WO2024015733A1 - Procédés et systèmes améliorés pour l'identification et la caractérisation de molécules de liaison à l'antigène à partir de cellules uniques - Google Patents

Procédés et systèmes améliorés pour l'identification et la caractérisation de molécules de liaison à l'antigène à partir de cellules uniques Download PDF

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WO2024015733A1
WO2024015733A1 PCT/US2023/069877 US2023069877W WO2024015733A1 WO 2024015733 A1 WO2024015733 A1 WO 2024015733A1 US 2023069877 W US2023069877 W US 2023069877W WO 2024015733 A1 WO2024015733 A1 WO 2024015733A1
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cell
nucleic acid
sequence
molecule
cells
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PCT/US2023/069877
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English (en)
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Payam Shahi
Wyatt James MCDONNELL
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10X Genomics, Inc.
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Publication of WO2024015733A1 publication Critical patent/WO2024015733A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/10—Oligonucleotides as tagging agents for labelling antibodies

Definitions

  • the present disclosure relates generally to the field of immunology, and particularly relates to improved methods and systems useful for identification and characterization of antigen-binding molecules (ABMs) obtained from biological samples, e.g., single 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.
  • reagents for antigen mapping e.g., reagents where antigens are operably coupled with nucleic acid barcode sequences that identify the antigens.
  • single-cell methods can be utilized to simultaneously identify a nucleotide sequence encoding at least a portion of an ABM expressed by a cell and detection of the antigen to which that ABM binds.
  • the immune system's ability to respond in an antigen-specific fashion against a plethora of foreign, non-self, and damage signals can introduce noise in such workflows and antigen- specific enrichment methods in a number of ways, including the non-specific binding of fluorophores, oligonucleotides, and molecular biology reagents (e.g. biotin, streptavidin) in such assays.
  • This can present a number of challenges in downstream analysis and confound interpretation of data. Further screening of immune receptors recovered by such discovery methods increases with the number of ABMs identified, so can be important to improve signal to noise in the data resulting from such workflows.
  • the present disclosure provides, inter alia, methods and systems useful for identification 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, e.g., single cells.
  • ABMs antigen-binding molecules
  • systems for the identification and/or characterization of these ABMs including without limitation determining sequences, complete and/or partial sequences, of ABMs.
  • the sequences comprise or essentially consist of CDR3s segments.
  • sequences are paired VH and VL chains from BCRs. In certain embodiments, these are paired sequences from TCRs.
  • ABSMs antigen-binding molecule
  • the methods identify uncharacterized ABM(s).
  • the disclosed methods include: (a) partitioning a reaction mixture, or a portion thereof, into a plurality of partitions, wherein the reaction mixture includes: (i) a target antigen operably coupled to a reporter oligonucleotide including a target antigen reporter barcode sequence, (ii) a plurality of cells, wherein the cells express a natural ligand of the target antigen, and (iii) a ligand blocking agent that blocks or inhibits binding of the target antigen with the natural ligand, wherein the partitioning provides a partition of the plurality of partition including: (i) a partitioned cell expressing an antigen-binding molecule (ABM), (ii) the target antigen, and (iii) a plurality of nucleic acid barcode molecules including a partition-specific barcode sequence; (b) a generating a first barcoded polynucleotide including (i) the partition barcode sequence or a reverse complement thereof and (ii) the target anti
  • Non- limiting exemplary embodiments of the methods disclosed herein can include one or more of the following features.
  • the reaction mixture is formed by a prior step of contacting the plurality of cells with the target antigen and the ligand blocking agent.
  • the target antigen is associated with a proliferative disease, an autoimmune disease, inflammatory disease, or an infectious disease.
  • the target antigen is associated with a proliferative disease selected from the group consisting of angiogenic diseases, metastatic diseases, tumorigenic diseases, neoplastic diseases, and cancers.
  • the cancer is a solid tumor, a soft tissue tumor, a metastatic lesion, or a hematological malignancy.
  • the cancer is lymphoma.
  • the lymphoma is B-cell lymphoma.
  • the target antigen is over-expressed or under-expressed in the lymphoma compared to a control non-lymphoma sample.
  • the partitioned cell has been engineered to express the ABM.
  • the ABM is a B cell receptor (BCR), an antibody (Ab), and T cell receptor (TCR), or a chimeric antigen receptor (CAR).
  • the engineered cell is a human embryonic kidney (HEK) cell, a Chinese hamster ovary cell (CHO cell), a baby hamster kidney cell (BHK), a mouse sertoli cell, a monkey kidney cell, a human cervical carcinoma cell (HeLa), a canine kidney cell (MDCK), a buffalo rat liver cell (BRL 3A), a human lung cell (W138), a human liver cell (Hep G2), a mouse mammary tumor (MMT 060562), a TRI cell, a FS4 cell, an African green monkey kidney cell (Vero cell), a human A549 cell, a human cervix cell, a human CHME5 cell, a human PER.C6 cell, a NS0 murine myeloma cell, a human epidermoid larynx cell, a human fibroblast cell, a human HUH-7 cell, a human MRC-5 cell, a human muscle cell, a human endothelial
  • the partitioned cell is an immune cell.
  • the immune cell is a B cell.
  • the B cell is a plasmablast, a plasma cell, a memory B cell, a regulatory B cell, or a lymphoplasmacytoid cell.
  • the ABM produced by the B cell is a B cell receptor (BCR), an antibody (Ab) or an antigen-binding fragment thereof.
  • the immune cell is a T cell.
  • the T cell is a CD 8+ T cytotoxic lymphocyte cell or a CD4+ T helper lymphocyte cell.
  • the CD8+ T cytotoxic lymphocyte cell is selected from the group consisting of naive CD 8+ T cells, central memory CD 8+ T cells, effector memory CD 8+ 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 ABM produced by the T cell is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).
  • the antigen is an immune checkpoint molecule, e.g., a cell surface receptor that regulates immune system.
  • the immune checkpoint molecule is an inhibitory checkpoint molecule or a stimulatory checkpoint molecule.
  • the inhibitory checkpoint molecule is selected from the group consisting of PD-1, CTLA-4, A2AR, B7-H3 (CD276), B7-H4, BTLA, CD5, CD39, CD73, CD96, CD132, CD328, CD329, IDO, KIR, LAG-3, TIM-3, TIGIT, and VISTA.
  • the stimulatory checkpoint molecule is selected from the group consisting of CD27, CD28, CD40, CD122, CD137 (4-1BB), CD226, ICOS (CD278), 0X40, and GITR.
  • the natural ligand of the antigen is a proteinaceous ligand.
  • the ligand is known or suspected to be expressed by the partitioned cell.
  • the ligand is known or expected to bind to the target antigen.
  • the blocking agent binds the target antigen and blocks or inhibits binding of the target antigen to the natural ligand. In some embodiments, the blocking agent binds the natural ligand and blocks or inhibits binding of the natural ligand to the target antigen. In some embodiments, the blocking agent is selected from the group consisting of peptides, oligopeptides, small organic molecules, peptidomimetics, and antibodies and antigen-binding fragments thereof. In some embodiments, the blocking agent binds the immune checkpoint molecule and does not modulate (e.g. , activate or inhibit) the immune checkpoint pathway.
  • the methods disclosed herein 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 disclosed herein 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. In some embodiments, the methods disclosed herein further include (i) identifying the immune cell having bound the target antigen based on the determined sequence of the second barcoded nucleic acid molecule or amplicon thereof, and (ii) identifying the ABM as expressed by the immune cell based on the determined sequence of the first barcoded nucleic acid molecule or amplicon thereof. In some embodiments, the methods further include determining a sequence of the third barcoded nucleic acid molecule or an amplicon thereof. In some embodiments, the methods further include determining a sequence of the fourth barcoded nucleic acid molecule or an amplicon thereof.
  • the target antigen is operably coupled to a detectable label.
  • the detectable label is configured for magnetic separation.
  • the detectable label includes a mass tag.
  • the detectable label includes a fluorophore molecule.
  • the fluorophore molecule is or includes phycoerythrin (PE), allophycocyanin (APC), Alexa Fluor 405, Pacific Blue, Alexa Fluor 488, Alexa Fluor 647, Alexa Fluor 700, DyLight 405, DyLight 550, DyLight 650, fluorescein isothiocyanate (FITC), peridinin chlorophyll protein (PerCP), StarBright Violet 440, StarBright Violet 515, StarBright 610, StarBright Violet 670, or StarBright Blue 700.
  • 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
  • PerCP peridinin chlorophyll protein
  • StarBright Violet 440 StarBright Violet 515, StarBright
  • the methods of the disclosure further include sorting of the immune cell according to its binding to the target antigen.
  • the sorting of the immune cell is based on detection of one or more detectable labels coupled to the target antigen.
  • systems useful for the identification and/or characterization of ABMs include: (a) a labelling reagent comprising a detectable label and a reporter oligonucleotide; and (b) a blocking agent that blocks or inhibits binding of a target antigen with a natural ligand of the target antigen.
  • Non-limiting exemplary embodiments of the systems disclosed herein can include one or more of the following features.
  • the systems of the disclosure further include instructions for coupling the target antigen to the labelling agent, and optionally instructions for use according to a method disclosed herein.
  • the reporter oligonucleotide comprises a target antigen reporter barcode sequence.
  • the systems of the disclosure further include a plurality of nucleic acid barcode molecules comprising a partition-specific barcode sequence and a capture sequence.
  • the systems further include a partitioning system for generating a partition.
  • the partitioning system comprises a microfluidic device.
  • the systems further include reagents for generating a first of a plurality of barcoded nucleic acid molecules formed by complementary base pairing of: (a) the capture sequence of the plurality of nucleic acid barcode molecules and (b) a capture handle sequence of an mRNA or DNA analyte comprising a nucleic acid sequence encoding at least a portion of the ABM.
  • the systems further include an analysis engine.
  • the systems further include a network.
  • the systems further include a sequencer.
  • 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. 7-8 schematically summarize the results from a competitive inhibition assay for detection of plasma CTLA-4 antibody from the mouse plasma samples.
  • FIG. 9 pictorially summarizes the results from an experiment demonstrating use of blocking natural ligands in high-throughput large molecule discovery and antigen mapping.
  • FIG. 10 shows percentages of cells falling into the high, medium, and low gates in the ligand-blocking experiment described in FIG. 9.
  • FIG. 11 schematically illustrates example labelling agents with nucleic acid molecules attached thereto.
  • FIGS. 12A-12D schematically depict exemplary workflow for processing nucleic acid molecules.
  • FIG. 12A schematically shows an example of labelling agents.
  • FIG. 12B schematically shows another example workflow for processing nucleic acid molecules.
  • FIG. 12C schematically shows another example workflow for processing nucleic acid molecules.
  • FIG. 12D schematically shows another example workflow for processing nucleic acid molecules.
  • FIG. 13 schematically shows another example of a barcode-carrying bead.
  • FIG. 14 shows an exemplary microfluidic channel structure for delivering barcode carrying beads to droplets.
  • the present disclosure provides, inter alia, methods and systems useful for identification 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, e.g., single cells.
  • ABSMs antigen-binding molecules
  • TCRs T cell receptors
  • Abs TCR-like antibodies
  • Characterization of AB Ms having desirable properties, e.g., that recognize and bind to cells displaying cancer-associated antigens or viral antigens can be useful in the development of new immunotherapies to treat cancers and/or infectious disease.
  • systems for the identification and/or characterization of these ABMs are also provided in some embodiments of the disclosure.
  • noise may be introduced in high throughput ABM discovery workflows and antigen-specific enrichment methods in a number of ways, including the nonspecific binding of fluorophores, oligonucleotides, and molecular biology reagents (e.g. biotin, streptavidin) in such assays. This can present a number of challenges in downstream analysis and confound interpretation of the data.
  • a cell includes one or more cells, including mixtures thereof.
  • a and/or B is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”.
  • adaptor(s) can be used synonymously.
  • An adaptor or tag can be coupled to a polynucleotide sequence to be “tagged” by any approach, including ligation, hybridization, or other approaches.
  • 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.
  • 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).
  • the term “bead,” as used herein, generally refers to a particle.
  • the bead may be a solid or semi-solid particle.
  • the bead may be a gel bead.
  • the gel bead may include a polymer matrix (e.g. , matrix formed by polymerization or cross-linking).
  • the polymer matrix may include one or more polymers (e.g., polymers having different functional groups or repeat units). Polymers in the polymer matrix may be randomly arranged, such as in random copolymers, and/or have ordered structures, such as in block copolymers. Cross-linking can be via covalent, ionic, or inductive, interactions, or physical entanglement.
  • the bead may be a macromolecule.
  • the bead may be formed of nucleic acid molecules bound together.
  • the bead may be formed via covalent or non-covalent assembly of molecules (e.g. , macromolecules), such as monomers or polymers.
  • Such polymers or monomers may be natural or synthetic.
  • Such polymers or monomers may be or include, for example, nucleic acid molecules (e.g., DNA or RNA).
  • the bead may be formed of a polymeric material.
  • the bead may be magnetic or non-magnetic.
  • the bead may be rigid.
  • the bead may be flexible and/or compressible.
  • the bead may be disruptable or dissolvable.
  • the bead may be a solid particle (e.g. , a metal-based particle including but not limited to iron oxide, gold or silver) covered with a coating comprising one or more polymers. Such coating may be disruptable or dissolvable.
  • 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.
  • sample generally refers to a biological sample of a subject.
  • the biological sample may comprise any number of macromolecules, for example, cellular macromolecules.
  • the sample may be a cell sample.
  • the sample may be a cell line or cell culture sample.
  • the sample can include one or more cells.
  • the sample can include one or more microbes.
  • the biological sample may be a nucleic acid sample or protein sample.
  • the biological sample may also be a carbohydrate sample or a lipid sample.
  • the biological sample may be derived from another sample.
  • the sample may be a tissue sample, such as a biopsy, core biopsy, needle aspirate, or fine needle aspirate.
  • the sample may be a fluid sample, such as a blood sample, urine sample, or saliva sample.
  • the sample may be a skin sample.
  • the sample may be a cheek swab.
  • the sample may be a plasma or serum sample.
  • the sample may be a cell-free or cell free sample.
  • a cell-free sample may include extracellular polynucleotides. Extracellular polynucleotides may be isolated from a bodily sample that may be selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool and tears.
  • sequence of nucleotide bases in one or more polynucleotides generally refers to methods and technologies for determining the sequence of nucleotide bases in one or more polynucleotides.
  • the polynucleotides can be, for example, nucleic acid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (e.g. , single stranded DNA). Sequencing can be performed by various systems currently available, such as, without limitation, a sequencing system by Illumina®, Pacific Biosciences (PacBio®), Oxford Nanopore®, or Life Technologies (Ion Torrent®).
  • sequencing may be performed using nucleic acid amplification, polymerase chain reaction (PCR) (e.g., digital PCR, quantitative PCR, or real time PCR), or isothermal amplification.
  • PCR polymerase chain reaction
  • Such systems may provide a plurality of raw genetic data corresponding to the genetic information of a subject (e.g., human), as generated by the systems from a sample provided by the subject.
  • sequencing reads also “reads” herein).
  • a read may include a string of nucleic acid bases corresponding to a sequence of a nucleic acid molecule that has been sequenced.
  • systems and methods provided herein may be used with proteomic information.
  • 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.
  • 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.
  • one aspect of the disclosure relates to new approaches and methods for the identification and/or characterization of antigen-binding molecules (ABMs), e.g. , antibodies (Abs), B cell receptors, T cell receptors (TCRs), and TCR-like antibodies, obtained from biological samples, such as cells, e.g., single cells.
  • ABSMs antigen-binding molecules
  • the methods and systems provided herein may identify and/or characterize an ABM by identifying it as having particular nucleic acid sequences(s) and/or having particular amino acid sequence(s).
  • the methods and systems provided herein may further, or alternatively, identify and/or characterize an ABM as binding to and/or having affinity for a target antigen, e.g., and further having specific binding to and/or affinity for the target antigen (e.g., on- target binding).
  • the disclosed methods include: (a) partitioning a reaction mixture, or a portion thereof, into a plurality of partitions, wherein the reaction mixture includes: (i) a target antigen operably coupled to a reporter oligonucleotide including a target antigen reporter barcode sequence, (ii) a plurality of cells, wherein the cells express a natural ligand of the target antigen, and (iii) a ligand blocking agent that blocks or inhibits binding of the target antigen with the natural ligand, wherein the partitioning provides a partition of the plurality of partition including: (i) a partitioned cell expressing an antigen-binding molecule (ABM), (ii) the target antigen, and (iii) a plurality of nucleic acid barcode molecules including a partition- specific barcode sequence; (b) a generating a first barcoded polynucleotide including (i) the partition barcode sequence or a reverse complement thereof and (ii
  • Non- limiting exemplary embodiments of the methods disclosed herein can include one or more of the following features.
  • the reaction mixture is formed by a prior step of contacting the plurality of cells with the target antigen and the ligand blocking agent.
  • the target antigen and the ligand blocking agent may be contacted with the plurality of cells simultaneously or sequentially.
  • the blocking agent is capable of binding the target antigen and further capable of blocking/inhibiting binding of the target antigen to the natural ligand
  • the target antigen may first be placed in contact with the blocking agent, and the cells expressing the natural ligand are then provided.
  • the cells expressing the natural ligand may first be placed in contact with the blocking agent, and the target antigen is then provided. In some embodiments, the cells expressing the natural ligand are simultaneously contacted with the target antigen and the ligand blocking agent.
  • the plurality of cells in step (a) comprises single cells.
  • Suitable methods, compositions, systems, and kits for single cell analysis of antigen-binding molecules produced by immune cells and/or antigen binding activity are disclosed in US20180105808A1, US20180179590A1, US20190338353A1, and US20190367969A1, each of which are incorporated by reference herein in their entirety.
  • Cells suitable for the methods and systems of the present disclosure can be: (i) cells that naturally express at least one ABM; (ii) cells that naturally do not endogenously express an ABM but have been engineered to heterologously express the ABM; or (iii) cells that naturally express a first ABM and have been engineered to express a second ABM heterologously.
  • Exemplary ABMs suitable for the methods and systems of the disclosure include B cell receptors (BCRs), antibodies (Abs), T cell receptors (TCRs), chimeric antigen receptors (CARs), and antigen-binding fragments of any thereof.
  • Examples of cells that may be suitably engineered for expression of ABMs include mammalian cells.
  • mammalian cells suitable for the methods and systems of the disclosure include human embryonic kidney (HEK) cell, Chinese hamster ovary cell (CHO cell), baby hamster kidney cell (BHK), mouse sertoli cell, monkey kidney cell, human cervical carcinoma cell (HeLa), canine kidney cell (MDCK), buffalo rat liver cell (BRL 3A), human lung cell (W138), human liver cell (Hep G2), mouse mammary tumor (MMT 060562), TRI cell, FS4 cell, African green monkey kidney cell (Vero cell), human A549 cell, human cervix cell, human CHME5 cell, human PER.C6 cell, NSO murine myeloma cell, human epidermoid larynx cell, human fibroblast cell, human HUH-7 cell, human MRC-5 cell, human muscle cell, human endothelial cell, human astrocyte cell, human macrophage cell
  • HEK human
  • the engineered cell is a HEK cell.
  • the HEK cell is a HEK 293 cell, a HEK 293T cell, a HEK 293S cell, or a HEK 293 EBNA cell.
  • the engineered cell is a CHO cell.
  • the partitioned cell naturally expresses an ABM.
  • the partitioned cell may be an immune cell.
  • the immune cell is a B cell.
  • B cells suitable for the methods and systems of the disclosure include plasmablast, plasma cell, memory B cell, regulatory B cell, and a lymphoplasmacytoid cell.
  • AB Ms produced by B cells include B cell receptors (BCR) and antibodies (Ab).
  • BCR B cell receptors
  • Ab antibodies
  • the ABM produced by the partitioned B cell is selected from BCR, antibody, or an antigen-binding fragment of any thereof.
  • the ABM may be an antibody, or an antigen-binding fragment thereof.
  • the ABM identified or characterized by the methods herein may be an antibody having an Immunoglobulin (Ig)A (e.g., IgAl or IgA2), IgD, IgE, IgG e.g., IgGl, IgG2, IgG3 and IgG4) or IgM constant region.
  • IgAl or IgA2 Immunoglobulin
  • IgG immunoglobulin
  • IgG immunoglobulin 2
  • IgM constant region e.g., IgGl, IgG2, IgG3 and IgG4
  • the ABM may be a fragment of the antibody that may be any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
  • An ABM that is a fragment of an antibody may be one of: (i) Fab fragments; (ii) F(ab')2 fragments; (hi) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) sdAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g. , an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FWR3-CDR3-FWR4 peptide.
  • CDR complementarity determining region
  • an antigen-binding fragment of an antibody may be an engineered molecule, such as a domain-specific antibody, single domain antibody, chimeric antibody, CDR-grafted antibody, diabody, triabody, tetrabody, minibody, nanobody (e.g., monovalent nanobodies, bivalent nanobodies, etc.), a small modular immunopharmaceutical (SMIP), or a shark immunoglobulin new antigen receptor (IgNAR) variable domain.
  • SMIP small modular immunopharmaceutical
  • IgNAR shark immunoglobulin new antigen receptor
  • the immune cell is a T cell.
  • the T cell is a CD 8+ T cytotoxic lymphocyte cell or a CD4+ T helper lymphocyte cell.
  • the CD8+ T cytotoxic lymphocyte cell is selected from the group consisting of naive CD 8+ T cells, central memory CD 8+ T cells, effector memory CD 8+ 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 ABM produced by the T cell is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).
  • the ABM characterized by the methods provided herein may be characterized by its having bound to, or having binding affinity for, a target antigen.
  • the target antigen may be any antigen for which the characterization and/or identification of an ABM capable of binding or as having an affinity thereto is desirable.
  • the target antigen may be of a length of at least 20 amino acid residues, at least 40 amino acid residues, at least 60 amino acid residues, at least 80 amino acid residues, at least 100 amino acid residues, at least 200 amino acid residues, at least 300 amino acid residues, at least 400 amino acid residues, at least 500 amino acid residues, at least 600 amino acid residues, at least 700 amino acids, at least 800 amino acid residues, at least 900 amino acid residues, at least 1000 amino acid residues, at least 1100 amino acid residues, at least 1200 amino acid residues, at least 1300 amino acid residues, up to 40 amino acid residues, up to 60 amino acid residues, up to 80 amino acid residues, up to 100 amino acid residues, up to 200 amino acid residues, up to 300 amino acid residues, up to 400 amino acid residues, up to 500 amino acid residues, up to 600 amino acid residues, up to 700 amino acids, up to 800 amino acid residues, up to 900 amino acid residues, up to
  • the target antigen may be an antigen that includes one domain, at least one domain, two domains, at least two domains, three domains, at least three domains, four domains, at least four domains, five domains, at least five domains, six domains, at least six domains, seven domains, at least seven domains, eight domains, at least eight domains, nine domains, at least nine domains, ten domains, at least ten domains, at least thirty domains, at least forty domains, at least fifty domains, at least sixty domains, at least seventy domains, at least eighty domains, at least ninety domains or at least one hundred domains.
  • the target antigen may be an antigen that includes at most two hundred domains, at most 175 domains, at most 150 domains, at most 125 domains, at most 100 domains, at most 75 domains, at most 50 domains, at most 25 domains, at most 20 domains, at most 15 domains, at most 10 domains, or at most 5 domains.
  • the target antigen may be a protein or peptide as expressed by a cell, e.g. , full-length target antigen that may or may not include its leader sequence and may or may not have undergone a similar cell-processing step.
  • target antigens suitable for the methods and systems of the disclosure.
  • suitable target antigens include, but are not limited to, antigens associated with proliferative diseases, autoimmune diseases, inflammatory diseases, and infectious diseases.
  • the target antigen is associated with an autoimmune disease.
  • autoimmune diseases suitable for the methods and systems of the disclosure include, but are not limited to, rheumatoid arthritis, osteoarthritis, Still’s disease, Familiar Mediterranean Fever, systemic sclerosis, multiple sclerosis, ankylosing spondylitis, Hashimoto’s tyroidism, systemic lupus erythematosus, Sjogren's syndrome, diabetic retinopathy, diabetic vasculopathy, diabetic neuralgia, insulitis, psoriasis, alopecia greata, warm and cold autoimmune hemolytic anemia (AIHA), pernicious anemia, acute inflammatory diseases, autoimmune adrenalitis, chronic inflammatory demyelinating polyneuropathy (CIDP), Lambert-Eaton syndrome, lichen sclerosis, Lyme disease, Graves disease, Behcet's disease, Meniere’s disease, reactive arthritis (Reiter's syndrome
  • the target antigen may be an antigen associated with an infectious agent, such as a viral, bacterial, parasitic, protozoal, or prion agent.
  • the viral agent may be severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East Respiratory Syndrome (MERS), human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis B virus (HCV), Cytomegalovirus (CMV), respiratory syncytial virus (RSV), human papillomavirus (HPV), Epstein-Barr virus (EBV), influenza virus, and Ebola virus.
  • SARS-CoV2 severe acute respiratory syndrome coronavirus 2
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • MERS Middle East Respiratory Syndrome
  • HAV human immunodeficiency virus
  • HBV hepatitis B virus
  • HCV hepatitis B virus
  • HCV Cytome
  • Additional infections suitable for the methods of the disclosure include infections with intracellular parasites such as Leishmania, Rickettsia, Chlamydia, Coxiella, Plasmodium, Brucella, mycobacteria, Listeria, Toxoplasma and Trypanosoma.
  • intracellular parasites such as Leishmania, Rickettsia, Chlamydia, Coxiella, Plasmodium, Brucella, mycobacteria, Listeria, Toxoplasma and Trypanosoma.
  • the target antigen is associated with an inflammatory disease.
  • inflammatory diseases suitable for the methods and systems of the disclosure include inflammatory diseases such as asthma, inflammatory bowel disease (IBD), chronic colitis, splenomegaly, and rheumatoid arthritis.
  • the target antigen is associated with a proliferative disease selected from the group consisting of angiogenic diseases, metastatic diseases, tumorigenic diseases, neoplastic diseases, and cancers.
  • exemplary proliferative diseases can include, without limitation, angiogenic diseases, a metastatic diseases, tumorigenic diseases, neoplastic diseases and cancers.
  • the proliferative disease is a cancer.
  • the term “cancer” generally refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. The aberrant cells may form solid tumors or constitute a hematological malignancy. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body.
  • the cancer may be a solid tumor, a soft tissue tumor, a metastatic lesion, or a hematological malignancy.
  • suitable cancers include ovarian cancer, renal cancer, breast cancer, prostate cancer, liver cancer, brain cancer, lymphoma, leukemia, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, lung cancer and the like.
  • cancers that can be suitable for the methods and systems of the present disclosure include, but are not limited to, acute myeloblastic leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelocytic leukemia (CML), adrenal cortical cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain cancers, central nervous system (CNS) cancers, peripheral nervous system (PNS) cancers, breast cancer, cervical cancer, colon and rectum cancer, endometrial cancer, esophagus cancer, Ewing's family of tumors (e.g.
  • Ewing's sarcoma eye cancer, transitional cell carcinoma, vaginal cancer, myeloproliferative disorders, nasal cavity and paranasal cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Non-Hodgkin's lymphoma, Hodgkin's lymphoma, childhood Non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, lung carcinoid tumors, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, rhabdomyo
  • cancers include, but are not limited to, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, mesothelioma, leukemia, lymphoma, brain cancer, prostate cancer, multiple myeloma, melanoma, bladder cancer, bone sarcomas, soft tissue sarcomas, retinoblastoma, renal tumors, neuroblastoma, and carcinomas.
  • the cancer is a multiply drug resistant cancer or a recurrent cancer. It is contemplated that the compositions and methods disclosed here are suitable for both non- metastatic cancers and metastatic cancers. Accordingly, in some embodiments, the cancer is a non-metastatic cancer.
  • the cancer is a metastatic cancer.
  • the target antigen may be associated with a tumor or a cancer.
  • it may be, for example, epidermal growth factor receptor (EGFR), CD38, platelet- derived growth factor receptor (PDGFR) alpha, insulin growth factor receptor (IGFR), CD20, CD19, CD47, or human epidermal growth factor receptor 2 (HER2).
  • the cancer is lymphoma. Both Hodgkin lymphoma and non-Hodgkin lymphoma are suitable. Exemplary Hodgkin lymphoma include, but are not limited to, odular sclerosing, mixed cellularity, lymphocyte rich, lymphocyte depleted, or lymphocyte -predominant. In some embodiments, the non-Hodgkin lymphoma may be follicular lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid tissue, marginal zone, diffuse large B cells, Burkitt, or mantle cells.
  • the lymphoma may be, specifically, a malignant tumor of B cell origin, which may be at least one selected from the group consisting of non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and acute lymphoblastic leukemia.
  • the lymphoma is B-cell lymphoma.
  • the target antigen may be over-expressed in the lymphoma compared to a control non- lymphoma sample.
  • the target antigen may be underexpressed in the lymphoma compared to a control non-lymphoma sample.
  • the antigen may be an immune checkpoint molecule, e.g., a cell surface receptor that regulates immune system. Both inhibitory checkpoint molecules and stimulatory checkpoint molecules can be suitable target antigens. Accordingly, in some embodiments, the target antigen may be an inhibitory checkpoint molecule.
  • Non-limiting examples of inhibitory checkpoint molecules suitable for the methods and systems of the disclosure include PD-1, CTLA-4, A2AR, B7-H3 (CD276), B7-H4, BTLA, CD5, CD39, CD73, CD96, CD132, CD328, CD329, IDO, KIR, LAG-3, TIM-3, TIGIT, and VISTA.
  • the inhibitory checkpoint molecule may be CTLA-4.
  • the inhibitory checkpoint molecule may be PD-1.
  • the target antigen may be a stimulatory checkpoint molecule.
  • stimulatory checkpoint molecules suitable for the methods and systems of the disclosure include CD27, CD28, CD40, CD122, CD137 (4-1BB), CD226, ICOS (CD278), 0X40, and GITR.
  • the partitioned cell(s) may express one or more natural ligands of the target antigen.
  • the natural ligand of the target antigen is a proteinaceous ligand, for example, a polypeptide or an oligopeptide.
  • the natural ligand is a secreted protein.
  • the natural ligand is cell-surface protein.
  • the ligand is known or suspected to be expressed by the partitioned cell.
  • the ligand is known or expected to bind to the target antigen.
  • the natural ligand comprises any one of the following: CD155, CD112 (PVRL2), HMG-1, CEACAM-1 (CD66a), LAG-3, class II MHC molecules, and TLR9 (CD289).
  • the blocking agent binds the target antigen and blocks or inhibits binding of the target antigen to the natural ligand. In some embodiments, the blocking agent binds the natural ligand and blocks or inhibits binding of the natural ligand to the target antigen. In some embodiments, the blocking agent is selected from the group consisting of peptides, oligopeptides, small organic molecules, peptidomimetics, and antibodies and antigen-binding fragments thereof. In particular embodiments, the blocking agent is a blocking antibody that binds the natural ligand. In some embodiments, the blocking agent binds the immune checkpoint molecule and does not modulate (e.g. , activate or inhibit) the immune checkpoint pathway.
  • the immune cell natural ligand can be TLR9.
  • the blocking agent can be or can comprise single- stranded salmon sperm DNA.
  • the methods disclosed herein 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 disclosed herein 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. In some embodiments, the methods disclosed herein further include (i) identifying the immune cell having bound the target antigen based on the determined sequence of the second barcoded nucleic acid molecule or amplicon thereof, and (ii) identifying the ABM as expressed by the immune cell based on the determined sequence of the first barcoded nucleic acid molecule or amplicon thereof. In some embodiments, the methods further include determining a sequence of the third barcoded nucleic acid molecule or an amplicon thereof. In some embodiments, the methods further include determining a sequence of the fourth barcoded nucleic acid molecule or an amplicon thereof.
  • the target antigen is operably coupled to a detectable label.
  • the detectable label is configured for magnetic separation.
  • the detectable label includes a mass tag.
  • the detectable label includes a fluorophore molecule.
  • 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
  • PerCP peridinin chlorophyll protein
  • StarBright Violet 440 StarBright
  • some embodiments of the methods of the disclosure may further include sorting of the immune cell according to its binding to the target antigen using one or more suitable cell separation and/or isolation techniques, for example, flow cytometry-based cell sorting techniques.
  • the sorting of the immune cell is based on detection of one or more detectable labels coupled to the target antigen.
  • the methods and systems 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 partition can be a droplet in an emulsion or a well.
  • a partition may comprise one or more other partitions.
  • 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 comprise one or more biological particles and/or macromolecular constituents thereof.
  • a partition may comprise one or more beads.
  • a partition may comprise one or more gel beads.
  • a partition may comprise 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, may be injected into the droplets previous to, subsequent to, or concurrently with droplet generation, such as via a 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 (
  • 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.
  • partitions such as droplets and/or emulsions as described herein.
  • 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., co-partitioned 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 may comprise, for example, micro-vesicles that have an outer barrier surrounding an inner fluid center or core.
  • the partitions may comprise a porous matrix that is capable of entraining and/or retaining materials within its matrix.
  • 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 may be provided in a water- in-oil emulsion or oil-in-water emulsion.
  • a variety of different vessels are described in, for example, U.S. Patent Application Publication No. 2014/0155295, which is entirely incorporated herein by reference for all purposes.
  • 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, which is entirely incorporated herein by reference for all purposes.
  • 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.
  • 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 may 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 may contain at most one biological particle (e.g. , bead, DNA, cell or cellular material).
  • the various parameters e.g. , fluid properties, particle properties, microfluidic architectures, etc.
  • 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.
  • 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 (or cells) 114 may be transported along channel segment 102 into junction 110, while a second fluid 116 that is immiscible with the aqueous fluid 112 is delivered to the junction 110 from each of channel segments 104 and 106 to create discrete droplets 118, 120 of the first aqueous fluid 112 flowing into channel segment 108, and flowing away from junction 110.
  • the channel segment 108 may be fluidically coupled to an outlet reservoir where the discrete droplets can be stored and/or harvested.
  • a discrete droplet generated may include an individual biological particle 114 (such as droplets 118).
  • a discrete droplet generated may include more than one individual biological particle 114 (not shown in FIG. 1).
  • a discrete droplet may contain no biological particle 114 (such as droplet 120).
  • Each discrete partition may 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 fluorosurfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets 118, 120. Examples of particularly useful partitioning fluids and fluorosurfactants are described, for example, in U.S. Patent Application Publication No. 2010/0105112, which is entirely incorporated herein by reference for all purposes.
  • the channel segments described herein may 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 may have other geometries.
  • a microfluidic 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 may 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.
  • Fluid may also or otherwise be controlled via applied pressure differentials, centrifugal force, electrokinetic pumping, vacuum, capillary or gravity flow, or the like.
  • the generated droplets may comprise two subsets of droplets: (1) occupied droplets 118, containing one or more biological particles 114, and (2) unoccupied droplets 120, not containing any biological particles 114.
  • Occupied droplets 118 may comprise singly occupied droplets (having one biological particle) and multiply occupied droplets (having more than one biological particle).
  • the majority of occupied partitions can include no more than one biological particle per occupied partition and some of the generated partitions can be unoccupied (of any biological particle). In some cases, though, some of the occupied partitions may include more than one biological particle.
  • the partitioning process may 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 Poissonian distribution may expectedly increase the number of partitions that include multiple biological particles. As such, where singly occupied partitions are to be obtained, at most about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less of the generated partitions can be unoccupied.
  • flows can be controlled so as to present a non- Poissonian distribution of single-occupied partitions while providing lower levels of unoccupied partitions (e.g., no more than about 50%, about 25%, or about 10% unoccupied).
  • unoccupied 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 and additional reagents, such as beads (e.g., gel beads) carrying nucleic acid barcode molecules (e.g., oligonucleotides).
  • additional reagents such as 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. Accordingly, as noted above, 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.
  • Microfluidic systems for partitioning are further described in U.S. Patent Application Pub. No. US 2015/0376609.
  • FIG. 14 shows an example of a microfluidic channel structure 1400 for delivering barcode carrying beads to droplets.
  • the channel structure 1400 can include channel segments 1401, 1402, 1404, 1406 and 1408 communicating at a channel junction 1410.
  • the channel segment 1401 may transport an aqueous fluid 1412 that includes a plurality of beads 1414 (e.g., with nucleic acid molecules, e.g.. nucleic acid barcode molecules or barcoded oligonucleotides, molecular tags) along the channel segment
  • the plurality of beads 1414 may be sourced from a suspension of beads.
  • the channel segment 1401 may be connected to a reservoir comprising an aqueous suspension of beads 1414.
  • the channel segment 1402 may transport the aqueous fluid 1412 that includes a plurality of biological particles 1416 along the channel segment
  • the plurality of biological particles 1416 may be sourced from a suspension of biological particles.
  • the channel segment 1402 may be connected to a reservoir comprising an aqueous suspension of biological particles 1416.
  • the aqueous fluid 1412 in either the first channel segment 1401 or the second channel segment 1402, or in both segments, can include one or more reagents, as further described below.
  • a second fluid 1418 that is immiscible with the aqueous fluid 1412 e.g., oil
  • the aqueous fluid 1412 can be partitioned as discrete droplets 1420 in the second fluid 1418 and flow away from the junction 1410 along channel segment 1408.
  • the channel segment 1408 may deliver the discrete droplets to an outlet reservoir fluidly coupled to the channel segment 1408, where they may be harvested.
  • the channel segments 1401 and 1402 may meet at another junction upstream of the junction 1410.
  • beads and biological particles may form a mixture that is directed along another channel to the junction 1410 to yield droplets 1420.
  • 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.
  • Droplet size may 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 may be adjusted to control droplet size.
  • 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 may 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.
  • 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 may 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 comprise biological particles.
  • the aqueous fluid 208 can have a substantially uniform concentration or frequency of biological particles.
  • the biological particles 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 may 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.
  • 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 may be upstream or downstream of the second separate channel introducing the biological particles.
  • the second fluid 210 can comprise an oil, such as a fluorinated oil, that includes a fluorosurfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets.
  • the second fluid 210 may not be subjected to and/or directed to any flow in or out of the reservoir 204.
  • the second fluid 210 may be substantially stationary in the reservoir 204.
  • the second fluid 210 may 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 may 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. Systems and methods for controlled partitioning are described further in PCT/US2018/047551.
  • biological particles e.g., cells
  • a cell bead can contain a biological particle (e.g., a cell) or macromolecular constituents (e.g., RNA, DNA, proteins, etc.) of a biological particle.
  • a cell bead may include a single cell or multiple cells, or a derivative of the single cell or multiple cells. For example after lysing and washing the cells, inhibitory components from cell lysates can be washed away and the macromolecular constituents can be bound as cell beads.
  • Cell beads may be or include a cell, cell derivative, cellular material and/or material derived from the cell in, within, or encased in a matrix, such as a polymeric matrix.
  • a cell bead may comprise a live cell.
  • the live cell may be capable of being cultured when enclosed in a gel or polymer matrix, or of being cultured when comprising a gel or polymer matrix.
  • the polymer or gel may be diffusively permeable to certain components and diffusively impermeable to other components (e.g., macromolecular constituents).
  • Cell beads can provide certain potential advantages of being more storable and more portable than droplet-based partitioned biological particles. Furthermore, in some cases, it may be desirable to allow biological particles to incubate for a select period of time before analysis, such as in order to characterize changes in such biological particles over time, either in the presence or absence of different stimuli (or reagents).
  • Suitable polymers or gels may include one or more of disulfide cross-linked polyacrylamide, agarose, alginate, polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate, PEG-thiol, PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronic acid, collagen, fibrin, gelatin, or elastin.
  • the polymer or gel may comprise any other polymer or gel.
  • Encapsulation of biological particles may be performed by a variety of processes. Such processes may combine an aqueous fluid containing the biological particles with a polymeric precursor material that may be capable of being formed into a gel or other solid or semi-solid matrix upon application of a particular stimulus to the polymer precursor.
  • the conditions sufficient to polymerize or gel the precursors may comprise any conditions sufficient to polymerize or gel the precursors.
  • Such stimuli can include, for example, thermal stimuli (e.g., either heating or cooling), photo-stimuli (e.g., through photo-curing), chemical stimuli (e.g., through crosslinking, polymerization initiation of the precursor (e.g. , through added initiators)), electromagnetic radiation, mechanical stimuli, or any combination thereof.
  • air knife droplet or aerosol generators may be used to dispense droplets of precursor fluids into gelling solutions in order to form cell beads that include individual biological particles or small groups of biological particles.
  • membranebased encapsulation systems may be used to generate cell beads comprising encapsulated biological particles as described herein.
  • Microfluidic systems of the present disclosure such as that shown in FIG. 1, may be readily used in encapsulating biological particles (e.g., cells) as described herein. Exemplary methods for encapsulating biological particles (e.g., cells) are also further described in U.S. Patent Application Pub. No. US 2015/0376609 and PCT/US2018/016019. In particular, and with reference to FIG.
  • nonaqueous fluid 116 may also include an initiator (not shown) to cause polymerization and/or crosslinking of the polymer precursor to form the bead that includes the entrained biological particles.
  • initiator not shown
  • polymer precursor/initiator pairs include those described in U.S. Patent Application Publication No. 2014/0378345, which is entirely incorporated herein by reference for all purposes.
  • encapsulated biological particles can be selectively releasable from the cell bead, such as through passage of time or upon application of a particular stimulus, that degrades the bead sufficiently to allow the biological particles (e.g., cell), or its other contents to be released from the bead, such as into a partition (e.g., droplet).
  • a particular stimulus that degrades the bead sufficiently to allow the biological particles (e.g., cell), or its other contents to be released from the bead, such as into a partition (e.g., droplet).
  • a partition e.g., droplet
  • the polymer or gel may be diffusively permeable to chemical or biochemical reagents.
  • the polymer or gel may be diffusively impermeable to macromolecular constituents of the biological particle. In this manner, the polymer or gel may act to allow the biological particle to be subjected to chemical or biochemical operations while spatially confining the macromolecular constituents to a region of the droplet defined by the polymer or gel.
  • the polymer or gel may be functionalized to bind to targeted analytes, such as nucleic acids, proteins, carbohydrates, lipids or other analytes.
  • the polymer or gel may be functionalized to bind to targeted analytes, such as nucleic acids, proteins, carbohydrates, lipids or other analytes.
  • the polymer or gel e.g., polymer gel matrix, hydrogel or hydrogel matrix, may be functionalized to couple or link to a plurality of capture agents.
  • the plurality of capture agents may, e.g. , covalently or non-covalently, couple or link to the backbone of the polymer. See, e.g. , U.S. Pat.
  • a first capture agent of a plurality of capture agents may be a polypeptide or aptamer that (i) couples or links to the backbone of the polymer, and (ii) binds a specific analyte (e.g., antibody or antigen-binding fragment thereof) secreted by the cell, e.g., B cell.
  • a first capture agent of a plurality of capture agents may be a polypeptide, e.g., antibody, or aptamer that couples/links to the backbone of the polymer and binds to a secreted antibody, e.g., at its Fc region.
  • the first capture agent of the plurality of capture agents may, rather than couple/link to the backbone of the polymer of the gel matrix, embed in/couple to the cell membrane.
  • the first capture agent e.g., polypeptide or aptamer
  • the first capture agent may (i) embed in the membrane of the cell and/or bind to a cell surface protein and (ii) bind the specific analyte, e.g., antibody or antigen-binding fragment thereof, thereby tethering the secreted analyte, e.g., antibody, to the cell.
  • the polymer or gel may be polymerized or gelled via a passive mechanism.
  • the polymer or gel may be stable in alkaline conditions or at elevated temperature.
  • the polymer or gel may have mechanical properties similar to the mechanical properties of the bead. For instance, the polymer or gel may be of a similar size to the bead.
  • the polymer or gel may have a mechanical strength (e.g. tensile strength) similar to that of the bead.
  • the polymer or gel may be of a lower density than an oil.
  • the polymer or gel may be of a density that is roughly similar to that of a buffer.
  • the polymer or gel may have a tunable pore size. The pore size may be chosen to, for instance, retain denatured nucleic acids.
  • the pore size may be chosen to maintain diffusive permeability to exogenous chemicals such as sodium hydroxide (NaOH) and/or endogenous chemicals such as inhibitors.
  • the polymer or gel may be biocompatible.
  • the polymer or gel may maintain or enhance cell viability.
  • the polymer or gel may be biochemically compatible.
  • the polymer or gel may be polymerized and/or depolymerized thermally, chemically, enzymatically, and/or optically.
  • 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, may be at least partially dissolvable, disruptable, and/or degradable.
  • a solid support, e.g., a bead may not be degradable.
  • 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 semisolid 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 micro wells 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, 30-75pm, 20-75pm, 40-85pm, 40-95pm, 20-100pm, I0-100pm, l -100pm, 20-250pm, or 20-500pm.
  • 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, glass-ceramics, 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 carbon-carbon 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, cofactors (e.g.
  • reagents for enzymatic reactions e.g., enzymes, co-factors, substrates, buffers
  • reagents for nucleic acid modification reactions such as polymerization, ligation, or digestion
  • reagents for template preparation e.g., tagmentation
  • sequencing platforms e.g., Nextera® for Illumina®
  • 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.
  • 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.
  • a partition e.g., droplet
  • 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 e.g., primer, barcoded oligonucleotide
  • the molecular tag molecules e.g., primer, e.g., barcoded oligonucleotide
  • the pre-defined concentration may be selected to facilitate certain reactions for generating a sequencing library, e.g. , amplification, within the partition.
  • the pre-defined 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 a primer sequence for messenger RNA).
  • TSO template switch oligonucleotide
  • 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 a primer sequence for messenger RNA.
  • 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
  • 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.
  • nucleic acid barcode molecules 302 can hybridize to the poly- A tail of a mRNA molecule. 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. Because the nucleic acid barcode molecule 302 comprises an anchoring sequence 314, it will more likely hybridize to and prime reverse transcription at the sequence end of the poly-A tail of the mRNA. 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 e.g., UMI segment
  • 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.
  • 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.
  • further processing may be performed, in the partitions or outside the partitions (e.g., in bulk).
  • the 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 operations described herein may be performed at any useful or convenient step.
  • 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 e.g., a targeted priming sequence
  • 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 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 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 single- stranded 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.
  • 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 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 may 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 may 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 may be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter. These nucleotides may be completely contiguous, i.e., in a single stretch of adjacent nucleotides, or they may be separated into two or more separate subsequences that are separated by 1 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 may be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer.
  • the barcode subsequence may 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 may 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 comprise 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 nucleic acids (e.g. , mRNA, the genomic DNA) from the individual biological particles within the partitions while attaching the associated barcode sequences, sequencing primers or primer recognition sites, hybridization or probing sequences, e.g., for identification of presence of the sequences or for pulling down barcoded nucleic acids, or any of a number of other potential functional sequences. Other mechanisms of co-partitioning oligonucleotides may also be employed, including, e.g.
  • coalescence of two or more droplets where one droplet contains oligonucleotides, or microdispensing of oligonucleotides (e.g., attached to a bead) into partitions, e.g., droplets within microfluidic systems.
  • beads are provided that each include large numbers of the above described nucleic acid barcode molecules releasably attached to the beads, where all of the nucleic acid barcode molecules attached to a particular bead will include a common nucleic acid barcode sequence, but where a large number of diverse barcode sequences are represented across the population of beads used.
  • hydrogel beads e.g., comprising polyacrylamide polymer matrices, are used as a solid support and delivery vehicle for the nucleic acid barcode molecules into the partitions, as they are capable of carrying large numbers of nucleic acid barcode molecules, and may 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.
  • the population of beads provides a diverse barcode sequence library that includes about 1,000 to about 10,000 different barcode sequences, about 5,000 to about 50,000 different barcode sequences, about 10,000 to about 100,000 different barcode sequences, about 50,000 to about 1,000,000 different barcode sequences, or about 100,000 to about 10,000,000 different barcode sequences.
  • 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.
  • the number of nucleic acid molecules including the barcode sequence on an individual bead is between about 1,000 to about 10,000 nucleic acid molecules, about 5,000 to about 50,000 nucleic acid molecules, about 10,000 to about 100,000 nucleic acid molecules, about 50,000 to about 1,000,000 nucleic acid molecules, about 100,000 to about 10,000,000 nucleic acid molecules, about 1,000,000 to about 1 billion nucleic acid molecules.
  • 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. In some embodiments, such different barcode sequences can be associated with a given bead.
  • 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 barcode molecules, at least about 5,000 nucleic acid barcode molecules, at least about 10,000 nucleic acid barcode molecules, at least about 50,000 nucleic acid barcode molecules, at least about 100,000 nucleic acid barcode molecules, at least about 500,000 nucleic acids, at least about 1,000,000 nucleic acid barcode molecules, at least about 5,000,000 nucleic acid barcode molecules, at least about 10,000,000 nucleic acid barcode molecules, at least about 50,000,000 nucleic acid barcode molecules, at least about 100,000,000 nucleic acid barcode molecules, at least about 250,000,000 nucleic acid barcode molecules and in some cases at least about 1 billion nucleic acid barcode molecules.
  • the resulting population of partitions provides a diverse barcode sequence library that includes about 1,000 to about 10,000 different barcode sequences, about 5,000 to about 50,000 different barcode sequences, about 10,000 to about 100,000 different barcode sequences, about 50,000 to about 1,000,000 different barcode sequences, or about 100,000 to about 10,000,000 different barcode sequences. Additionally, each partition of the population can include between about 1,000 to about 10,000 nucleic acid barcode molecules, about 5,000 to about 50,000 nucleic acid barcode molecules, about 10,000 to about 100,000 nucleic acid barcode molecules, about 50,000 to about 1,000,000 nucleic acid barcode molecules, about 100,000 to about 10,000,000 nucleic acid barcode molecules, about 1,000,000 to about 1 billion nucleic acid barcode molecules.
  • a mixed, but known set of barcode sequences may 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 may be releasable from the beads upon the application of a particular stimulus to the beads.
  • the stimulus may be a photo-stimulus, e.g., through cleavage of a photo-labile linkage that releases the nucleic acid molecules.
  • a thermal stimulus may 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 may be degraded for release of the attached nucleic acid molecules through exposure to a reducing agent, such as DTT.
  • biological particles may be partitioned along with lysis reagents in order to release the contents of the biological particles within the partition.
  • 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.
  • biological particles may be partitioned along with other reagents, as will be described further below.
  • the methods and systems of the present disclosure may comprise microfluidic devices and methods of use thereof, which may be used for co-partitioning biological particles with reagents. Such systems and methods are described in U.S. Patent Publication No. US/20190367997, which is herein incorporated by reference in its entirety for all purposes.
  • the lysis reagents can facilitate the release of the contents of the biological particles within the partition.
  • the contents released in a partition may remain discrete from the contents of other partitions.
  • the channel segments of the microfluidic devices described elsewhere herein may 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 may have various 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 may be controlled to control the partitioning of the different elements into droplets.
  • Fluid may be directed 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 may 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 may 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 may be used to lyse cells, although these may be less desirable for emulsion based systems where the surfactants can interfere with stable emulsions.
  • lysis solutions may include non-ionic surfactants such as, for example, TritonX-100 and Tween 20.
  • lysis solutions may include ionic surfactants such as, for example, sarcosyl and sodium dodecyl sulfate (SDS).
  • Electroporation, thermal, acoustic or mechanical cellular disruption may also be used in certain cases, e.g., non-emulsion-based partitioning such as encapsulation of biological particles that may 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 may 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 co-partitioned with the biological particles, including, for example, DNase and RNase inactivating agents or inhibitors, such as proteinase K, chelating agents, such as EDTA, and other reagents employed in removing or otherwise reducing negative activity or impact of different cell lysate components on subsequent processing of nucleic acids.
  • DNase and RNase inactivating agents or inhibitors such as proteinase K
  • chelating agents such as EDTA
  • the biological particles may be exposed to an appropriate stimulus to release the biological particles or their contents from a co-partitioned bead.
  • a chemical stimulus may be co-partitioned along with an encapsulated biological particle to allow for the degradation of the bead and release of the cell or its contents into the larger partition.
  • this stimulus may be the same as the stimulus described elsewhere herein for release of nucleic acid molecules (e.g., oligonucleotides) from their respective bead.
  • this may 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.
  • compositions, and systems for encapsulating cells also referred to as a “cell bead”
  • a cell bead For a description of methods, compositions, and systems for encapsulating cells (also referred to as a “cell bead”), see, e.g., U.S. Pat. 10,428,326 and U.S. Pat. Pub. 20190100632, which are each incorporated by reference in their entirety.
  • Additional reagents may also be co-partitioned with the biological particle, such as endonucleases to fragment a biological particle’s DNA, DNA polymerase enzymes and dNTPs used to amplify the biological particle’s nucleic acid fragments and to attach the barcode molecular tags to the amplified fragments.
  • Other enzymes may be co-partitioned, including without limitation, polymerase, transposase, ligase, proteinase K, DNAse, etc.
  • Additional reagents may 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. In some cases, template switching can be used to append a predefined nucleic acid sequence to the cDNA. Template switching is further described in PCT/US2017/068320, which is hereby incorporated by reference in its entirety. Template switching oligonucleotides may comprise a hybridization region and a template region. Template switching oligonucleotides are further described in PCT/US2017/068320, which is hereby incorporated by reference in its entirety.
  • reagents described in this disclosure may be encapsulated in, or otherwise coupled to, a droplet, or bead, with any chemicals, particles, and elements suitable for sample processing reactions involving biomolecules, such as, but not limited to, nucleic acid molecules and proteins.
  • a bead or droplet used in a sample preparation reaction for DNA sequencing may comprise 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, and oligonucleotides.
  • the macromolecular components e.g., macromolecular constituents of biological particles, such as RNA, DNA, or proteins
  • the macromolecular component contents of individual biological particles can be provided with unique identifiers such that, upon characterization of those macromolecular components they may be attributed as having been derived from the same biological particle or particles.
  • unique identifiers such that, upon characterization of those macromolecular components they may be attributed as having been derived from the same biological particle or particles.
  • the ability to attribute characteristics to individual biological particles or groups of biological particles is provided by the assignment of unique identifiers specifically to an individual biological particle or groups of biological particles.
  • Unique identifiers e.g., in the form of nucleic acid barcodes can be assigned or associated with individual biological particles or populations of biological particles, in order to tag or label the biological particle’s macromolecular components (and as a result, its characteristics) with the unique identifiers. These unique identifiers can then be used to attribute the biological particle’s components and characteristics to an individual biological particle or group of biological particles. In some aspects, this is performed by copartitioning the individual biological particle or groups of biological particles with the unique identifiers, such as described above (with reference to FIGS. 1 or 2).
  • additional beads can be used to deliver additional reagents to a partition.
  • the flow and frequency of the different beads into the channel or junction may be controlled to provide for a certain ratio of beads from each source, while ensuring a given pairing or combination of such beads into a partition with a given number of biological particles (e.g. , one biological particle and one bead per partition).
  • subsequent operations can include generation of amplification products, purification (e.g., via solid phase reversible immobilization (SPRI)), further processing (e.g., shearing, ligation of functional sequences, and subsequent amplification (e.g., via PCR)). These operations may occur in bulk (e.g., outside the partition). In the case where a partition is a droplet in an emulsion, the emulsion can be broken and the contents of the droplet pooled for additional operations.
  • SPRI solid phase reversible immobilization
  • a partition which may be a well.
  • the well may be a well of a plurality of wells of a substrate, such as a microwell of a microwell array or plate, or the well may be a microwell or microchamber of a device (e.g., microfluidic device) comprising a substrate.
  • the well may be a well of a well array or plate, or the well may be a well or chamber of a device (e.g., fluidic device).
  • a well of a fluidic device is fluidically connected to another well of the fluidic device.
  • the wells or microwells may assume an “open” configuration, in which the wells or micro wells 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 may assume a “closed” or “sealed” configuration, in which the microwells are not accessible on a planar face of the substrate.
  • the wells or microwells may be configured to toggle between “open” and “closed” configurations.
  • an “open” microwell or set of microwells may 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.
  • a membrane e.g., semi-permeable membrane
  • an oil e.g., fluorinated oil to cover an aqueous solution
  • a lid e.g., a lid
  • the well may have a volume of less than 1 milliliter (mL).
  • the well may be configured to hold a volume of at most 1000 microliters (pL), at most 100 uL, 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 may 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 may 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 may 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 may be of a plurality of wells that have varying volumes and may be configured to hold a volume appropriate to accommodate any of the partition volumes described herein.
  • a microwell array or plate comprises a single variety of microwells.
  • a microwell array or plate comprises a variety of microwells.
  • the microwell array or plate may comprise one or more types of microwells within a single microwell array or plate.
  • the types of microwells may 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 may comprise any number of different types of microwells.
  • the microwell array or plate may comprise 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 may 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 comprises different types of microwells that are located adjacent to one another within the array or plate. For instance, a micro well with one set of dimensions may be located adjacent to and in contact with another microwell with a different set of dimensions. Similarly, microwells of different geometries may be placed adjacent to or in contact with one another.
  • the adjacent microwells may be configured to hold different articles; for example, one micro well may be used to contain a cell, cell bead, or other sample (e.g., cellular components, nucleic acid molecules, etc.) while the adjacent microwell may be used to contain a droplet, bead, or other reagent.
  • the adjacent microwells may 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 may be used in the systems, compositions, and methods described herein.
  • any suitable number of partitions e.g., wells or droplets
  • wells 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 may comprise both unoccupied wells (e.g., empty wells) and occupied wells.
  • a well may comprise any of the reagents described herein, or combinations thereof. These reagents may include, for example, barcode molecules, enzymes, adapters, and combinations thereof.
  • the reagents may be physically separated from a sample (e.g., a cell, cell bead, or cellular components, e.g., proteins, nucleic acid molecules, etc.) that is placed in the well. This physical separation may be accomplished by containing the reagents within, or coupling to, a bead that is placed within a well.
  • the physical separation may 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 may be, for example, an oil, wax, membrane (e.g., semi-permeable membrane), or the like.
  • the well may be sealed at any point, for example, after addition of the bead, after addition of the reagents, or after addition of either of these components.
  • the sealing of the well may 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 well-based array) comprising the cell (or cells) may be subjected to freeze-thaw cycling to process the cell (or cells), e.g., cell lysis.
  • the well containing the cell may be subjected to freezing temperatures (e.g., 0°C, below 0°C, -5°C, -10°C, -15°C, -20°C, -25°C, - 30°C, -35°C, -40°C, -45°C, -50°C, -55°C, -60°C, -65°C, -70°C, -80°C, or -85°C). Freezing may be performed in a suitable manner, e.g., sub-zero freezer or a dry ice/ethanol bath.
  • 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
  • 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 may comprise free reagents and/or reagents encapsulated in, or otherwise coupled to or associated with, beads, or droplets.
  • kits may comprise instructions for use, a microwell array or device, and reagents (e.g., beads).
  • the kit may comprise 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 comprises a bead, or droplet that comprises a set of reagents that has a similar attribute (e.g., a set of enzymes, a set of minerals, a set of oligonucleotides, a mixture of different barcode molecules, or a mixture of identical barcode molecules).
  • a bead or droplet comprises a heterogeneous mixture of reagents.
  • the heterogeneous mixture of reagents can comprise all components necessary to perform a reaction.
  • such mixture can comprise 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 droplet or bead, or within a solution within a partition (e.g., microwell) of the system.
  • FIG. 5 schematically illustrates an example of a microwell array.
  • the array can be contained within a substrate 500.
  • the substrate 500 comprises a plurality of wells 502.
  • the wells 502 may 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 may comprise a cell or cellular components (e.g., nucleic acid molecules) is copartitioned with a bead 504, which may comprise a nucleic acid barcode molecule coupled thereto.
  • the wells 502 may 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.
  • gravity or other loading technique e.g., centrifugation, liquid handler, acoustic loading, optoelectronic, etc.
  • at least one of the wells 502 contains a single sample molecule 506 (e.g., cell) and a single bead 504.
  • Reagents may be loaded into a well either sequentially or concurrently.
  • reagents are introduced to the device either before or after a particular operation.
  • reagents (which may be provided, in certain instances, in droplets, or beads) are introduced sequentially such that different reactions or operations occur at different steps.
  • the reagents (or droplets, or beads) may also be loaded at operations interspersed with a reaction or operation step.
  • beads comprising reagents for fragmenting polynucleotides e.g., restriction enzymes) and/or other enzymes (e.g., transposases, ligases, polymerases, etc.) may be loaded into the well or plurality of wells, followed by loading of droplets, or beads comprising reagents for attaching nucleic acid barcode molecules to a sample nucleic acid molecule.
  • Reagents may 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 may be useful in performing multi-step operations or reactions.
  • the nucleic acid barcode molecules and other reagents may be contained within a bead, or droplet. These beads, or droplets may be loaded into a partition (e.g. , a microwell) before, after, or concurrently with the loading of a cell, such that each cell is contacted with a different bead, or droplet.
  • a partition e.g. , a microwell
  • This technique may be used to attach a unique nucleic acid barcode molecule to nucleic acid molecules obtained from each cell.
  • the sample nucleic acid molecules may be attached to a support.
  • the partition e.g., microwell
  • the partition may comprise a bead which has coupled thereto a plurality of nucleic acid barcode molecules.
  • the sample nucleic acid molecules, or derivatives thereof, may couple or attach to the nucleic acid barcode molecules on the support.
  • the resulting barcoded nucleic acid molecules may then be removed from the partition, and in some instances, pooled and sequenced.
  • the nucleic acid barcode sequences may be used to trace the origin of the sample nucleic acid molecule. For example, polynucleotides with identical barcodes may be determined to originate from the same cell or partition, while polynucleotides with different barcodes may be determined to originate from different cells or partitions.
  • the samples or reagents may 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
  • the samples may be loaded into the well or microwell using an external force, e.g., gravitational force, electrical force, magnetic force, or using mechanisms to drive the sample or reagents into the well, e.g., via pressure-driven flow, centrifugation, optoelectronics, acoustic loading, electrokinetic pumping, vacuum, capillary flow, etc.
  • a fluid handling system may be used to load the samples or reagents into the well.
  • the loading of the samples or reagents may 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 may be modified to accommodate a useful sample or reagent distribution; for instance, the size and spacing of the microwells may be adjusted such that the sample or reagents may be distributed in a super-Poissonian fashion.
  • the microwell array or plate comprises pairs of microwells, in which each pair of microwells is configured to hold a droplet (e.g. , comprising a single cell) and a single bead (such as those described herein, which may, in some instances, also be encapsulated in a droplet).
  • a droplet e.g. , comprising a single cell
  • a single bead such as those described herein, which may, in some instances, also be encapsulated in a droplet.
  • the droplet and the bead may be loaded simultaneously or sequentially, and the droplet and the bead may 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 comprising 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 comprise reagents that may react with an agent in the droplet of the other microwell of the pair.
  • one droplet can comprise 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 may be released from the bead into the partition e.g., the microwell or microwell pair that are in contact), and further processing may 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 may comprise lysis reagents for lysing the cell upon droplet merging.
  • a droplet or bead may be partitioned into a well.
  • the droplets may be selected or subjected to pre-processing prior to loading into a well.
  • the droplets may comprise cells, and only certain droplets, such as those containing a single cell (or at least one cell), may be selected for use in loading of the wells.
  • Such a pre-selection process may 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 may be useful in obtaining or preventing cell doublet or multiplet formation prior to or during loading of the microwell.
  • the wells can comprise nucleic acid barcode molecules attached thereto.
  • the nucleic acid barcode molecules may 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 may 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 comprise 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 comprise a unique molecular identifier for individual molecule identification.
  • the nucleic acid barcode molecules may be configured to attach to or capture a nucleic acid molecule within a sample or cell distributed in the well.
  • the nucleic acid barcode molecules may comprise a capture sequence that may be used to capture or hybridize to a nucleic acid molecule (e.g., RNA, DNA) within the sample.
  • the nucleic acid barcode molecules may 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 may comprise a chemical cross-linker which may be cleaved upon application of a stimulus (e.g. , photo-, magnetic, chemical, biological, stimulus).
  • a stimulus e.g. , photo-, magnetic, chemical, biological, stimulus.
  • the nucleic acid barcode molecules which may be hybridized or configured to hybridize to a sample nucleic acid molecule, 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 barcode molecules attached to a bead or droplet 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 may be used to identify the cell or partition from which a nucleic acid molecule originated.
  • Characterization of samples within a well may 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 may be useful in measuring sample profiles in fixed spatial locations.
  • imaging of each microwell and the contents contained therein may 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 may be used to characterize live cells in the wells, including, but not limited to: dynamic live-cell tracking, cell-cell interactions (when two or more cells are copartitioned), cell proliferation, etc.
  • imaging may be used to characterize a quantity of amplification products in the well.
  • a well may be loaded with a sample and reagents, simultaneously or sequentially.
  • the well may be subjected to washing, e.g., to remove excess cells from the well, microwell array, or plate. Similarly, washing may be performed to remove excess beads or other reagents from the well, microwell array, or plate.
  • the cells may be lysed in the individual partitions to release the intracellular components or cellular analytes. Alternatively, the cells may be fixed or permeabilized in the individual partitions.
  • the intracellular components or cellular analytes may couple to a support, e.g., on a surface of the micro well, on a solid support (e.g.
  • the intracellular components or cellular analytes may be transferred to individual droplets or other partitions for barcoding.
  • the intracellular components or cellular analytes e.g., nucleic acid molecules
  • the bead may be collected and further processed, e.g., subjected to nucleic acid reaction such as reverse transcription, amplification, or extension, and the nucleic acid molecules thereon may be further characterized, e.g., via sequencing.
  • the intracellular components or cellular analytes may be barcoded in the well (e.g., using a bead comprising nucleic acid barcode molecules that are releasable or on a surface of the microwell comprising nucleic acid barcode molecules).
  • the barcoded nucleic acid molecules or analytes may be further processed in the well, or the barcoded nucleic acid molecules or analytes may 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).
  • the well or microwell array or plate
  • the well may be sealed (e.g. , using an oil, membrane, wax, etc.), which enables storage of the assay or selective introduction of additional reagents.
  • FIG. 6 schematically shows an example workflow for processing nucleic acid molecules within a sample.
  • a substrate 600 comprising a plurality of microwells 602 may be provided.
  • a sample 606 which may comprise 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 comprising nucleic acid barcode molecules.
  • the sample 606 may be processed within the partition.
  • the cell may be subjected to conditions sufficient to lyse the cells and release the analytes contained therein.
  • the bead 604 may be further processed.
  • processes 620a and 620b schematically illustrate different workflows, depending on the properties of the bead 604.
  • the bead comprises nucleic acid barcode molecules that are attached thereto, and sample nucleic acid molecules (e.g., RNA, DNA) may attach, e.g., via hybridization of ligation, to the nucleic acid barcode molecules. Such attachment may occur on the bead.
  • sample nucleic acid molecules e.g., RNA, DNA
  • the beads 604 from multiple wells 602 may be collected and pooled. Further processing may be performed in process 640. For example, one or more nucleic acid reactions may 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 may be appended to each end of the nucleic acid molecule.
  • further characterization such as sequencing may be performed to generate sequencing reads.
  • the sequencing reads may yield information on individual cells or populations of cells, which may be represented visually or graphically, e.g., in a plot 655.
  • the bead comprises nucleic acid barcode molecules that are releasably attached thereto, as described below.
  • the bead may degrade or otherwise release the nucleic acid barcode molecules into the well 602; the nucleic acid barcode molecules may then be used to barcode nucleic acid molecules within the well 602. Further processing may be performed either inside the partition or outside the partition. For example, one or more nucleic acid reactions may 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 may be appended to each end of the nucleic acid molecule.
  • further characterization such as sequencing may be performed to generate sequencing reads.
  • the sequencing reads may yield information on individual cells or populations of cells, which may be represented visually or graphically, e.g., in a plot 655.
  • a sample may derive from any useful source including any subject, such as a human subject.
  • a sample may comprise material (e.g., one or more biological particles) 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, may be obtained for analysis as described herein. For example, a first sample may be obtained from a subject at a first time and a second sample may be obtained from the subject at a second time later than the first time.
  • the first time may be before a subject undergoes a treatment regimen or procedure (e.g., to address a disease or condition), and the second time may be during or after the subject undergoes the treatment regimen or procedure.
  • a first sample may be obtained from a first bodily location or system of a subject (e.g., using a first collection technique) and a second sample may be obtained from a second bodily location or system of the subject (e.g. , using a second collection technique), which second bodily location or system may be different than the first bodily location or system.
  • multiple samples may be obtained from a subject at a same time from the same or different bodily locations.
  • Different samples may undergo the same or different processing (e.g., as described herein).
  • a first sample may undergo a first processing protocol and a second sample may undergo a second processing protocol.
  • a portion of a sample may undergo a first processing protocol and a second portion of the sample may undergo a second processing protocol.
  • a sample may be a biological sample, such as a cell sample (e.g., as described herein).
  • a sample may include one or more biological particles, such as one or more cells and/or cellular constituents, such as one or more cell nuclei.
  • a sample may be a tissue sample.
  • a sample may comprise a plurality of biological particles, such as a plurality of cells and/or cellular constituents.
  • Biological particles (e.g., cells or cellular constituents, such as cell nuclei) of a sample may be of a single type or a plurality of different types.
  • cells of a sample may include one or more different types or blood cells.
  • Cells and cellular constituents of a sample may be of any type.
  • a cell or cellular constituent may be a vertebral, mammalian, fungal, plant, bacterial, or other cell type.
  • the cell is a mammalian cell, such as a human cell.
  • the cell may be, for example, a stem cell, liver cell, nerve cell, bone cell, blood cell, reproductive cell, skin cell, skeletal muscle cell, cardiac muscle cell, smooth muscle cell, hair cell, hormone- secreting cell, or glandular cell.
  • the cell may be, for example, an erythrocyte (e.g., red blood cell), a megakaryocyte (e.
  • platelet precursor e.g., platelet precursor
  • a monocyte e.g., white blood cell
  • a leukocyte e.g., a B cell
  • a T cell such as a helper, suppressor, cytotoxic, or natural killer T cell
  • an osteoclast a dendritic cell
  • a connective tissue macrophage e.g., an epidermal Langerhans cell
  • a microglial cell e.g., a granulocyte, a hybridoma cell, a mast cell, a natural killer cell, a reticulocyte, a hematopoietic stem cell, a myoepithelial cell, a myeloid-derived suppressor cell, a platelet, a thymocyte, a satellite cell, an epithelial cell, an endothelial cell, an epididymal cell, a kidney cell, a liver cell, an adipocyte, a lipocyte, or a neuron cell
  • a biological sample may 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), may 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 may comprise the use of microfluidics (e.g., to separate biological particles of different sizes, types, charges, or other features).
  • a sample comprising one or more cells may 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 may be processed to separate and/or sort groups of cells and/or cellular constituents, such as to separate and/or sort cells and/or cellular constituents of different types.
  • Examples of cell separation include, but are not limited to, separation of white blood cells or immune cells from other blood cells and components, separation of circulating tumor cells from blood, and separation of bacteria from bodily cells and/or environmental materials.
  • a separation process may comprise 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 may comprise, 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
  • BAS buoyancy- activated cell sorting
  • a flow cytometry method may 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 may comprise 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 may interrogate the cells and/or cellular constituents and scattered light and/or fluorescence may 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 may be labeled with an electric charge (e.g. , using an electrical charging ring), which charge may be used to separate such droplets from droplets including other cells and/or cellular constituents.
  • FACS may comprise labeling cells and/or cellular constituents with fluorescent markers (e.g., using internal and/or external biomarkers). Cells and/or cellular constituents may then be measured and identified one by one and sorted based on the emitted fluorescence of the marker or absence thereof.
  • MACS may 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 may use microbubbles (e.g., glass microbubbles) labeled with antibodies to target cells of interest.
  • Cells and/or cellular components coupled to microbubbles may float to a surface of a solution, thereby separating target cells and/or cellular components from other components of a sample.
  • Cell separation techniques may be used to enrich for populations of cells of interest (e.g. , prior to partitioning, as described herein).
  • a sample comprising a plurality of cells including a plurality of cells of a given type may be subjected to a positive separation process.
  • the plurality of cells of the given type may 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 may then be subjected to subsequent partition-based analysis (e.g., as described herein) or other downstream analysis.
  • the fluorescent marker may be removed prior to such analysis or may be retained.
  • the fluorescent marker may comprise an identifying feature, such as a nucleic acid barcode sequence and/or unique molecular identifier.
  • a first sample comprising 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 comprising a second plurality of cells including a second plurality of cells of the given type may be subjected to a positive separation process.
  • the first and second samples may 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 may be from a first subject and the second sample may be from a second subject different than the first subject.
  • the first plurality of cells of the first sample may 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 may 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 may include a first identifying feature, such as a first barcode, while the second plurality of fluorescent markers may 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 may 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 may 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 may 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 may then be combined for subsequent analysis.
  • the encoded identifying features of the different fluorescent markers may be used to identify cells originating from the first sample and cells originating from the second sample.
  • the first and second identifying features may 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.
  • a sample may be a fixed sample.
  • a sample may comprise a plurality of fixed samples, such as a plurality of fixed cells or fixed nuclei.
  • a sample may comprise a fixed tissue. Fixation of cell or cellular constituent, or a tissue comprising a plurality of cells or nuclei, may comprise application of a chemical species or chemical stimulus.
  • the term “fixed” as used herein with regard to biological samples generally refers to the state of being preserved from decay and/or degradation.
  • fixation generally refers to a process that results in a fixed sample, and in some instances can include contacting the biomolecules within a biological sample with a fixative (or fixation reagent) for some amount of time, whereby the fixative results in covalent bonding interactions such as crosslinks between biomolecules in the sample.
  • a “fixed biological sample” may generally refer to a biological sample that has been contacted with a fixation reagent or fixative. For example, a formaldehyde-fixed biological sample has been contacted with the fixation reagent formaldehyde.
  • “Fixed cells” or “fixed tissues” generally refer to cells or tissues that have been in contact with a fixative under conditions sufficient to allow or result in the formation of intra- and inter-molecular covalent crosslinks between biomolecules in the biological sample.
  • a fixation reagent e.g., paraformaldehyde or PF A
  • provision of the fixation reagent such as formaldehyde, may result in covalent aminal crosslinks within RNA, DNA, and/or protein molecules.
  • the widely used fixative reagent paraformaldehyde or PFA, fixes tissue samples by catalyzing crosslink formation between basic amino acids in proteins, such as lysine and glutamine. Both intramolecular and inter-molecular crosslinks can form in the protein. These crosslinks can preserve protein secondary structure and also eliminate enzymatic activity in the preserved tissue sample.
  • fixation reagents include but are not limited to aldehyde fixatives (e.g., formaldehyde, also commonly referred to as “paraformaldehyde,” “PFA,” and “formalin”; glutaraldehyde; etc.), imidoesters, NHS (N-Hydroxysuccinimide) esters, and the like.
  • aldehyde fixatives e.g., formaldehyde, also commonly referred to as “paraformaldehyde,” “PFA,” and “formalin”; glutaraldehyde; etc.
  • imidoesters e.g., imidoesters, NHS (N-Hydroxysuccinimide) esters, and the like.
  • fixation reagents include, for example, organic solvents such as alcohols (e.g., methanol or ethanol), ketones (e.g., acetone), and aldehydes (e.g., paraformaldehyde, formaldehyde (e.g., formalin), or glutaraldehyde).
  • organic solvents such as alcohols (e.g., methanol or ethanol), ketones (e.g., acetone), and aldehydes (e.g., paraformaldehyde, formaldehyde (e.g., formalin), or glutaraldehyde).
  • cross-linking agents may also be used for fixation including, without limitation, disuccinimidyl suberate (DSS), dimethylsuberimidate (DMS), formalin, and dimethyladipimidate (DMA), dithio-bis(-succinimidyl propionate) (DSP), disuccinimidyl tartrate (DST), and ethylene glycol bis(succinimidyl succinate) (EGS).
  • a cross-linking agent may be a cleavable cross-linking agent (e.g., thermally cleavable, photocleavable, etc.), hi some cases, more than one fixation reagent can be used in combination when preparing a fixed biological sample.
  • Changes to a characteristic or a set of characteristics of a cell or cellular constituents may be at least partially reversible (e.g., via rehydration or decrosslinking).
  • changes to a characteristic or set of characteristics of a cell or cellular constituents may be substantially irreversible.
  • the methods described herein may comprise templated ligation.
  • a templated ligation process may comprise contacting a nucleic acid molecule (e.g., an RNA molecule) with a probe molecule.
  • the probe molecule may interact with one or more other probe molecules, for example, comprising a barcode sequence, to generate a probe-barcode complex.
  • An extension reaction may be performed on at least a portion of the probe-barcode complex to generate a nucleic acid product that comprises the barcode sequence and is associated with a sequence of the nucleic acid molecule.
  • the methods described herein may allow barcoding of the nucleic acid molecule without performing reverse transcription on the nucleic acid molecule.
  • the methods herein may comprise ligation- mediated reactions.
  • a method may comprise contacting a nucleic acid molecule (e.g., an RNA molecule) with a first probe molecule, comprising a first sequence and a second sequence, under conditions sufficient for the first sequence to hybridize to a sequence of the nucleic acid molecule.
  • a second probe molecule comprising a third sequence may hybridize to the second sequence of the first probe molecule.
  • the first probe or the second probe molecule may comprise a barcode sequence (e.g. , as described herein).
  • the second probe molecule may be a nucleic acid molecule (e.g., as described herein).
  • a splint molecule may be used to link the first and second probe molecules.
  • a fourth sequence of the splint molecule may hybridize to the second sequence of the first probe molecule and a fifth sequence of the splint molecule may hybridize to the third sequence of the second probe molecule.
  • a first probe molecule with a first reactive moiety and a second probe molecule with a second reactive moiety may be used.
  • a first sequence of the first probe molecule may hybridize to a first sequence of a nucleic acid molecule and a second sequence of the second probe molecule may hybridize to a second sequence of the nucleic acid molecule.
  • the first and second sequences of the nucleic acid molecule may be adjacent or may be separated by a gap of one or more nucleotides, which gap may optionally be filled (e.g., using a polymerase).
  • the first reactive moiety of the first probe molecule and the second reactive moiety of the second probe molecule may be subjected to conditions sufficient for the first and second reactive moieties to react to provide a linking moiety.
  • a click chemistry reaction involving an alkyne moiety and an azide moiety may be used to provide a triazole linking moiety.
  • an iodide moiety may be chemically ligated to a phosphorothioate moiety to form a phosphorothioate bond
  • an acid may be ligated to an amine to form an amide bond
  • a phosphate may be ligated to an amine to form a phosphoramidate bond.
  • the probes may be subjected to an enzymatic ligation reaction, using a ligase, e.g., SplintR ligases, T4 ligases, KOD ligases, PBCV1 enzymes, etc. to form a probe-linked nucleic acid molecule. Where the two probes are nonadj acent, gap regions between the probes may be filled prior to ligation. In some instances, ribonucleotides or deoxyribonucleotides are ligated between the first and second probes.
  • a ligase e.g., SplintR ligases, T4 ligases, KOD ligases, PBCV1 enzymes, etc.
  • a third probe molecule e.g. , a nucleic acid barcode molecule
  • the third probe molecule may comprise a barcode sequence.
  • a splint molecule may be used to link the first and third probe molecules.
  • the first and second probe molecules may be linked to one another such that a loop or “padlock” is formed after hybridization of the first sequence of the first probe molecule to the first sequence of the nucleic acid molecule and the second sequence of the second probe molecule to the second sequence of the nucleic acid molecule.
  • a linkage between the first and second probe molecules may be generated after hybridization of the first and second probe molecules to the nucleic acid molecule, such as via reaction between two reactive moieties to form a linking moiety.
  • the first and second probe molecules may be linked to one another before the first and second probe molecules hybridize to the nucleic acid molecule.
  • All or a portion of the templated ligation processes described herein may be performed within a partition (e.g., as described herein). Alternatively, one or more such processes may be performed within a bulk solution.
  • one or more probe molecules may be subjected to conditions sufficient to hybridize to a nucleic acid molecule (e.g., a nucleic acid molecule included in a biological particle such as a cell) within a bulk solution.
  • the nucleic acid molecule may be partitioned within various reagents e.g., as described herein) including a nucleic acid barcode molecule, such as a nucleic acid barcode molecule releasably coupled to a bead (e.g., as described herein).
  • the nucleic acid barcode molecule may hybridize to a sequence of a probe molecule hybridized to the nucleic acid molecule, thereby generated a barcode-linked nucleic acid molecule.
  • Templated ligation processes may permit indirect barcoding of a nucleic acid molecule without the use of reverse transcription. Details of such processes and additional schemes are included in, for example, International Patent Publication No. WO2019/165318.
  • the methods provided herein may comprise the use of a targeting process to, e.g., enrich selected nucleic acid molecules within a sample.
  • An exemplary target enrichment method may comprise providing a plurality of barcoded nucleic acid molecules and hybridizing barcoded nucleic acid molecules comprising targeted regions of interest to oligonucleotide probes (“baits”) which are complementary to the targeted regions of interest (or to regions near or adjacent to the targeted regions of interest). Baits may be attached to a capture molecule, including without limitation a biotin molecule.
  • the capture molecule e.g., biotin
  • the capture molecule can be used to selectively pull down the targeted regions of interest (for example, with magnetic streptavidin beads) to thereby enrich the resultant population of barcoded nucleic acid molecules for those containing the targeted regions of interest.
  • Another exemplary enrichment method may comprise providing a plurality of barcoded nucleic acid molecules comprising a plurality of different barcode sequences, identifying a barcode sequence of the plurality of different barcode sequences, and enriching barcoded nucleic acid molecules comprising the barcode sequence.
  • Enriching may comprise performing a nucleic acid extension reaction using a barcoded nucleic acid molecule comprising the barcode sequence and a primer comprising a sequence specific for the barcode sequence to generate an enriched plurality of barcoded nucleic acid molecules comprising the barcode sequence of interest. Details of such processes and additional schemes are included in, for example, International Patent Application No. PCT/US2020/012413 herein entirely incorporated by reference for all purposes.
  • 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 cell features may be used to characterize biological particles and/or cell features.
  • cell features include cell surface features.
  • Cell surface features may 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 may 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 may 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.
  • the labelling agents 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 may comprise a barcode sequence that permits identification of the labelling agent.
  • a labelling agent that is specific to one type of cell feature e.g., a first cell surface feature
  • a labelling agent that is specific to a different cell feature e.g., a second cell surface feature
  • a labelling agent that is specific to a different cell feature e.g., a second cell surface feature
  • a library of potential cell feature labelling agents may 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 may be characterized by the presence of a different oligonucleotide sequence label.
  • an antibody capable of binding to a first protein may have associated with it a first reporter oligonucleotide sequence
  • an antibody capable of binding to a second protein may have a different reporter oligonucleotide sequence associated with it.
  • the presence of the particular oligonucleotide sequence may be indicative of the presence of a particular antibody or cell feature which may be recognized or bound by the particular antibody.
  • Labelling agents capable of binding to or otherwise coupling to one or more biological particles may be used to characterize a biological particle as belonging to a particular set of biological particles.
  • labeling agents may be used to label a sample of cells or a group of cells.
  • a group of cells may be labeled as different from another group of cells.
  • a first group of cells may originate from a first sample and a second group of cells may originate from a second sample.
  • Labelling agents may allow the first group and second group to have a different labeling agent (or reporter oligonucleotide associated with the labeling agent). This may, for example, facilitate multiplexing, where cells of the first group and cells of the second group may be labeled separately and then pooled together for downstream analysis.
  • the downstream detection of a label may indicate analytes as belonging to a particular group.
  • a reporter oligonucleotide may be linked to an antibody or an epitope binding fragment thereof, and labeling a biological particle may comprise 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 biological particle.
  • the binding affinity between the antibody or the epitope binding fragment thereof and the molecule present on the surface may be within a desired range to ensure that the antibody or the epitope binding fragment thereof remains bound to the molecule.
  • the binding affinity may 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 may 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
  • a reporter oligonucleotide may be coupled to a cellpenetrating peptide (CPP), and labeling cells may comprise delivering the CPP coupled reporter oligonucleotide into a biological particle.
  • Labeling biological particles may comprise delivering the CPP conjugated oligonucleotide into a cell and/or cell bead by the cellpenetrating peptide.
  • a cell-penetrating peptide that can be used in the methods provided herein can comprise at least one non- functional cysteine residue, which may be either free or derivatized to form a disulfide link with an oligonucleotide that has been modified for such linkage.
  • Non-limiting examples of cell-penetrating peptides that can be used in embodiments herein include penetratin, transportan, plsl, TAT(48-60), pVEC, MTS, and MAP.
  • Cellpenetrating 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 cell-penetrating peptide may be an arginine-rich peptide transporter.
  • the cell-penetrating peptide may be Penetratin or the Tat peptide.
  • a reporter oligonucleotide may be coupled to a fluorophore or dye, and labeling cells may comprise subjecting the fluorophore-linked barcode molecule to conditions suitable for binding the fluorophore to the surface of the biological particle.
  • fluorophores can interact strongly with lipid bilayers and labeling biological particles may comprise subjecting the fluorophore-linked barcode molecule to conditions such that the fluorophore binds to or is inserted into a membrane of the biological particle.
  • 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, which is hereby incorporated by reference in its entirety for all purposes
  • a reporter oligonucleotide may be coupled to a lipophilic molecule, and labeling biological particles may comprise delivering the nucleic acid barcode molecule to a membrane of the biological particle 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 biological particle may be such that the biological particle 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 may enter into the intracellular space and/or a cell nucleus.
  • a reporter oligonucleotide may be part of a nucleic acid molecule comprising 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 may be incubated with the library of labelling agents, that may 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 may be washed from the cells, and the cells may 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 may include the cell or cells, as well as the bound labelling agents and their known, associated reporter oligonucleotides.
  • labelling agents may 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 may be washed from the cells
  • a labelling agent that is specific to a particular cell feature may 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 may 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.
  • libraries of labelling agents may be associated with a particular cell feature as well as be used to identify analytes as originating from a particular biological particle, population, or sample.
  • the biological particles may be incubated with a plurality of libraries and a given biological particle may comprise multiple labelling agents.
  • a cell may comprise coupled thereto a lipophilic labeling agent and an antibody.
  • the lipophilic labeling agent may indicate that the cell is a member of a particular cell sample, whereas the antibody may indicate that the cell comprises a particular analyte.
  • the reporter oligonucleotides and labelling agents may allow multianalyte, multiplexed analyses to be performed.
  • these reporter oligonucleotides may comprise 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 may 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 may be achieved through any of a variety of direct or indirect, covalent or non-covalent associations or attachments.
  • oligonucleotides may 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, which is entirely incorporated herein by reference for all purposes. Likewise, protein and peptide biotinylation techniques have been developed and are readily available. See, e.g., U.S. Pat. No. 6,265,552, which is entirely incorporated herein by reference for all purposes.
  • click reaction chemistry such as a Methyltetrazine-PEG5-NHS Ester reaction, a TCO-PEG4-NHS Ester reaction, or the like, may 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 may 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 comprising a barcode sequence that identifies the label agent.
  • the labelling agent may be directly coupled (e.g., covalently bound) to a hybridization oligonucleotide that comprises 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 may 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 may 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
  • the labelling agent can comprise a reporter oligonucleotide and a label.
  • a label can be fluorophore, a radioisotope, a molecule capable of a colorimetric reaction, a magnetic particle, or any other suitable molecule or compound capable of detection.
  • the label can be conjugated to a labelling agent (or reporter oligonucleotide) either directly or indirectly (e.g., the label can be conjugated to a molecule that can bind to the labelling agent or reporter oligonucleotide).
  • a label is conjugated to an oligonucleotide that is complementary to a sequence of the reporter oligonucleotide, and the oligonucleotide may be allowed to hybridize to the reporter oligonucleotide.
  • FIG. 11 describes exemplary labelling agents (1110, 1120, 1130) comprising reporter oligonucleotides (1140) attached thereto.
  • Labelling agent 1110 e.g. , any of the labelling agents described herein
  • reporter oligonucleotide 1140 may comprise barcode sequence 1142 that identifies labelling agent 1110.
  • Reporter oligonucleotide 1140 may also comprise one or more functional sequences 1141 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 1140 conjugated to a labelling agent comprises a functional sequence 1141 (e.g. , a primer sequence), a barcode sequence that identifies the labelling agent (e.g. , 1110, 1120, 1130), and reporter capture handle 1143.
  • Reporter capture handle 1143 can be configured to hybridize to a complementary sequence, such as a complementary sequence present on a nucleic acid barcode molecule 1190 (not shown), such as those described elsewhere herein.
  • nucleic acid barcode molecule 1190 is attached to a support (e.g.
  • a bead such as a gel bead
  • nucleic acid barcode molecule 1190 may be attached to the support via a releasable linkage (e.g., comprising a labile bond), such as those described elsewhere herein.
  • reporter oligonucleotide 1140 comprises one or more additional functional sequences, such as those described above.
  • the labelling agent 1110 is a protein or polypeptide (e.g., an antigen or prospective antigen) comprising reporter oligonucleotide 1140.
  • Reporter oligonucleotide 1140 comprises barcode sequence 1142 that identifies polypeptide 1110 and can be used to infer the presence of an analyte, e.g. , a binding partner of polypeptide 1110 (i.e., a molecule or compound to which polypeptide 1110 can bind).
  • the labelling agent 1110 is a lipophilic moiety (e.g.
  • reporter oligonucleotide 1140 comprising reporter oligonucleotide 1140, where the lipophilic moiety is selected such that labelling agent 1110 integrates into a membrane of a cell or nucleus.
  • Reporter oligonucleotide 1140 comprises barcode sequence 1142 that identifies lipophilic moiety 1110 which in some instances is used to tag cells e.g., groups of cells, cell samples, etc.) and may be used for multiplex analyses as described elsewhere herein.
  • the labelling agent is an antibody 1120 (or an epitope binding fragment thereof) comprising reporter oligonucleotide 1140.
  • Reporter oligonucleotide 1140 comprises barcode sequence 1142 that identifies antibody 1120 and can be used to infer the presence of, e.g., a target of antibody 1120 (i.e., a molecule or compound to which antibody 1120 binds).
  • labelling agent 1130 comprises an MHC molecule 1131 comprising peptide 1132 and reporter oligonucleotide 1140 that identifies peptide 1132.
  • the MHC molecule is coupled to a support 1133.
  • support 1133 may be or comprise a polypeptide, such as streptavidin, avidin, neutravidin, or a polysaccharide, such as dextran.
  • support 1133 further comprises a detectable label, e.g., a detectable label described herein, e.g., a fluorescent label.
  • reporter oligonucleotide 1140 may be directly or indirectly coupled to MHC labelling agent 1130 in any suitable manner.
  • reporter oligonucleotide 1140 may be coupled to MHC molecule 1131, support 1133, or peptide 1132.
  • labelling agent 1130 comprises a plurality of MHC molecules described herein, (e.g. is an MHC multimer, which may be coupled to a support (e.g., 1133)).
  • reporter oligonucleotide 1140 and MHC molecule 1130 are attached to the polypeptide or polysaccharide of support 1133. In some embodiments, reporter oligonucleotide 1140 and MHC molecule 1130 are attached to the detectable label of support 1133. In some embodiments, reporter oligonucleotide 1140 and an antigen (e.g., protein, polypeptide) are attached to polypeptide or polysaccharide of support 1133. In some embodiments, reporter oligonucleotide 1140 and an antigen (e.g. , protein, polypeptide) are attached to the detectable label of support 1133.
  • an antigen e.g., protein, polypeptide
  • Class I and/or Class II MHC multimers that can be utilized with the compositions, methods, and systems disclosed herein, e.g., MHC tetramers, MHC pentamers (MHC assembled via a coiled-coil domain, e.g., Pro5® MHC Class I Pentamers, (Prolmmune, Ltd.), MHC octamers, MHC dodecamers, MHC decorated dextran molecules (e.g., MHC Dextramer® (Immudex)), etc.
  • MHC tetramers MHC pentamers (MHC assembled via a coiled-coil domain
  • Pro5® MHC Class I Pentamers Pro5® MHC Class I Pentamers
  • MHC octamers MHC dodecamers
  • MHC decorated dextran molecules e.g., MHC Dextramer® (Immudex)
  • exemplary labelling agents including antibody and MHC -based labelling agents, reporter oligonu
  • FIG. 13 illustrates another example of a barcode carrying bead.
  • analysis of multiple analytes may comprise nucleic acid barcode molecules as generally depicted in FIG. 13.
  • nucleic acid barcode molecules 1310 and 1320 are attached to support 1330 via a releasable linkage 1340 (e.g. , comprising a labile bond) as described elsewhere herein.
  • Nucleic acid barcode molecule 1310 may comprise adapter sequence 1311, barcode sequence 1312 and capture sequence 1313.
  • Nucleic acid barcode molecule 1320 may comprise adapter sequence 1321, barcode sequence 1312, and capture sequence 1323, wherein capture sequence 1323 comprises a different sequence than capture sequence 1313.
  • adapter 1311 and adapter 1321 comprise the same sequence.
  • adapter 1311 and adapter 1321 comprise different sequences.
  • support 1330 is shown comprising nucleic acid barcode molecules 1310 and 1320, any suitable number of barcode molecules comprising common barcode sequence 1312 are contemplated herein.
  • support 1330 further comprises nucleic acid barcode molecule 1350.
  • Nucleic acid barcode molecule 1350 may comprise adapter sequence 1351, barcode sequence 1312 and capture sequence 1353, wherein capture sequence 1353 comprises a different sequence than capture sequence 1313 and 1323.
  • nucleic acid barcode molecules e.g., 1310, 1320, 1350
  • nucleic acid barcode molecules 1310, 1320 or 1350 may interact with analytes as described elsewhere herein, for example, as depicted in FIGs. 12A-C.
  • 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 1223 may be complementary to a capture handle sequence 1213 of a reporter oligonucleotide.
  • Cells may be contacted with one or more reporter oligonucleotide 1220 conjugated labelling agents 1210 e.g., polypeptide, antibody, or others described elsewhere herein).
  • the cells may be further processed prior to barcoding.
  • processing steps may include one or more washing and/or cell sorting steps.
  • a cell that is bound to labelling agent 1210 which is conjugated to oligonucleotide 1220 and support 1230 (e.g. , a bead, such as a gel bead) comprising nucleic acid barcode molecule 1290 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 comprises at most a single cell bound to labelling agent 1210.
  • reporter oligonucleotide 1220 conjugated to labelling agent 1210 comprises a first adapter sequence 1211 (e.g., a primer sequence), a barcode sequence 1212 that identifies the labelling agent 1210 (e.g., the polypeptide, antibody, or peptide of a pMHC molecule or complex), and an capture handle sequence 1213.
  • Capture handle sequence 1213 may be configured to hybridize to a complementary sequence, such as a capture sequence 1223 present on a nucleic acid barcode molecule 1290.
  • oligonucleotide 1220 comprises 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 may be generated in reactions (e.g., via a nucleic acid reaction, such as nucleic acid extension, ligation or any combination thereof) that include the reagents and steps described in FIGs. 12A-12D.
  • capture handle sequence 1213 may then be hybridized to complementary sequence, such as capture sequence 1223 to generate (e.g., via a nucleic acid reaction, such as nucleic acid extension or ligation) a barcoded nucleic acid molecule comprising cell (e.g., partition specific) barcode sequence 1222 (or a reverse complement thereof) and reporter barcode sequence 1212 (or a reverse complement thereof).
  • a nucleic acid reaction such as nucleic acid extension or ligation
  • the nucleic acid barcode molecule 1290 e.g., partition- specific barcode molecule
  • 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, which is hereby entirely incorporated by reference for all purposes. 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 may be performed.
  • the workflow may comprise a workflow as generally depicted in any of FIGs. 12A-12D, or a combination of workflows for an individual analyte, as described elsewhere herein.
  • the workflow may comprise a workflow as generally depicted in any of FIGs. 12A-12D, or a combination of workflows for an individual analyte, as described elsewhere herein.
  • multiple analytes can be analyzed.
  • analysis of an analyte comprises a workflow as generally depicted in FIG. 12A.
  • a nucleic acid barcode molecule 1290 may be co-partitioned with the one or more analytes.
  • nucleic acid barcode molecule 1290 is attached to a support 1230 (e.g., a bead, such as a gel bead), such as those described elsewhere herein.
  • nucleic acid barcode molecule 1290 may be attached to support 1230 via a releasable linkage 1240 (e.g., comprising a labile bond), such as those described elsewhere herein.
  • Nucleic acid barcode molecule 1290 may comprise a functional sequence 1221 and optionally comprise other additional sequences, for example, a barcode sequence 1222 (e.g., common barcode, partition-specific barcode, or other functional sequences described elsewhere herein), and/or a UMI sequence (not shown).
  • the nucleic acid barcode molecule 1290 may include other additional sequences, for example, a barcode sequence 1222 (e.g., common barcode, partition- specific barcode, or other functional sequences described elsewhere herein), and/or a UMI sequence.
  • the nucleic acid barcode molecule 1290 may comprise a capture sequence 1223 that may be complementary to another nucleic acid sequence, such that it may hybridize to a particular sequence, e.g. , capture handle sequence 1213.
  • 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 polyT sequence (FIG. 12C).
  • capture sequence 1223 may comprise a poly-T sequence and may be used to hybridize to mRNA.
  • nucleic acid barcode molecule 1290 comprises capture sequence 1223 complementary to a sequence of RNA molecule 1260 from a cell.
  • capture sequence 1223 comprises a sequence specific for an RNA molecule.
  • Capture sequence 1223 may comprise a known or targeted sequence or a random sequence.
  • a nucleic acid extension reaction may be performed, thereby generating a barcoded nucleic acid product comprising capture sequence 1223, the functional sequence 1221, barcode sequence 1222, any other functional sequence, and a sequence corresponding to the RNA molecule 1260.
  • the capture sequence 1223 of the nucleic acid barcode molecule 1290 includes non-templated nucleotides appended to its 3’ end and is configured to couple (e.g., hybridize) to a capture handle sequence 1213 of a reporter oligonucleotide 1220 conjugated to labelling agent 1210 e.g., polypeptide such as an antigen, antibody, or others described elsewhere herein).
  • labelling agent 1210 e.g., polypeptide such as an antigen, antibody, or others described elsewhere herein.
  • the capture sequence 1223 of the nucleic acid barcode molecule 1290 may include non-templated guanines appended to its 3’ end.
  • the nucleic acid barcode molecule 1290 may further include a template switch oligonucleotide (TSO).
  • TSO template switch oligonucleotide
  • the nucleic acid barcode molecule 1290 may further include a unique molecule identifier (UMI).
  • UMI unique molecule identifier
  • the hybridization of the capture sequence 1223 to the capture handle sequence 1213 extends reverse transcription of the hybridization product into the reporter oligonucleotide 1220 to generate a barcoded nucleic acid product including the capture sequence 1223, the capture handle sequence 1213, the functional sequences 1221 1211, and the reporter barcode sequence (e.g., UMI and/or TSO) 1212 1222.
  • UMI unique molecule identifier
  • capture sequence 1223 may be complementary to an overhang sequence or an adapter sequence that has been appended to an analyte.
  • primer 1250 comprises a sequence complementary to a sequence of nucleic acid molecule 1260 (such as an RNA encoding for a BCR sequence) from a biological particle.
  • primer 1250 comprises one or more sequences 1251 that are not complementary to RNA molecule 1260.
  • Sequence 1251 may 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 1250 comprises a poly-T sequence.
  • primer 1250 comprises a sequence complementary to a target sequence in an RNA molecule. In some instances, primer 1250 comprises a sequence complementary to a region of an immune molecule, such as the constant region of a TCR or BCR sequence.
  • Primer 1250 is hybridized to nucleic acid molecule 1260 and complementary molecule 1270 is generated (see Panel 1202).
  • complementary molecule 1270 may be cDNA generated in a reverse transcription reaction.
  • an additional sequence may be appended to complementary molecule 1270.
  • the reverse transcriptase enzyme may be selected such that several non-templated bases 1280 (e.g., a poly-C sequence) are appended to the cDNA.
  • Nucleic acid barcode molecule 1290 comprises a sequence 1224 complementary to the non-templated bases, and the reverse transcriptase performs a template switching reaction onto nucleic acid barcode molecule 1290 to generate a barcoded nucleic acid molecule comprising cell (e.g., partition specific) barcode sequence 1222 (or a reverse complement thereof) and a sequence of complementary molecule 1270 (or a portion thereof).
  • sequence 1223 comprises a sequence complementary to a region of an immune molecule, such as the constant region of a TCR or BCR sequence. Sequence 1223 is hybridized to nucleic acid molecule 1260 and a complementary molecule 1270 is generated.
  • complementary molecule 1270 may be generated in a reverse transcription reaction generating a barcoded nucleic acid molecule comprising cell (e.g., partition specific) barcode sequence 1222 (or a reverse complement thereof) and a sequence of complementary molecule 1270 (or a portion thereof). Additional methods and compositions suitable for barcoding cDNA generated from mRNA transcripts including those encoding V(D).T 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. filed June 26, 2015. and U.S. Patent Publication No. 2019/0367969, , each of which applications is lierein entirely incorporated by reference for all purposes.
  • 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).
  • the systems of the disclosure include: (a) a labelling reagent comprising a detectable label and a reporter oligonucleotide; and (b) a blocking agent that blocks or inhibits binding of a target antigen with a natural ligand of the target antigen.
  • Non-limiting exemplary embodiments of the systems disclosed herein can include one or more of the following features.
  • the systems of the disclosure further include instructions for coupling the target antigen to the labelling agent, and optionally instructions for use according to a method disclosed herein.
  • the reporter oligonucleotide comprises a target antigen reporter barcode sequence.
  • the systems of the disclosure further include a plurality of nucleic acid barcode molecules comprising a partition-specific barcode sequence and a capture sequence.
  • the systems further include a partitioning system for generating a partition.
  • the partitioning system comprises a microfluidic device.
  • the systems further include reagents for generating a first of a plurality of barcoded nucleic acid molecules formed by complementary base pairing of: (a) the capture sequence of the plurality of nucleic acid barcode molecules and (b) a capture handle sequence of an mRNA or DNA analyte comprising a nucleic acid sequence encoding at least a portion of the ABM.
  • the systems further include an analysis engine.
  • the systems further include a network.
  • the systems further include a sequencer.
  • Sample preparation Twenty BALB/c mice were immunized using human CTLA-4 antigen according to the following schedule: (1): day 0 prime, 50 pg of CTLA-4 subcutaneous in complete Freund’s adjuvant; day 14 boost, 25 ug of CTLA-4 subcutaneous in incomplete Freund’s adjuvant; day 28 boost, 25 ug of CTLA-4 subcutaneous in incomplete Freund’s adjuvant; day 42 boost, 25 ug of CTLA-4 subcutaneous in incomplete Freund’s adjuvant; day 51 boost, 25 ug of CTLA-4 subcutaneous with no adjuvant; day 53 boost, 25 pg of CTLA-4 subcutaneous with no adjuvant; day 59 final boost, 25 ug of CTLA-4 subcutaneous with no adjuvant.
  • each TotalSeqC reagent was mixed with biotinylated antigen at a IX molar excess of antigen to streptavidin (STA). The reactions were quenched with biotin, and then centrifuged at 2500g at 4°C, then used immediately for cell labeling.
  • STA streptavidin

Abstract

La présente invention concerne d'une manière générale le domaine de l'immunologie, et concerne en particulier des procédés et des systèmes améliorés utiles pour l'identification et la caractérisation de molécules de liaison à l'antigène (ABM) obtenues à partir d'échantillons biologiques, par exemple, des cellules uniques.
PCT/US2023/069877 2022-07-13 2023-07-10 Procédés et systèmes améliorés pour l'identification et la caractérisation de molécules de liaison à l'antigène à partir de cellules uniques WO2024015733A1 (fr)

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