Systems and methods for metabolome analysis

Abstract

The present disclosure provides systems and methods for detecting and analyzing the metabolome of cells. Such methods and systems may include or utilize barcoding of nucleic acid molecules and sequencing of nucleic acid molecules.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Patent Application No. 16 / 435,393, filed on June 7, 2019, U.S. Patent Application No. 16 / 434,099, filed on June 6, 2019, U.S. Provisional Patent Application No. 62 / 756,495, filed on November 6, 2018, and U.S. Provisional Patent Application No. 62 / 711,351, filed on July 27, 2018, each of which is incorporated herein by reference in its entirety. Background Art

[0003] Samples can be processed for various purposes, such as identifying the type of part within the sample. The sample can be a biological sample. Biological samples can be processed, for example, to detect a disease (e.g., cancer) or to identify a specific substance. There are various methods for processing samples, such as polymerase chain reaction (PCR) and sequencing.

[0004] Biological samples can be processed in various reaction environments, such as partitions. The partitions can be wells or droplets. Droplets or wells can be used to process biological samples in a manner that enables them to be distributed and processed individually. For example, such droplets can be separated from other droplet fluids, thereby enabling precise control of the corresponding environment in the droplet.

[0005] The biological samples in the partitions can be subjected to various processes, such as chemical processes or physical processes. The samples in the partitions can be subjected to heating or cooling, or chemical reactions, for example, to obtain substances that can be processed qualitatively or quantitatively.

[0006] Partitioning of cells and / or cellular material can be used to analyze nucleic acid molecules endogenous to the cells. Summary of the invention

[0007] Although the distribution of cells and / or cellular materials can be used for the analysis of endogenous nucleic acids, the analysis of other cellular components (so that the analysis can be associated with specific cells) remains challenging. Such analysis includes the analysis of cellular metabolites. The metabolite array (metabolome) of the dissected cell can be used to assess many cellular features, including cellular processes, functions and vigor. Therefore, it is recognized herein that there is a need for improved systems and methods for detecting and analyzing cellular compounds. In particular, the systems and methods described herein can be used for detecting and analyzing the metabolome of cells.

[0008] One aspect of the present disclosure provides a method for processing or analyzing metabolites from cells. The method includes: (a) generating a partition containing the following: (i) the metabolite, (ii) one or more beads (e.g., a single bead, a plurality of beads) containing a plurality of nucleic acid cell barcode molecules, each of which contains a cell barcode sequence and a capture sequence, the cell barcode sequence uniquely corresponding to the cell, and (iii) a molecular complex capable of coupling to the metabolite, wherein the molecular complex contains a molecular complex capture sequence and a molecular complex identifier sequence, wherein the molecular complex capture sequence cannot be approached by the capture sequence when the metabolite is not coupled to the molecular complex; (b) in the partition, providing a molecular complex sufficient to couple the metabolite to the molecule The invention relates to a method for synthesizing a nucleic acid molecule comprising: synthesizing a nucleic acid molecule comprising: a nucleic acid molecule having a molecular complex and a cell barcode sequence; and a method for synthesizing a nucleic acid molecule comprising: synthesizing a nucleic acid molecule having a molecular complex and a cell barcode sequence; and ...

[0009] In some embodiments, the method further comprises sequencing at least a portion of the nucleic acid molecule or a derivative thereof to identify the molecular complex identifier sequence and the cell barcode sequence. In some embodiments, the method further comprises using the molecular complex identifier sequence and the cell barcode sequence to identify the metabolite as being derived from the cell. In some embodiments, the molecular complex is a riboswitch. In some embodiments, the partition comprises the cell. In some embodiments, the method further comprises releasing the metabolite from the cell after (a).

[0010] In some embodiments, the method further comprises releasing the nucleic acid cell barcode molecule from the bead before (c). In some embodiments, the nucleic acid cell barcode molecule is released from the bead after exposure to a chemical stimulus in the partition. In some embodiments, (c) is performed in the partition. In some embodiments, the method further comprises releasing the nucleic acid molecule or its derivative from the partition or recovering the nucleic acid molecule or its derivative from the partition. In some embodiments, in (c), the nucleic acid molecule is synthesized using one or more of a nucleic acid amplification reaction, a reverse transcription reaction, and a template switching reaction.

[0011] In some embodiments, the method further comprises subjecting the nucleic acid molecule to one or more additional reactions. In some embodiments, the one or more additional reactions comprise a primer extension reaction. In some embodiments, the one or more additional reactions comprise adding one or more functional sequences to the nucleic acid molecule, wherein the one or more functional sequences are configured to allow the nucleic acid molecule or its derivative to be attached to a flow cell of a sequencer.

[0012] In some embodiments, the beads are gel beads. In some embodiments, the nucleic acid cell barcode molecules are attached to the beads when the nucleic acid molecules are synthesized. In some embodiments, the partitions comprise additional molecular complexes capable of coupling to additional metabolites different from the metabolites. In some embodiments, the beads comprise additional multiple nucleic acid cell barcode molecules, each of which comprises the cell barcode sequence and an additional capture sequence capable of coupling to an additional molecular complex capture sequence of the additional molecular complex when the additional metabolite is coupled to the additional molecular complex. In some embodiments, the method further comprises: in the partition, allowing the additional metabolite to couple to the additional molecular complex so as to (iii) make the additional molecular complex capture sequence accessible to the additional capture sequence of a given additional nucleic acid cell barcode molecule among the additional multiple nucleic acid cell barcode molecules, and (iv) allowing the additional capture sequence to couple to the additional molecular complex capture sequence; and in the case where the additional capture sequence is coupled to the additional molecular complex capture sequence, using the given additional nucleic acid cell barcode molecule and the additional molecular complex to synthesize an additional nucleic acid molecule, the additional nucleic acid molecule comprising (3) a third nucleic acid sequence corresponding to the additional molecular complex identifier sequence, and (4) a fourth additional nucleic acid sequence corresponding to the cell barcode sequence, wherein the third nucleic acid sequence and the fourth nucleic acid sequence allow the additional metabolite to be identified as corresponding to the cell.

[0013] In some embodiments, the plurality of nucleic acid cell barcode molecules comprises at least 100,000 nucleic acid cell barcode molecules. In some embodiments, the partition is a droplet in a plurality of droplets. In some embodiments, the partition is a hole in a plurality of wells. In some embodiments, the metabolite is selected from the group consisting of: adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), guanosine triphosphate (GTP), guanosine diphosphate (GDP), guanosine monophosphate (GMP), ribose, glucose, mannose, glycerol, phosphatidylcholine, phosphorylcholine, glycerylphosphorylcholine, phosphatidylserine, phosphatidylethanolamine, diglyceride, nicotinamide adenine dinucleotide phosphate, nicotinamide adenine dinucleotide, glycine, glutamine, aspartic acid, citric acid, glycerol, acetone, acetoacetic acid and lysine.

[0014] In some embodiments, each nucleic acid cell barcode molecule in the plurality of nucleic acid cell barcode molecules comprises an identifier sequence separate from the cell barcode sequence, and wherein the identifier sequence is different for each nucleic acid cell barcode molecule in the plurality of nucleic acid cell barcode molecules. In some embodiments, wherein in (a), each nucleic acid cell barcode molecule in the plurality of nucleic acid cell barcode molecules comprises the cell barcode sequence, wherein the bead is from a plurality of beads, and wherein the cell barcode sequence is different from the cell barcode sequence of nucleic acid cell barcode molecules of other beads in the plurality of beads.

[0015] In some embodiments, wherein the beads contain a plurality of additional nucleic acid cell barcode molecules, each of the plurality of additional nucleic acid cell barcode molecules comprises the cell barcode sequence and a binding sequence, the binding sequence being capable of coupling to an additional nucleic acid molecule different from the molecular complex. In some embodiments, the additional nucleic acid molecules are selected from the group consisting of: ribonucleic acid molecules of the cell, deoxyribonucleic acid molecules of the cell, and editing nucleic acid molecules capable of participating in a gene editing reaction. In some embodiments, the method further comprises exposing the protein of the cell to an antibody coupled to the additional nucleic acid molecule before (a), wherein the antibody binds to the protein. In some embodiments, the partition comprises the additional nucleic acid molecule, and the method further comprises allowing a given additional nucleic acid cell barcode molecule in the additional plurality of nucleic acid cell barcode molecules to bind to the additional nucleic acid molecule via the binding sequence. In some embodiments, the method further comprises using the additional nucleic acid molecule and the given additional nucleic acid cell barcode molecule of the additional plurality of nucleic acid cell barcode molecules to synthesize a reporter nucleic acid molecule, the reporter nucleic acid molecule comprising (3) a third nucleic acid sequence corresponding to the sequence of the additional nucleic acid molecule, and (4) a fourth nucleic acid sequence corresponding to the cell barcode sequence. In some embodiments, the method further comprises sequencing at least a portion of the nucleic acid molecule or a derivative thereof to identify the substance as associated with the cell.

[0016] Another aspect of the present disclosure provides a non-transitory computer-readable medium comprising machine-executable code that, when executed by one or more computer processors, implements any of the methods herein above or elsewhere herein.

[0017] Another aspect of the present disclosure provides a system comprising one or more computer processors and a computer memory coupled thereto. The computer memory comprises machine executable code, which when executed by one or more computer processors implements any method described above or elsewhere herein.

[0018] Additional aspects and advantages of the present disclosure will be apparent to those skilled in the art from the following detailed description, wherein only exemplary embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure is capable of other different embodiments, and its several details can be modified in various obvious aspects, all without departing from the present disclosure. Therefore, the drawings and descriptions are to be regarded as illustrative rather than restrictive in nature.

[0019] Incorporated by Reference

[0020] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent the publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The novel features of the present invention are described in detail in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description and the following drawings (also referred to herein as "Figures") which illustrate exemplary embodiments in which the principles of the present invention are utilized, in which:

[0022] Figure 1 An example of a microfluidic channel structure for dispensing individual bioparticles or analyte carriers is shown;

[0023] Figure 2 An example of a microfluidic channel structure for delivering barcode-carrying beads to droplets is shown;

[0024] Figure 3 An example of a microfluidic channel structure for co-dispensing biological particles or analyte carriers and reagents is shown;

[0025] Figure 4 An example of a microfluidic channel structure for controlled dispensing of beads into discrete droplets is shown;

[0026] Figure 5 An example of a microfluidic channel structure for increasing droplet generation throughput is shown;

[0027] Figure 6 Another example of a microfluidic channel structure for increasing droplet generation throughput is shown;

[0028] Fig. 7A A cross-sectional view of another example of a microfluidic channel structure having geometric features for controlled dispensing is shown. Figure 7B Shows Fig. 7A A perspective view of the channel structure;

[0029] Figure 8 An example of a barcode-carrying bead is illustrated;

[0030] Fig. 9 A computer system is shown that is programmed or otherwise configured to implement the methods provided herein;

[0031] Fig.10Schematic illustration of exemplary molecular complexes, riboswitches, and their functions upon binding of metabolites;

[0032] Fig.11A Schematically illustrates an exemplary method for detecting and analyzing cellular metabolites;

[0033] Fig. 11B An exemplary scheme for generating barcoded sequencing constructs derived from molecular complexes is schematically illustrated;

[0034] Fig. 11C schematically illustrates an exemplary scheme for adding additional sequences to a barcoded sequencing construct; and

[0035] Fig.12 Exemplary beads comprising nucleic acid cellular barcode molecules that can be used to detect and analyze a variety of different types of substances are schematically illustrated. DETAILED DESCRIPTION

[0036] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided merely for example. Various changes, variations and substitutions will now be made by those skilled in the art without departing from the present invention. It should be understood that a plurality of alternatives to the embodiments of the present invention described herein may be employed.

[0037] Where values ​​are referred to as ranges, it should be understood that such disclosure includes disclosure of all possible sub-ranges within such ranges, as well as specific values ​​within such ranges, whether or not a specific value or a specific sub-range is explicitly stated.

[0038] As used herein, the terms "a," "an," and "the" generally refer to either the singular or the plural referent, unless the context clearly dictates otherwise.

[0039] As used herein, the term "barcode" generally refers to a label or identifier that conveys or is capable of conveying information about an analyte. A barcode may be part of an analyte. A barcode may be independent of an analyte. A barcode may be a label or combination of labels attached to an analyte (e.g., a nucleic acid molecule) in addition to an endogenous feature of the analyte (e.g., the size or terminal sequence of the analyte). A barcode may be unique. A barcode may have a variety of different formats. For example, a barcode may include: a polynucleotide barcode; a random nucleic acid and / or amino acid sequence; and a synthetic nucleic acid and / or amino acid sequence. A barcode may be attached to an analyte in a reversible or irreversible manner. A barcode may be added to a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample, for example, before, during, and / or after sample sequencing. A barcode may allow identification and / or quantification of individual sequencing reads.

[0040] As used herein, the term "real time" may refer to a response time of less than about 1 second, one tenth of a second, one hundredth of a second, milliseconds, or less. The response time may be greater than 1 second. In some cases, real time may refer to simultaneous or substantially simultaneous processing, detection, or identification.

[0041] As used herein, the term "subject" generally refers to an animal, such as a mammal (e.g., a human) or an avian (e.g., a bird), or other organism, such as a plant. For example, a subject can be a vertebrate, a mammal, a rodent (e.g., a mouse), a primate, an ape, or a human. Animals can include, but are not limited to, farm animals, sports animals, and pets. A subject can be a healthy or asymptomatic individual, an individual who is sick or suspected of being sick (e.g., cancer) or susceptible to a disease, and / or an individual who is in need of treatment or suspected of being in need of treatment. A subject can be a patient. A subject can be a microorganism (e.g., a bacterium, a fungus, an archaea, a virus).

[0042] As used herein, the term "genome" generally refers to genomic information from a subject, which can be, for example, at least a portion or all of the subject's genetic information. The genome can be encoded in DNA or RNA. The genome can include coding regions (e.g., regions encoding proteins) and non-coding regions. The genome can include the sequences of all chromosomes together in an organism. For example, the human genome generally has a total of 46 chromosomes. The sequences of all chromosomes together can form the human genome.

[0043] The terms "aptamer", "adaptor" and "tag" can be used synonymously. The aptamer or tag can be coupled to the polynucleotide sequence to be "labeled" by any method, including ligation, hybridization or other methods.

[0044] As used herein, the term "sequencing" generally refers to methods and techniques for determining the sequence of nucleotide bases in one or more polynucleotides. A polynucleotide can be, for example, a nucleic acid molecule, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (e.g., single-stranded DNA). Sequencing can be performed by a variety of currently available systems, such as, but not limited to, Pacific Biosciences Oxford or Life Technologies (Ion ) sequencing system. Alternatively or in addition, nucleic acid amplification, polymerase chain reaction (PCR) (e.g., digital PCR, quantitative PCR, or real-time PCR) or isothermal amplification can be used. Such systems can provide a plurality of original genetic data corresponding to the genetic information of a subject (e.g., human), such as generated by a sample provided by the system from a subject. In some instances, such systems provide sequencing reads (also referred to herein as "reads"). Reads can include a string of nucleic acid bases corresponding to a sequence of nucleic acid molecules that have been sequenced. In some cases, the systems and methods provided herein can be used together with proteomics information.

[0045] As used herein, the term "bead" generally refers to a particle. A bead can be a solid or semi-solid particle. A bead can be a gel bead. A gel bead can include a polymer matrix (e.g., a matrix formed by polymerization or cross-linking). A polymer matrix can include one or more polymers (e.g., polymers with different functional groups or repeating units). The polymers in the polymer matrix can be randomly arranged, such as in a random copolymer, and / or have an ordered structure, such as in a block copolymer. Crosslinking can be via covalent, ionic or inductive interactions or physical entanglement. A bead can be a macromolecule. A bead can be formed by nucleic acid molecules bound together. A bead can be formed via covalent or non-covalent assembly of molecules (e.g., macromolecules), such as monomers or polymers. Such polymers or monomers can be natural or synthetic. Such polymers or monomers can be or include, for example, nucleic acid molecules (e.g., DNA or RNA). A bead can be formed by a polymeric material. A bead can be magnetic or non-magnetic. A bead can be rigid. A bead can be flexible and / or compressible. A bead can be breakable or soluble. The beads may be solid particles (eg, metal-based particles, including but not limited to iron oxide, gold, or silver) covered with a coating comprising one or more polymers. Such coatings may be disruptable or dissolvable.

[0046] As used herein, the term "sample" generally refers to a biological sample of a subject. A biological sample may contain many macromolecules, such as cellular macromolecules. A sample may be a cell sample. A sample may be a cell line or a cell culture sample. A sample may include one or more cells. A sample may include one or more microorganisms. A biological sample may be a nucleic acid sample or a protein sample. A biological sample may also be a carbohydrate sample or a lipid sample. A biological sample may be derived from another sample. A sample may be a tissue sample, such as a biopsy, a core biopsy, a needle aspirate, or a fine needle aspirate. A sample may be a fluid sample, such as a blood sample, a urine sample, or a saliva sample. A sample may be a skin sample. A sample may be a cheek swab. A sample may be a plasma or serum sample. A sample may be a cell-free or cell-free sample. A cell-free sample may include extracellular polynucleotides. Extracellular polynucleotides may be separated from a body sample that may be selected from the group consisting of: blood, plasma, serum, urine, saliva, mucosal excretions, sputum, feces, and tears.

[0047] As used herein, the term "biological particle" or "analyte carrier" generally refers to a discrete biological system derived from a biological sample. A biological particle or analyte carrier may contain or carry a target analyte (e.g., biological analyte) therein. In some embodiments, the analyte carrier itself is a target analyte. A biological particle or analyte carrier may be a macromolecule. A biological particle or analyte carrier may be a small molecule. A biological particle or analyte carrier may be a virus. A biological particle or analyte carrier may be a cell or a derivative of a cell. A biological particle or analyte carrier may be an organelle. A biological particle or analyte carrier may be a rare cell from a cell colony. A biological particle or analyte carrier may be any type of cell, including but not limited to prokaryotic cells, eukaryotic cells, bacteria, fungi, plants, mammals or other animal cell types, mycoplasmas, normal tissue cells, tumor cells, or any other cell type, whether derived from a single cell or a multicellular organism. A biological particle or analyte carrier may be a component of a cell. A biological particle or analyte carrier may be or may include DNA, RNA, organelles, proteins, or any combination thereof. The bioparticle or analyte carrier can be or can include a matrix (e.g., a gel or polymer matrix) containing cells or one or more components from cells (e.g., cell beads), such as DNA, RNA, organelles, proteins from cells, or any combination thereof. The bioparticle or analyte carrier can be obtained from the tissue of the subject. The bioparticle or analyte carrier can be a hardened cell. Such a hardened cell may or may not include a cell wall or a cell membrane. The bioparticle or analyte carrier can include one or more components of the cell, but may not include other components of the cell. An example of such a component is a nucleus or an organelle. The cell can be a living cell. The living cell can be cultured, such as when loaded into a gel or polymer matrix, or when containing a gel or polymer matrix. As used herein, the terms "bioparticle" and "analyte carrier" can be used interchangeably.

[0048] As used herein, the term "macromolecule component" generally refers to a macromolecule contained in or from a bioparticle or analyte carrier. The macromolecule component may include a nucleic acid. In some cases, the bioparticle or analyte carrier may be a macromolecule. The macromolecule component may include DNA. The macromolecule component may include RNA. RNA may be coding or non-coding. RNA may be, for example, messenger RNA (mRNA), ribosomal RNA (rRNA) or transfer RNA (tRNA). RNA may be a transcript. RNA may be a small RNA having a length of less than 200 nucleic acid bases, or a large RNA having a length of greater than 200 nucleic acid bases. Small RNA may include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), micro RNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), Piwi interacting RNA (piRNA), small RNA derived from tRNA (tsRNA) and RNA derived from small rDNA (srRNA). RNA may be double-stranded RNA or single-stranded RNA. RNA may be circular RNA. The macromolecule component may include a protein. The macromolecule component may include a peptide. The macromolecular component may comprise a polypeptide.

[0049] As used herein, the term "molecular tag" generally refers to a molecule that can bind to a macromolecular component. A molecular tag can bind to a macromolecular component with high affinity. A molecular tag can bind to a macromolecular component with high specificity. A molecular tag can comprise a nucleotide sequence. A molecular tag can comprise a nucleic acid sequence. A nucleic acid sequence can be at least a portion or all of a molecular tag. A molecular tag can be a nucleic acid molecule or can be a portion of a nucleic acid molecule. A molecular tag can be an oligonucleotide or a polypeptide. A molecular tag can comprise a DNA aptamer. A molecular tag can be or comprise a primer. A molecular tag can be or comprise a protein. A molecular tag can comprise a polypeptide. A molecular tag can be a barcode.

[0050] As used herein, the term "partition" generally refers to a space or volume that can be suitable for accommodating one or more substances or performing one or more reactions. A partition can be a physical compartment, such as a droplet or a hole. A partition can separate a space or volume from another space or volume. A droplet can be a first phase (e.g., an aqueous phase) in a second phase (e.g., an oil) that is immiscible with the first phase. A droplet can be a first phase in a second phase that is not separated from the first phase, such as a capsule or a liposome in an aqueous phase. A partition can include one or more other (internal) partitions. In some cases, a partition can be a virtual compartment that can be defined and identified across multiple and / or remote physical compartments by a marker (e.g., a marker library). For example, a physical compartment can include multiple virtual compartments.

[0051] Cellular metabolome analysis

[0052] Systems and methods for analyzing a cell metabolome are provided. Metabolites of a cell can be assigned to a partition so that their cell of origin can be identified. The partition can also include a molecular complex that can be uniquely coupled to the metabolite and have a nucleic acid sequence that identifies the molecular complex and the metabolite therefrom. Such sequences can be determined and used to identify the metabolite as originating from the cell.

[0053] In one aspect, the present disclosure provides a method for processing or analyzing metabolites from cells. The method comprises: (a) generating a partition comprising: (i) the metabolite, (ii) one or more beads (e.g., a single bead, a plurality of beads) comprising a plurality of nucleic acid cell barcode molecules, each of which comprises a cell barcode sequence and a capture sequence, wherein the cell barcode sequence uniquely corresponds to the cell, and (iii) a molecular complex capable of coupling to the metabolite, wherein the molecular complex comprises a molecular complex capture sequence and a molecular complex identifier sequence, wherein the molecular complex capture sequence cannot be approached by the capture sequence when the metabolite is not coupled to the molecular complex; (b) in the partition, providing a molecular complex sufficient to couple the metabolite to the molecular complex; conditions of the molecular complex so as to (i) make the capture sequence of the molecular complex accessible to the capture sequence of a given nucleic acid cell barcode molecule among the multiple nucleic acid cell barcode molecules, and (ii) allow the capture sequence to be coupled to the molecular complex capture sequence; and (c) when the capture sequence is coupled to the molecular complex capture sequence, use the given nucleic acid cell barcode molecule and the molecular complex to synthesize a nucleic acid molecule, the nucleic acid molecule comprising (1) a first nucleic acid sequence corresponding to the molecular complex identifier sequence, and (2) a second nucleic acid sequence corresponding to the cell barcode sequence, wherein the first nucleic acid sequence and the second nucleic acid sequence allow the metabolite and the cell to be identified.

[0054] In some cases, the plurality of nucleic acid cell barcode molecules comprises at least about 1,000 nucleic acid cell barcode molecules, at least about 5,000 nucleic acid cell barcode molecules, at least about 10,000 nucleic acid cell barcode molecules, at least about 50,000 nucleic acid cell barcode molecules, at least about 100,000 nucleic acid cell barcode molecules, at least about 500,000 nucleic acid cell barcode molecules, at least about 1,000,000 nucleic acid cell barcode molecules, at least about 5,000,000 nucleic acid cell barcode molecules, at least about 10,000,000 nucleic acid cell barcode molecules, at least about 100,000,000 nucleic acid cell barcode molecules, at least about 1,000,000,000 nucleic acid cell barcode molecules, or more. In some cases, the nucleic acid cell barcode molecules comprise minicell barcode sequences.

[0055] Each of the plurality of nucleic acid cell barcode molecules may include an identifier sequence separate from a cell barcode sequence, wherein the identifier sequence is different for each of the plurality of nucleic acid cell barcode molecules. In some cases, such an identifier sequence is a unique molecular identifier sequence (UMI) as described elsewhere herein. As described elsewhere herein, a UMI sequence can uniquely identify a barcoded specific nucleic acid molecule, which can identify a specific nucleic acid molecule analyzed, count the specific nucleic acid molecule analyzed, and the like. In addition, in some cases, including (a) each of the plurality of nucleic acid cell barcode molecules may include a cell barcode sequence, and the beads may be from a plurality of beads, such as a population of barcoded beads described elsewhere herein. Each cell barcode sequence may be different from the cell barcode sequence of the nucleic acid barcode molecules of other beads in the plurality of beads. In this case, a population of barcoded beads may be obtained, each of which includes a different cell barcode sequence.

[0056] The method can process or analyze any suitable metabolite from the cell. Non-limiting examples of such metabolites include adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), guanosine triphosphate (GTP), guanosine diphosphate (GDP), guanosine monophosphate (GMP), ribose, glucose, mannose, glycerol, phosphatidylcholine, phosphorylcholine, glycerylphosphorylcholine, phosphatidylserine, phosphatidylethanolamine, diglyceride, nicotinamide adenine dinucleotide phosphate, nicotinamide adenine dinucleotide, glycine, glutamine, aspartic acid, citric acid, glycerol, acetone, acetoacetic acid and lysine.

[0057] In general, a molecular complex at least partially comprises a nucleic acid component that provides a capture sequence and a molecular complex identifier sequence. However, in some cases, a molecular complex comprises multiple nucleic acid components, and each molecular complex capture sequence and molecular complex identifier sequence are on different nucleic acid components of the molecular complex. The molecular complex identifier sequence can be determined and used to identify metabolites, and the molecular complex capture sequence can be used to help generate a barcoded molecule corresponding to the molecular complex (e.g., via its molecular complex identifier sequence).

[0058] An example of a molecular complex is a riboswitch. A riboswitch may refer to a regulatory segment in a nucleic acid molecule (e.g., a ribonucleic acid molecule (RNA), a messenger RNA, etc.) that can bind to a substance (including a small molecule, a metabolite, etc.), resulting in a change in the production of a protein encoded by the nucleic acid molecule in a cell. Additional details about riboswitches are provided in Ruff and Strobel, RNA, November 2014; 20(11): 1775-88 and Butler et al., Chem Biol. March 25, 2011; 18(3): 293-298, both of which are incorporated herein by reference in their entirety for all purposes. A specific riboswitch may be uniquely bound to a specific substance (e.g., a specific metabolite) and may also have a unique sequence in other riboswitches. Such a riboswitch identifier sequence may be determined and used to determine the binding of a specific substance. In addition, when the substance binds to its respective riboswitch, the riboswitch can change its secondary structure and / or tertiary structure, so that one or more sequences of the riboswitch that are inaccessible in the substance-free state become accessible in the substance-bound state. Such sequences can be used as capture sequences that can bind to nucleic acid barcode molecules, and a complementary sequence corresponding to the riboswitch (including the riboswitch identifier sequence) is added to the nucleic acid barcode molecule via one or more reactions (e.g., one or more primer extension reactions).

[0059] exist Fig.10 Schematically depicting an exemplary riboswitch in a substance-free configuration and a substance-bound configuration. As shown, when the riboswitch 1001 is not bound to its corresponding substance 1003 (e.g., a metabolite) that binds to the riboswitch, the riboswitch 1001 comprises an inaccessible capture sequence 1002. The capture sequence 1002 cannot bind to other oligonucleotides in its inaccessible configuration. When the substance 1003 is bound to the riboswitch 1001, the capture sequence 1002 becomes accessible. As shown in the example, the riboswitch also includes a riboswitch identifier sequence 1004, which is 5' of the accessible capture sequence. The barcode nucleic acid molecule can be bound to the accessible capture sequence and extended to add a sequence complementary to the riboswitch identifier sequence 1004 to the nucleic acid barcode molecule.

[0060] In some cases, the riboswitch may also include an aptamer sequence in addition to the capture sequence. When not bound to a ligand, the aptamer sequence may engage with the capture sequence, and when bound to a ligand, the aptamer sequence is disengaged from the capture sequence. The aptamer sequence may also provide one or more binding sites for a ligand such as a metabolite. In some instances, the aptamer sequence and the capture sequence are configured in a 5' to 3' order (e.g., 5'-aptamer sequence-capture sequence-3'). In other instances, they are configured in an opposite configuration (e.g., 5'-capture sequence-aptamer sequence-3'). In addition, the aptamer sequence may include an identifier sequence for the riboswitch.

[0061] The riboswitch can comprise any suitable number of nucleotides. For example, the riboswitch can comprise at least about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more nucleotides. In some examples, a riboswitch can comprise up to about 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or fewer nucleotides. In addition, the aptamer sequence of the riboswitch can comprise at least about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more nucleotides. In some examples, the aptamer sequence of a riboswitch can comprise up to about 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or fewer nucleotides.In addition, the capture sequence of a riboswitch can comprise at least about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more nucleotides. In some examples, the capture sequence of a riboswitch can comprise up to about 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or fewer nucleotides.

[0062] In addition, in some cases, the partition comprises cells (which may be complete), and after the partition is generated in (a), the method may further include releasing metabolites from the cells. Can be released by any suitable strategy (including cell lysis). In such cases, the partition may include a cell lysis reagent. In some cases, the partition may include a cell bead, which comprises at least a portion of a cell or its cellular material and comprises metabolites. Cell beads and their available applications are described in U.S. Patent Application No. 15 / 887,947, which is incorporated herein by reference in its entirety for all purposes. In the case of using cell beads, metabolites can be released from cells before generating the partition in (a). In some cases, such as when implementing a hole when completing the method, releasing metabolites from cells may occur before distribution, so that the hole implemented in the partition contains cells or a cell extract comprising metabolites.

[0063] Prior to synthesizing the nucleic acid molecule, in (c), the nucleic acid cell barcode molecule can be released from the bead, for example after exposing the bead to a chemical stimulus in a partition as described elsewhere herein. However, in some cases, the nucleic acid cell barcode molecule is attached to the bead, such that the synthesized nucleic acid molecule is coupled to the bead.

[0064] In addition, in (c), nucleic acid molecules can be synthesized using one or more of nucleic acid amplification reactions, reverse transcription reactions, and template switching reactions as described elsewhere herein. Any suitable type of nucleic acid amplification can be used for synthesis, including amplification-based barcoding methods, including any method described elsewhere herein and those methods described in U.S. Patent Publication No. 2014 / 0378349, U.S. Patent Publication No. 2016 / 0257984, and U.S. Patent Publication No. 2015 / 0376609, which are incorporated herein by reference in their entirety for all purposes.

[0065] The method can also include subjecting the nucleic acid molecule to one or more additional reactions, which can occur in the partition and / or can occur once the nucleic acid molecule or a derivative of the nucleic acid molecule (e.g., a subsequent nucleic acid molecule, which comprises at least a portion of the nucleic acid molecule or its complement, etc.) is released or recovered from the partition. Therefore, the method can also include releasing or recovering the nucleic acid molecule from the partition, such as performing the synthesis of the nucleic acid molecule as in (c) in the partition. When the partition is a droplet in a plurality of droplets (e.g., a droplet of an emulsion as described elsewhere herein), the removal or recovery of the nucleic acid molecule or its derivative can be achieved by droplet destruction (e.g., destroying the emulsion in which the droplet is located). When the partition is a hole in a plurality of holes, the nucleic acid molecule or its derivative can be removed from the hole, for example, using standard fluid transport techniques (e.g., pipetting or other forced fluid flows).

[0066] In some cases, one or more additional reactions may include a primer extension reaction or multiple primer extension reactions. Such reactions include nucleic acid amplification reactions, such as polymerase chain reaction (PCR). Primer extension reactions can be used to add additional sequences to nucleic acid molecules or their derivatives by using primers and / or sequences associated with primers as templates, by initiating primer binding sites on nucleic acid molecules or their derivatives and further extending nucleic acid molecules. Primer extension reactions can also be used to increase the copy number of nucleic acid molecules or their derivatives. Depending on the specific construct to be used for analysis, any number of primer extension reactions can be implemented.

[0067] In some cases, the one or more additional reactions may include a ligation reaction (or a ligation reaction combined with one or more primer extension reactions). Additional sequences may be appended to the nucleic acid molecule or derivative thereof by the action of a ligase.

[0068] In addition, one or more additional reactions can add one or more functional sequences to nucleic acid molecules, which can make nucleic acid molecules or derivatives thereof suitable for nucleic acid sequencers and sequencing analysis. For example, one or more functional sequences can be configured to allow sequencing primers to hybridize with nucleic acid molecules or derivatives thereof, can be configured to sample markers, or can be configured to allow nucleic acid molecules or derivatives thereof to be connected to a flow cell of a sequencer (e.g., an Illumina sequencer or a sequencer of other types in which oligonucleotides for sequencing are fixed).

[0069] In addition, the method can also include sequencing at least a portion of the nucleic acid molecule or its derivative to identify the molecular complex identifier sequence and the cell barcode sequence. The molecular complex identifier sequence can be determined and used to counter-determine specific metabolites bound to the molecular complex. Since the bound molecular complexes are those that can interact with the nucleic acid cell barcode molecules, the molecular complex identifier sequence can uniquely identify specific metabolites present in the partition (and thus identify potential cells). In addition, the cell barcode sequence can be used to identify specific partitions (and thereby identify their contents, including cellular material from specific cells), thereby identifying potential cells from which metabolites are derived. Any suitable platform can be used for sequencing, including next-generation sequencing platforms, such as Illumina.

[0070] exist FIG. 11A to FIG. 11C An exemplary method for processing or analyzing metabolites from cells is schematically depicted in FIG. Fig.11A , a barcode bead 1101 in a plurality of barcode beads 1107 comprises a plurality of nucleic acid cell barcode molecules 1102. Each of the nucleic acid cell barcode molecules 1102 comprises a functional sequence 1103 that allows the nucleic acid cell barcode molecule 1102 to be connected to a flow cell (or other component) of a sequencer, a cell barcode sequence 1104, and a functional sequence 1105 that allows a sequencing primer to bind to the nucleic acid cell barcode molecule 1102. The nucleic acid cell barcode molecule also comprises a capture sequence 1106 that hybridizes with a molecular complex capture sequence. Although not shown in FIG. 11 , the nucleic acid cell barcode molecule may also comprise an identifier sequence such as a UMI.

[0071] The barcoded beads 1101 are provided to a dual-junction microfluidic device in the form of an aqueous mixture, wherein the beads are mixed at the first junction of the microfluidic device with a water stream 1109 containing a molecular complex 1108 that can bind to a specific metabolite, a reagent necessary for barcoding, cells 1111, one or more cell lysis reagents, and one or more reagents capable of releasing nucleic acid cell barcode molecules 1102 from the beads. The resulting mixture is then transferred to the second junction of the microfluidic device, where the mixture is contacted with an immiscible stream (e.g., a stream containing an oil such as a fluorinated oil and optionally a surfactant), thereby generating droplets. The droplets contain the molecular complex 1108, the barcoded beads 1101, the cells 1110, the cell lysis reagent, and the reagents required for barcoding. The cell lysis reagent lyses 1112, the cells 1110, so that the contents of the cells 1110 (including metabolites) are released into the droplets.

[0072] Metabolites 1113 released from cells 1110 then bind to molecular complex 1108, which changes the conformation of the molecular complex (e.g. Fig.10 1106 ), and makes its molecular complex capture sequence accessible to the capture sequence 1106 of the nucleic acid cell barcode molecule 1102. With the help of a corresponding releasing agent (e.g., such as a reducing agent, which degrades the disulfide bonds of the beads, destroys the disulfide bonds between the beads and the nucleic acid cell barcode molecule 1102, etc.), the nucleic acid cell barcode molecule 1102 is released from the barcoded bead. A given nucleic acid cell barcode molecule in the released nucleic acid cell barcode molecule 1102 is bound to the molecular complex 1108 for barcoding the molecular complex identifier sequence of the molecular complex 1108.

[0073] like Fig. 11B As schematically depicted in , barcoding of the molecular complex identifier sequence adds a sequence complementary to a given nucleic acid cell barcode molecule. Fig. 11B , a given nucleic acid cell barcode molecule is bound to a molecular complex capture sequence 1114 via its capture sequence 1106. The given nucleic acid cell barcode molecule is extended by the action of a polymerase to add a sequence complementary to the sequence of the molecular complex identifier sequence 1115 of the molecular complex 1108. After extension, a terminal transferase adds three cytosine nucleotides to the extended nucleic acid molecule, which allows the extended nucleic acid molecule to participate in a template switching reaction. A template switch oligonucleotide 1116 containing a functional sequence (e.g., a sequencing primer binding site) is then hybridized to the terminal cytosine nucleotide, and the extended nucleic acid molecule is then further extended. The resulting product can then be released from the droplet (e.g., by destroying the emulsion containing the droplet) and further amplified to produce a double-stranded product 1117.

[0074] Additional sequences (e.g., additional functional sequences, sample marker sequences, etc.) may be added to the double-stranded nucleic acid product 1117. In some cases, such sequences may be added by binding to a template containing the additional sequences through additional rounds of primer extension reactions and / or amplification. Fig. 11C An example is schematically shown in FIG. Fig. 11C , nucleic acid molecule 1118 can add a first sequence to a strand of double-stranded product 1117 by hybridization and extension / amplification. This added first sequence can then be hybridized with another nucleic acid molecule 1119 to add a second sequence by hybridization and extension / amplification. Any number of additional sequences can be added. Similarly, nucleic acid molecule 1120 can be used to add additional sequences to the other end of product 1117 by hybridization and extension. The completed product 1121 can then be separated and sequenced for analysis. Sequencing analysis can determine the cell barcode sequence and the molecular complex identifier sequence, thereby identifying the metabolites and the cell of origin.

[0075] Although not in Fig. 11C As shown in , there are other schemes for adding additional sequences, including, for example, ligation. For example, any of nucleic acid molecules 1118, 1119, and 1120 can be added to the chain of product 1117 by ligation. In some cases, shearing of product 1117 can be completed first before ligation.

[0076] Moreover, despite Fig.11A and Fig. 11B The example shown in FIG. 1 implements the droplets as partitions, but the method shown can be performed in a similar manner in another type of partition, such as a well. In such a case, Fig. 11C As shown, cells, barcoded beads, and all other reagents can be provided to the wells for cell lysis and barcoding, and the contents can be removed from the wells and processed in batches after barcoding to add additional sequences to the barcoded products and / or amplified products.

[0077] The method can also be used to process or analyze a variety of different metabolites. Determine that different metabolites can be used to illustrate the metabolome of the cell. Therefore, the partition can include other molecular complexes that can be coupled to other metabolites that are different from metabolites. In some cases, the bead comprises other multiple nucleic acid cell barcode molecules, each nucleic acid cell barcode molecule comprises a cell barcode sequence and other capture sequences of other molecular complex capture sequences that can be coupled to other molecular complexes when other metabolites are coupled to other molecular complexes. In some cases, the other capture sequence is identical to the capture sequence. In other cases, the other capture sequence is a different capture sequence.

[0078] In the case of achieving an additional molecular complex that is associated with an additional metabolite, the method may also include allowing the additional metabolite to couple to the additional molecular complex so that (iii) the additional molecular complex capture sequence is accessible to the additional capture sequence of a given additional nucleic acid cell barcode molecule in the additional plurality of nucleic acid cell barcode molecules, and (iv) allowing the additional capture sequence to couple to the additional molecular complex capture sequence. In the case of the additional capture sequence being coupled to the additional molecular complex capture sequence, the given additional nucleic acid cell barcode molecule and the additional molecular complex may be used to synthesize an additional nucleic acid molecule, the additional nucleic acid molecule comprising (3) a third nucleic acid sequence corresponding to the additional molecular complex identifier sequence, and (4) a fourth additional nucleic acid sequence corresponding to the cell barcode sequence. The third nucleic acid sequence and the fourth nucleic acid sequence may allow additional metabolites to be identified together with the cell.

[0079] As an example, you can modify FIG. 11A to FIG. 11C The exemplary method shown in , so that each droplet is provided with a library of different molecular complexes. Each synthesized nucleic acid molecule will contain a cell barcode sequence and a sequence corresponding to a specific molecular complex, where its corresponding metabolite is present in the cell. The identification of the molecular complex identifier sequences of various molecular complexes can then be used to identify a series of metabolites present in the cell. The resulting analysis can then be used to construct the metabolome of the cell.

[0080] Metabolite processing and analysis can be combined with other analyses in the partition, non-limiting examples of which include transcript analysis, cell surface feature analysis, cell protein analysis, and analysis of cell gene editing processes and related nucleic acid molecules. Suitable methods for multi-analyte analysis in partitions are described in U.S. Patent Application No. 15 / 720,085 and PCT International Application No. PCT / US2017 / 068320, each of which is incorporated herein by reference in its entirety for all purposes.

[0081] In multi-analyte analysis, the barcoded beads used for analysis may include barcode molecules that can capture different analytes. Therefore, the beads may include multiple additional nucleic acid cell barcode molecules, each nucleic acid cell barcode molecule including a cell barcode sequence and a binding sequence, the binding sequence being capable of coupling to additional nucleic acid molecules different from the molecular complex (e.g., cellular ribonucleic acid molecules (e.g., messenger ribonucleic acid (mRNA), deoxyribonucleic acid (e.g., genomic deoxyribonucleic acid (gDNA)) and editing nucleic acid molecules capable of participating in gene editing reactions). The partitions may include additional nucleic acid molecules, and the method may also include allowing a given additional nucleic acid cell barcode molecule in the additional plurality of nucleic acid barcode molecules to bind to the additional nucleic acid molecules via the binding sequence. For example, a given additional nucleic acid cell barcode molecule may be bound to a nucleic acid molecule coupled to an agent (e.g., an antibody) that binds to a cellular protein (whether on the cell surface or inside the cell). In such cases, prior to distribution, the protein may be exposed to an antibody coupled to an additional nucleic acid molecule, wherein the antibody binds to the protein.

[0082] In addition, the additional nucleic acid molecules and a given additional nucleic acid cell barcode molecule in the additional plurality of nucleic acid cell barcode molecules can be used to synthesize a reporter nucleic acid molecule, the reporter nucleic acid molecule comprising (3) a third nucleic acid sequence corresponding to the sequence of the additional nucleic acid molecule, and (4) a fourth nucleic acid sequence corresponding to the cell barcode sequence. At least a portion of the reporter nucleic acid molecule or a derivative thereof can be sequenced to identify the additional nucleic acid molecule as originating from a cell. In the case where the additional nucleic acid molecule is coupled to an agent that binds to a cellular protein (e.g., an antibody), a barcoded nucleic acid molecule can be generated from the additional nucleic acid molecule, sequenced, and the antibody and its target protein can be identified as originating from a specific cell.

[0083] Fig.12 An exemplary barcoded bead that can be provided to a partition for multi-analyte analysis is schematically depicted in FIG. Fig.12 , barcode bead 1201 comprises two nucleic acid cell barcode molecules 1202 and 1203. Nucleic acid cell barcode molecule 1202 comprises a functional sequence 1204 that allows nucleic acid cell barcode molecule 1202 to be attached to sequencer components; a cell barcode sequence 1205; a UMI sequence 1206; a functional sequence that can bind to a sequencing primer 1207 and a capture sequence 1208. Capture sequence 1208 can bind to a specific cellular analyte, such as mRNA, gDNA, or a nucleic acid molecule coupled to an agent that binds to a cellular protein, etc. Barcoded molecules can be generated in partitions and sequenced (or derivatives sequenced) to identify target molecules as originating from potential cells from which cellular material in the partitions was provided with barcoded beads 1201.

[0084] Additionally, nucleic acid cellular barcode molecule 1203 comprises sequences 1204, 1205, and 1207 as in nucleic acid cellular barcode molecules, and further comprises a capture sequence 1209. In some cases, capture sequence 1209 is the same as capture sequence 1208. In other cases, capture sequence 1209 is a different sequence than capture sequence 1208. Capture sequence 1209 can be coupled to a molecular complex having an accessible binding sequence (e.g., due to metabolite binding), and processing / analysis of the metabolites performed as described elsewhere herein.

[0085] Systems and methods for sample partitioning

[0086] In one aspect, the systems and methods described herein provide for partitioning, deposition or distribution of one or more particles (e.g., bioparticles or analyte carriers, macromolecular components of bioparticles or analyte carriers, beads, reagents, etc.) into discrete compartments or partitions (interchangeably referred to herein as partitions), wherein each partition maintains its own contents separate from the contents of other partitions. A partition may be a droplet in an emulsion. A partition may include one or more other partitions.

[0087] Partitions may include one or more particles. Partitions may include one or more types of particles. For example, partitions of the present disclosure may include one or more biological particles or analyte carriers and / or their macromolecular components. Partitions may include one or more gel beads. Partitions may include one or more cell beads. Partitions may include a single gel bead, a single cell bead, or both a single cell bead and a single gel bead. Partitions may include one or more reagents. Alternatively, partitions may be unoccupied. For example, partitions may not include beads. Cell beads may be, for example, one or more biological particles or analyte carriers and / or their macromolecular components encapsulated in a gel or polymer matrix via polymerization of droplets containing biological particles or analyte carriers and precursors capable of polymerization or gelation. A unique identifier (e.g., a barcode) may be injected into a droplet, for example, via a microcapsule (e.g., a bead) before, after, or simultaneously with the generation of the droplet, as described elsewhere herein. A microfluidic channel network (e.g., on a chip) may be used to generate partitions as described herein. Alternative mechanisms may also be employed in the dispensing of individual bioparticles or analyte carriers, including porous membranes through which an aqueous mixture of cells is extruded into a non-aqueous fluid.

[0088] Partitions can flow in fluid streams. Partitions can include, for example, microbubbles, which have an external barrier around the center or core of the internal fluid. In some cases, partitions can include a porous matrix capable of entraining and / or retaining materials in its matrix. Partitions can be droplets of the first phase in the second phase, wherein the first phase and the second phase are immiscible. For example, partitions can be droplets of aqueous fluids in a non-aqueous continuous phase (e.g., an oil phase). In another example, partitions can be droplets of non-aqueous fluids in an aqueous phase. In some examples, partitions can be provided in the form of water-in-oil emulsions or oil-in-water emulsions. A variety of different containers are, for example, described in U.S. Patent Application Publication No. 2014 / 0155295, which is incorporated herein by reference in its entirety for all purposes. An emulsion system for producing stable droplets in a non-aqueous or oily continuous phase is described in, for example, U.S. Patent Application Publication No. 2010 / 0105112, which is incorporated herein by reference in its entirety for all purposes.

[0089] In the case of droplets in an emulsion, in a non-limiting example, the distribution of individual particles into discrete partitions can be achieved by introducing a particle stream flowing in an aqueous fluid into a flowing non-aqueous fluid stream so that droplets are generated at the junction of the two streams. Fluid properties (e.g., fluid flow rate, fluid viscosity, etc.), particle properties (e.g., volume fraction, particle size, particle concentration, etc.), microfluidic architectures (e.g., channel geometry, etc.), and other parameters can be adjusted to control the occupancy of the resulting partitions (e.g., the number of biological particles or analyte carriers per partition, the number of beads per partition, etc.). For example, the partition occupancy can be controlled by providing a water flow with a certain particle concentration and / or flow rate. In order to produce a single biological particle or analyte carrier partition, the relative flow rate of the immiscible fluid can be selected so that the partition can contain less than one biological particle or analyte carrier per partition to ensure that those partitions occupied are mainly occupied individually. In some cases, a partition in a plurality of partitions can contain at most one biological particle (e.g., bead, DNA, cell, or cell material). In some embodiments, various parameters (e.g., fluid properties, particle properties, microfluidic structure, etc.) can be selected or adjusted so that most of the partitions are occupied, for example, allowing only a small portion of the partitions to be unoccupied. The flow and channel architecture can be controlled to ensure a given amount of single occupied partitions, unoccupied partitions below a certain level, and / or multiple occupied partitions below a certain level.

[0090] Figure 1An example of a microfluidic channel structure 100 for dispensing individual biological particles or analyte carriers is shown. The channel structure 100 may include channel segments 102, 104, 106, and 108 connected at a channel junction 110. In operation, a first aqueous fluid 112 including a suspended biological particle or analyte carrier (or cell) 114 may be transported along the channel segment 102 to the junction 110, and a second fluid 116 immiscible with the aqueous fluid 112 is delivered from each of the channel segments 104 and 106 to the junction 110 to produce discrete droplets 118, 120 of the first aqueous fluid 112, the first aqueous fluid flows into the channel segment 108, and flows away from the junction 110. The channel segment 108 may be coupled to an outlet tank fluid, and the discrete droplets may be stored and / or harvested in the outlet tank. The generated discrete droplets may include individual biological particles or analyte carriers 114 (e.g., droplets 118). The generated discrete droplets may include more than one individual bioparticle or analyte carrier 114 (in Figure 1 14). The discrete droplets may not contain a biological particle or analyte carrier 114 (e.g., droplet 120). Each discrete partition may keep its own contents (e.g., a single biological particle or analyte carrier 114) separate from the contents of other partitions.

[0091] The second fluid 116 may comprise an oil, such as a fluorinated oil, including a fluorinated surfactant for stabilizing the resulting droplets, such as inhibiting subsequent coalescence of the resulting droplets 118, 120. Examples of particularly useful dispensing fluids and fluorinated surfactants are described, for example, in U.S. Patent Application Publication No. 2010 / 0105112, which is incorporated herein by reference in its entirety for all purposes.

[0092] As will be appreciated, channel sections as described herein can be coupled to any of various fluid sources or receiving components, including fluid components of storage tanks, pipelines, manifolds or other systems.As will be appreciated, microfluidic channel structure 100 can have other geometric structures.For example, microfluidic channel structure can have more than one channel joint.For example, microfluidic channel structure can have 2,3,4 or 5 channel sections, each of which carries particles (for example, bioparticles or analyte carriers, cell beads and / or gel beads), and these channel sections meet at channel joints. Fluid can be guided to flow along one or more channels or storage tanks via one or more fluid flow units. Fluid flow units can include compressors (for example, positive pressure is provided), pumps (for example, negative pressure is provided), actuators, etc., to control the flow of fluid. Fluid can also or otherwise be controlled by applied pressure differential, centrifugal force, electric pumping, vacuum, capillary flow or gravity flow, etc.

[0093] The generated droplets may include two subsets of droplets: (1) occupied droplets 118, which contain one or more biological particles or analyte carriers 114, and (2) unoccupied droplets 120, which do not contain any biological particles or analyte carriers 114. The occupied droplets 118 may include singly occupied droplets (having one biological particle or analyte carrier) and multiple occupied droplets (having more than one biological particle or analyte carrier). As described elsewhere herein, in some cases, each occupied partition in the majority of occupied partitions may include no more than one biological particle or analyte carrier, and some of the generated partitions may be unoccupied (not occupied by any biological particle or analyte carrier). However, in some cases, some occupied partitions may include more than one biological particle or analyte carrier. In some cases, the allocation process can be controlled so that less than about 25% of the occupied partitions contain more than one bioparticle or analyte carrier, and in many cases, less than about 20% of the occupied partitions have more than one bioparticle or analyte carrier, and in some cases, less than about 10% or even less than about 5% of the occupied partitions include more than one bioparticle or analyte carrier per partition.

[0094] In some cases, it may be useful to minimize the generation of an excessive number of empty partitions, for example to reduce costs and / or improve efficiency. Although such minimization can be achieved by providing a sufficient number of bioparticles or analyte carriers (e.g., bioparticles or analyte carriers 114) at the distribution junction 110 to ensure that at least one bioparticle or analyte carrier is encapsulated in a partition, a Poissonian distribution can be expected to increase the number of partitions that include multiple bioparticles or analyte carriers. Therefore, in the case of individually occupied partitions, 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 may be unoccupied.

[0095] In some cases, the flow rate of one or more of the biological particles or analyte carriers (e.g., in channel segment 102) or other fluids directed into the distribution junction (e.g., in channel segments 104, 106) can be controlled so that in many cases, no more than about 50% of the generated partitions, no more than about 25% of the generated partitions, or no more than about 10% of the generated partitions are unoccupied. These flows can be controlled to present a non-Poisson distribution of a single occupied partition while providing a lower level of unoccupied partitions. The above range of unoccupied partitions can be achieved while still providing any of the above individual occupancies. For example, in many cases, the use of the systems and methods described herein can produce a resulting partition having a plurality of occupancies of less than about 25%, less than about 20%, less than about 15%, less than about 10%, and in many cases, less than about 5%, while the unoccupied partitions are less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less.

[0096] As will be appreciated, the above occupancy ratios also apply to partitions that include both biological particles or analyte carriers and additional reagents, including but not limited to microcapsules or beads (e.g., gel beads) carrying barcoded nucleic acid molecules (e.g., oligonucleotides) (relative to Figure 2 An occupied partition (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the occupied partition) can include both a microcapsule (e.g., a bead) comprising a barcoded nucleic acid molecule and a bioparticle or analyte carrier.

[0097] On the other hand, in addition to or in place of droplet-based distribution, bioparticles or analyte carriers can be encapsulated in microcapsules, which include shells, layers or porous substrates, wherein one or more independent bioparticles or analyte carriers or small groups of bioparticles or analyte carriers are entrained. Microcapsules can include other reagents. The encapsulation of bioparticles or analyte carriers can be carried out by a variety of methods. Such methods can be combined with aqueous fluids containing bioparticles or analyte carriers and polymer precursor materials, which can form gels or other solid or semisolid matrices when specific stimulation is applied to polymer precursors. Such stimulation can include, for example, thermal stimulation (for example, heating or cooling), light stimulation (for example, by photocuring), chemical stimulation (for example, by crosslinking, polymerization initiation of precursors (for example, by added initiators)), mechanical stimulation or their combination.

[0098] Encapsulation of microcapsules containing bioparticles or analyte carriers can be performed by a variety of methods. For example, droplets of a precursor fluid can be dispensed into a gelling solution using an air knife droplet or an aerosol generator to form microcapsules containing individual bioparticles or analyte carriers or small groups of bioparticles or analyte carriers. Similarly, a membrane-based encapsulation system can be used to generate microcapsules containing encapsulated bioparticles or analyte carriers as described herein. As described herein, the microfluidic systems of the present disclosure, e.g. Figure 1 The microfluidic system shown in can be easily used to encapsulate cells. Specifically, and ref. Figure 1 , an aqueous fluid 112 containing (i) a biological particle or analyte carrier 114 and (ii) a polymer precursor material (not shown) flows into a channel junction 110, where the aqueous fluid is distributed into droplets 118, 120 by a non-aqueous fluid stream 116. In the case of an encapsulation method, the non-aqueous fluid 116 may also include an initiator (not shown) to cause polymerization and / or cross-linking of the polymer precursor to form a microcapsule including the entrained biological particle or analyte carrier. Examples of polymer precursor / initiator pairs include those described in U.S. Patent Application Publication No. 2014 / 0378345, which is incorporated herein by reference in its entirety for all purposes.

[0099] For example, where the polymer precursor material comprises a linear polymer material (e.g., linear polyacrylamide, PEG, or other linear polymer material), the activator may comprise a crosslinker or a chemical that activates the crosslinker within the formed droplet. Similarly, for polymer precursors comprising polymerizable monomers, the activator may comprise a polymerization initiator. For example, in some cases, where the polymer precursor comprises a mixture of acrylamide monomers and N,N'-bis-(acryloyl)cystamine (BAC) comonomers, a reagent such as tetraethylmethylenediamine (TEMED) may be provided within the second fluid stream 116 in channel segments 104 and 106 that may initiate copolymerization of acrylamide and BAC into a crosslinked polymer network or hydrogel.

[0100] After the second fluid stream 116 contacts the first fluid stream 112 at the junction 110, during droplet formation, TEMED can diffuse from the second fluid 116 into the aqueous fluid 112 comprising linear polyacrylamide, which will activate the cross-linking of the polyacrylamide within the droplets 118, 120, thereby resulting in the formation of gel (e.g., hydrogel) microcapsules as solid or semi-solid beads or particles entraining the cells 114. Although described in terms of polyacrylamide encapsulation, other "activatable" encapsulation compositions can also be employed in the context of the methods and compositions described herein. For example, the formation of alginate droplets followed by exposure to divalent metal ions (e.g., Ca 2+ions), can be used as an encapsulation method using the method. Likewise, agarose droplets can also be transformed into capsules by temperature-based gelation (e.g., upon cooling, etc.).

[0101] In some cases, the encapsulated bioparticles or analyte carriers can be selectively released from the microcapsules, such as over time or when a specific stimulus is applied, the microcapsules are sufficiently degraded to allow the bioparticles or analyte carriers (e.g., cells) or other contents thereof to be released from the microcapsules, such as into partitions (e.g., droplets). For example, in the case of the above-mentioned polyacrylamide polymers, degradation of the microcapsules can be achieved by introducing appropriate reducing agents such as DTT to cleave the disulfide bonds that cross-link the polymer matrix. See, for example, U.S. Patent Application Publication No. 2014 / 0378345, which is incorporated herein by reference in its entirety for all purposes.

[0102] The bioparticle or analyte carrier can be subjected to other conditions sufficient to polymerize or gel the precursor. The conditions sufficient to polymerize or gel the precursor can include exposure to heating, cooling, electromagnetic radiation and / or light. The conditions sufficient to polymerize or gel the precursor can include any conditions sufficient to polymerize or gel the precursor. After polymerization or gelation, a polymer or gel can be formed around the bioparticle or analyte carrier. The polymer or gel can be diffusely permeable to chemical or biochemical reagents. The polymer or gel can be non-diffusible permeable to the macromolecular components of the bioparticle or analyte carrier. In this way, the polymer or gel can play a role in allowing the bioparticle or analyte carrier to undergo chemical or biochemical operations while spatially limiting the macromolecular components to the droplet region defined by the polymer or gel. The polymer or gel may include one or more of the following: 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 include any other polymer or gel.

[0103] The polymer or gel can be functionalized to bind to a target analyte, such as a nucleic acid, protein, carbohydrate, lipid or other analyte. The polymer or gel can be polymerized or gelled by a passive mechanism. The polymer or gel can be stable under alkaline conditions or high temperatures. The polymer or gel can have mechanical properties similar to those of beads. For example, the polymer or gel can have a size similar to that of beads. The polymer or gel can have a mechanical strength (e.g., tensile strength) similar to that of beads. The density of the polymer or gel can be lower than that of oil. The density of the polymer or gel can be roughly similar to that of a buffer. The polymer or gel can have an adjustable pore size. The pore size can be selected to, for example, retain denatured nucleic acids. The pore size can be selected to maintain diffusion permeability to exogenous chemicals (e.g., sodium hydroxide (NaOH)) and / or endogenous chemicals (e.g., inhibitors). The polymer or gel can be biocompatible. The polymer or gel can maintain or enhance cell viability. The polymer or gel can be biochemically compatible. The polymer or gel can be polymerized and / or depolymerized by thermal, chemical, enzymatic and / or optical means.

[0104] The polymer may comprise poly(acrylamide-co-acrylic acid) cross-linked via disulfide bonds. The preparation of the polymer may comprise a two-step reaction. In the first activation step, the poly(acrylamide-co-acrylic acid) may be exposed to an acylating agent to convert the carboxylic acid into an ester. For example, the poly(acrylamide-co-acrylic acid) may be exposed to 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM). The polyacrylamide-co-acrylic acid may be exposed to other salts of 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine. In the second cross-linking step, the ester formed in the first step may be exposed to a disulfide cross-linking agent. For example, the ester may be exposed to cystamine (2,2'-disulfide bis(ethylamine)). After these two steps, the bioparticle or analyte carrier may be surrounded by polyacrylamide chains linked together by disulfide bridges. In this way, biological particles or analyte carriers can be enclosed in a gel or matrix (e.g., a polymer matrix) or include the gel or matrix to form "cell beads". Cell beads can contain biological particles or analyte carriers (e.g., cells) or macromolecular components of biological particles or analyte carriers (e.g., RNA, DNA, proteins, etc.). Cell beads can include one cell or multiple cells, or derivatives of the one or multiple cells. For example, after lysing and washing the cells, the inhibitory components from the cell lysate can be washed away, and the macromolecular components can be combined into cell beads. The systems and methods disclosed herein are applicable to both cell beads (and / or droplets or other partitions) containing biological particles or analyte carriers and cell beads (and / or droplets or other partitions) containing macromolecular components of biological particles or analyte carriers.

[0105] Compared with the biological particles or analyte carriers distributed based on droplets, the encapsulated biological particles or analyte carriers can provide some potential advantages that are easier to store and more portable. In addition, in some cases, it may be useful to allow the biological particles or analyte carriers to hatch a selected period of time before analysis, for example, to characterize the change of such biological particles or analyte carriers over time in the presence or absence of different stimuli. In such cases, encapsulation can allow longer incubation than distributing in emulsion droplets, but in some cases, the biological particles or analyte carriers of droplet partitions can also be hatched for different time periods, for example, at least 10 seconds, at least 30 seconds, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours or at least 10 hours or more. The encapsulation of biological particles or analyte carriers can constitute the partitions of biological particles, and other reagents are distributed in biological particles or analyte carriers together. Alternatively or in addition, the encapsulated biological particles or analyte carriers can be easily deposited in other partitions (for example, droplets) as described above.

[0106] Beads

[0107] The partition can include one or more unique identifiers, such as bar codes. The bar code can be delivered in advance, subsequently or simultaneously to the partition that holds the bioparticles or analyte carriers that are divided or allocated. For example, the bar code can be injected into the droplet before, after or simultaneously with the droplet generation. The bar code is delivered to a specific partition allowing the characteristics of the individual bioparticles or analyte carriers to be subsequently attributed to the specific partition. The bar code can be delivered to the partition by any suitable mechanism, for example, on a nucleic acid molecule (e.g., an oligonucleotide). The barcoded nucleic acid molecules can be delivered to the partition by a microcapsule. In some cases, the microcapsule can include beads. Beads are described in more detail below.

[0108] In some cases, the barcoded nucleic acid molecule can be initially associated with the microcapsule and then released from the microcapsule. The release of the barcoded nucleic acid molecule can be passive (e.g., by diffusing out of the microcapsule). Additionally or alternatively, release from the microcapsule can be after applying a stimulus that allows the barcoded nucleic acid molecule to dissociate or release from the microcapsule. Such a stimulus can destroy the microcapsule, which is a barcoded nucleic acid molecule coupled to the microcapsule or in the microcapsule or an interaction between the two. Such a stimulus can include, for example, thermal stimulation, light stimulation, chemical stimulation (e.g., pH changes or the use of a reducing agent), mechanical stimulation, radiation stimulation, biological stimulation (e.g., enzymes) or any combination thereof.

[0109] Figure 2An example of a microfluidic channel structure 200 for delivering beads carrying barcodes to droplets is shown. The channel structure 200 may include channel segments 201, 202, 204, 206, and 208 connected at a channel junction 210. In operation, the channel segment 201 may transport an aqueous fluid 212 including a plurality of beads 214 (e.g., containing nucleic acid molecules, oligonucleotides, molecular tags) along the channel segment 201 to the junction 210. The plurality of beads 214 may be derived from a bead suspension. For example, the channel segment 201 may be connected to a reservoir containing an aqueous suspension of beads 214. The channel segment 202 may transport an aqueous fluid 212 including a plurality of biological particles or analyte carriers 216 along the channel segment 202 to the junction 210. The plurality of biological particles or analyte carriers 216 may be derived from a suspension of biological particles or analyte carriers. For example, channel segment 202 can be connected to a storage tank containing an aqueous suspension of biological particles or analyte carrier 216. In some cases, the aqueous fluid 212 in the first channel segment 201 or the second channel segment 202 or both segments can include one or more reagents, as described in further detail below. A second fluid 218 (e.g., oil) that is immiscible with the aqueous fluid 212 can be delivered to the junction 210 from each of channel segments 204 and 206. When the aqueous fluid 212 from each of channel segments 201 and 202 meets the second fluid 218 from each of channel segments 204 and 206 at the channel junction 210, the aqueous fluid 212 can be distributed as discrete droplets 220 in the second fluid 218 and flow away from the junction 210 along the channel segment 208. The channel segment 208 can deliver the discrete droplets to an outlet storage tank connected to the channel segment 208 fluid, and the discrete droplets can be harvested in the outlet storage tank.

[0110] Alternatively, channel segments 201 and 202 may meet at another junction upstream of junction 210. At this junction, the beads and the biological particles or analyte carriers may form a mixture, which is directed along another channel to junction 210 to obtain droplets 220. The mixture may provide beads and biological particles or analyte carriers in an alternating manner, such that, for example, a droplet contains a single bead and a single biological particle or analyte carrier.

[0111] Beads, bioparticles or analyte carriers and droplets can flow along the channel with a substantially regular flow curve (e.g., with a regular flow rate). Such regular flow curves can allow droplets to include one or more beads (e.g., single beads, multiple beads) and single bioparticles or analyte carriers. Such regular flow curves can allow droplets to have an occupancy rate greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% (e.g., droplets with beads and bioparticles or analyte carriers). Such conventional flow patterns and devices that can be used to provide such conventional flow patterns are provided in, for example, U.S. Patent Publication No. 2015 / 0292988, which is incorporated herein by reference in its entirety.

[0112] The second fluid 218 may comprise an oil, such as a fluorinated oil, which includes a fluorinated surfactant for stabilizing the resulting droplets, such as inhibiting subsequent coalescence of the resulting droplets 220 .

[0113] The generated discrete droplets may include individual biological particles or analyte carriers 216. The generated discrete droplets may include beads 214 carrying barcodes or other reagents. The generated discrete droplets may include both individual biological particles or analyte carriers and beads (e.g., droplets 220) carrying barcodes. In some cases, the discrete droplets may include more than one individual biological particle or analyte carrier or no biological particle or analyte carrier. In some cases, the discrete droplets may include more than one bead or no beads. The discrete droplets may be unoccupied (e.g., no beads, no biological particle or analyte carrier).

[0114] Advantageously, dispensing the bioparticle or analyte carrier and the barcoded beads into discrete droplets can effectively allow the attribution of barcodes to macromolecular components of the bioparticle or analyte carrier within a partition.The contents of a partition can be kept discrete from the contents of other partitions.

[0115] As will be appreciated, channel sections as described herein can be coupled to any of various fluid sources or receiving components, including fluid components of storage tanks, pipelines, manifolds or other systems.As will be appreciated, microfluidic channel structure 200 can have other geometric structures.For example, microfluidic channel structure can have more than one channel joint.For example, microfluidic channel structure can have 2,3,4 or 5 channel sections, each channel section carries beads, and these channel sections meet at channel joints.Fluid can be guided to flow along one or more channels or storage tanks via one or more fluid flow units.Fluid flow unit can include compressor (for example, positive pressure is provided), pump (for example, negative pressure is provided), actuator, etc., to control the flow of fluid.Fluid can also or otherwise be controlled by applied pressure difference, centrifugal force, electric pumping, vacuum, capillary flow or gravity flow, etc.

[0116] The beads can be porous, non-porous, solid, semi-solid, semi-fluid, fluid and / or combinations thereof. In some cases, the beads can be soluble, rupturable and / or degradable. In some cases, the beads may not be degradable. In some cases, the beads can be gel beads. The gel beads can be hydrogel beads. The gel beads can be formed by molecular precursors such as polymers or monomeric substances. Semi-solid beads can be liposome beads. Solid beads can contain metals, including iron oxide, gold and silver. In some cases, beads can be silica beads. In some cases, beads can be rigid. In other cases, beads can be flexible and / or compressible.

[0117] The beads may have any suitable shape. Examples of bead shapes include, but are not limited to, spherical, non-spherical, elliptical, rectangular, amorphous, circular, cylindrical, and variations thereof.

[0118] The beads may have a uniform size or a non-uniform size. In some cases, the diameter of the beads may be at least about 10 nanometers (nm), 100 nm, 500 nm, 1 micrometer (μm), 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1 mm or more. In some cases, the diameter of the beads may be less than about 10 nm, 100 nm, 500 nm, 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1 mm or less. In some cases, the diameter of the beads may be in the range of about 40-75 μm, 30-75 μm, 20-75 μm, 40-85 μm, 40-95 μm, 20-100 μm, 10-100 μm, 1-100 μm, 20-250 μm, or 20-500 μm.

[0119] In certain aspects, the beads can be provided as a population of beads or a plurality of beads having a relatively monodisperse size distribution. In the case of providing a relatively consistent amount of reagent within a partition, maintaining relatively consistent bead characteristics (e.g., size) can contribute to overall consistency. Specifically, the beads described herein can have a size distribution having a coefficient of variation of less than 50%, less than 40%, less than 30%, less than 20% in its cross-sectional dimension, and in some cases less than 15%, less than 10%, less than 5%, or less.

[0120] The beads may comprise natural materials and / or synthetic materials. For example, the beads may comprise natural polymers, synthetic polymers, or both natural polymers and synthetic polymers. Examples of natural polymers include proteins and sugars, such as deoxyribonucleic acid, rubber, cellulose, starch (e.g., amylose, amylopectin), proteins, enzymes, polysaccharides, silk, polyhydroxyalkyl esters, chitosan, dextran, collagen, carrageenan, psyllium ovata, gum arabic, agar, gelatin, shellac, karaya gum, xanthan gum, corn gum, guar gum, karaya gum, agarose, alginic acid, alginate, or their natural polymers. Examples of synthetic polymers include acrylics, nylons, silicones, spandex, viscose rayon, polycarboxylates, polyvinyl acetates, polyacrylamides, polyacrylates, polyethylene glycols, polyurethanes, polylactic acid, silica, polystyrene, polyacrylonitrile, polybutadiene, polycarbonate, polyethylene, polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethylene oxide), poly(ethylene terephthalate), polyethylene, polyisobutylene, poly(methyl methacrylate), poly(oxymethylene), polyoxymethylene, polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinyl acetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidene chloride), poly(vinylidene fluoride), poly(vinyl fluoride), and / or combinations thereof (e.g., copolymers). Beads may also be formed from materials other than polymers, including lipids, micelles, ceramics, glass-ceramics, composites, metals, other inorganic materials, and the like.

[0121] In some cases, beads can contain molecular precursors (for example, monomers or polymers), which can form polymer networks by polymerization of molecular precursors. In some cases, precursors can be polymerized substances, which can be further polymerized via, for example, chemical crosslinking. In some cases, precursors can include one or more of acrylamide or methacrylamide monomers, oligomers or polymers. In some cases, beads can include prepolymers, which are oligomers that can be further polymerized. For example, polyurethane beads prepared using prepolymers can be used. In some cases, beads can contain separate polymers that can be further polymerized together. In some cases, beads can be produced by polymerization of different precursors so that they include mixed polymers, copolymers and / or block copolymers. In some cases, beads can be included in covalent bonds or ionic bonds between polymer precursors (for example, monomers, oligomers, linear polymers), nucleic acid molecules (for example, oligonucleotides), primers and other entities. In some cases, covalent bonds can be carbon-carbon bonds, thioether bonds or carbon-heteroatom bonds.

[0122] The crosslinking may be permanent or reversible, depending on the specific crosslinking agent used. Reversible crosslinking may allow the polymer to linearize or dissociate under appropriate conditions. In some cases, reversible crosslinking may also allow reversible attachment of materials bound to the bead surface. In some cases, the crosslinking agent may form disulfide bonds. In some cases, the chemical crosslinking agent that forms disulfide bonds may be cystamine or modified cystamine.

[0123] In some cases, disulfide bonds can be formed between molecular precursor units (e.g., monomers, oligomers, or linear polymers) or precursors incorporated into beads and nucleic acid molecules (e.g., oligonucleotides). Cystamine (including modified cystamine), for example, is an organic reagent containing disulfide bonds that can be used as a cross-linking agent between individual monomers or polymer precursors of beads. Polyacrylamide can be polymerized in the presence of cystamine or a substance containing cystamine (e.g., modified cystamine) to produce polyacrylamide gel beads containing disulfide bonds (e.g., chemically degradable beads containing a chemically reducible cross-linking agent). When the beads are exposed to a reducing agent, the disulfide bonds can allow the beads to degrade (or dissolve).

[0124] In some cases, chitosan (a linear polysaccharide polymer) can be cross-linked with glutaraldehyde via hydrophilic chains to form beads. Cross-linking of chitosan polymers can be achieved by chemical reactions initiated by heat, pressure, pH changes, and / or radiation.

[0125] In some cases, beads can include an acrylamide moiety that can be used in some aspects to attach one or more nucleic acid molecules (e.g., barcode sequences, barcoded nucleic acid molecules, barcoded oligonucleotides, primers, or other oligonucleotides) to beads. In some cases, the acrylamide moiety can refer to an acrylamide analog produced by the reaction of acrylamide with one or more substances, such as acrylamide with other monomers and crosslinking agents during polymerization. The acrylamide moiety can be modified to form a chemical bond with a substance to be attached, such as a nucleic acid molecule (e.g., barcode sequence, barcoded nucleic acid molecule, barcoded oligonucleotides, primers, or other oligonucleotides). The acrylamide moiety can be modified with a thiol group that can form a disulfide bond or can be modified with a group that already contains a disulfide bond. Thiol or disulfide (via disulfide exchange) can be used as an anchor point for the substance to be attached, or another part of the acrylamide moiety can be used for attachment. In some cases, the attachment may be reversible, such that when the disulfide bond is broken (eg, in the presence of a reducing agent), the attached substance is released from the bead. In other cases, the acrylamido moiety may contain reactive hydroxyl groups that can be used for attachment.

[0126] Functionalization of beads for attachment of nucleic acid molecules (e.g., oligonucleotides) can be achieved by a variety of different methods including activation of chemical groups within the polymer, incorporation of active or activatable functional groups in the polymer structure, or attachment at the prepolymer or monomer stage during bead generation.

[0127] For example, the precursor (e.g., monomer, cross-linking agent) that is polymerized to form a bead can include an acrylamide moiety so that when the bead is generated, the bead also includes an acrylamide moiety. The acrylamide moiety can be attached to a nucleic acid molecule (e.g., an oligonucleotide), which can include a primer sequence (e.g., a primer for amplifying a target nucleic acid, a random primer, a primer sequence for messenger RNA) and / or one or more barcode sequences. The more than one barcode sequence can include the same sequence for all nucleic acid molecules coupled to a given bead and / or different sequences in all nucleic acid molecules coupled to a given bead. Nucleic acid molecules can be incorporated into beads.

[0128] In some cases, the nucleic acid molecule can include a functional sequence, e.g., a functional sequence for attachment to a sequencing flow cell, e.g., for In some cases, the nucleic acid molecule may include a barcode sequence. In some cases, the primer may also include a unique molecular identifier (UMI). In some cases, the primer may include an R1 primer sequence for Illumina sequencing. In some cases, the primer may include an R2 primer sequence for Illumina sequencing. Examples of such nucleic acid molecules (e.g., oligonucleotides, polynucleotides, etc.) and uses thereof, such as those that can be used with the compositions, devices, methods, and systems disclosed herein, are provided in U.S. Patent Publications Nos. 2014 / 0378345 and 2015 / 0376609 (each of which is incorporated herein by reference in its entirety).

[0129] Figure 8 An example of a bead carrying a barcode is illustrated. A nucleic acid molecule 802, such as an oligonucleotide, can be coupled to a bead 804 via a releasable linkage 806, such as a disulfide linker. The same bead 804 can be coupled to one or more other nucleic acid molecules 818, 820 (e.g., via a releasable linkage). The nucleic acid molecule 802 can be or include a barcode. As described elsewhere herein, the structure of a barcode can include a number of sequence elements. The nucleic acid molecule 802 can include a functional sequence 808 that can be used for subsequent processing. For example, the functional sequence 808 can include one or more of the following: a sequencer-specific flow cell attachment sequence (e.g., for P5 sequence of sequencing system) sequencing primer sequence (for example, R1 sequence of sequencing system). Nucleic acid molecule 802 can include a barcode sequence 810 for barcoding a sample (e.g., DNA, RNA, protein, etc.). In some cases, barcode sequence 810 can be bead-specific, so that barcode sequence 810 is shared by all nucleic acid molecules (e.g., including nucleic acid molecule 802) coupled to the same bead 804. Alternatively or in addition, barcode sequence 810 can be partition-specific, so that barcode sequence 810 is shared by all nucleic acid molecules coupled to one or more beads assigned to the same partition. Nucleic acid molecule 802 can include specific primer sequence 812, such as mRNA-specific primer sequence (e.g., poly-T sequence), targeted primer sequence and / or random primer sequence. Nucleic acid molecule 802 can include anchor sequence 814 to ensure that specific primer sequence 812 hybridizes at sequence end (e.g., sequence end of mRNA). For example, anchor sequence 814 may include a random short nucleotide sequence, such as a 1-mer, 2-mer, 3-mer, or longer sequence, which can ensure that the poly-T segment is more likely to hybridize at the sequence end of the poly-A tail of the mRNA.

[0130] The nucleic acid molecule 802 can include a unique molecular identification sequence 816 (e.g., a unique molecular identifier (UMI)). In some cases, the unique molecular identification sequence 816 can include about 5 to about 8 nucleotides. Alternatively, the unique molecular identification sequence 816 can include less than about 5 or more than about 8 nucleotides. The unique molecular identification sequence 816 can be a unique sequence that is different between individual nucleic acid molecules (e.g., 802, 818, 820, etc.) coupled to a single bead (e.g., bead 804). In some cases, the unique molecular identification sequence 816 can be a random sequence (e.g., a random N-mer sequence). For example, the UMI can provide a unique identifier of the captured starting mRNA molecule to allow quantification of the amount of RNA originally expressed. As will be appreciated, Figure 8 Nucleic acid molecules 802, 818, 820 are shown coupled to the surface of a bead 804, and a single bead can be coupled to any number of individual nucleic acid molecules, such as from one to tens to thousands or even millions of individual nucleic acid molecules. The corresponding barcodes of the individual nucleic acid molecules can include both common sequence segments or relative common sequence segments (e.g., 808, 810, 812, etc.) and variable or unique sequence segments (e.g., 816) between different individual nucleic acid molecules coupled to the same bead.

[0131] In operation, biological particles or analyte carriers (e.g., cells, DNA, RNA, etc.) can be co-distributed together with barcoded beads 804. Barcoded nucleic acid molecules 802, 818, 820 can be released from beads 804 in partitions. For example, in the case of analyzing sample RNA, a poly-T segment (e.g., 812) of one of the released nucleic acid molecules (e.g., 802) can hybridize with the poly-A tail of an mRNA molecule. Reverse transcription can produce a cDNA transcript of mRNA, but the transcript includes each of the sequence segments 808, 810, 816 of the nucleic acid molecule 802. Because the nucleic acid molecule 802 includes an anchor sequence 814, it will be more likely to hybridize with the sequence end of the poly-A tail of the mRNA and initiate reverse transcription. In any given partition, the cDNA transcript of a single mRNA molecule may include a common barcode sequence segment 810. However, transcripts produced by different mRNA molecules within a given partition may differ at unique molecular identifier sequence 812 segments (e.g., UMI segments). Advantageously, even after any subsequent amplification of the contents of a given partition, the number of different UMIs can indicate the amount of mRNA derived from a given partition and therefore from a biological particle or analyte carrier (e.g., a cell). As described above, the transcripts can be amplified, purified, and sequenced to identify the sequence of the cDNA transcript of the mRNA, as well as sequencing the barcode segments and UMI segments. Although poly-T primer sequences are described, other targeted or random primer sequences can also be used to initiate the reverse transcription reaction. Similarly, although described as releasing barcoded oligonucleotides into partitions, in some cases, nucleic acid molecules bound to beads (e.g., gel beads) can be used to hybridize and capture mRNA on a solid phase of the beads, for example, to facilitate separation of RNA from other cellular contents.

[0132] In some cases, precursors containing functional groups that are reactive or capable of being activated to become reactive can be polymerized with other precursors to generate gel beads containing activated or activatable functional groups. The functional groups can then be used to attach additional substances (e.g., disulfide linkers, primers, other oligonucleotides, etc.) to the gel beads. For example, some precursors containing carboxylic acid (COOH) groups can be copolymerized with other precursors to form gel beads that also contain COOH functional groups. In some cases, acrylic acid (a substance containing free COOH groups), acrylamide, and bis(acryloyl)cystamine can be copolymerized together to produce gel beads containing free COOH groups. The COOH groups of the gel beads can be activated (e.g., via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) or 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM)) to make them reactive (e.g., reactive to amine functional groups when activated using EDC / NHS or DMTMM). The activated COOH groups can then react with a suitable substance containing a moiety to be attached to the beads (e.g., a substance containing an amine functional group in which the carboxylic acid group is activated to be reactive with the amine functional group).

[0133] Beads containing disulfide bonds in their polymer network can be functionalized with additional substances by reducing some of the disulfide bonds to free thiols. Disulfide bonds can be reduced by the action of, for example, a reducing agent (e.g., DTT, TCEP, etc.) to produce free thiol groups without the beads dissolving. The free thiols of the beads can then react with the free thiols of the substance or substances containing another disulfide bond (e.g., by thiol-disulfide exchange) so that the substance can be attached to the beads (e.g., through the disulfide bonds generated). In some cases, the free thiols of the beads can react with any other suitable groups. For example, the free thiols of the beads can react with substances containing acrylamide moieties. The free thiol groups of the beads can react with acrylamide groups by Michael addition chemistry so that the substances containing acrylamide groups are attached to the beads. In some cases, uncontrolled reactions can be prevented by including a thiol capping agent (e.g., N-ethylmaleimide or iodoacetic acid).

[0134] The activation of disulfide bonds in the beads can be controlled so that only a small number of disulfide bonds are activated. For example, this can be controlled by controlling the concentration of the reducing agent used to generate free thiol groups and / or the concentration of the reagent used to form disulfide bonds in the bead polymerization. In some cases, a low concentration of reducing agent molecules (e.g., less than or equal to about 1:100,000,000,000, less than or equal to about 1:10,000,000,000, less than or equal to about 1:1,000,000,000, less than or equal to about 1:100,000,000, less than or equal to about 1:10,000,000, less than or equal to about 1:1,000,000,000, less than or equal to about 1:100,000,000, less than or equal to about 1:1,000,000, less than or equal to about 1:100,000, less than or equal to about 1:10,000,000) can be used for reduction. Controlling the number of disulfide bonds reduced to free thiols can be useful to ensure the structural integrity of the beads during functionalization. In some cases, optically active agents such as fluorescent dyes can be coupled to the beads via their free thiol groups and used to quantify the amount of free thiols present in the beads and / or to track the beads.

[0135] In some cases, it may be advantageous to add a part to the gel beads after the gel beads are formed. For example, adding oligonucleotides (for example, oligonucleotides with barcodes) after the gel beads are formed can avoid the loss of material during the chain transfer termination, which can occur during the polymerization process. Moreover, smaller precursors (for example, monomers or cross-linking agents that do not include side chain groups and connected parts) can be used for polymerization, and can be due to the viscosity effect, hinder its growth chain end to a minimum. In some cases, the functionalization after the gel beads are synthesized can minimize the exposure of the material (for example, oligonucleotide) to load potential damage factors (for example, free radicals) and / or chemical environment. In some cases, the gel produced can have an upper critical solution temperature (UCST), which allows beads to be swollen and collapsed by temperature drive. Such functionality can contribute to oligonucleotides (for example, primers) to infiltrate beads during the functionalization of beads with oligonucleotides subsequently. Functionalization after generation can also be used to control the load ratio of material in beads, so that the variability of load ratio is minimized. Substance loading can also be performed in a batch process, so that multiple beads can be functionalized with substances in a single batch.

[0136] The beads injected or otherwise introduced into the partitions may contain a releasably, cleavably or reversibly attached barcode. The beads injected or otherwise introduced into the partitions may contain an activatable barcode. The beads injected or otherwise introduced into the partitions may be degradable, rupturable or soluble beads.

[0137] The barcode can be releasably, cleavably or reversibly attached to the beads, so that the barcode can be released or releasable by the cleavage of the bond between the barcode molecule and the beads, or released by the degradation of the base beads themselves, thereby allowing the barcode to be approached or accessible by other reagents, or both. In a non-limiting example, the cleavage can be achieved by the reduction of disulfide bonds, the use of restriction enzymes, photoactivated cleavage, or the cleavage and / or reaction of other types of stimulation (e.g., chemical, thermal, pH, enzyme stimulation, etc.), such as described elsewhere herein. Releasable barcodes can sometimes be referred to as activatable because they can be used for reaction once released. Therefore, for example, an activatable barcode can be activated by releasing the barcode from the beads (or other suitable types of partitions described herein). Other activatable structures are also contemplated in the context of the methods and systems.

[0138] In addition to or as an alternative to a cleavable bond between a bead and an associated molecule, such as a barcode containing a nucleic acid molecule (e.g., a barcoded oligonucleotide), the bead can be degradable, rupturable, or soluble spontaneously or upon exposure to one or more stimuli (e.g., temperature change, pH change, exposure to a particular chemical or phase, exposure to light, a reducing agent, etc.). In some cases, the bead can be soluble such that the material components of the bead are dissolved when exposed to a particular chemical or environmental change, such as a temperature change or a pH change. In some cases, the gel bead can be degraded or dissolved under high temperature and / or alkaline conditions. In some cases, the bead can be thermally degradable such that the bead degrades when the bead is exposed to an appropriate temperature change (e.g., heat). Degradation or dissolution of a bead bound to a substance (e.g., a nucleic acid molecule, e.g., a barcoded oligonucleotide) can cause the substance to be released from the bead.

[0139] As can be appreciated from the above disclosure, degradation of beads can refer to the dissociation of bound or entrained substances from the beads with or without structural degradation of the physical beads themselves. For example, degradation of beads can involve cleavage of cleavable bonds by one or more substances and / or methods described elsewhere herein. In another example, entrained substances can be released from the beads by osmotic pressure differentials due to, for example, changes in the chemical environment. For example, changes in the pore size of the beads due to osmotic pressure differentials can generally occur without structural degradation of the beads themselves. In some cases, the increase in pore size due to osmotic swelling of the beads can allow the release of entrained substances within the beads. In other cases, osmotic shrinkage of the beads can allow the beads to better retain entrained substances due to pore size shrinkage.

[0140] Degradable beads can be introduced into partitions, such as emulsion droplets or wells, so that when an appropriate stimulus is applied, the beads degrade within the partitions and any associated substances (e.g., oligonucleotides) are released within the droplets. Free substances (e.g., oligonucleotides, nucleic acid molecules) can interact with other reagents contained within the partitions. For example, polyacrylamide beads containing cystamine and connected to a barcode sequence via a disulfide bond can be combined with a reducing agent in a droplet of an oil-in-water emulsion. Within the droplet, the reducing agent can break various disulfide bonds, resulting in degradation of the beads and release of the barcode sequence into the internal aqueous environment of the droplet. In another example, heating a droplet containing a bead-bound barcode sequence in an alkaline solution can also result in degradation of the beads and release of the attached barcode sequence into the internal aqueous environment of the droplet.

[0141] Any suitable number of molecular tag molecules (e.g., primers, barcoded oligonucleotides) can be associated with the beads so that upon release from the beads, a predetermined concentration of molecular tag molecules (e.g., primers, such as barcoded oligonucleotides) is present in the partition. Such predetermined concentrations can be selected to facilitate certain reactions, such as amplification, in generating sequencing libraries within the partition. In some cases, the predetermined concentration of primers can be limited by the process of generating beads with nucleic acid molecules (e.g., oligonucleotides).

[0142] In some cases, beads can be non-covalently loaded with one or more reagents. For example, by subjecting beads to conditions sufficient to swell the beads, allowing enough time for the reagent to diffuse into the inside of the beads, and subjecting the beads to conditions sufficient to deswell the beads, beads can be non-covalently loaded. The swelling of beads can be completed by, for example, placing the beads in a thermodynamically favorable solvent, subjecting the beads to higher or lower temperatures, subjecting the beads to higher or lower ion concentrations and / or subjecting the beads to an electric field. The swelling of beads can be completed by various swelling methods. The deswelling of beads can be completed by, for example, transferring beads in a thermodynamically favorable solvent, subjecting the beads to lower temperatures or high temperatures, subjecting the beads to lower or higher ion concentrations and / or removing the beads from an electric field. The deswelling of beads can be completed by various deswelling methods. Transferring beads can cause the pores in the beads to shrink. Then shrinkage can hinder the reagent in the beads from diffusing out from the inside of the beads. The obstruction may be caused by the spatial interaction between the reagent and the inside of the beads. The transfer can be accomplished using microfluidics. For example, the transfer can be accomplished 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 can be adjusted by varying the polymer composition of the beads.

[0143] In some cases, the acrylamide moiety attached to the precursor, another substance attached to the precursor, or the precursor itself can contain an unstable bond, such as a chemically sensitive bond, a heat-sensitive bond, or a light-sensitive bond, such as a disulfide bond, a UV-sensitive bond, etc. Once the acrylamide moiety or other moiety containing an unstable bond is incorporated into the bead, the bead may also contain an unstable bond. An unstable bond can, for example, be used to reversibly connect (e.g., covalently connect) a substance (e.g., a barcode, a primer, etc.) to the bead. In some cases, a thermally unstable bond can include an attachment based on nucleic acid hybridization, for example, where an oligonucleotide is hybridized to a complementary sequence attached to the bead, such that thermal melting of the hybrid releases the oligonucleotide (e.g., a sequence containing a barcode) from the bead or microcapsule.

[0144] Adding multiple types of unstable bonds to gel beads can lead to the generation of beads that can respond to various stimuli. Each type of unstable bond may be sensitive to relevant stimuli (e.g., chemical stimuli, light, temperature, enzymes, etc.), so that the release of substances attached to the beads via each unstable bond can be controlled by applying appropriate stimuli. Such functionality can be used for the controlled release of substances from gel beads. In some cases, another substance containing an unstable bond can be connected to the gel beads after the gel beads are formed by, for example, the activated functional groups of the gel beads as described above. As will be appreciated, the barcodes releasably, cleavably or reversibly attached to the beads described herein include barcodes released by the cleavage of the bond between the barcode molecule and the beads, or released by the degradation of the base beads themselves, thereby allowing the barcode to be approached or accessible by other reagents, or both.

[0145] Releasable barcodes as described herein may sometimes be referred to as activatable because they are available for reaction once released. Thus, for example, 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 contemplated in the context of the methods and systems.

[0146] In addition to thermally cleavable bonds, disulfide bonds, and UV-sensitive bonds, other non-limiting examples of labile bonds that can be coupled to the precursor or bead include ester bonds (e.g., cleavable by acid, base, or hydroxylamine), vicinal diol bonds (e.g., cleavable via sodium periodate), Diels-Alder bonds (e.g., cleavable via heat), sulfone bonds (e.g., cleavable via base), silyl ether bonds (e.g., cleavable via acid), glycosidic bonds (e.g., cleavable via amylase), peptide bonds (e.g., cleavable via protease), or phosphodiester bonds (e.g., cleavable via nucleases (e.g., DNase)). The bonds can be cleaved by other enzymes that target nucleic acid molecules, such as restriction enzymes (e.g., restriction endonucleases), as described further below.

[0147] Substances may be encapsulated in beads during bead generation (e.g., during precursor polymerization). Such substances may or may not participate in polymerization. Such substances may enter the polymerization reaction mixture so that after bead formation, the resulting beads contain the substance. In some cases, such substances may be added to the gel beads after formation. Such substances may include, for example, nucleic acid molecules (e.g., oligonucleotides), reagents for nucleic acid amplification reactions (e.g., primers, polymerases, dNTPs, cofactors (e.g., ionic cofactors), buffers) (including those described herein), reagents for enzymatic reactions (e.g., enzymes, cofactors, substrates, buffers), reagents for nucleic acid modification reactions (e.g., polymerization, ligation, or digestion), and / or reagents for template preparation (e.g., labeling) for one or more sequencing platforms (e.g., for of ). Such substances may include one or more enzymes described herein, including but not limited to polymerases, reverse transcriptases, restriction enzymes (e.g., endonucleases), transposases, ligases, proteinase K, DNA enzymes, etc. Such substances may include one or more reagents described elsewhere herein (e.g., cleavage agents, inhibitors, inactivators, chelators, stimulants). The capture of such substances may be controlled by the density of the polymer network generated during the polymerization of the precursor, the control of the ionic charge in the gel beads (e.g., by ionic substances connected to polymeric substances), or by the release of other substances. The encapsulated substances may be released from the beads when the beads are degraded and / or by applying a stimulus that enables the substances to be released from the beads. Alternatively or in addition, the substances may be distributed in the partitions (e.g., droplets) during or after the partition formation. Such substances may include but are not limited to the above-mentioned substances that may also be encapsulated in the beads.

[0148] Degradable beads can contain one or more substances with unstable bonds, such that when the beads / substances are exposed to an appropriate stimulus, the bonds break and the beads degrade. The unstable bonds can be chemical bonds (e.g., covalent bonds, ionic bonds) or can be another type of physical interaction (e.g., van der Waals interactions, dipole-dipole interactions, etc.). In some cases, the cross-linking agent used to produce the beads can contain unstable bonds. When exposed to appropriate conditions, the unstable bonds can break and the beads degrade. For example, when polyacrylamide gel beads containing cystamine cross-linking agent are exposed to a reducing agent, the disulfide bonds of cystamine can break and the beads degrade.

[0149] Compared with non-degradable beads, when appropriate stimulation is applied to the beads, degradable beads can be used to release attached substances (e.g., nucleic acid molecules, barcode sequences, primers, etc.) from the beads more quickly. For example, for substances bound to the inner surface of porous beads, or in the case of encapsulated substances, when the beads are degraded, the substances may have greater mobility and accessibility to other substances in solution. In some cases, the substance can also be connected to the degradable beads via a degradable linker (e.g., a disulfide linker). The degradable linker can respond to the same stimulation with the degradable beads, or two degradable substances can respond to different stimulations. For example, the barcode sequence can be connected to the polyacrylamide beads containing cystamine through a disulfide bond. When the barcoded beads are exposed to a reducing agent, the beads will degrade and when the disulfide bonds between the barcode sequence and the beads and the disulfide bonds of the cystamine in the beads are broken, the barcode sequence is released.

[0150] As will be appreciated from the above disclosure, when referred to as degradation of the beads, in many cases as mentioned above, the degradation may refer to the dissociation of bound or entrained material from the beads with or without structural degradation of the physical beads themselves. For example, entrained material may be released from the beads by an osmotic pressure differential resulting from, for example, a change in the chemical environment. For example, changes in the pore size of the beads due to an osmotic pressure differential may generally occur without structural degradation of the beads themselves. In some cases, an increase in pore size due to osmotic swelling of the beads may allow for the release of entrained material within the beads. In other cases, osmotic shrinkage of the beads may allow the beads to better retain entrained material due to the shrinkage in pore size.

[0151] In the case of providing degradable beads, it may be beneficial to avoid exposing such beads to one or more stimuli that cause such degradation before a given time, for example, to avoid premature degradation of the beads and problems caused by such degradation, including, for example, poor flow characteristics and aggregation. For example, in the case where the beads contain reducible cross-linking groups, such as disulfide groups, it may be useful to avoid contacting such beads with reducing agents such as DTT or other disulfide cleavage reagents. In such cases, treatment of the beads described herein without a reducing agent (e.g., DTT) will be provided in some cases. Because reducing agents are often provided in commercial enzyme preparations, enzyme preparations without reducing agents (or without DTT) can be provided when treating the beads described herein. Examples of such enzymes include, for example, polymerase preparations, reverse transcriptase preparations, ligase preparations, and many other enzyme preparations that can be used to treat the beads described herein. The term "reducing agent-free" or "DTT-free" preparation can refer to a preparation of such materials for degrading beads having a lower limit of less than about 1 / 10, less than about 1 / 50, or even less than about 1 / 100. For example, for DTT, a formulation without a reducing agent can have less than about 0.01 millimolar (mM), 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or even less than about 0.0001 mM DTT. In many cases, the amount of DTT is undetectable.

[0152] Many chemical triggers can be used to trigger the degradation of beads. Examples of these chemical changes can include, but are not limited to, pH-mediated changes in the integrity of components within the beads, degradation of bead components through cleavage of crosslinks, and depolymerization of bead components.

[0153] In some embodiments, beads may be formed by materials comprising degradable chemical crosslinkers such as BAC or cystamine. The degradation of such degradable crosslinkers can be accomplished by many mechanisms. In some instances, beads may be contacted with chemical degradation agents that can induce oxidation, reduction or other chemical changes. For example, a chemical degradation agent may be a reducing agent, such as dithiothreitol (DTT). Other examples of reducing agents may include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), tris(2-carboxyethyl)phosphine (TCEP) or a combination thereof. Reducing agents may degrade the disulfide bonds formed between the gel precursors that form beads, and thus the beads may be degraded. In other cases, changes in solution pH, such as an increase in pH, may trigger degradation of beads. In other cases, exposure to an aqueous solution such as water may trigger hydrolytic degradation, and thus the degradation of beads may be triggered. In some cases, any combination of stimuli may trigger degradation of beads. For example, changes in pH may make chemical reagents (e.g., DTT) effective reducing agents.

[0154] When a thermal stimulus is applied, the beads can also be induced to release their contents. Changes in temperature can cause various changes in the beads. For example, heat can cause solid beads to liquefy. Changes in heat can cause beads to melt, causing a portion of the beads to degrade. In other cases, heat can increase the internal pressure of the bead components, causing the beads to rupture or burst. Heat can also act on thermosensitive polymers used as materials for constructing beads.

[0155] Any suitable reagent can degrade beads. In some embodiments, the change of temperature or pH can be used to degrade the thermosensitive or pH-sensitive bonds in beads. In some embodiments, chemical degradation agents can be used to degrade the chemical bonds in beads by oxidation, reduction or other chemical changes. For example, chemical degradation agents can be reducing agents, such as DTT, wherein DTT can degrade the disulfide bonds formed between the cross-linking agent and the gel precursor, thereby degrading beads. In some embodiments, reducing agents can be added to degrade beads, which may or may not cause the beads to release their contents. The example of reducing agent can include dithiothreitol (DTT), β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), tris (2-carboxyethyl) phosphine (TCEP) or a combination thereof. Reducing agent can be present in a concentration of 0.1mM, 0.5mM, 1mM, 5mM, 10mM. The reducing agent may be present at a concentration of at least about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, or above 10 mM. The reducing agent may be present at a concentration of up to about 10 mM, 5 mM, 1 mM, 0.5 mM, 0.1 mM, or less.

[0156] Any suitable number of molecular tag molecules (e.g., primers, barcoded oligonucleotides) can be associated with the beads so that after release from the beads, a predetermined concentration of molecular tag molecules (e.g., primers, such as barcoded oligonucleotides) is present in the partition. Such predetermined concentrations can be selected to facilitate certain reactions, such as amplification, that generate sequencing libraries within the partition. In some cases, the predetermined concentration of primers can be limited by the process of generating beads with oligonucleotides.

[0157] although Figure 1 and Figure 2It has been described above in terms of providing a substantially single occupied partition, but in some cases, multiple occupied partitions may be provided, for example, containing two, three, four or more cells and / or microcapsules (e.g., beads) containing barcoded nucleic acid molecules (e.g., oligonucleotides) within a single partition. Thus, as described above, the flow characteristics of the fluid containing the biological particles or analyte carriers and / or beads and the distribution fluid may be controlled to provide such multiple occupied partitions. Specifically, the flow parameters may be controlled to provide a given occupancy of greater than about 50% of a partition, greater than about 75%, and in some cases greater than about 80%, 90%, 95%, or more.

[0158] In some cases, other microcapsules can be used to deliver other reagents to the partitions. In such cases, it may be advantageous to introduce different beads from different bead sources (e.g., containing different related reagents) into such a common channel or droplet generation junction (e.g., junction 210) through different channel entrances that pass into the common channel or droplet generation junction. In such cases, the flow rate and frequency of different beads flowing into the channel or junction can be controlled to provide a certain ratio of microcapsules from each source while ensuring a given pairing or combination of such beads entering the partition with a given number of biological particles or analyte carriers (e.g., one biological particle or analyte carrier and one bead per partition).

[0159] The partitions described herein can comprise small volumes, for example, less than about 10 microliters (μL), 5 μL, 1 μL, 900 picoliters (pL), 800 pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 20 pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less.

[0160] For example, in the case of droplet-based partitioning, the total volume of the droplets may be less than about 1000 pL, 900 pL, 800 pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 20 pL, 10 pL, 1 pL or less. In the case of co-dispensing with microcapsules, it will be appreciated that the volume of sample fluid within the partition, e.g., including co-dispensed bioparticles or analyte carriers and / or beads, may be less than about 90% of the above volume, less than about 80% of the above volume, less than about 70% of the above volume, less than about 60% of the above volume, less than about 50% of the above volume, less than about 40% of the above volume, less than about 30% of the above volume, less than about 20% of the above volume, or less than about 10% of the above volume.

[0161] As described elsewhere herein, material distribution can produce a group or multiple partitions. In such cases, any suitable number of partitions can be produced 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 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 produced or otherwise provided. Moreover, the multiple partitions can include unoccupied partitions (e.g., empty partitions) and occupied partitions.

[0162] Reagents

[0163] According to certain aspects, the bioparticle or analyte carrier can be distributed together with a lysis reagent to release the contents of the bioparticle or analyte carrier in the partition. In such cases, the lysis reagent can be contacted with the bioparticle or analyte carrier suspension at the same time or just before the bioparticle or analyte carrier is introduced into the distribution junction / droplet generation area (e.g., junction 210), such as through one or more additional channels upstream of the channel junction. According to other aspects, in addition or alternatively, the bioparticle or analyte carrier can be partitioned together with other reagents, as will be further described below.

[0164] Figure 3 An example of a microfluidic channel structure 300 for co-dispensing biological particles or analyte carriers and reagents is shown. Channel structure 300 may include channel segments 301, 302, 304, 306, and 308. Channel segments 301 and 302 are connected at a first channel junction 309. Channel segments 302, 304, 306, and 308 are connected at a second channel junction 310.

[0165] In an exemplary operation, the channel segment 301 can transport an aqueous fluid 312 including a plurality of biological particles or analyte carriers 314 along the channel segment 301 to the junction 310. Alternatively or additionally, the channel segment 301 can also transport beads (eg, gel beads). The beads can contain barcode molecules.

[0166] For example, channel segment 301 can be connected to a reservoir containing an aqueous suspension of biological particles or analyte carriers 314. Upstream of second junction 310 and just before reaching the second junction, channel segment 301 can meet channel segment 302 at first junction 309. Channel segment 302 can transport a plurality of reagents 315 (e.g., lysing agents) suspended in aqueous fluid 312 along channel segment 302 into first junction 309. For example, channel segment 302 can be connected to a reservoir containing reagents 315. After first junction 309, aqueous fluid 312 in channel segment 301 can carry both biological particles or analyte carriers 314 and reagents 315 to second junction 310. In some cases, aqueous fluid 312 in channel segment 301 can include one or more reagents, which can be the same as or different from reagent 315. A second fluid 316 (e.g., oil) that is immiscible with the aqueous fluid 312 can be delivered from each of the channel segments 304 and 306 to the second junction 310. When the aqueous fluid 312 from the channel segment 301 meets the second fluid 316 from each of the channel segments 304 and 306 at the second channel junction 310, the aqueous fluid 312 can be distributed as discrete droplets 318 in the second fluid 316 and flow away from the second junction 310 along the channel segment 308. The channel segment 308 can deliver the discrete droplets 318 to an outlet reservoir fluidly coupled to the channel segment 308, where the discrete droplets can be harvested.

[0167] The second fluid 316 may comprise an oil, such as a fluorinated oil, which includes a fluorinated surfactant for stabilizing the resulting droplets, such as inhibiting subsequent coalescence of the resulting droplets 318 .

[0168] The generated discrete droplets may include individual biological particles or analyte carriers 314 and / or one or more reagents 315. In some cases, the generated discrete droplets may include beads (not shown) carrying barcodes, such as via other microfluidics described elsewhere herein. In some cases, the discrete droplets may be unoccupied (e.g., no reagents, no biological particles or analyte carriers).

[0169] Advantageously, when the lysis reagent and the biological particle or analyte carrier are co-dispensed, the lysis reagent can promote the release of the contents of the biological particle or analyte carrier within the partition. The released contents in the partition can remain discrete from the contents of other partitions.

[0170] As will be appreciated, channel sections as herein described can be coupled to any one of various fluid sources or receiving components, including fluid components of storage tanks, pipelines, manifolds or other systems.As will be appreciated, microfluidic channel structure 300 can have other geometric structures.For example, microfluidic channel structure can have more than two channel joints.For example, microfluidic channel structure can have 2,3,4,5 channel sections or more, each channel section carries the same or different types of beads, reagents and / or bioparticles or analyte carriers, and these channel sections meet at channel joints.The fluid flow in each channel section can be controlled to control the distribution of different elements in the droplet.Fluid can be guided to flow along one or more channels or storage tanks via one or more fluid flow units.Fluid flow unit can include compressor (for example, positive pressure is provided), pump (for example, negative pressure is provided), actuator, etc., to control the flow of fluid.Fluid can also or otherwise be controlled by pressure difference, centrifugal force, electric pumping, vacuum, capillary flow or gravity flow, etc. applied.

[0171] The example of lysing agent includes bioactive reagent, for example, lysing enzyme for cracking different cell types (for example Gram-positive or negative bacteria, plant, yeast, mammal etc.), for example lysozyme, colorless peptidase, lysostaphin, labiase, kitalase, lysing enzyme, and multiple other lysing enzymes that can be obtained from, for example, Sigma-Aldrich, Inc. (St Louis, MO), and other commercially available lysing enzymes. Other lysing agents can be additionally or alternatively co-distributed with bioparticles or analyte carriers to cause the contents of bioparticles or analyte carriers to be released into partitions. For example, in some cases, lysing solution based on surfactant can be used for lysing cells. In some cases, lysing solution can include nonionic surfactants, for example TritonX-100 and Tween 20. In some cases, lysing solution can include ionic surfactants, for example, as sodium lauryl sarcosinate and sodium dodecyl sulfate (SDS). Electroporation, thermal, acoustic, or mechanical cell disruption may also be used in some cases, for example, non-emulsion-based dispensing, such as bioparticle or analyte carrier encapsulation may be used in addition to or instead of droplet dispensing, where any pore size of the encapsulant is small enough to retain nucleic acid fragments of a given size after cell disruption.

[0172] As a substitute or supplement of the lysing agent co-assigned with the above-mentioned bioparticles or analyte carrier, other reagents may also be co-assigned with the bioparticles or analyte carrier, including for example DNA enzyme and RNA enzyme inactivator or inhibitor (such as proteinase K), chelating agent (such as EDTA), and for eliminating or otherwise reducing the negative activity of different cell lysate components or other reagents of the impact on nucleic acid subsequent treatment. In addition, in the case of encapsulated bioparticles or analyte carriers, bioparticles or analyte carriers may be exposed to appropriate stimulation so that bioparticles or analyte carriers or their content are released from the co-assigned microcapsules. For example, in some cases, chemical stimulation may be co-assigned together with the encapsulated bioparticles or analyte carriers to allow microcapsule degradation and cell or its content to be released into larger partitions. In some cases, this stimulation may be identical with the stimulation described elsewhere herein for nucleic acid molecules (such as, oligonucleotides) released from their respective microcapsules (such as, beads). In alternative aspects, this may be different and non-overlapping stimulations, so as to allow the encapsulated bioparticles or analyte carriers to be released into the same partition at a time different from nucleic acid molecules being released into the partition.

[0173] Other reagents can also be co-assigned with the biological particles or analyte carriers, such as endonucleases for fragmenting the DNA of biological particles or analyte carriers, DNA polymerases and dNTPs for amplifying nucleic acid fragments of biological particles or analyte carriers and attaching barcode molecular tags to amplified fragments. Other enzymes can be co-assigned, including but not limited to polymerases, transposases, ligases, proteinases K, DNA enzymes, etc. Additional reagents can also include reverse transcriptases, including enzymes with terminal transferase activity, primers and oligonucleotides, and conversion oligonucleotides (also referred to herein as "conversion oligos" or "template conversion oligonucleotides") that can be used for template switching. In some cases, template switching can be used to increase the length of cDNA. In some cases, template switching can be used to attach a predetermined nucleic acid sequence to cDNA. In the example of template switching, cDNA can be generated by reverse transcription of a template such as cell mRNA, wherein a reverse transcriptase with terminal transferase activity can add additional nucleotides, such as poly-C, to cDNA in a template-independent manner. The switching oligonucleotide can include a sequence complementary to the additional nucleotides such as poly-G. The additional nucleotides (e.g., poly-C) on the cDNA can be hybridized with the additional nucleotides (e.g., poly-G) on the switching oligonucleotide, so that the switching oligonucleotide can be used as a template by a reverse transcriptase to further extend the cDNA. The template switching oligonucleotide can include a hybridization region and a template region. The hybridization region can include any sequence that can hybridize with the target. In some cases, as previously described, the hybridization region includes a series of G bases to complement the C bases protruding at the 3' end of the cDNA molecule. The G bases of the series can include 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G bases or more than 5 G bases. The template sequence can include any sequence to be incorporated into the cDNA. In some cases, the template region includes at least 1 (e.g., at least 2, 3, 4, 5 or more) tag sequences and / or functional sequences. The switching oligomer can comprise deoxyribonucleic acid; ribonucleic acid; modified nucleic acid including 2-aminopurine, 2,6-diaminopurine (2-amino-dA), reverse dT, 5-methyl dC, 2'-deoxyinosine, super T (5-hydroxybutyryl-2'-deoxyuridine), super G (8-aza-7-deazaguanosine), locked nucleic acid (LNA), unlocked nucleic acid (UNA, such as UNA-A, UNA-U, UNA-C, UNA-G), iso-dG, iso-dC, 2' fluoro bases (e.g., fluoro-C, fluoro-U, fluoro-A and fluoro-G) or any combination.

[0174] 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 1 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or 250 nucleotides or longer.

[0175] In some cases, the length of the toggle oligonucleotide can be at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 ,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137 7, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194 , 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or 250 nucleotides.

[0176] Once the contents of the cell are released into their respective partitions, the macromolecular components contained therein (e.g., macromolecular components of biological particles or analyte carriers, such as RNA, DNA or protein) can be further processed in the partitions. According to the methods and systems described herein, unique identifiers can be provided for the macromolecular component contents of individual biological particles or analyte carriers, so that when characterizing those macromolecular components, they can be attributed to the same one or more biological particles or analyte carriers. The ability to attribute features to individual biological particles or analyte carriers or multiple groups of biological particles or analyte carriers is provided by specifically assigning unique identifiers to individual biological particles or analyte carriers or multiple groups of biological particles or analyte carriers. Unique identifiers (e.g., in the form of nucleic acid barcodes) can be assigned to individual biological particles or analyte carriers or biological particles or analyte carrier colonies or associated therewith, so as to mark or label the macromolecular components of biological particles or analyte carriers (and therefore, mark or label their features) with unique identifiers. Then, these unique identifiers can be used to attribute the components and features of biological particles or analyte carriers to individual biological particles or analyte carriers or a group of biological particles or analyte carriers.

[0177] In some aspects, this is done by assigning individual bioparticles or analyte carriers or groups of bioparticles or analyte carriers with unique identifiers, such as described above (see Figure 2 ). In some aspects, the unique identifier is provided in the form of a nucleic acid molecule (e.g., an oligonucleotide) comprising a nucleic acid barcode sequence that can be attached or otherwise associated with the nucleic acid content of a separate biological particle or analyte carrier, or with other components of a biological particle or analyte carrier, and in particular with fragments of those nucleic acids. The nucleic acid molecules are distributed so that the nucleic acid barcode sequences contained therein are the same between nucleic acid molecules in a given partition, but between different partitions, the nucleic acid molecules can and do have different barcode sequences, or at least present a large number of different barcode sequences in all partitions in a given analysis. In some aspects, only one nucleic acid barcode sequence can be associated with a given partition, but in some cases, two or more different barcode sequences can be present.

[0178] In the sequence of a nucleic acid molecule (e.g., an oligonucleotide), a nucleic acid barcode sequence may include about 6 to about 20 nucleotides. A nucleic acid barcode sequence may include about 6 to about 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides. In some cases, the length of the barcode sequence may be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of the 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. In some cases, the length of the 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 can be completely continuous, i.e., in a single adjacent nucleotide segment, or they can be separated into two or more separated subsequences separated by 1 or more nucleotides. In some cases, the length of the separated barcode subsequence can be about 4 to about 16 nucleotides. In some cases, the length of the barcode sequence can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the length of the barcode sequence can be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the length of the barcode sequence can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[0179] The co-distributed nucleic acid molecules may also include other functional sequences that can be used to process nucleic acids from the co-distributed bioparticles or analyte carriers. These sequences include, for example, targeted or random / universal amplification primer sequences for amplifying genomic DNA from separate bioparticles or analyte carriers within a partition while attaching associated barcode sequences, sequencing primers or primer recognition sites, hybridization or detection sequences (e.g., for identifying the presence of a sequence or for pulling down barcoded nucleic acids), or any of many other potential functional sequences. Other mechanisms for co-distributing oligonucleotides may also be employed, including, for example, coalescence of two or more droplets, one of which contains an oligonucleotide, or microdistributing an oligonucleotide into a partition (e.g., a droplet within a microfluidic system).

[0180] In one example, microcapsules, such as beads, are provided, each of which includes a large number of the above-described barcoded nucleic acid molecules (e.g., barcoded oligonucleotides) releasably attached to the beads, wherein all nucleic acid molecules attached to a particular bead will include the same nucleic acid barcode sequence, but wherein a large number of different barcode sequences are present in the population of beads used. In some embodiments, hydrogel beads (e.g., comprising a polyacrylamide polymer matrix) are used as a solid support and vehicle for delivering nucleic acid molecules to partitions because they are able to carry a large number of nucleic acid molecules and can be configured to release nucleic acid molecules when exposed to a specific stimulus as described elsewhere herein. In some cases, the population of beads provides a diverse barcode sequence library comprising at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000,000 different barcode sequences, at least about 5,000,000 different barcode sequences, or at least about 10,000,000 different barcode sequences or more. Additionally, a large number of attached nucleic acid (e.g., oligonucleotide) molecules can be provided per bead. Specifically, the number of molecules including a barcode sequence among the nucleic acid molecules on a single 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 nucleic acid molecules of a given bead can include the same (or common) barcode sequence, different barcode sequences, or a combination of both. The nucleic acid molecules of a given bead can include multiple sets of nucleic acid molecules. The nucleic acid molecules of a given collection may include the same barcode sequence. The same barcode sequence may be different from the barcode sequence of the nucleic acid molecules of another collection.

[0181] Moreover, when the bead population is partitioned, the resulting bead population can also include a diverse barcode library comprising 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. In addition, each partition of the population can include at least about 1,000 nucleic acid molecules, at least about 5,000 nucleic acid molecules, at least about 10,000 nucleic acid molecules, at least about 50,000 nucleic acid molecules, at least about 100,000 nucleic acid molecules, at least about 500,000 nucleic acids, at least about 1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acid molecules, at least about 10,000,000 nucleic acid molecules, at least about 50,000,000 nucleic acid molecules, at least about 100,000,000 nucleic acid molecules, at least about 250,000,000 nucleic acid molecules, and in some cases at least about 1 billion nucleic acid molecules.

[0182] In some cases, multiple different barcodes are incorporated within a given partition, whether attached to a single bead or multiple beads within the partition. For example, in some cases, a mixed but known set of barcode sequences can provide greater identity assurance in subsequent processing, for example, by providing stronger addressing or attribution of a barcode to a given partition as a duplicate or independent confirmation of output from a given partition.

[0183] Nucleic acid molecules (e.g., oligonucleotides) can be released from beads when a specific stimulus is applied to the beads. In some cases, the stimulus can be a light stimulus, such as releasing nucleic acid molecules by cracking light-labile bonds. In other cases, thermal stimulation can be used, wherein the increase in the ambient temperature of the beads will result in the breaking of bonds or the additional release of nucleic acid molecules from the beads. In other cases, chemical stimulation can be used, which cracks the bonds between nucleic acid molecules and beads, or otherwise causes nucleic acid molecules to be released from beads. In one case, such compositions include the above-mentioned polyacrylamide matrix for encapsulating bioparticles or analyte carriers, and can be degraded by exposure to a reducing agent (e.g., DTT) to release attached nucleic acid molecules.

[0184] In some aspects, systems and methods for controlled dispensing are provided. Droplet size can be controlled by adjusting certain geometric features in a channel structure (e.g., a microfluidic channel structure). For example, the expansion angle, width, and / or length of the channel can be adjusted to control droplet size.

[0185] Figure 4 An example of a microfluidic channel structure for controlled distribution of beads into discrete droplets is shown. Channel structure 400 may include a channel segment 402 connected to a reservoir 404 at a channel junction 406 (or intersection). Reservoir 404 may be a chamber. As used herein, any reference to a "reservoir" may also refer to a "chamber". During operation, an aqueous fluid 408 including suspended beads 412 may be transported along channel segment 402 to junction 406 to meet with a second fluid 410 in reservoir 404 that is immiscible with aqueous fluid 408 to produce droplets 416, 418 of aqueous fluid 408 flowing into reservoir 404. At the junction 406 where the aqueous fluid 408 and the second fluid 410 meet, droplets may be formed based on factors such as the fluid dynamics at the junction 406, the flow rates of the two fluids 408, 410, fluid properties, and certain geometric parameters of the channel structure 400 (e.g., w, h0, α, etc.). Multiple droplets may be collected in the reservoir 404 by continuously injecting the aqueous fluid 408 from the channel segment 402 through the junction 406.

[0186] The generated discrete droplets may include a bead (e.g., as in occupied droplet 416). Alternatively, the generated discrete droplets may include more than one bead. Alternatively, the generated discrete droplets may not include any beads (e.g., as in unoccupied droplet 418). In some cases, the generated discrete droplets may contain one or more biological particles or analyte carriers, as described elsewhere herein. In some cases, the generated discrete droplets may contain one or more reagents, as described elsewhere herein.

[0187] In some cases, aqueous fluid 408 can have a substantially uniform concentration or frequency of beads 412. Figure 4 The beads 412 are introduced into the channel segment 402 (not shown). The frequency of the beads 412 in the channel segment 402 can be controlled by controlling the frequency of the introduction of the beads 412 into the channel segment 402 and / or the relative flow rates of the fluids in the channel segment 402 and the separate channels. In some cases, beads can be introduced into the channel segment 402 from a plurality of different channels and the frequency controlled accordingly.

[0188] In some cases, the aqueous fluid 408 in the channel segment 402 may contain biological particles or analyte carriers (e.g., see Figure 1 and Figure 2In some cases, aqueous fluid 408 can have biological particles or analyte carriers that concentration or frequency are substantially uniform. As beads, biological particles or analyte carriers can be introduced into channel section 402 from independent passages. The frequency or concentration of biological particles or analyte carriers in aqueous fluid 408 in channel section 402 can be controlled by controlling the relative flow rate of fluid in frequency and / or channel section 402 and independent passages by which biological particles or analyte carriers are introduced into channel section 402. In some cases, biological particles or analyte carriers can be introduced into channel section 402 from a plurality of different passages, and therefore control frequency. In some cases, beads can be introduced into channel section 402 by the first independent passage and biological particles or analyte carriers can be introduced into wherein by the second independent passage. The first independent passage introducing beads can be in the upstream or downstream of the second independent passage introducing biological particles or analyte carriers.

[0189] The second fluid 410 may comprise an oil, such as a fluorinated oil, which includes a fluorinated surfactant for stabilizing the resulting droplets, such as inhibiting subsequent coalescence of the resulting droplets.

[0190] In some cases, the second fluid 410 may not be subjected to and / or directed to flow into and out of the reservoir 404. For example, the second fluid 410 may be substantially stationary in the reservoir 404. In some cases, the second fluid 410 may be subjected to flow within the reservoir 404, but not flow into and out of the reservoir 404, such as by applying pressure to the reservoir 404 and / or being affected by the oncoming flow of the aqueous fluid 408 at the junction 406. Alternatively, the second fluid 410 may be subjected to and / or directed to flow into or out of the reservoir 404. For example, the reservoir 404 may be a channel that directs the second fluid 410 from upstream to downstream, thereby transporting the generated droplets.

[0191] The channel structure 400 at or near the junction 406 can have certain geometric features that at least partially determine the size of the droplets formed by the channel structure 400. The channel segment 402 can have a height h0 and a width w at or near the junction 406. For example, the channel segment 402 can include a rectangular cross-section that leads to a reservoir 404 having a wider cross-section (e.g., in terms of width or diameter). Alternatively, the cross-section of the channel segment 402 can be other shapes, such as circular, trapezoidal, polygonal, or any other shape. The top and bottom walls of the reservoir 404 at the junction 406 can be inclined at an expansion angle α. The expansion angle α increases the length of the tongue (the portion where the aqueous fluid 408 leaves the channel segment 402 at the junction 406 and enters the reservoir 404 before the droplet is formed) and promotes a decrease in the curvature of the droplet formed in the middle. The droplet size decreases as the expansion angle increases. The radius R of the resulting droplet dIt can be predicted by the following equations for the aforementioned geometric parameters h0, w and α:

[0192]

[0193] For example, for a channel structure of w=21 μm, h=21 μm, and α=3°, the predicted droplet size is 121 μm. In another example, for a channel structure of w=25 μm, h=25 μm, and α=5°, the predicted droplet size is 123 μm. In another example, for a channel structure of w=28 μm, h=28 μm, and α=7°, the predicted droplet size is 124 μm.

[0194] In some cases, the expansion angle α can be between about 0.5° to about 4°, about 0.1° to about 10°, or about 0° to about 90°. For example, the expansion angle can be at least about 0.01°, 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 6°, 7°, 8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 85° or more. In some cases, the expansion angle can be at most about 89°, 88°, 87°, 86°, 85°, 84°, 83°, 82°, 81°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01° or less. In some cases, the width w can be in the range of about 100 microns (μm) to about 500 μm. In some cases, the width w can be in the range of about 10 μm to about 200 μm. Alternatively, the width can be less than about 10 μm. Alternatively, the width can be greater than about 500 μm. In some cases, the flow rate of aqueous fluid 408 entering junction 406 can be between about 0.04 microliters (μL) / minute (min) to about 40 μL / min. In some cases, the flow rate of aqueous fluid 408 entering junction 406 can be between about 0.01 microliters (μL) / minute (min) to about 100 μL / min. Alternatively, the flow rate of aqueous fluid 408 entering junction 406 can be less than about 0.01 μL / min. Alternatively, the flow rate of aqueous fluid 408 entering junction 406 can be greater than about 40 μL / min, such as 45 μL / min, 50 μL / min, 55 μL / min, 60 μL / min, 65 μL / min, 70 μL / min, 75 μL / min, 80 μL / min, 85 μL / min, 90 μL / min, 95 μL / min, 100 μL / min, 110 μL / min, 120 μL / min, 130 μL / min, 140 μL / min, 150 μL / min, or more. At lower flow rates, such as about less than or equal to 10 microliters / minute, the droplet radius can be independent of the flow rate of aqueous fluid 408 entering junction 406.

[0195] In some cases, at least about 50% of the droplets generated may be of uniform size. In some cases, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the droplets generated may be of uniform size. Alternatively, less than about 50% of the droplets generated may be of uniform size.

[0196] The throughput of droplet generation can be increased by increasing the number of generation points, such as increasing the number of junctions (e.g., junctions 406) between an aqueous fluid 408 channel segment (e.g., channel segment 402) and reservoir 404. Alternatively or in addition, the throughput of droplet generation can be increased by increasing the flow rate of the aqueous fluid 408 in the channel segment 402.

[0197] Figure 5 An example of a microfluidic channel structure for increasing the throughput of droplet generation is shown. The microfluidic channel structure 500 can include a plurality of channel segments 502 and a reservoir 504. Each of the plurality of channel segments 502 can be in fluid communication with the reservoir 504. The channel structure 500 can include a plurality of channel junctions 506 between the plurality of channel segments 502 and the reservoir 504. Each channel junction can be a droplet generation point. Figure 4 The channel segment 402 from the channel structure 400 and any description of its components may correspond to a given channel segment in the plurality of channel segments 502 in the channel structure 500 and any description of its corresponding components. The reservoir 404 from the channel structure 400 and any description of its components may correspond to the reservoir 504 from the channel structure 500 and any description of its corresponding components.

[0198] Each of the plurality of channel segments 502 may include an aqueous fluid 508 containing suspended beads 512. The reservoir 504 may include a second fluid 510 that is immiscible with the aqueous fluid 508. In some cases, the second fluid 510 may not be subjected to and / or directed to flow into and out of the reservoir 504. For example, the second fluid 510 may be substantially stationary in the reservoir 504. In some cases, the second fluid 510 may be subjected to flow within the reservoir 504, but not flow into and out of the reservoir 504, such as by applying pressure to the reservoir 504 and / or being affected by the incoming flow of the aqueous fluid 508 at the junction. Alternatively, the second fluid 510 may be subjected to and / or directed to flow into or out of the reservoir 504. For example, the reservoir 504 may be a channel that directs the second fluid 510 from upstream to downstream, thereby transporting the generated droplets.

[0199] In operation, an aqueous fluid 508 including suspended beads 512 can be transported along the plurality of channel segments 502 to the plurality of junctions 506 to meet with a second fluid 510 in a reservoir 504 to produce droplets 516, 518. Droplets can be formed from each channel segment at each respective junction with the reservoir 504. At the junction where the aqueous fluid 508 and the second fluid 510 meet, droplets can be formed based on factors such as fluid dynamics at the junction, flow rates of the two fluids 508, 510, fluid properties, and certain geometric parameters of the channel structure 500 as described elsewhere herein (e.g., w, h0, α, etc.). Multiple droplets can be collected in the reservoir 504 by continuously injecting the aqueous fluid 508 from the plurality of channel segments 502 through the plurality of junctions 506. With the parallel channel configuration of the channel structure 500, throughput can be significantly increased. For example, the frequency of droplet generation of a channel structure having five inlet channel sections containing aqueous fluid 508 can be five times that of a channel structure having one inlet channel section, provided that the fluid flow rate in the channel section is substantially the same. The fluid flow rate in different inlet channel sections can be substantially the same or can be substantially different. The channel structure can have as many parallel channel sections as are actually allowed with the storage tank size. For example, the channel structure can have at least about 2,3,4,5,6,7,8,9,10,20,30,40,50,60,70,80,90,100,150,500,250,300,350,400,450,500,600,700,800,900,1000,1500,5000 or more parallel or substantially parallel channel sections.

[0200] The geometric parameters w, h0 and α may be uniform or non-uniform for each channel segment in the plurality of channel segments 502. For example, each channel segment may have the same or different widths at or near its respective channel junction with the storage tank 504. For example, each channel segment may have the same or different heights at or near its respective channel junction with the storage tank 504. In another example, the storage tank 504 may have the same or different expansion angles at different channel junctions with the plurality of channel segments 502. When the geometric parameters are uniform, it is beneficial that the droplet size can be controlled to be uniform even if the throughput increases. In some cases, the geometric parameters of the plurality of channel segments 502 may be changed accordingly to provide different droplet size distributions.

[0201] In some cases, at least about 50% of the droplets generated may be of uniform size. In some cases, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the droplets generated may be of uniform size. Alternatively, less than about 50% of the droplets generated may be of uniform size.

[0202] Figure 6 Another example of a microfluidic channel structure for increasing the throughput of droplet generation is shown. The microfluidic channel structure 600 may include a plurality of channel segments 602 arranged in a generally circular shape around the periphery of a reservoir 604. Each of the plurality of channel segments 602 may be in fluid communication with the reservoir 604. The channel structure 600 may include a plurality of channel junctions 606 between the plurality of channel segments 602 and the reservoir 604. Each channel junction may be a droplet generation point. Figure 2 The channel segment 402 from the channel structure 400 and any description of its components may correspond to a given channel segment in the plurality of channel segments 602 in the channel structure 600 and any description of its corresponding components. The reservoir 404 from the channel structure 400 and any description of its components may correspond to the reservoir 604 from the channel structure 600 and any description of its corresponding components.

[0203] Each of the plurality of channel segments 602 may include an aqueous fluid 608 containing suspended beads 612. The reservoir 604 may include a second fluid 610 that is immiscible with the aqueous fluid 608. In some cases, the second fluid 610 may not be subjected to and / or directed to flow into and out of the reservoir 604. For example, the second fluid 610 may be substantially stationary in the reservoir 604. In some cases, the second fluid 610 may be subjected to flow within the reservoir 604, but not into and out of the reservoir 604, such as by applying pressure to the reservoir 604 and / or being affected by the incoming flow of the aqueous fluid 608 at the junction. Alternatively, the second fluid 610 may be subjected to and / or directed to flow into or out of the reservoir 604. For example, the reservoir 604 may be a channel that directs the second fluid 610 from upstream to downstream, thereby transporting the generated droplets.

[0204] In operation, an aqueous fluid 608 including suspended beads 612 can be transported along the plurality of channel segments 602 to the plurality of junctions 606 to meet with a second fluid 610 in a reservoir 604 to produce a plurality of droplets 616. Droplets can be formed from each channel segment at each respective junction with the reservoir 604. At the junction where the aqueous fluid 608 and the second fluid 610 meet, droplets can be formed based on factors such as fluid dynamics at the junction, flow rates of the two fluids 608, 610, fluid properties, and certain geometric parameters of the channel structure 600 as described elsewhere herein (e.g., width and height of the channel segment 602, expansion angle of the reservoir 604, etc.). The plurality of droplets can be collected in the reservoir 604 by continuously injecting the aqueous fluid 608 from the plurality of channel segments 602 through the plurality of junctions 606. With a substantially parallel channel configuration of the channel structure 600, throughput can be significantly increased. The passage structure can have as many substantially parallel passage sections as actual and storage tank size allows.For example, the passage structure can have at least about 2,3,4,5,6,7,8,9,10,20,30,40,50,60,70,80,90,100,150,200,250,300,350,400,450,500,600,700,800,900,1000,1500,5000 or more parallel or substantially parallel passage sections.Described multiple passage sections can be for example substantially evenly spaced around the edge or the periphery of storage tank.Alternately, the interval of described multiple passage sections can be uneven.

[0205] The reservoir 604 may have an expansion angle α at or near each channel junction (in Figure 6 604). Each of the plurality of channel segments 602 may have a width w and a height h0 at or near a channel junction. The geometric parameters w, h0, and α may be uniform or non-uniform for each of the plurality of channel segments 602. For example, each channel segment may have the same or different widths at or near its respective channel junction with the storage tank 604. For example, each channel segment may have the same or different heights at or near its respective channel junction with the storage tank 604.

[0206] The storage tank 604 may have the same or different expansion angles at different channel junctions with the plurality of channel segments 602. For example, a circular storage tank (such as Figure 6) can have a conical, dome-shaped, or hemispherical top plate (e.g., top wall) to provide the same or substantially the same expansion angle for each channel segment 602 at or near the plurality of channel connectors 606. When the geometric parameters are uniform, advantageously, the resulting droplet size can be controlled to be uniform even if the throughput increases. In some cases, the geometric parameters of the plurality of channel segments 602 can be varied accordingly to generate different droplet size distributions.

[0207] In some cases, at least about 50% of the droplets generated may have a uniform size. In some cases, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the droplets generated may have a uniform size. Alternatively, less than about 50% of the droplets generated may have a uniform size. The beads and / or bioparticles or analyte carriers injected into the droplets may or may not have a uniform size.

[0208] Fig. 7A A cross-sectional view of another example of a microfluidic channel structure having geometric features for controlled dispensing is shown. Channel structure 700 can include a channel segment 702 that communicates with a reservoir 704 at a channel junction 706 (or intersection). In some cases, one or more of channel structure 700 and its components correspond to one or more of channel structure 100 and its components. Figure 7B Shows Fig. 7A A perspective view of the channel structure 700 is shown.

[0209] The aqueous fluid 712 including the plurality of particles 716 can be transported along the channel segment 702 to the junction 706 to meet the second fluid 714 (e.g., oil, etc.) that is immiscible with the aqueous fluid 712 in the reservoir 704 to produce droplets 720 of the aqueous fluid 712 flowing into the reservoir 704. At the junction 706 where the aqueous fluid 712 and the second fluid 714 meet, droplets can be formed based on factors such as fluid dynamics at the junction 706, flow rates of the two fluids 712, 714, fluid properties, and certain geometric parameters (e.g., Δh, etc.) of the channel structure 700. Multiple droplets can be collected in the reservoir 704 by continuously injecting the aqueous fluid 712 from the channel segment 702 through the junction 706.

[0210] The generated discrete droplets may contain one or more particles of the plurality of particles 716. As described elsewhere herein, the particles may be any particles, such as beads, cell beads, gel beads, biological particles or analyte carriers, macromolecular components of biological particles or analyte carriers, or other particles. Alternatively, the generated discrete droplets may not include any beads.

[0211] In some cases, the aqueous fluid 712 can have particles 716 that are substantially uniform in concentration or frequency. Figure 4 ), particles 716 (e.g., beads) can be introduced into channel segment 702 from a separate channel (not shown in FIG. 7 ). The frequency of particles 716 in channel segment 702 can be controlled by controlling the frequency of introducing particles 716 into channel segment 702 and / or the relative flow rates of fluids in channel segment 702 and the separate channels. In some cases, particles 716 can be introduced into channel segment 702 from a plurality of different channels, and the frequency can be controlled accordingly. In some cases, different particles are introduced via separate channels. For example, a first separate channel can introduce beads into channel segment 702 and a second separate channel can introduce biological particles or analyte carriers therein. The first separate channel into which beads are introduced can be upstream or downstream of the second separate channel into which biological particles or analyte carriers are introduced.

[0212] In some cases, the second fluid 714 may not be subjected to and / or directed to flow into and out of the reservoir 704. For example, the second fluid 714 may be substantially stationary in the reservoir 704. In some cases, the second fluid 714 may be subjected to flow within the reservoir 704, but not flow into and out of the reservoir 704, such as by applying pressure to the reservoir 704 and / or being affected by the oncoming flow of the aqueous fluid 712 at the junction 706. Alternatively, the second fluid 714 may be subjected to and / or directed to flow into or out of the reservoir 704. For example, the reservoir 704 may be a channel that directs the second fluid 714 from upstream to downstream, thereby transporting the generated droplets.

[0213] The channel structure 700 at or near the junction 706 may have certain geometric features that at least partially determine the size and / or shape of the droplets formed by the channel structure 700. The channel section 702 may have a first cross-sectional height h1, and the reservoir 704 may have a second cross-sectional height h2. The first cross-sectional height h1 and the second cross-sectional height h2 may be different, so that at the junction 706, there is a height difference Δh. The second cross-sectional height h2 may be greater than the first cross-sectional height h1. In some cases, the reservoir may gradually increase the cross-sectional height thereafter, for example, the farther away from the junction 706. In some cases, at the junction 706, the cross-sectional height of the reservoir may increase according to the expansion angle β. The height difference Δh and / or the expansion angle β may increase the length of the tongue (the portion where the aqueous fluid 712 leaves the channel section 702 at the junction 706 and enters the reservoir 704 before the droplet is formed) and promote a reduction in the curvature of the droplet formed in the middle. For example, the droplet size may decrease as the height difference increases and / or the expansion angle increases.

[0214] The height difference Δh may be at least about 1 μm. Alternatively, the height difference may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 μm or more. Alternatively, the height difference may be at most about 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 μm or less. In some cases, the expansion angle β can be between about 0.5° to about 4°, about 0.1° to about 10°, or about 0° to about 90°. For example, the expansion angle can be at least about 0.01°, 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 6°, 7°, 8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 85°, or more. In some cases, the expansion angle can be at most about 89°, 88°, 87°, 86°, 85°, 84°, 83°, 82°, 81°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less.

[0215] In some cases, the flow rate of aqueous fluid 712 entering junction 706 can be between about 0.04 microliters (μL) / minute (min) to about 40 μL / min. In some cases, the flow rate of aqueous fluid 712 entering junction 706 can be between about 0.01 microliters (μL) / minute (min) to about 100 μL / min. Alternatively, the flow rate of aqueous fluid 712 entering junction 706 can be less than about 0.01 μL / min. Alternatively, the flow rate of aqueous fluid 712 entering junction 706 can be greater than about 40 μL / min, such as 45 μL / min, 50 μL / min, 55 μL / min, 60 μL / min, 65 μL / min, 70 μL / min, 75 μL / min, 80 μL / min, 85 μL / min, 90 μL / min, 95 μL / min, 100 μL / min, 110 μL / min, 120 μL / min, 130 μL / min, 140 μL / min, 150 μL / min, or more. At lower flow rates, such as about less than or equal to 10 microliters / minute, the droplet radius can be independent of the flow rate of aqueous fluid 712 entering junction 706. Second fluid 714 can be substantially stationary in reservoir 704. Alternatively, the second fluid 714 may be flowing, such as at a flow rate as described above for the aqueous flow 712 .

[0216] In some cases, at least about 50% of the droplets generated may be of uniform size. In some cases, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the droplets generated may be of uniform size. Alternatively, less than about 50% of the droplets generated may be of uniform size.

[0217] Although Fig. 7A and 7B A height difference Δh is illustrated as changing suddenly (e.g., increasing in a step) at the junction 706, but the height difference may also increase gradually (e.g., from about 0 μm to a maximum height difference). Alternatively, the height difference may gradually decrease (e.g., taper) from the maximum height difference. As used herein, a gradual increase or decrease in the height difference may refer to a continuous increase or decrease in the height difference, wherein the angle between any one difference segment of the height profile and the adjacent difference segment of the height profile is greater than 90°. For example, at the junction 706, the bottom wall of the channel and the bottom wall of the tank may meet at an angle greater than 90°. Alternatively or in addition, the top wall of the channel (e.g., top plate) and the top wall of the tank (e.g., top plate) may meet at an angle greater than 90°. The gradual increase or decrease may be linear or nonlinear (e.g., exponential, sinusoidal, etc.). Alternatively or in addition, the height difference may variably increase and / or decrease linearly or nonlinearly. Although Fig. 7A and 7B The expansion tank cross-sectional height is described as linear (e.g., constant expansion angle β), but the cross-sectional height can be expanded non-linearly. For example, the tank can be at least partially defined by a dome-like (e.g., hemispherical) shape with a variable expansion angle. The cross-sectional height can be expanded in any shape.

[0218] For example, the channel network described as above or elsewhere herein can be connected with suitable fluid components fluid.For example, the inlet channel section is connected with the suitable material source fluid that they will be delivered to the channel connector.These sources can include any of various fluid components, from the simple storage tank that is defined in the main structure of the microfluidic device or is connected to the main structure of the microfluidic device to the fluid conduit of the fluid conveying fluid such as from the device outer source, manifold, fluid flow unit (for example, actuator, pump, compressor).Equally, the outlet channel section (for example, channel section 208, storage tank 604, etc.) can be connected with the receiving container or conduit fluid for the cell that has been distributed to carry out subsequent treatment.Equally, this can be the storage tank that is defined in the main body of the microfluidic device, or it can be the fluid conduit for the cell that has been distributed to be delivered to subsequent operation, instrument or parts.

[0219] The methods and systems described herein can be used to greatly increase the efficiency of cell applications and / or applications that receive droplet-based inputs. For example, after sorting occupied cells and / or cells of appropriate size, subsequent operations that can be performed can include generating amplification products, purification (e.g., by solid phase reversible fixation (SPRI)), further processing (e.g., shearing, connecting functional sequences and subsequent amplification (e.g., by PCR)). These operations can be performed in large quantities (e.g., outside the partition). In the case where the partition is a droplet in an emulsion, the emulsion can be destroyed and the contents of the droplets can be combined for additional operations. Other reagents that can be co-distributed with barcoded beads can include oligonucleotides that block ribosomal RNA (rRNA) and nucleases that digest genomic DNA from cells. Alternatively, rRNA removers can be applied to additional processing operations. The configuration of the constructs produced by such methods can help minimize (or avoid) sequencing of the 5' end of the poly-T sequence and / or polynucleotide sequence during sequencing. Amplification products, such as the first amplification product and / or the second amplification product, can be sequenced for sequence analysis. In some cases, amplification can be performed using the Partial Hairpin Amplification for Sequencing (PHASE) method.

[0220] A variety of applications require the assessment of the presence and quantification of different bioparticle or analyte carrier or organism types within a population of bioparticles or analyte carriers, including, for example, microbiome analysis and characterization, environmental testing, food safety testing, epidemiological analysis (e.g., in tracking contamination), etc.

[0221] Computer Systems

[0222] The present disclosure provides computer systems programmed to implement the methods of the present disclosure. Fig. 9 An exemplary computer system 901 is shown, which can be programmed or otherwise configured to, for example, process and / or analyze metabolites, control the addition of reagents to a reaction mixture, control the generation of partitions, control the addition of reagents to partitions, provide conditions sufficient for reactions, acquire and process sequencing data, output sequencing results to a user, provide an interface for user input to control devices coupled to a computer processor. The computer system 901 can regulate various aspects of the present disclosure, such as regulating fluid flow, delivery of reagents, generation of partitions, regulating reaction conditions, etc. The computer system 901 can be an electronic device of a user or a computer system remotely located relative to the electronic device. The electronic device can be a mobile electronic device.

[0223] The computer system 901 includes a central processing unit (CPU, also referred to herein as a "processor" and "computer processor") 905, which can be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system 901 also includes a memory or storage location 910 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 915 (e.g., a hard disk), a communication interface 920 (e.g., a network adapter) for communicating with one or more other systems, and peripheral devices 925, such as cache memory, other memory, data storage, and / or an electronic display adapter. The memory 910, the storage unit 915, the interface 920, and the peripheral devices 925 communicate with the CPU 905 through a communication bus (solid line), such as a motherboard. The storage unit 915 can be a storage unit (or data repository) for storing data. The computer system 901 can be operably coupled to a computer network ("network") 930 by means of the communication interface 920. The network 930 can be the Internet, an intranet and / or an extranet, or an intranet and / or an extranet that communicates with the Internet. The network 930 is a radio communication and / or data network in some cases. The network 930 may include one or more computer servers that may implement distributed computing, such as cloud computing. The network 930, in some cases with the aid of the computer system 901, may implement a peer-to-peer network that may enable a device to couple with the computer system 901 to function as a client or server.

[0224] The CPU 905 may execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a storage location, such as a memory 910. The instructions may be directed to the CPU 905, which may then be programmed or otherwise configured to implement the methods of the present disclosure. Examples of operations performed by the CPU 905 may include fetching instructions, decoding, executing, and writing back.

[0225] CPU 905 may be part of a circuit, such as an integrated circuit. One or more other components of system 901 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0226] The storage unit 915 can store folders, such as drivers, program libraries, and saved programs. The storage unit 915 can store user data, such as user preferences and user programs. In some cases, the computer system 901 may include one or more additional data storage units, which are outside the computer system 901, such as located on a remote server, and the remote server communicates with the computer system 901 via an intranet or the Internet.

[0227] The computer system 901 can communicate with one or more remote computer systems via the network 930. For example, the computer system 901 can communicate with a remote computer system of a user (e.g., an operator). Examples of remote computer systems include personal computers (e.g., portable PCs), touch screen tablets or tablet PCs (e.g., iPad, GalaxyTab), phones, smartphones (e.g. iPhone, Android Launcher, ) or a personal digital assistant. A user can access the computer system 901 via the network 930.

[0228] The methods described herein may be implemented with the aid of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 901, such as the memory 910 or the electronic storage unit 915. The machine executable or machine readable code may be provided in the form of software. During use, the code may be executed by the processor 905. In some cases, the code may be retrieved from the storage unit 915 and stored on the memory 910 for ready access by the processor 905. In some cases, the electronic storage unit 915 may be excluded and the machine executable instructions may be stored on the memory 910.

[0229] The code may be precompiled and configured for use with a machine having a processor adapted to execute the code, or may be compiled during runtime. The code may be provided in a programming language selected so that the code can be executed in a precompiled or as compiled manner.

[0230] Aspects of the systems and methods provided herein, such as computer system 901, can be embodied in programming. Aspects of this technology can be considered as "products" or "articles", usually in the form of machine (or processor) executable code and / or related data carried or embodied on a machine-readable medium. Machine executable code can be stored in an electronic storage unit, such as a memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. "Storage" type media can include any or all tangible memories of a computer, processor, etc., or its related modules, such as various semiconductor memories, tape drives, disk drives, etc., which can provide non-temporary storage for software programming at any time. The software as a whole or in part can sometimes communicate through the Internet or various other telecommunications networks. Such communications, for example, can load software from one computer or processor to another computer or processor, such as from a management server or host to a computer platform of an application server. Therefore, another type of medium that can load software elements includes light waves, radio waves, and electromagnetic waves, such as across the physical interface between local devices, through wired and optical fixed networks and via various air links. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., can also be considered as media that carry software. As used herein, unless limited to non-transitory tangible "storage" media, terms such as computer or machine "readable media" refer to any medium that participates in providing instructions to a processor for execution.

[0231] Thus, machine-readable media, such as computer executable code, can take many forms, including but not limited to tangible storage media, carrier media, or artificial transmission media. Non-volatile storage media include, for example, optical or magnetic disks, such as any storage device in any computer shown in the drawings, etc., which can be used to implement databases, etc. Volatile storage media include dynamic memory, such as the main memory of such computer platforms. Tangible transmission media include coaxial cables; copper wires and optical fibers, including wires that comprise busbars within a computer system. Carrier transmission media can take the form of electrical or electromagnetic signals, or sound or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer-readable media include, for example: floppy disks, floppy disks, hard disks, magnetic tape, any other magnetic media, CD-ROM, DVD or DVD-ROM, any other optical media, punched paper tape, any other physical storage medium with a pattern of holes, RAM, ROM, PROM and EPROM, FLASH-EPROM, any other memory chip or memory card, carrier waves conveying data or instructions, cables or links conveying such carrier waves, or any other medium from which a computer can read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0232] The computer system 901 may include or communicate with an electronic display 935, the electronic display including a user interface (UI) 940, the user interface is used to provide, for example, monitoring of sample preparation, monitoring of reactions and / or reaction conditions, monitoring of sequencing, results of sequencing, and allow user input for sample preparation, reaction, sequencing and / or sequencing analysis. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0233] The methods and systems of the present disclosure may be implemented with the aid of one or more algorithms. The algorithms may be implemented by software when executed by the central processor 905. The algorithms may, for example, implement sample preparation protocols, reaction protocols, sequencing protocols, data analysis protocols, and system or device operation protocols.

[0234] The devices, systems, compositions and methods disclosed herein can be used for various applications, such as processing a single analyte (e.g., RNA, DNA, or protein) or multiple analytes (e.g., DNA and RNA, DNA and protein, RNA and protein, or RNA, DNA and protein) from a cell. For example, a bioparticle or analyte carrier (e.g., a cell or cell bead) is distributed in a partition (e.g., a droplet) and multiple analytes from the bioparticle or analyte carrier are processed for subsequent processing. Multiple analytes can be from cells. This enables, for example, simultaneous proteomic, transcriptomic, and genomic analysis of cells.

[0235] Example

[0236] Example 1: GEM generation and barcoding.

[0237] Prepare cell suspension, wherein the vigor is greater than 80% as measured by trypan blue staining. Before the cell suspension is loaded on the microfluidic chip, it can be optionally filtered by a 40 μm filter. Then prepare a premix, the premix comprises a reverse transcriptase (RT) reagent mixture, RT primer, DTT, RT enzyme mixture and a certain volume of cells according to the expected cell recovery number, and the volume reaches 100 μL. The premix can include a riboswitch, which can be combined with a specific metabolite from the cell and expose the capture sequence on the riboswitch. The premix of 90 μL is loaded into the first channel of the microfluidic chip. 40 μL gel beads (for example, each bead comprises a single barcode sequence) comprising a barcode sequence are loaded into the second channel of this chip. 270 μL of distribution oil is loaded into the third channel of the chip. In order to generate a gel bead emulsion (GEM) containing cells and barcoded gel beads, a specially designed gasket is attached to the chip, and the loaded chip is then transferred to the controller. The instrument will recognize the chip, and the user can initialize the generation of GEMs by pressing a button. A given GEM can contain one or more sequences for analysis, including, for example, RNA (e.g., mRNA) from cells in the GEM and / or riboswitch sequences in the GEM (e.g., riboswitch capture sequences). After the instrument generates the GEMs, 100 μl of the GEMs are aspirated from the recovery well and carefully dispensed into one or more PCR tubes.

[0238] Then can carry out gel bead emulsion-reverse transcriptase (GEM-RT) reaction.Described one or more tubes are loaded into thermal cycler, and tube is incubated at 53 ℃ for 45 minutes, then incubated at 85 ℃ for 5 minutes, and reaction volume is 125 μ L.After incubation, described one or more tubes can be stored at 4 ℃ or make it carry out another reaction.After thermal cycle, given GEM may comprise the barcoded cDNA generated from the analyte sequence in GEM, including the cDNA corresponding to cellular RNA or riboswitch sequence, as discussed above.Barcode can be obtained from those coupled with gel beads in GEM or released from gel beads.Barcoding to analyte including cellular RNA and riboswitch sequence can be carried out as discussed elsewhere herein, and in some cases by means of template switching reaction.

[0239] Example 2: Post-GEM-RT clean-up and cDNA amplification

[0240] After generating the barcoded nucleic acids in Example 1, a recovery reaction is performed. 125 μL of perfluorooctanol (a recovery agent) is added to each tube containing the product obtained in Example 1. The perfluorooctanol is allowed to interact with the GEM for 1 minute without interference such as pipetting or vortexing. 125 μL of the recovery agent and oil are slowly removed from the bottom of the tube and discarded. A silane bead cleaning mixture is prepared by mixing 4 μL of DynaBead MyOne silane, 5 μL of 1M DTT, 182 μL of sample cleaning buffer, and 9 μL of nuclease-free water for each sample. 200 μL of the silane cleaning mixture is added to each sample, mixed thoroughly, and then incubated at room temperature for 10 minutes. The samples are then placed in a magnetic separator. The supernatant is clarified and then the supernatant can be removed and discarded. 300 μL of freshly prepared 80% ethanol is added to each sample while the sample is still placed in the magnetic separator. The ethanol mixture is allowed to stand for 30 seconds, then removed and discarded. Add 200 μL of 80% ethanol, let stand again for 30 seconds, then remove and discard. Then centrifuge the sample and return it to the magnetic separator. Discard any residual ethanol in the sample. Allow the sample to air dry for 1 minute. Then remove the sample from the magnetic separator and add 35.5 μL of elution solution. Mix the sample well and incubate at room temperature for 1 minute. Return the sample to the magnetic separator and allow the solution to clarify. Transfer 35 μL of the supernatant containing the purified GEM-RT product to a new test tube.

[0241] In order to amplify this purified cDNA, a cDNA amplification reaction was performed. This cDNA amplification reaction was performed by adding 50 μL of amplification premix (which may contain enzymes and nucleotides), 5 μL of cDNA amplification additive mixture, 2 μL of cDNA primer mixture, and 8 μL of nuclease-free water to 35 μL of purified cDNA obtained from the GEM-RT reaction. The cDNA amplification reaction was thoroughly mixed, briefly centrifuged, capped, and then loaded into a thermal cycler. The following program was run with a cap temperature of 105°C and a total reaction volume of 100 μL:

[0242]

[0243] Once the cDNA is amplified, the cDNA corresponding to, for example, a riboswitch sequence or cellular RNA can be isolated or purified using SPRIselect reagent for size selection to obtain the appropriate fragment size corresponding to the desired cDNA. After cDNA amplification, 60 μL of SPRIselect reagent is added to each sample to achieve a 0.6-fold ratio. The samples are then mixed thoroughly, incubated at room temperature for 5 minutes, and then placed in a magnetic separator. Once the solution is substantially clear, 150 μL of supernatant is collected in a separate tube and stored at 4°C. The supernatant of this size selection reaction will contain sequences corresponding to the size of the riboswitch sequence. The precipitate is then washed twice with 200 μL of 80% ethanol each time and air-dried for 2 minutes. The samples are then removed from the magnetic separator and 40.5 μL of elution buffer is added to each sample. Each sample is mixed with a pipette and then incubated at room temperature for 2 minutes. The samples are placed back on the magnetic separator for 4 minutes to clarify the solution. 40 μL of each sample is then transferred to a new tube for downstream processing.

[0244] Example 3: Cell RNA library selection and preparation

[0245] Can select corresponding to the cDNA of cellular RNA, for example mRNA, then prepare for downstream sequencing reaction.Can produce this cDNA by using the material and scheme discussed in embodiment 1, then as discussed in embodiment 2, carry out cDNA amplification.For example, can produce cDNA corresponding to gene expression by GEM comprising gel beads, and described gel beads comprise the capture sequence of mRNA.As mentioned in embodiment 2, can carry out size selection to cDNA by using SPRIselect reagent, make can separate the cDNA (for example, the cDNA relevant to riboswitch) with the cDNA relevant to cellular RNA with the cDNA that is irrelevant to cellular RNA.Prepare these cDNA for downstream sequencing reaction by the process of fragmentation, end repair and A tailing of cDNA.In order to carry out fragmentation, end repair and A tailing, can use the following incubation scheme to prepare thermal cycler:

[0246]

[0247] The protocol begins, waiting for the user to initiate a move through the first pre-cooling step. A fragmentation mix is ​​then prepared using 5 μL of fragmentation buffer and 10 μL of fragmentation enzyme blend per reaction. 15 μL of this fragmentation mix is ​​dispensed into each well of a PCR8 tube strip that has been placed on ice. 17.5 μL of purified, size-selected cDNA corresponding to cellular RNA and 17.5 μL of nuclease-free water are added to each well of the PCR tube strip. The solution is mixed thoroughly, then placed in the thermal cycler and the thermal cycler is allowed to proceed to the "Fragmentation" step at the user's prompt. After the thermal cycler protocol is completed, 30 μL of SPRIselect reagent is added to each sample to a 0.6x ratio. The samples are mixed thoroughly, incubated at room temperature for 5 minutes, and then placed in a magnetic separator. Once the solution is clear, the supernatant is removed and transferred to a new tube. The tube with the magnetic beads can be discarded. 10 μL of SPRIselect reagent is then added to each sample to a 0.8x ratio. The samples are mixed thoroughly and incubated at room temperature for 5 minutes. After incubation, the samples were placed on a magnetic separator and the solution was clarified. The supernatant was then removed and discarded. The precipitate was then washed twice with 200 μL 80% ethanol each time, followed by air drying for 2 minutes. The samples were removed from the magnetic separator, and 50.5 μL elution buffer was added to each sample and mixed thoroughly with a pipette. After the samples were incubated at room temperature for 2 minutes, the samples were placed back on the magnetic separator for 4 minutes to clarify the solution. 50 μL of each sample was transferred to a new tube for use in the next step. Next, 17.5 μL of nuclease-free water, 20 μL of connection buffer, 10 μL of DNA ligase, and 2.5 μL of Adapter Mix were used to prepare the adapter connection mixture. 50 μL of the adapter connection mixture was added to 50 μL of each sample (fragmented, end-repaired, and A-tailed size selected) to a total volume of 100 μL. The solution was thoroughly mixed and briefly centrifuged, then incubated at 20°C for 15 minutes in a thermal cycler with a lid temperature of 30°C. After incubation and ligation of the adapters, 80 μL of SPRIselect reagent was added to each sample to achieve a 0.8x ratio. The samples were then mixed thoroughly, incubated at room temperature for 5 minutes, and then placed in a magnetic separator. Once the solution was substantially clear, the supernatant was removed and discarded. The precipitate was then washed twice with 200 μL of 80% ethanol each time and air-dried for 2 minutes. The samples were then removed from the magnetic separator and 30.5 μL of elution buffer was added to each sample. Each sample was mixed with a pipette and then incubated at room temperature for 2 minutes. The samples were placed back on the magnetic separator for 4 minutes to allow the solution to clarify. 30 μL of each sample was then transferred to a new tube for processing via sample marker PCR. The sample marker PCR will add a marker to each sample.Prepare the sample tag PCR (SI-PCR) reaction mixture by mixing 8 μL of nuclease-free water, 50 μL of amplification premix, and 2 μL of SI-PCR primers. Add this 60 μL solution to each test tube containing 30 μL of each sample. Add 10 μL of sample tag to the samples. In order to keep track of the tags, it is highly recommended to record the tag assignment for each sample. After the samples are thoroughly mixed and briefly centrifuged, incubate each sample in a hot lid thermal cycler using the following protocol:.

[0248]

[0249] After incubation in the thermal cycler, the samples can be stored at 4°C overnight or at -20°C until the next step is started. To clean up and perform dual size selection, 60 μL of SPRIselect reagent is added to each sample to achieve a 0.6x ratio. The samples are then mixed thoroughly and incubated at room temperature for 5 minutes. After incubation, the samples are placed in a magnetic separator until the solution is clear. Each supernatant is then removed and transferred to a new tube. Tubes with remaining beads can be discarded. 20 μL of SPRIselect reagent is added to each supernatant in a new tube to achieve a 0.8x ratio. These samples are mixed thoroughly and then incubated at room temperature for 5 minutes. The samples are then placed on a magnetic separator and the solution is clarified. The supernatant is then removed and discarded. The beads are washed twice using 200 μL of 80% ethanol for each wash, followed by air drying for 2 minutes. The samples are then removed from the magnetic separator and 35.5 μL of elution solution is added. After mixing with a pipette followed by incubation at room temperature for 2 minutes, the sample was placed back on the magnetic separator for 4 minutes to clarify the solution. 35 μL of each sample was transferred to a new tube to store overnight at 4°C or for long-term storage at -20°C. In addition, qualitative analysis of the cDNA library and estimation of concentration and size can be performed by running purified samples on an Agilent Bioanalyzer High Sensitivity Chip for quality control and quantification.

[0250] Example 4: Riboswitch library selection and preparation

[0251] The materials and protocols of Example 1 can be used to select and prepare a library of riboswitches associated with certain metabolites found in cells. As seen in Example 1, the generated GEMs contain capture sequences that bind to riboswitches in the presence of metabolites, which facilitates the determination of the cell metabolome. cDNAs corresponding to these riboswitches can be generated by a reverse transcriptase reaction as described in Example 1, and amplified and purified as described in Example 2. In order to clean up and perform size selection of cDNAs corresponding to riboswitches, 60 μL of SPRIselect reagent was added to each 150 μL supernatant sample obtained in Example 2 to a 1.2-fold ratio. The samples were mixed thoroughly and incubated at room temperature for 5 minutes. The samples were then placed in a magnetic separator until the solution was clear. The supernatant was then removed and discarded. The precipitate was washed twice with 200 μL of 80% ethanol for each wash, followed by air drying for 2 minutes. The sample was removed from the magnetic separator and 40.5 μL of elution solution was added. The samples were mixed thoroughly using a pipette and incubated at room temperature for 2 minutes. The samples were then returned to the magnetic separator and allowed to sit for 4 minutes to allow the solution to become clear. 40 μL of sample was transferred to a new tube. To label the samples, a sample labeling PCR (SI-PCR) mixture was prepared by mixing 8 μL of nuclease-free water, 50 μL of amplification mix, and 2 μL of SI-PCR primers for each sample. 60 μL of sample PCR labeling mixture was added to each PCR tube, and 5 μL of purified riboswitch cDNA product was added to each tube. 25 μL of nuclease-free water and 10 μL of sample label were then added to each sample. It is recommended to record the label assignment for proper labeling. The samples were mixed thoroughly, centrifuged briefly, and then incubated in a hot lid thermal cycler using the following protocol:

[0252]

[0253] The samples after thermal cycling can be stored at 4°C overnight or at -20°C until the purification step begins. For cleanup and size selection, 100 μL of SPRIselect reagent was added to each sample to achieve a 1.0x ratio. The samples were mixed well and then incubated at room temperature for 5 minutes. The samples were then placed on a magnetic separator and the samples were clarified. The supernatant was then removed and discarded. The beads were washed twice using 200 μL of 80% ethanol for each wash and then air dried for 2 minutes. The samples were then removed from the magnetic separator and 35.5 μL of elution solution was added. After mixing with a pipette and then incubating at room temperature for 2 minutes, the samples were placed back on the magnetic separator for 4 minutes to clarify the solution. 35 μL of each sample was transferred to a new tube. 28 μL of SPRIselect reagent was added to each sample to achieve a 0.8x ratio. The samples were then mixed well and incubated for 5 minutes, then placed on a magnetic separator and the solution was clarified. The supernatant was then removed and discarded. Each washing uses 200 μ L of 80% ethanol, and the beads are washed twice, followed by air drying for 2 minutes. Then the sample is taken out from the magnetic separator, and 35.5 μ L of elution solution is added. After mixing with a pipette and then incubating at room temperature for 2 minutes, the sample is placed back on the magnetic separator for 4 minutes to clarify the solution. 35 μ L of each sample is transferred to a new tube. After incubation in a thermal cycler, the sample can be stored overnight at 4°C or stored for a long time at -20°C. In addition, the qualitative analysis of the cDNA library and the estimation of concentration and size can be carried out by running the purified sample on an Agilent bioanalyzer high sensitivity chip (Agilent Bioanalyzer High Sensitivity Chip), to carry out quality control and quantification.

[0254] Example 5: Library sequencing

[0255] The cellular RNA and riboswitch libraries generated by Example 3 and Example 4, respectively, can both contain standard Illumina paired-end constructs starting and ending with P5 and P7. Reads 1 and 2 can use standard Illumina sequencing primer sites used in paired-end sequencing. Read 1 can be used to sequence barcodes and unique molecular identifiers. Read 2 can be used to sequence insert sequences or riboswitch sequences. Optionally, but preferably, 8 cycles of flag reading are performed. Taking the above examples as examples, it is possible to purify two different libraries. One library can contain gene expression information related to the cellular RNA of each single cell successfully captured in the partition (Example 3). Another library as discussed in Example 4 is a riboswitch library, which can contain metabolome information from each single cell successfully captured in the partition.

[0256] Thus, both gene expression and metabolome information can be traced back to the same cell using barcoding and labeling as described elsewhere herein, and then processed accordingly using software tools. The information obtained in these and many other embodiments of the present disclosure can be very useful in understanding the current state of a cell or cell population. Gene expression information is highly informative, but since many of the functions performed by cells are determined by the metabolites present, the present disclosure can allow researchers to more thoroughly classify and reclassify cells based on various molecular patterns.

[0257] Although preferred embodiments of the present invention have been shown and described herein, it is obvious to those skilled in the art that such embodiments are provided only for example. It is not intended that the present invention be limited by the specific examples provided in this specification. Although the present invention has been described with reference to the foregoing description, the description and illustration of the embodiments herein are not intended to be interpreted in a restrictive sense. Various modifications, changes and substitutions will now be made by those skilled in the art without departing from the present invention. In addition, it should be understood that all aspects of the present invention are not limited to the specific description, configuration or relative proportion depending on various conditions and variables set forth herein. It should be understood that the present invention can be implemented using multiple alternatives of the embodiments of the present invention described herein. Therefore, it is expected that the present invention will also cover any such alternatives, modifications, changes or equivalents. It is intended that the following claims define the scope of the present invention, and therefore encompass methods and structures and their equivalents within the scope of these claims.

Claims

1. A method for processing or analyzing metabolites from cells, the method comprising: (a) generating a partition comprising: (i) the metabolite, (ii) a bead comprising a plurality of nucleic acid cellular barcode molecules, each of the plurality of nucleic acid cellular barcode molecules comprising a cellular barcode sequence and a capture sequence, the cellular barcode sequence uniquely corresponding to the cell, and (iii) a riboswitch capable of coupling to the metabolite, wherein the riboswitch comprises a riboswitch capture sequence and a riboswitch identifier sequence, wherein the riboswitch capture sequence is inaccessible to the capture sequence when the metabolite is not coupled to the riboswitch; (b) providing conditions in the partition sufficient to allow coupling of the metabolite to the riboswitch so as to (i) render the riboswitch capture sequence accessible to the capture sequence of a given nucleic acid cellular barcode molecule in the plurality of nucleic acid cellular barcode molecules and (ii) permit coupling of the capture sequence to the riboswitch capture sequence; as well as (c) using the given nucleic acid cellular barcode molecule and the riboswitch to synthesize a nucleic acid molecule comprising (1) a first nucleic acid sequence corresponding to the riboswitch identifier sequence, and (2) a second nucleic acid sequence corresponding to the cellular barcode sequence, where the capture sequence is coupled to the riboswitch capture sequence, wherein the first nucleic acid sequence and the second nucleic acid sequence allow the metabolite to be identified as corresponding to the cell.

2. The method of claim 1, further comprising sequencing at least a portion of the nucleic acid molecule or a derivative thereof to identify the riboswitch identifier sequence and the cellular barcode sequence.

3. The method of claim 2, further comprising using the riboswitch identifier sequence and the cellular barcode sequence to identify the metabolite as originating from the cell. The method of claim 1 , wherein the partitions comprise the cells.

5. The method of claim 4, further comprising releasing the metabolite from the cell after (a).

6. The method of claim 1, further comprising releasing the given nucleic acid cellular barcode molecule from the bead before (c).

7. The method of claim 6, wherein said releasing the nucleic acid cellular barcode molecules from the beads comprises exposing the beads to a chemical stimulus in the partitions.

8. The method of claim 1, wherein (c) is performed in the partition.

9. The method of claim 8, further comprising releasing the nucleic acid molecule or its derivative from the partition or recovering the nucleic acid molecule or its derivative from the partition.

10. The method of claim 1, wherein in (c), the nucleic acid molecule is synthesized using one or more reactions selected from the group consisting of a nucleic acid amplification reaction, a reverse transcription reaction, and a template switching reaction.

11. The method of claim 10, further comprising subjecting the nucleic acid molecule to one or more additional reactions.

12. The method of claim 11, wherein the one or more additional reactions comprise a primer extension reaction.

13. The method of claim 11, wherein the one or more additional reactions comprise adding one or more functional sequences to the nucleic acid molecule, wherein the one or more functional sequences are configured to allow the nucleic acid molecule or a derivative thereof to attach to a flow cell of a sequencer.

14. The method of claim 1, wherein the beads are gel beads.

15. The method of claim 1, wherein: The nucleic acid cellular barcode molecules are attached to the beads as the nucleic acid molecules are synthesized.

16. The method of claim 1, wherein the partition comprises an additional riboswitch capable of coupling to an additional metabolite different from the metabolite.

17. The method of claim 16, wherein the bead comprises an additional plurality of nucleic acid cellular barcode molecules, each of the additional plurality of nucleic acid cellular barcode molecules comprising the cellular barcode sequence and an additional capture sequence capable of coupling to an additional riboswitch capture sequence of the additional riboswitch when the additional metabolite is coupled to the additional riboswitch.

18. The method of claim 17, further comprising: providing, in the partition, conditions sufficient to allow coupling of the additional metabolite to the additional riboswitch, so as to (iii) render the additional riboswitch capture sequence accessible to the additional capture sequence of a given additional nucleic acid cellular barcode molecule in the additional plurality of nucleic acid cellular barcode molecules, and (iv) permit coupling of the additional capture sequence to the additional riboswitch capture sequence; as well as Where the additional capture sequence is coupled to the additional riboswitch capture sequence, the given additional nucleic acid cellular barcode molecule and the additional riboswitch are used to synthesize an additional nucleic acid molecule comprising (3) a third nucleic acid sequence corresponding to the additional riboswitch identifier sequence, and (4) a fourth additional nucleic acid sequence corresponding to the cellular barcode sequence, wherein the third nucleic acid sequence and the fourth nucleic acid sequence allow the additional metabolite to be identified as corresponding to the cell.

19. The method of claim 1, wherein the plurality of nucleic acid cellular barcode molecules comprises at least 100,000 nucleic acid cellular barcode molecules.

20. The method of claim 1, wherein the partition is a droplet of a plurality of droplets.

21. The method of claim 1, wherein the partition is a hole in a plurality of holes.

22. The method of claim 1, wherein the metabolite is selected from the group consisting of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), guanosine triphosphate (GTP), guanosine diphosphate (GDP), guanosine monophosphate (GMP), ribose, glucose, mannose, glycerol, phosphatidylcholine, phosphorylcholine, glycerylphosphorylcholine, phosphatidylserine, phosphatidylethanolamine, diglyceride, nicotinamide adenine dinucleotide phosphate, nicotinamide adenine dinucleotide, glycine, glutamine, aspartic acid, citric acid, acetone, acetoacetate, and lysine.

23. The method of claim 1, wherein a nucleic acid cellular barcode molecule of the plurality of nucleic acid cellular barcode molecules comprises an identifier sequence separate from the cellular barcode sequence, and wherein the identifier sequence is different for each nucleic acid cellular barcode molecule in at least the subset of the plurality of nucleic acid cellular barcode molecules.

24. The method of claim 1, wherein in (a), a nucleic acid cellular barcode molecule among the plurality of nucleic acid cellular barcode molecules comprises the cellular barcode sequence, wherein the bead is from a plurality of beads, and wherein the cellular barcode sequence is different from the cellular barcode sequence of the nucleic acid cellular barcode molecules of other beads in the plurality of beads.

25. The method of claim 1, wherein the bead comprises a plurality of additional nucleic acid cellular barcode molecules, each of the plurality of additional nucleic acid cellular barcode molecules comprising the cellular barcode sequence and a binding sequence capable of coupling to an additional nucleic acid molecule different from the riboswitch.

26. The method of claim 25, wherein the additional nucleic acid molecule is selected from the group consisting of: a ribonucleic acid molecule (RNA) of the cell, a deoxyribonucleic acid molecule (DNA) of the cell, and an editing nucleic acid molecule capable of participating in a gene editing reaction.

27. The method of claim 25, further comprising exposing the protein of the cell to an antibody coupled to the additional nucleic acid molecule prior to (a), wherein the antibody binds to the protein.

28. The method of claim 25, wherein the partition comprises the additional nucleic acid molecules, and the method further comprises allowing a given additional nucleic acid cellular barcode molecule of the additional plurality of nucleic acid cellular barcode molecules to bind to the additional nucleic acid molecules via the binding sequence.

29. The method of claim 28, further comprising using the additional nucleic acid molecule and the given additional nucleic acid cellular barcode molecule among the additional plurality of nucleic acid cellular barcode molecules to synthesize a reporter nucleic acid molecule, wherein the reporter nucleic acid molecule comprises (3) a third nucleic acid sequence corresponding to the sequence of the reporter nucleic acid molecule, and (4) a fourth nucleic acid sequence corresponding to the cellular barcode sequence.

30. The method of claim 29, further comprising sequencing at least a portion of the reporter nucleic acid molecule or a derivative thereof to identify the additional nucleic acid molecule as corresponding to the cell.

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