 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.
  • lysis solutions can include non-ionic surfactants such as, for example, TritonX-100 and Tween 20.
  • 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.
  • 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.
  • reagents can also be copartitioned 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.
  • DNase and RNase inactivating agents or inhibitors such as proteinase K
  • chelating agents such as EDTA
  • the biological particles can be exposed to an appropriate stimulus to release the biological particles or their contents from a co-partitioned microcapsule (e.g., bead).
  • a chemical stimulus can be co-partitioned along with an encapsulated biological particle to allow for the degradation of the microcapsule (e.g., bead) and release of the cell or its contents into the larger partition.
  • this stimulus can be the same as the stimulus described elsewhere herein for release of nucleic acid molecules (e.g., oligonucleotides) from their respective microcapsule (e.g., bead).
  • 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.
  • 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.
  • the biological particles e.g., labelled immune cells, B cells, or T cells
  • 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.
  • reverse transcriptase enzymes including enzymes with terminal transferase activity
  • primers and oligonucleotides include primers and oligonucleotides, and switch oligonucleotides (also referred to herein as “switch oligos” or “template switching oligonucleotides”) which can be used for template switching.
  • switch oligonucleotides also referred to herein as “switch oligos” or “template switching oligonucleotides” which can be used for template switching.
  • template switching can be used to increase the length of a cDNA.
  • template switching can
  • 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.
  • the macromolecular components e.g., macromolecular constituents of biological particles, such as RNA, DNA, proteins, or secreted antibodies or antigen-binding fragments thereof
  • the macromolecular component contents of individual biological particles e.g., labelled immune cells, B cells, or T cells
  • 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 tbe 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.
  • 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).
  • 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 arc 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 functional sequences useful in the processing of the nucleic acids from the co-partitioned biological particles. 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,
  • 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.
  • microcapsules such as beads
  • barcoded nucleic acid molecules e.g., barcoded oligonucleotides
  • 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.
  • 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.
  • each bead can be provided with large numbers of nucleic acid (e.g., oligonucleotide) molecules attached.
  • 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.
  • 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.
  • 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.
  • 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.
  • the nucleic acid molecules are releasable from the beads upon the application of a particular stimulus to the beads.
  • the stimulus can be a photostimulus, e.g., through cleavage of a photo-labile linkage that releases the nucleic acid molecules.
  • 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.
  • 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.
  • 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.
  • 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.
  • channel architecture e.g., microfluidics channel architecture
  • 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.”
  • 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.
  • 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.
  • 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.
  • 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.
  • the beads can be introduced into the channel segment 202 from a plurality of different channels, and the frequency controlled accordingly.
  • 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.
  • biological particles e.g., described with reference to FIG. 1
  • the aqueous fluid 208 can have a substantially uniform concentration or frequency of biological particles.
  • the biological particles e.g., labelled immune cells, B cells, or T cells
  • the frequency or concentration of the biological particles in the aqueous fluid 208 in the channel segment 202 can be controlled by
  • the biological particles can be introduced into the channel segment 202 from a plurality of different channels, and the frequency controlled accordingly.
  • 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.
  • 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.
  • 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.
  • the second fluid 210 cannot be subjected to and/or directed to any flow in or out of the reservoir 204.
  • the second fluid 210 can be substantially stationary in the reservoir 204.
  • 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.
  • the second fluid 210 can be subjected and/or directed to flow in or out of the reservoir 204.
  • the reservoir 204 can be a channel directing the second fluid 210 from upstream to downstream, transporting the generated droplets.
  • 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.
  • 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).
  • 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:
  • 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.
  • 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)).
  • SPRI solid phase reversible immobilization
  • further processing e.g., shearing, ligation of functional sequences
  • subsequent amplification e.g., via PCR
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • first sample can undergo a first processing protocol and a second sample can undergo a second processing protocol.
  • 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.
  • 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.
  • cells of a sample can include one or more different types of blood cells.
  • a biological sample can include a plurality of cells having different dimensions and features.
  • 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.
  • 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, permeabilization, isolation, agitation, heating, and/or other processes.
  • a sample may be filtered to remove a contaminant or other materials.
  • a filtration process can include the use of microfluidics (e.g., to separate analyte carriers of different sizes, types, charges, or other features).
  • 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).
  • 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.
  • 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).
  • 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
  • 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
  • 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.
  • FACS fluorescence-activated cell sorting
  • MCS magnetic-activated cell sorting
  • AFS buoyancy-activated cell sorting
  • 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.
  • 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
  • droplets e.g., aqueous droplets
  • Droplets including cells and/or cellular constituents of interest e.g., as determined via optical detection
  • an electric charge e.g., using an electrical charging ring
  • 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-scale 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the sample 606 can be processed within the partition.
  • the cell can be subjected to conditions sufficient to lyse the cells and release the analytes contained therein.
  • the bead 604 can be further processed.
  • processes 620a and 620b schematically illustrate different workflows, depending on the properties of the bead 604.
  • 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.
  • sample nucleic acid molecules e.g., RNA, DNA
  • the beads 604 from multiple wells 602 can be collected and pooled. Further processing can be performed in process 640.
  • one or more nucleic acid reactions can be performed, such as reverse transcription, nucleic acid extension, amplification, ligation, transposition, etc.
  • adapter sequences are ligated to the nucleic acid molecules, or derivatives thereof, as described elsewhere herein.
  • sequencing primer sequences can be appended to each end of the nucleic acid molecule.
  • 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.
  • 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.
  • sequencing primer sequences can be appended to each end of the nucleic acid molecule.
  • 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.
  • steps (b) and (c) of the methods described herein are performed in multiplex format.
  • 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 AB Ms produced by the additional immune cells.
  • the present disclosure provides methods and systems for multiplexing, and otherwise increasing throughput of samples for analysis.
  • 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.
  • 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.
  • 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.
  • 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 described herein.
  • the labelling agents e.g., any of the labelling compositions 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.
  • the reporter oligonucleotide can include a barcode sequence that permits identification of the labelling agent.
  • a labelling agent that is specific to one type of cell feature can have a first reporter oligonucleotide coupled thereto
  • a labelling agent that is specific to a different cell feature e.g., a second cell surface feature
  • 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.
  • a library of potential cell feature labelling agents can be provided, where the respective cell feature labelling agents are 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.
  • different members of the library can be characterized by the presence of a different oligonucleotide sequence label.
  • an antibody capable of binding to a first protein can have associated with it a first reporter oligonucleotide sequence
  • 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.
  • Uabelling 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.
  • labeling agents can be used to label a sample of cells or a group of cells. Tn this way, a group of cells can be labeled as different from another group of cells.
  • 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.
  • 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.
  • 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.
  • the pretreatment is performed prior to the step of contacting the immune cells with the antigens.
  • 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.
  • 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 n
  • the dissociation constant can be less than about 10 pM.
  • 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.
  • 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 pcnctratin, 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.
  • a reporter oligonucleotide can be coupled to a fluorophore 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.
  • 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.
  • the fluorophore is a water-soluble, organic fluorophore.
  • 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.
  • 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.
  • 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.
  • 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.
  • the cells 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.
  • labelling agents e.g., receptors, proteins, etc.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • CMOs cholesterol-modified oligonucleotides
  • anti-calcium channel antibodies include anti-KCNN4 antibodies, anti-BK channel beta 3 antibodies, anti-alB calcium channel antibodies, and anti-CACNAlA antibodies.
  • 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.
  • 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.
  • 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.
  • the reporter oligonucleotides and labelling agents can allow multi-analyte, multiplexed analyses to be performed.
  • 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.
  • 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.
  • 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; 31(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.
  • 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.
  • a labelling agent is indirectly (e.g., via hybridization) coupled to a reporter oligonucleotide including a barcode sequence that identifies the label agent.
  • 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.
  • the reporter oligonucleotides are releasable from the labelling agent, such as upon application of a stimulus.
  • 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.
  • 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).
  • UMI unique molecular identifier
  • a sequencer specific flow cell attachment sequence such as an P5, P7, or partial P5 or P7 sequence
  • a primer or primer binding sequence such as an Rl, R2, or partial R1 or R2 sequence.
  • the labelling agent e.g., an antigen, an antigen fragment, an antibody, an antibody fragment
  • the labelling agent 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.
  • a labelling agent e.g., an antigen, an antigen fragment, an antibody, an antibody fragment
  • a labelling agent e.g., an antigen, an antigen fragment, an antibody, an antibody fragment
  • a labelling agent e.g., an antigen, an antigen fragment, an antibody, an antibody fragment
  • a labelling agent e.g., an antigen, an antigen fragment, an antibody, an antibody fragment
  • a labelling agent e.g., an antigen, an antigen fragment, an antibody, an antibody fragment
  • a labelling agent e.g., an antigen, an antigen fragment, an antibody, an antibody fragment
  • an octamer e.g., an antigen, an antigen fragment, an antibody, an antibody fragment
  • a labelling agent e.g., an antigen, an antigen fragment, an antibody, an antibody fragment
  • a labelling agent e.g., an antigen, an antigen fragment, an antibody, an antibody fragment
  • a labelling agent e.g., an antigen, an antigen fragment, an antibody, an antibody fragment
  • a labelling agent is presented as a 104- mer.
  • the labelling agent can include a reporter oligonucleotide and a label (e.g., detectable label).
  • a label e.g., detectable label
  • 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).
  • 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.
  • 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
  • Reporter oligonucleotide 740 can include barcode sequence 742 that identifies labelling agent 710.
  • Reporter oligonucleotide 740 can also include one or more functional sequences741 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).
  • UMI unique molecular identifier
  • sequencer specific flow cell attachment sequence such as an P5, P7, or partial P5 or P7 sequence
  • primer or primer binding sequence such as an Rl, R2, or partial R1 or R2 sequence
  • reporter oligonucleotide 740 conjugated to a labelling agent 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.
  • 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).
  • a support e.g., a bead, such as a gel bead
  • 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).
  • reporter oligonucleotide 740 includes one or more additional functional sequences, such as those described above.
  • 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).
  • 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.
  • 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 (i.e., a molecule or compound to which antibody 720 binds).
  • labelling agent 730 includes an MHC molecule 731 including peptide 732 and reporter oligonucleotide 740 that identifies peptide 732.
  • the MHC molecule is coupled to a support 733.
  • support 733 can be or comprise a polypeptide, such as avidin, neutravidin, streptavidin, or a polysaccharide, such as dextran.
  • support 733 further comprises a detectable label, e.g., a detectable label described herein, e.g., a fluorescent label.
  • reporter oligonucleotide 740 can be directly or indirectly coupled to MHC labelling agent 730 in any suitable manner.
  • reporter oligonucleotide 740 can be coupled to MHC molecule 731, support 733, or peptide 732.
  • 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)).
  • 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.
  • an antigen e.g., protein, polypeptide
  • 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® (Tmmudex)), etc.
  • MHC tetramers MHC pentamers
  • MHC pentamers MHC assembled via a coiled-coil domain
  • MHC octamers MHC dodecamers
  • MHC decorated dextran molecules e.g., MHC Dextramer® (Tmmudex)
  • exemplary labelling agents including antibody and MHC-bascd labelling agents, reporter oligonucleotides, and methods of use, see, e.g., U.
  • 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, 10A-10B).
  • 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, 10A-10B).
  • a support e.g., a bead, such as a gel bead
  • 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.
  • reporter oligonucleotide 740 includes one or more additional functional sequences, such as those described above.
  • 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.
  • 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.
  • the antigen of interest comprises a ligand that can be bound by the binding region of support 750.
  • 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).
  • the support 750 is coupled to, complexed with, or bound to a ligand 751.
  • support 750 can be a polypeptide.
  • the polypeptide can be streptavidin.
  • the polypeptide can be avidin.
  • support 750 can be a polysaccharide.
  • 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.
  • the ligand 751 may be biotin and the support 750 may be a streptavidin support.
  • the ligand 751 is coupled to or conjugated to antigen 753 via a linker 752.
  • the partitioned cells are contacted with one or more biotinylated antigens.
  • the antigens can include Avitag biotinylation site and/or a His tag. Protein biotinylation techniques are available.
  • reporter oligonucleotide 740 can be directly or indirectly coupled to labelling agent 760 in any suitable manner.
  • reporter oligonucleotide 740 can be coupled to the antigen 753, support 750, anchor sequence 745, or ligand 751.
  • 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.
  • 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.
  • Exemplary barcode molecules attached to a support is shown in FIG. 9.
  • analysis of multiple analytes can include nucleic acid barcode molecules as generally depicted in FIG. 9.
  • 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 capture sequence 923, wherein capture sequence 923 includes a different sequence than capture sequence 913.
  • adapter 911 and adapter 921 include the same sequence.
  • adapter 911 and adapter 921 include different sequences.
  • 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.
  • support 930 further includes nucleic acid barcode molecule 950.
  • Nucleic acid barcode molecule 950 can include adapter sequence 951, barcode sequence 912 and capture sequence 953, wherein capture sequence 953 includes a different sequence than capture sequence 913 and 923.
  • nucleic acid barcode molecules e.g., 910, 920, 950
  • nucleic acid barcode molecules 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-10D.
  • methods include analysis of multiple analytes, e.g., without limitation nucleic acids, including without limitation DNA, whole transcriptome RNA, and/or target nucleic acid sequences; cell features, e.g. without limitation cell surface proteins, membrane lipids; and one or more analytes using labelling agents described herein.
  • capture sequence 1023 can be complementary to a capture handle sequence 1013 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).
  • the cells can be further processed prior to barcoding.
  • processing steps can include one or more washing and/or cell sorting steps.
  • 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).
  • the partition includes at most a single cell bound to labelling agent 1010.
  • reporter oligonucleotide 1020 conjugated to labelling agent 1010 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).
  • reporter oligonucleotide 1020 includes one or more additional functional sequences, such as those described elsewhere herein.
  • Barcoded nucleic acid molecules can be generated in various reactions that include any of the labeling compositions of the invention under suitable conditions and in the presence of reagents that permit nucleic acid reactions.
  • barcoded nucleic acid molecules can be generated in reactions (e.g., via a nucleic acid reaction, such as nucleic acid extension, reverse transcription, ligation or any combination thereof) that include the reagents and steps described in FIGS. 10A-10D.
  • capture handle sequence 1013 can 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).
  • cell barcode for example, common barcode, e.g., partition- specific barcode
  • reporter barcode sequence 1012 or a reverse complement thereof.
  • the nucleic acid barcode molecule 1090 e.g., partition-specific barcode molecule
  • the nucleic acid barcode molecule 1090 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. While in some embodiments, a poly-T capture sequence is described, other targeted and/or random capture sequences may also be used in capturing a capture handle sequence, and/or for priming a reverse transcription reaction.
  • analysis of multiple analytes can be performed.
  • the workflow can include a workflow as generally depicted in any of FIGS. 10A-10D, or a combination of workflows for an individual analyte, as described elsewhere herein.
  • multiple analytes can be analyzed.
  • analysis of an analyte 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.
  • 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.
  • 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 functional 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.
  • 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.
  • the capture sequence is configured to couple to the capture handle sequence of a reporter oligonucleotide by complementarity base pairing.
  • the capture sequence is configured to couple to an mRNA analyte, wherein the capture sequence configured to couple to the mRNA analyte includes a poly-T sequence (FIG. 10C).
  • capture sequence 1023 can include a poly-T sequence and can be used to hybridize to mRNA.
  • nucleic acid barcode molecule 1090 includes capture sequence 1023 complementary to a sequence of RNA molecule 1060 from a cell.
  • 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.
  • 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.
  • the capture sequence 1023 of the nucleic acid barcode molecule 1090 includes non-templated nucleotides appended to its 3’ end and is configured to couple (e.g., hybridize) to a capture handle sequence 1013 of a reporter oligonucleotide 1020 conjugated to labelling agent 1010 (e.g., polypeptide such as an antigen, antibody, or others described elsewhere herein).
  • labelling agent 1010 e.g., polypeptide such as an antigen, antibody, or others described elsewhere herein.
  • the capture sequence 1023 of the nucleic acid barcode molecule 1090 may include non-templated guanines appended to its 3’ end.
  • the nucleic acid barcode molecule 1090 may further include a template switch oligonucleotide (TSO).
  • TSO template switch oligonucleotide
  • the nucleic acid barcode molecule 1090 may further include a unique molecule identifier (UMI).
  • UMI unique molecule identifier
  • the hybridization of the capture sequence 1023 to the capture handle sequence 1013 extends reverse transcription of the hybridization product into the reporter oligonucleotide 1020 to generate a barcoded nucleic acid product including the capture sequence 1023, the capture handle sequence 1013, the functional sequences 1021 1011, and the reporter barcode sequence (e.g., UMI and/or TSO) 1012 1022.
  • UMI unique molecule identifier
  • capture sequence 1023 can be complementary to an overhang sequence or an adapter sequence that has been appended to an analyte.
  • capture sequence 1023 is complementary to a sequence that has been appended to a nucleic acid molecule derived from an analyte of interest.
  • 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.
  • 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.
  • 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.
  • 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.
  • primer 1050 includes a poly-T sequence.
  • primer 1050 includes a sequence complementary to a target sequence in an RNA molecule.
  • 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.
  • complementary molecule 1070 can be cDNA generated in a reverse transcription reaction.
  • an additional sequence can be appended to complementary molecule 1070.
  • 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.
  • 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).
  • 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.
  • 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).
  • cell barcode e.g., common barcode or partition- specific barcode
  • 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.
  • nucleic acid reactions e.g., nucleic acid extension, reverse transcription, ligation or any combination thereof
  • barcode capture reaction(s) are carried in a partition.
  • nucleic acid reactions, which occur after barcodes are captured in a partition are carried out outside of the partition.
  • nucleic acid reactions are carried out in bulk.
  • biological particles e.g., cells, nuclei
  • a plurality of samples e.g., a plurality of subjects
  • biological particles 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).
  • Gene expression data can reflect the underlying genome and mutations and structural variants therein.
  • 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.
  • ABM e.g., antibodies or B cell receptors
  • ABM can be used to infer germline alleles from unrelated individuals, which information may be used for demultiplexing.
  • barcoding of a nucleic acid molecule may be done using a combinatorial approach.
  • one or more nucleic acid molecules (which may be comprised in a biological particle, e.g., without limitation a cell, e.g., without limitation a fixed cell, a permeabilized cell, organelle, fixed nucleus, permeabilized 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 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • combinatorial barcoding comprising three operations, each with 100 partitions, may yield up to 10 6 unique barcode combinations.
  • the combinatorial barcode approach may be helpful in determining whether a partition contained only one cell or more than one cell.
  • 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.
  • combinatorial barcoding may be achieved in the same compartment.
  • 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.
  • 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.
  • 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.
  • a partition is a volume wherein diffusion of contents beyond the volume is inhibited
  • the partition contains a diffusion resistant material.
  • a 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.
  • 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.
  • 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.
  • 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.
  • further processing e.g., generation of barcoded nucleic acid molecules, may be performed in the diffusion resistant partition or in bulk.
  • 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.
  • 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.
  • some embodiments of the present disclosure relate to methods for preparing labelling compositions.
  • the methods for preparing labelling compositions disclosed herein that involve intramolecular ligation using a splint nucleic acid molecule (e.g., a splint oligonucleotide).
  • the splint nucleic acid molecule may comprise a first annealing sequence and a second annealing sequence positioned adjacently to each other.
  • the splint nucleic acid molecule may comprise one, two, three, or more intervening nucleotide residues between the first and second annealing sequences.
  • the splint nucleic acid molecule may comprise modified oligonucleotides.
  • Modified nucleotides include, but are not limited to, 2-Aminopurine, 2,6-Diaminopurine (2- Amino-dA), inverted dT, 5-Methyl dC, 2’ -deoxyinosine, Super T (5-hydroxybutynl-2’- deoxyuridine), Super G (8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlocked nucleic acids (UNAs, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, and 2’ Fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and Fluoro G).
  • the methods for preparing labelling compositions disclosed herein include: (a) providing a splint oligonucleotide comprising a first annealing region and a second annealing region; (b) hybridizing the provided splint oligonucleotide with: (i) a core support attached an anchor nucleic acid molecule comprising a sequence capable of hybridizing to (e.g., having sufficient sequence complementarity to) the first annealing region of the splint oligonucleotide; and (ii) a reporter oligonucleotide comprising (1) a reporter barcode sequence, (2) a capture handle sequence, and (3) a sequence capable of hybridizing to (e.g., having sufficient sequence complementarity to) the second annealing region of the splint oligonucleotide; and (c) ligating the anchor nucleic acid molecule and the reporter oligonucleotide hybridized adjacent
  • the methods for preparing labelling compositions disclosed herein include: (a) providing a reaction mixture including (i) a core support, (ii) a detectable label (e.g., a fluorophore (“FL”) comprising a plurality of sites for chemical conjugation, (ii) a core support (“core”) configured for attachment to a chemical conjugation site of the detectable label, and (iii) a nucleic acid molecule configured for attachment to a chemical conjugation site of the detectable label, wherein the nucleic acid molecule includes: (a) a reporter oligonucleotide including (i) a reporter barcode sequence and (ii) a capture handle sequence; and (b) carrying out a 3-way conjugation reaction to generate a labelling composition comprising a detectable label directly attached to the core support and the nucleic acid molecule (see, e.g., FIG. 11D).
  • a detectable label e.g., a fluorophore (“FL”) comprising a
  • the first annealing region is adjacent to the second annealing region.
  • the splint nucleic acid molecule includes one, two, three, or more intervening nucleotide residues between the first and second annealing sequences.
  • sufficient sequence complementarity generally refers to a level of sequence complementarity sufficient that permits an annealing region splint oligonucleotide to specifically bind to its cognate target sequence (e.g., a cognate target sequence in the anchor nucleic acid molecule or a cognate target sequence in the reporter oligonucleotide) and form a double- strand hybrid.
  • cognate target sequence e.g., a cognate target sequence in the anchor nucleic acid molecule or a cognate target sequence in the reporter oligonucleotide
  • the complementarity of the annealing region splint oligonucleotide and its target cognate sequence in the anchor nucleic acid molecule or in the reporter oligonucleotide is 100%, at least 99%, or at least 98%, or at least 97%, or at least 96%, or at least 95%, or at least 94%, or at least 93%, or at least 92%, or at least 91%, or at least 90%, or at least 89%, or at least 88%, or at least 87%, or at least 86%, or at least 85%, or at least 84%, or at least 83%, or at least 82%, or at least 81%, or at least 80%, or at least 79%, or at least 78%, or at least 77%, or at least 76%, or at least 75%, or at least 74%, or at least 73%, or at least 72%, or at least 71%, or at least 70%.
  • the complementarity of the annealing region splint oligonucleotide and its target cognate sequence in the anchor nucleic acid molecule or in the reporter oligonucleotide is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%).
  • Non-limiting exemplary embodiments of the methods of preparing labelling compositions disclosed herein can include one or more of the following features.
  • the methods further include (d) coupling an antigen to the core support.
  • the antigen is coupled the core support wherein the reporter barcode sequence identifies the antigen.
  • the target antigen is an oligopeptide, a protein, a polysaccharide, a lipid, a liposome, an infectious agent, or a target MHC molecule complex.
  • the target MHC molecule complex is a peptide-MHC (pMHC) molecule complex.
  • the core support is further coupled to a detectable label.
  • detectable labels suitable for the methods disclosed herein include fluorophores, magnetic particles, and mass tags.
  • the detectable label includes a fluorophore molecule.
  • the fluorophore is or includes one or more of the following: phycoerythrin (PE), allophycocyanin (APC), Alexa Fluor 405, Pacific Blue, Alexa Fluor 488, Alexa Fluor 647, Alexa Fluor 700, DyEight 405, DyLight 550, DyEight 650, fluorescein isothiocyanate (FITC), peridinin chlorophyll protein (PerCP), StarBright Violet 440, StarBright Violet 515, StarBright 610, StarBright Violet 670, and StarBright Blue 700.
  • PE phycoerythrin
  • APC allophycocyanin
  • Alexa Fluor 405 Pacific Blue
  • Alexa Fluor 488 Alexa Fluor 647
  • Alexa Fluor 700 Alexa Fluor 700
  • DyEight 405 DyLight 550
  • DyEight 650 fluorescein isothiocyanate
  • PerCP peridinin chlorophyll protein
  • StarBright Violet 440 StarBright Violet 5
  • the core support includes a biotin-binding agent.
  • the biotin-binding agent is or includes a biotin-binding protein selected from streptavidin, avidin, deglycosylated avidin (e.g., Neu tr AvidinTM), traptavidin, tamavidin, xenavidin, bradavidin, AVR2, AVR4, and variants, mutants, derivatives, and homologs of any thereof.
  • the anchor nucleic acid molecule and the reporter oligonucleotide hybridized adjacently on the splint oligonucleotide may be operably linked to one another to form a continuous oligonucleotide attached to the core support.
  • the anchor nucleic acid molecule and the reporter oligonucleotide hybridized adjacently on the splint oligonucleotide may be covalently linked to one another via an enzymatic ligation reaction, e.g., using a ligase to generate a continuous ligation product attached to the core support.
  • Non-limiting examples of ligases suitable for the methods of the disclosure include SplintR® ligase, Taq DNA ligase, Ecoli DNA ligase, T3 DNA ligase, 9°NTM DNA Ligase, Tth DNA Ligase, Tma DNA Ligase, Tfi DNA Ligase, and Tsc DNA ligase, T7 DNA ligase, T4 DNA ligase, Acanthocystis turfacea chlorella virus 1 (ATCV-l) ligase, Mu polymerase, PBCV1 enzyme, T4 RNA ligase 2, and Thermoccocus Kodakarensis RNA Ligase (KODlRnl).
  • the ligase used for intramolecular ligation using a splint oligonucleotide is a T4 DNA ligase, e.g., an ATCV1 ligase.
  • This Example describes the results of experiments evaluating performance of some labelling reagents of the disclosure in an exemplary antigen mapping workflow.
  • Test 1 two exemplary labelling compositions having the configuration as depicted in FIG. 11A were prepared by directly conjugating a core support (e.g., a biotin-binding agent, e.g., streptavidin) and a reporter oligonucleotide comprising a reporter barcode sequence to a first detectable label (e.g., a fluorophore, e.g., PE).
  • a core support e.g., a biotin-binding agent, e.g., streptavidin
  • a reporter oligonucleotide comprising a reporter barcode sequence
  • a first detectable label e.g., a fluorophore, e.g., PE
  • pMHC complex reagents were prepared using CMV peptide (e.g., NLVPMVATV, SEQ ID NO: 3), biotinylated MHC, and either the Test 1, Test 2, Control 1, or Control 2 compositions described above. Tn addition, the pMHC complex reagents were incubated with the capture probe comprising a second detectable label spectrally distinct from the first detectable label (e.g., comprising Alexa488) to hybridize the capture probe to the pMHC complex reagents.
  • CMV peptide e.g., NLVPMVATV, SEQ ID NO: 3
  • biotinylated MHC e.g., NLVPMVATV, SEQ ID NO: 3
  • the pMHC complex reagents were incubated with the capture probe comprising a second detectable label spectrally distinct from the first detectable label (e.g., comprising Alexa488) to hybridize the capture probe to the pMHC complex reagents
  • Human CMV expanded T cells (Cellero, HLA-A*0201, Donor 401, Catalog number 1049, Lot number 4982DE20) were stained with the prepared pMHC complex reagents hybridized to the Alexa 488 capture probe according to manufacturer's (Tetramer Shop) instructions. After staining, excess capture probe was removed by washing.
  • Stained cells were sorted by FACS, gating according to the first detectable label, e.g., gating on PE+ events), and gating using low, medium, and high levels of signal from the second detectable label (e.g., Alexa 488), which correspond to low, medium, and high amounts of the reporter oligonucleotide attached to the Test 1, Test 2, Control 1, or Control 2 labelling compositions.
  • the second detectable label e.g., Alexa 488
  • Higher Alexa 488 signal indicates greater labelling of the reagent with reporter oligonucleotide, while lower signal is indicative of reporter oligonucleotide loss during sorting (see, e.g., FIGS.12A-12D).
  • the core support e.g., biotin- binding agent, e.g., streptavidin
  • This Example describes the results of experiments performed to test the impact of barcoded pMHC multimer reagent molecules: cell ratios during cell labeling, on an end to end high-throughput single-cell workflow for pMHC antigen/TCR mapping.
  • Multimer reagent assembly A PE-Streptavidin-barcode conjugate was mixed with biotinylated allele specific MHC monomer (HLA-A02:01) in a suitable reaction buffer and either loaded with CMV peptide, Flu peptide, or HIV peptide (as negative control).
  • the assembled multimer reagents each included reporter oligonucleotides comprising different reporter barcode sequences that identify the peptide-MHC (such reporter barcode sequences are also referred to in this Example as antigen barcodes).
  • FIG. 13A shows flow cytometry plots from the cell labeling experiments, with forward side scatter shown on the Y axis and PE signal on the X axis. As shown, separation of PE positive vs. PE negative populations was clear under all conditions, with improved separation with greater barcoded multimer reagent/cell ratios.
  • FIG. 13B shows a barcode rank plot of antigen UMI counts (Y axis, log scale) by cell barcode (X axis, log scale).
  • FIG. 13C shows a plot of antigen barcode UMI counts by cell density.
  • This Example describes the results of experiments performed to test the impact of (1) the number of barcoded antigen reagent molecules/cell ratios during cell labeling and (2) antigen: barcoded streptavidin ratios during barcoded reagent assembly, on an end to end high throughput single-cell workflow for antigcn/BCR mapping.
  • Splenocyte preparation splenocytes from transgenic mice that recognize gpl20 antigen were prepared by obtaining spleen samples from the mice, filtering the samples through a 70 pm filter, washing with cold buffer (e.g., PBS + 10% serum), centrifugation (e.g., at 300 g for 5 minutes), and lysis with ACK lysis buffer. Splenocytes were washed prior to cell counting.
  • cold buffer e.g., PBS + 10% serum
  • centrifugation e.g., at 300 g for 5 minutes
  • ACK lysis buffer e.g., ACK lysis buffer
  • FIG. 14A shows flow cytometry plots from the cell labeling experiments using barcoded antigen molecules assembled at a 4:1 antigen to conjugate ratio. Forward side scatter is shown on the Y axis and PE signal on the X axis. As shown, separation and discrimination of PE positive vs. PE negative populations improved with greater barcoded antigen reagent molecule/cell ratios. Similar results were obtained using barcoded antigen molecules at the 1:1 antigen to conjugate ratio (data not shown).
  • FIG. 14B shows a barcode rank plot of antigen UMI counts (Y axis, log scale) by cell barcode (X axis, log scale).
  • FIG. 14C shows a plot of antigen barcode UMI counts by cell density.
  • FIG. 14D shows median antigen barcode UMI counts per cell.
  • This Example describes the results of experiments performed to test the impact of (1) the number of barcoded antigen reagent molecules/cell ratios during cell labeling and (2) antigen: barcoded streptavidin ratios during barcoded reagent assembly, in an end to end high throughput single-cell workflow for antigen/BCR mapping, using a different transgenic mouse model.
  • Splenocyte preparation splenocytes from transgenic mice that recognize the foreign antigen hen egg lysozyme (HEL) were prepared by obtaining spleen samples from the mice, filtering the samples through a 70 pm filter, washing with cold buffer (e.g., PBS + 10% serum), centrifugation (e.g., at 300 g for 5 minutes), and lysis with ACK lysis buffer. Splenocytes were washed prior to cell counting. HEL splenocytes were mixed at 1:1 ratio with non-transgenic splenocytes.
  • HEL foreign antigen hen egg lysozyme
  • FIG. 15 shows flow cytometry results from the experiment. Little to no PE positive cells were detected at the 25k or 50k molecules/cell ratios. At both the 100k and 200k molecules/cell ratios, PE positive cell populations were robustly detected with clear separation. Greater separation and discrimination was observed at the 200k: 1 ratio.

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Abstract

La présente divulgation concerne de manière générale des compositions et des procédés utiles pour caractériser les molécules de liaison à l'antigène (ABM) produites par les cellules immunitaires, par exemple, les lymphocytes B et les lymphocytes T. La présente invention concerne également des réactifs et des kits pour l'identification et/ou la caractérisation des ABM.
PCT/US2023/066653 2022-05-06 2023-05-05 Réactifs pour caractériser des molécules de liaison à l'antigène à partir de cellules immunitaires WO2023215861A1 (fr)

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