Patent Publication Number: US-2023144760-A1

Title: Systems and methods for processing-nucleic acid molecules from a single cell using sequential co-partitioning and composite barcodes

Description:
CROSS-REFERENCE 
     This application is a continuation of U.S. Application Pat. No. 16/708, 214, filed Dec. 9, 2019, which claims the benefit of U.S. Provisional Pat. Application No. 62/777,402, filed Dec. 10, 2018, which are incorporated by reference herein in their entirety for all purposes. 
    
    
     BACKGROUND 
     A sample may be processed for various purposes, such as identification of a type of moiety within the sample. The sample may be a biological sample. Biological samples may be processed, such as for detection of a disease (e.g., cancer) or identification of a particular species. There are various approaches for processing samples, such as polymerase chain reaction (PCR) and sequencing. 
     Biological samples may be processed within various reaction environments, such as partitions. Partitions may be wells or droplets. Droplets or wells may be employed to process biological samples in a manner that enables the biological samples to be partitioned and processed separately. For example, such droplets may be fluidically isolated from other droplets, enabling accurate control of respective environments in the droplets. 
     Biological samples in partitions may be subjected to various processes, such as chemical processes or physical processes. Samples in partitions may be subjected to heating or cooling, or chemical reactions, such as to yield species that may be qualitatively or quantitatively processed. 
     SUMMARY 
     To gain further insight into the biology and pathology of cells, it may be useful to capture and analyze multiple different types of biomolecules, such as ribonucleic acid (RNA) molecules and deoxyribonucleic acid (DNA) molecules, from a single cell. However, different types of biomolecules may require, or be compatible with, different types of chemistry or processing mechanisms, and obtaining one type of biomolecule from a cell may undesirably alter or destroy another type of biomolecule, or otherwise render it unobtainable. Therefore, recognized herein is a need to address at least the abovementioned problems. 
     In an aspect, provided is a method for barcoding nucleic acid molecules of a cell, comprising: (a) co-partitioning a cell bead and a first bead in a first partition, wherein the cell bead is generated from the cell, wherein the cell comprises a first type of nucleic acid molecule and a second type of nucleic acid molecule, wherein the first type and the second type are different, and wherein the first bead comprises a first set of barcode molecules each comprising a first nucleic acid barcode sequence; (b) subjecting the first partition to first conditions sufficient to permit (i) a first target nucleic acid molecule of the first type to couple to a first barcode molecule of the first set of barcode molecules to generate a first barcoded nucleic acid molecule comprising the first nucleic acid barcode sequence, and (ii) a second barcode molecule of the first set of barcode molecules to associate with the cell bead; (c) co-partitioning the cell bead associated with the second barcode molecule or derivative thereof and a second bead in a second partition, wherein the second bead comprises a second set of barcode molecules, each comprising a second nucleic acid barcode sequence; and (d) subjecting the second partition to second conditions sufficient to permit (i) a second target nucleic acid molecule of the second type to couple to a third barcode molecule of the second set of barcode molecules to generate a second barcoded nucleic acid molecule comprising the second nucleic acid barcode sequence, and (ii) a fourth barcode molecule of the second set of barcode molecules to couple to the second barcode molecule to generate a composite barcode molecule comprising the first nucleic acid barcode sequence and the second nucleic acid barcode sequence. 
     In some embodiments, in (b), the second barcode molecule attaches to the cell bead. 
     In some embodiments, the method further comprises, subsequent to (b), releasing or recovering the first barcoded nucleic acid molecule or derivative thereof and the cell bead associated with the second barcode molecule or derivative thereof from the first partition. 
     In some embodiments, the method further comprises, subsequent to (d), releasing or recovering the second barcoded nucleic acid molecule or derivative thereof and the composite barcode molecule from the second partition. 
     In some embodiments, the method further comprises, sequencing the first barcoded nucleic acid molecule or derivative thereof, the second barcoded nucleic acid molecule or derivative thereof, and the composite barcode molecule or derivative thereof to generate sequencing reads, and using the sequencing reads to associate the first target nucleic acid molecule and the second target nucleic acid molecule as having originated from the cell or cell bead. 
     In some embodiments, prior to (a), the cell is alive. In some embodiments, the first conditions comprise lysing the cell. 
     In some embodiments, prior to (a), the cell is lysed. 
     In some embodiments, prior to (a), target nucleic acid molecules of the first type are coupled to the cell bead. In some embodiments, the target nucleic acid molecules of the first type are coupled to the cell bead via one or more capture nucleic acid molecules configured to capture the target nucleic acid molecules of the first type. In some embodiments, the first type of nucleic acid molecule comprises messenger ribonucleic acid (mRNA) and the one or more capture nucleic acid molecules comprise a dT sequence. 
     In some embodiments, the cell bead comprises a first layer of polymer network and a second layer of polymer network. In some embodiments, the first layer of polymer network and the second layer of polymer network are not covalently bound. In some embodiments, the first layer of polymer network and the second layer of polymer network are interpenetrating. In some embodiments, the first layer of polymer network is configured to dissociate by a first dissolution mechanism and the second layer of polymer network is configured to dissociate by a second dissolution mechanism, wherein the first dissolution mechanism and the second dissolution mechanism are different. 
     In some embodiments, the first dissolution mechanism comprises a reduction or oxidation reaction. In some embodiments, the second dissolution mechanism comprises a reduction or oxidation reaction. 
     In some embodiments, the first dissolution mechanism is configured to release target nucleic acid molecules of the first type from the cell bead. In some embodiments, the first dissolution mechanism is configured to release the target nucleic acid molecules of the first type from the cell bead but not target nucleic acid molecules of the second type. In some embodiments, the second dissolution mechanism is configured to release target nucleic acid molecules of the second type from the cell bead. 
     In some embodiments, the first conditions comprise applying the first dissolution mechanism. In some embodiments, the second conditions comprise applying the second dissolution mechanism. 
     In some embodiments, the first layer of polymer network comprises disulfide crosslinking bonds. 
     In some embodiments, the second layer of polymer network comprises dihydroxybutane. 
     In some embodiments, (c) comprises using a magnetic field to capture the cell bead associated with the second barcode molecule. 
     In some embodiments, the first type of nucleic acid molecule is selected from the group consisting of deoxyribonucleic acid and ribonucleic acid. In some embodiments, the second type of nucleic acid molecule is selected from the group consisting of deoxyribonucleic acid and ribonucleic acid. 
     In some embodiments, the first conditions and the second conditions are different. In some embodiments, the first conditions comprise applying a first dissolution mechanism and the second conditions comprise applying a second dissolution mechanism, wherein the first dissolution mechanism and the second dissolution mechanism are different. 
     In some embodiments, the first dissolution mechanism comprises a reduction or oxidation reaction. In some embodiments, the second dissolution mechanism comprises a reduction or oxidation reaction. In some embodiments, the first dissolution mechanism is configured to release target nucleic acid molecules of the first type from the cell bead. In some embodiments, the first dissolution mechanism is configured to release the target nucleic acid molecules of the first type from the cell bead but not target nucleic acid molecules of the second type. 
     In some embodiments, the first partition is a droplet. In some embodiments, the second partition is a droplet. 
     In some embodiments, the first bead is a gel bead. In some embodiments, the second bead is a gel bead. 
     Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein. 
     Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein. 
     Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. 
     INCORPORATION BY REFERENCE 
     All publications, patents, and patent applications mentioned in this specification are herein incorporated 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 publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which: 
         FIG.  1    shows an example of a microfluidic channel structure for partitioning individual biological particles. 
         FIG.  2    shows an example of a microfluidic channel structure for delivering barcode carrying beads to droplets. 
         FIG.  3    shows an example of a microfluidic channel structure for co-partitioning biological particles and reagents. 
         FIG.  4    shows an example of a microfluidic channel structure for the controlled partitioning of beads into discrete droplets. 
         FIG.  5    shows an example of a microfluidic channel structure for increased droplet generation throughput. 
         FIG.  6    shows another example of a microfluidic channel structure for increased droplet generation throughput. 
         FIG.  7 A  shows a cross-section view of another example of a microfluidic channel structure with a geometric feature for controlled partitioning.  FIG.  7 B  shows a perspective view of the channel structure of  FIG.  7 A . 
         FIG.  8    illustrates an example of a barcode-carrying bead. 
         FIG.  9    illustrates an example of a method for barcoding nucleic acid molecules in a cell encapsulated in a cell bead. 
         FIG.  10    illustrates an example of a method for barcoding nucleic acid molecules in a cell that is encapsulated and lysed in a cell bead. 
         FIG.  11    illustrates an example of a method for barcoding nucleic acid molecules in a cell that is encapsulated and lysed in a cell bead comprising an interpenetrating network. 
         FIG.  12    shows a computer system that is programmed or otherwise configured to implement methods provided herein. 
     
    
    
     DETAILED DESCRIPTION 
     While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed. 
     Where values are described as ranges, it will be understood that such disclosure includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated. 
     The term “barcode,” as used herein, generally refers to a label, or identifier, that conveys or is capable of conveying information about an analyte. A barcode can be part of an analyte. A barcode can be independent of an analyte. A barcode can be a tag attached to an analyte (e.g., nucleic acid molecule) or a combination of the tag in addition to an endogenous characteristic of the analyte (e.g., size of the analyte or end sequence(s)). A barcode may be unique. Barcodes can have a variety of different formats. For example, barcodes can include: polynucleotide barcodes; random nucleic acid and/or amino acid sequences; and synthetic nucleic acid and/or amino acid sequences. A barcode can be attached to an analyte in a reversible or irreversible manner. A barcode can be added to, for example, a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before, during, and/or after sequencing of the sample. Barcodes can allow for identification and/or quantification of individual sequencing-reads. 
     The term “real time,” as used herein, can refer to a response time of less than about 1 second, a tenth of a second, a hundredth of a second, a millisecond, or less. The response time may be greater than 1 second. In some instances, real time can refer to simultaneous or substantially simultaneous processing, detection or identification. 
     The term “subject,” as used herein, generally refers to an animal, such as a mammal (e.g., human) or avian (e.g., bird), or other organism, such as a plant. For example, the subject can be a vertebrate, a mammal, a rodent (e.g., a mouse), a primate, a simian or a human. Animals may include, but are not limited to, farm animals, sport animals, and pets. A subject can be a healthy or asymptomatic individual, an individual that has or is suspected of having a disease (e.g., cancer) or a pre-disposition to the disease, and/or an individual that is in need of therapy or suspected of needing therapy. A subject can be a patient. A subject can be a microorganism or microbe (e.g., bacteria, fungi, archaea, viruses). 
     The term “genome,” as used herein, generally refers to genomic information from a subject, which may be, for example, at least a portion or an entirety of a subject’s hereditary information. A genome can be encoded either in DNA or in RNA. A genome can comprise coding regions (e.g., that code for proteins) as well as non-coding regions. A genome can include the sequence of all chromosomes together in an organism. For example, the human genome ordinarily has a total of 46 chromosomes. The sequence of all of these together may constitute a human genome. 
     The terms “adaptor(s)”, “adapter(s)” and “tag(s)” may be used synonymously. An adaptor or tag can be coupled to a polynucleotide sequence to be “tagged” by any approach, including ligation, hybridization, or other approaches. 
     The term “sequencing,” as used herein, generally refers to methods and technologies for determining the sequence of nucleotide bases in one or more polynucleotides. The polynucleotides can be, for example, nucleic acid molecules such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (e.g., single-stranded DNA). Sequencing can be performed by various systems currently available, such as, without limitation, a sequencing system by Illumina®, Pacific Biosciences (PacBio®), Oxford Nanopore®, or Life Technologies (Ion Torrent®). Alternatively or in addition, sequencing may be performed using nucleic acid amplification, polymerase chain reaction (PCR) (e.g., digital PCR, quantitative PCR, or real time PCR), or isothermal amplification. Such systems may provide a plurality of raw genetic data corresponding to the genetic information of a subject (e.g., human), as generated by the systems from a sample provided by the subject. In some examples, such systems provide sequencing reads (also “reads” herein). A read may include a string of nucleic acid bases corresponding to a sequence of a nucleic acid molecule that has been sequenced. In some situations, systems and methods provided herein may be used with proteomic information. 
     The term “bead,” as used herein, generally refers to a particle. The bead may be a solid or semi-solid particle. The bead may be a gel bead. The gel bead may include a polymer matrix (e.g., matrix formed by polymerization or cross-linking). The polymer matrix may include one or more polymers (e.g., polymers having different functional groups or repeat units). Polymers in the polymer matrix may be randomly arranged, such as in random copolymers, and/or have ordered structures, such as in block copolymers. Cross-linking can be via covalent, ionic, or inductiveinteractions, or physical entanglement. The bead may comprise a macromolecule. The bead may comprise a sol-gel. The bead may be formed of nucleic acid molecules bound together. The bead may be formed via covalent or non-covalent assembly of molecules (e.g., macromolecules), such as monomers or polymers. Such polymers or monomers may be natural or synthetic. Such polymers or monomers may be or include, for example, nucleic acid molecules (e.g., DNA or RNA). The bead may be formed of a polymeric material. The bead may be magnetic or non-magnetic. The bead may be rigid. The bead may be flexible and/or compressible. The bead may be disruptable or dissolvable. The bead may be a solid particle (e.g., a metal-based particle including but not limited to iron oxide, gold or silver) covered with a coating comprising one or more polymers. Such coating may be disruptable or dissolvable. 
     The term “sample,” as used herein, generally refers to a biological sample of a subject. The biological sample may comprise any number of macromolecules, for example, cellular macromolecules. The sample may be a cell sample. The sample may be a cell line or cell culture sample. The sample can include one or more cells. The sample can include one or more microbes. The biological sample may be a nucleic acid sample or protein sample. The biological sample may also be a carbohydrate sample or a lipid sample. The biological sample may be derived from another sample. The sample may be a tissue sample, such as a biopsy, core biopsy, needle aspirate, or fine needle aspirate. The sample may be a fluid sample, such as a blood sample, urine sample, or saliva sample. The sample may be a skin sample. The sample may be a cheek swab. The sample may be a plasma or serum sample. The sample may be a cell-free sample. A cell-free sample may include extracellular polynucleotides. Extracellular polynucleotides may be isolated from a bodily sample that may be selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal excretions, sputum, stool and tears. 
     The term “biological particle,” as used herein, generally refers to a discrete biological system derived from a biological sample. The biological particle may be a macromolecule. The biological particle may be a small molecule. The biological particle may be a virus. The biological particle may be a cell or derivative of a cell. The biological particle may be an organelle. The biological particle may be a rare cell from a population of cells. The biological particle may be any type of cell, including without limitation prokaryotic cells, eukaryotic cells, bacterial, fungal, plant, mammalian, or other animal cell type, mycoplasmas, normal tissue cells, tumor cells, or any other cell type, whether derived from single cell or multicellular organisms. The biological particle may be a constituent of a cell. The biological particle may be or may include DNA, RNA, organelles, proteins, or any combination thereof. The biological particle may be or may include a matrix (e.g., a gel or polymer matrix) comprising a cell or one or more constituents from a cell (e.g., cell bead), such as DNA, RNA, organelles, proteins, or any combination thereof, from the cell. The biological particle may be obtained from a tissue of a subject. The biological particle may be a hardened cell. Such hardened cell may or may not include a cell wall or cell membrane. The biological particle may include one or more constituents of a cell, but may not include other constituents of the cell. An example of such constituents is a nucleus or an organelle. A cell may be a live cell. The live cell may be capable of being cultured, for example, being cultured when enclosed in a gel or polymer matrix, or cultured when comprising a gel or polymer matrix. 
     The term “macromolecular constituent,” as used herein, generally refers to a macromolecule contained within or from a biological particle. The macromolecular constituent may comprise a nucleic acid. In some cases, the biological particle may be a macromolecule. The macromolecular constituent may comprise DNA. The macromolecular constituent may comprise RNA. The RNA may be coding or non-coding. The RNA may be messenger RNA (mRNA), ribosomal RNA (rRNA) or transfer RNA (tRNA), for example. The RNA may be a transcript. The RNA may be small RNA that are less than  200  nucleic acid bases in length, or large RNA that are greater than 200 nucleic acid bases in length. Small RNAs may include 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), small nucleolar RNA (snoRNAs), Piwi-interacting RNA (piRNA), tRNA-derived small RNA (tsRNA) and small rDNA-derived RNA (srRNA). The RNA may be double-stranded RNA or single-stranded RNA. The RNA may be circular RNA. The macromolecular constituent may comprise a protein. The macromolecular constituent may comprise a peptide. The macromolecular constituent may comprise a polypeptide. 
     The term “molecular tag,” as used herein, generally refers to a molecule capable of binding to a macromolecular constituent. The molecular tag may bind to the macromolecular constituent with high affinity. The molecular tag may bind to the macromolecular constituent with high specificity or selectivity. The molecular tag may comprise a nucleotide sequence. The molecular tag may comprise a nucleic acid sequence. The nucleic acid sequence may be at least a portion or an entirety of the molecular tag. The molecular tag may be a nucleic acid molecule or may be part of a nucleic acid molecule. The molecular tag may be an oligonucleotide or a polypeptide. The molecular tag may comprise a DNA aptamer. The molecular tag may be or comprise a primer. The molecular tag may be, or comprise, a protein. The molecular tag may comprise a polypeptide. The molecular tag may be a barcode. 
     The term “partition,” as used herein, generally, refers to a space or volume that may be suitable to contain one or more species or conduct one or more reactions. A partition may be a physical compartment, such as a droplet or well. The partition may isolate space or volume from another space or volume. The droplet may be a first phase (e.g., aqueous phase) in a second phase (e.g., oil) immiscible with the first phase. The droplet may be a first phase in a second phase that does not phase separate from the first phase, such as, for example, a capsule or liposome in an aqueous phase. A partition may comprise one or more other (inner) partitions. In some cases, a partition may be a virtual compartment that can be defined and identified by an index (e.g., indexed libraries) across multiple and/or remote physical compartments. For example, a physical compartment may comprise a plurality of virtual compartments. 
     The terms “dissolution,” “disassociation” and “dissociation” may be used interchangeably herein and generally refer to the separation, disconnection, and/or splitting of one or more species from the same or different one or more species. 
     The terms “a,” “an,” and “the,” as used herein, generally refers to singular and plural references unless the context clearly dictates otherwise. 
     Methods for DNA and RNA Barcoding 
     Provided herein are methods for barcoding nucleic acid molecules of a cell. The method can comprise co-partitioning a cell bead and a first bead in a first partition, wherein the cell bead is generated from the cell, wherein the cell comprises a first type of nucleic acid molecule and a second type of nucleic acid molecule, wherein the first type and the second type are different, and wherein the first bead comprises a first set of barcode molecules, each comprising a first nucleic acid barcode sequence. The first partition can then be subject to first conditions sufficient to permit (i) a first target nucleic acid molecule of the first type to couple to a first barcode molecule of the first set of barcode molecules to generate a first barcoded nucleic acid molecule comprising the first nucleic acid barcode sequence, and (ii) a second barcode molecule of the first set of barcode molecules to associate with the cell bead. Then, the cell bead associated with the second barcode molecule or derivative thereof can be co-partitioned with a second bead in a second partition, wherein the second bead comprises a second set of barcode molecules, each comprising a second nucleic acid barcode sequence. The second partition can then be subject to second conditions sufficient to permit (i) a second target nucleic acid molecule of the second type to couple to a third barcode molecule of the second set of barcode molecules to generate a second barcoded nucleic acid molecule comprising the second nucleic acid barcode sequence, and (ii) a fourth barcode molecule of the second set of barcode molecules to couple to the second barcode molecule to generate a composite barcode molecule comprising the first nucleic acid barcode sequence and the second nucleic acid barcode sequence. 
     In some embodiments, a cell selected for generation of a cell bead is a eukaryotic cell (e.g., fungal, yeast, insect, and mammalian cells). Non-limiting examples of mammalian cells include HEK293 cells, HeLa cells, CHO cells, COS cells, Jurkat cells, U20 cells, U251 cells, MCF7 cells, SKBR3 cells, NS0 hybridoma cells, baby hamster kidney (BHK) cells, MDCK cells, NIH-3T3 fibroblast cells, and any other immortalized cell line derived from a mammalian cell or mammalian cells derived from primary cell culture. In some embodiments, a cell selected for generation of a cell bead is a prokaryotic cell (e.g., archaeal cells, bacterial cells). A cell may be alive or dead (e.g., fixed, permeabilized, or lysed) when the cell bead is generated. 
     The nucleic acid molecule may be, for example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof, described elsewhere herein. For example, the first type of nucleic acid molecule may be DNA and the second type of nucleic acid molecule may be RNA, or vice versa. In another example, the first type of nucleic acid molecule may comprise messenger RNA (mRNA) and the second type of nucleic acid molecule may comprise non-mRNA molecules. 
     A cell bead can be any cell bead, such as a bead that contains biological particles (e.g., a cell) or macromolecular constituents (e.g., RNA, DNA, proteins, etc.) of biological particles. See, e.g., U.S. Pat. Pub. 2018/0216162 and U.S. Pat. Pub. 2019/0100632, which are incorporated by reference in their entirety, for exemplary cell bead compositions, generation systems, and methods. A cell bead may include a single cell or multiple cells, or a derivative of the single cell or multiple cells. For example, after lysing and washing the cells, inhibitory components from cell lysates can be washed away and the macromolecular constituents can be bound as cell beads. Systems and methods disclosed herein can be applicable to both cell beads (and/or droplets or other partitions) containing biological particles and cell beads (and/or droplets or other partitions) containing macromolecular constituents of biological particles. 
     In some cases, a cell bead may comprise capture nucleic acid molecules configured to couple to a barcode nucleic acid molecule. See, e.g., U.S. Pat. Pub. 2019/0100632 and U.S. Pat. Application 16/434,095 filed Jun. 6, 2019, which are both incorporated by reference in their entirety, for exemplary cell bead polymers, functionalization strategies, and nucleic acid molecules for generating cell beads comprising, e.g., nucleic acid molecules. In some cases, the capture nucleic acid molecules may comprise barcode nucleic acid molecules (e.g., barcode RNA molecule, barcode DNA molecule, or barcode RNA-DNA molecule) that comprise a sequence that can hybridize with other barcode or non-barcode nucleic acid molecules. The capture nucleic acid molecules or barcode nucleic acid molecules may be bound to the cell bead through, in non-limiting examples, covalent or ionic bonds, or through hydrophobic (e.g., van der Waals) interactions. 
     A bead may be porous, non-porous, solid, semi-solid, semi-fluidic, fluidic, and/or a combination thereof. In some instances, a bead may be dissolvable, disruptable, and/or degradable. In some cases, a bead may not be degradable. In some cases, the bead may be a gel bead. A gel bead may be a hydrogel bead and comprise derivatives or variants thereof, described elsewhere herein. 
     In some cases, the beads (e.g., cell bead, gel bead) may comprise one or more crosslinking types that are dissolvable or dissociable. In some instances, as described elsewhere herein, nucleic acid barcode molecules attached to a bead may comprise one or more labile bonds releasable upon application of a stimulus. For example, a bead may comprise disulfide bonds, where upon exposure to a reducing agent, the bead may dissolve and/or dissociate. In another example, a bead may comprise dihydroxybutane crosslinks. Dissolution of the bead may occur by exposure to oxidation reagents, e.g., sodium periodate. 
     In some cases, a cell bead may be co-partitioned with a first bead (e.g., a gel bead as described elsewhere herein) comprising a first set of barcode nucleic acid molecules. Upon or following partitioning, the first set of barcode nucleic acid molecules from the first bead may be released, e.g., via a first dissolution or releasing mechanism. Dissolution mechanisms may comprise reducing agents (e.g., DTT, TCEP, etc.) and/or oxidizing agents (e.g., sodium periodate) or derivatives or variations thereof, as described elsewhere herein. The first set of barcode nucleic acid molecules may each comprise a first barcode sequence, for example, common to the first bead. 
     A subset of the first set of barcode nucleic acid molecules may enter the cell bead and associate (e.g., hybridize) with the bound capture nucleic acid molecules. The subset of the first set of barcode nucleic acid molecule may henceforth be bound to the cell bead. For example, in some instances, the capture nucleic acid molecules bound to the cell bead (which may be releasable or non-releasable, see, e.g., U.S. Pat. Pub. 2019/0100632, which is incorporated by reference in its entirety) may comprise a sequence complementary to a sequence on the nucleic acid barcode molecules on the first bead. Furthermore, the capture nucleic acid molecules may comprise one or more functional sequences such as a barcode sequence, a unique molecular identifier (UMI), a primer sequence or primer binding sequence (e.g., a sequencing primer sequence (or partial sequencing primer sequence) such as an R1 and/or R2 sequence), a sequence configured to attach to the flow cell of a sequencer (e.g., P5 and/or P7). In some cases, upon or following partitioning, the cell may be lysed to release intracellular contents (e.g., proteins, mRNA) into the cell bead. In some cases, the intracellular contents may exit the cell bead and enter another region of the first partition (e.g., droplet). See, e.g., See, e.g., U.S. Pat. 10,011,872, which is incorporated by reference in its entirety, for exemplary molecules and methods for analyzing protein molecules and other analytes using, e.g., barcoded labelling agents. In some cases, the cell nucleus may remain intact. In some cases, cell lysis may not maintain the nucleus intact. In some cases, a first type of nucleic acid molecule (e.g., DNA) from the cell may be retained in the cell bead while a portion or all of a second type of nucleic acid molecule (e.g., RNA) exits the cell bead and/or is released into the partition (e.g., droplet). In some cases, one or more types of nucleic acid molecules may be retained in the cell bead. In some cases, a portion of one or more types of nucleic acid molecules may be retained in the cell bead. 
     Cellular nucleic acid molecules released into the first partition may be barcoded, e.g., via hybridization with another subset of the first set of barcode nucleic acid molecules released from the first gel bead, to generate a first set of barcoded nucleic acid molecules. The barcoded nucleic acid molecules may then be collected from the partition while maintaining the cell bead intact. 
     The cell bead may be retrieved from the first partition and prepared for a second partitioning. Retrieval of the cell bead may occur through a sorting mechanism. In one non-limiting example, the cell bead can be magnetic and/or comprise magnetic particles. The cell bead may be retrieved by application of a magnetic field. Other non-limiting examples of retrieval mechanisms comprise biochemical mechanisms (e.g., incorporation of antigens and retrieval via antibodies) and/or physical mechanisms (e.g., optical tweezers). 
     In some cases, in a second partition, the cell bead may be co-partitioned with a second gel bead comprising a second set of barcode nucleic acid molecules. The second set of barcode nucleic acid molecules may be a different type of nucleic acid molecule than the first set of barcode nucleic acid molecules. The second set of barcode nucleic acid molecules may each comprise a second barcode sequence, for example, common to the second gel bead. The second barcode sequence may be different from the first barcode sequence. Upon or following partitioning, the second set of barcode nucleic acid molecules from the second gel bead may be released via a second dissolution mechanism. Simultaneously or thereafter, the cell bead may also be dissolved. Biomolecules retained in the cell bead may be released (e.g., intracellular and/or intra-nuclear proteins, DNA) into the second partition. The first set of barcode nucleic acid molecules bound to the cell bead may also be released. The released nucleic acid molecules (e.g., DNA) and the first set of barcode nucleic acid molecules, may be barcoded, e.g., via hybridization, with the second set of barcode nucleic acid molecules. The first set of barcode nucleic acid molecules bound to the cell bead may be barcoded with the second set of barcoded nucleic acid molecules to generate composite barcode molecules. The composite barcode molecules may each comprises a composite barcode sequence including both the first barcode sequence and the second barcode sequence. The released contents of the cell in the second partition (e.g., DNA, proteins) may be barcoded with the second set of barcoded nucleic acid molecules to generate a second set of barcoded nucleic acid molecules. The nucleic acid molecules released from the cell bead in the first partition and the second partition, respectively, may be of different types (e.g., RNA, DNA). 
     The components of the second partition may be collected. The second set of barcoded nucleic acid molecules or derivative thereof and the composite barcode molecules may be collected from the second partition. 
     Subsequent to collection, the barcoded nucleic acid molecules and/or derivatives thereof may be sequenced. For example, the first set of barcoded molecules from the first partition, the second set of barcoded molecule from the second partition, and the composite barcode molecules from the second partition may be sequenced. The sequencing reads generated may be used to associate the nucleic acid molecules with the origin of the nucleic acid molecules (e.g., from the cell or cell bead). For example, the first barcode sequence may be used to associate the first type of nucleic acid molecules to a cell, cell bead, or partition; the second barcode sequence may be used to associate the second type of nucleic acid nucleic acid molecules to the same or different cell, cell bead, or partition, and the composite barcode sequence may be used to associate the first type of nucleic acid molecule and the second type of nucleic acid molecule as arising from the same cell or cell bead. 
     In some embodiments, cell lysis may occur in the cell bead prior to co-partitioning. The intracellular contents (e.g., RNA, proteins) may be captured within and/or coupled to the surface of the cell bead by capture molecules (e.g., capture nucleic acid molecules) bound to the polymer matrix of the cell bead. In one non-limiting example, cellular mRNA may be captured by poly-T nucleic acids that are attached to the polymer matrix. Attachment of the capture molecules to the polymer matrix may occur through a variety of cross-links or binding mechanisms, e.g., acrydite-conjugated nucleic acids, NHS-ester linkages of antibodies, and may include variants or derivatives thereof, described elsewhere herein. 
       FIG.  9    illustrates an example of a method  900  for barcoding nucleic acid molecules. As shown in panel A, a cell bead  902  may comprise a single cell  904 . Capture nucleic acid molecules (not shown), which may optionally comprise a barcode sequence, may be bound to the cell bead  902 . Panel B shows a first partition (e.g., droplet)  906  comprising the cell bead  902  and a gel bead  910  comprising a first set of barcode nucleic acid molecules  914 , each of which comprise a first barcode sequence. The first set of barcode nucleic acid molecules may each comprise a first barcode sequence. The first partition can comprise, or be subjected to, conditions sufficient to permit the first set of barcode nucleic acid molecules (e.g., barcode RNA molecules)  914  to be released from the gel bead  910 . The first partition may also comprise, or be subject to, conditions sufficient to permit release of intracellular contents from the cell bead  902 , such as a first type of nucleic acid molecule (e.g., RNA). For example, such conditions may comprise lysis of the cell  904 . The conditions sufficient to release the first type of nucleic acid molecules from the cell bead  902  may retain the integrity of the cell bead  902 . In some instances, the conditions sufficient to release the first type of nucleic acid molecules from the cell bead  902  may be the same conditions sufficient to release the first set of barcode nucleic acid molecules  914  from the first gel bead  910 . The first type of nucleic acid molecules (e.g., RNA) may also associate with the first set of barcode nucleic acid molecules  914  from the gel bead  910  and form a first set of barcoded nucleic acid molecules  912  (e.g., barcoded RNA). The released first set of barcode nucleic acid molecules  914  may also enter the cell bead  902  and associate with the capture nucleic acid molecules in the cell bead, forming a second set of barcoded nucleic acid molecules  916  that are associated with the cell bead  902 . The cell bead  902  comprising the second set of barcoded nucleic acid molecules  916  may be collected. Also, the first set of barcoded nucleic acid molecules (e.g., barcoded RNA molecules)  912  may be collected and may be sequenced. Panel C shows a second partition (e.g., droplet)  918  comprising the cell bead  902  (e.g., collected from the first partition) and a second gel bead  922  comprising a second set of barcode nucleic acid molecules (e.g., barcode DNA molecules)  924 , each comprising a second barcode sequence. The second set of barcode nucleic acid molecules may each comprise a second barcode sequence, which may be the same or different than the first barcode sequence. The second partition may comprise, or be subject to, conditions sufficient to permit the second set of barcode nucleic acid molecules  924  to be released from the gel bead  922 . The second partition may also comprise, or be subject to, conditions sufficient to release a second type of nucleic acid molecule (e.g., DNA)  920  from the cell bead  902 . In some instances, the conditions sufficient to release the second type of nucleic acid molecule  920  from the cell bead  902  may comprise disassociation of the cell bead  902 . In some instances, the conditions sufficient to release the second type of nucleic acid molecule  920  from the cell bead  902  may be the same conditions sufficient to release the second set of barcode nucleic acid molecules  924  from the second gel bead  922 . The released second set of barcode nucleic acid molecules  924  may associate with the released second type of nucleic acid molecule (e.g., DNA)  920  from the cell bead  902 , forming a third set of barcoded nucleic acid molecule (e.g., barcoded DNA not shown). The released second set of barcode nucleic acid molecules  924  may also associate with the released, second set of barcoded nucleic acid molecule  916 , forming a set of composite barcoded molecules (not shown). The method  900  may also comprise collecting the first set of barcoded nucleic acid molecules (e.g., barcoded RNA), comprising the first barcode sequence, the third set of barcoded nucleic acid molecules (e.g., barcoded DNA), comprising the second barcode sequence, and the set of composite barcoded molecules, comprising both the first barcode sequence and the second barcode sequence. Upon sequencing, the first barcode sequence may be identified and used to associate the first type of nucleic acid molecules (e.g., RNA) to a cell, cell bead, or partition; the second barcode sequence may be identified and used to associate the second type of nucleic acid nucleic acid molecules (e.g., DNA) to the same or different cell, cell bead, or partition, and the composite barcode sequence may be identified and used to associate the first type of nucleic acid molecule and the second type of nucleic acid molecule as arising from the same cell or cell bead. 
       FIG.  10    illustrates another example of a method  1000  for barcoding nucleic acid molecules. As shown in Panel A, a cell bead  1002  may comprise a single cell  1004 . Capture nucleic acid molecules (which optionally may comprise a barcode sequence, not shown) may be bound to the cell bead  1002 . In some cases, the cell is lysed prior to co-partitioning, but the nucleus may remain intact. The intracellular contents, such as nucleic acids including the first type of nucleic acid molecule (e.g., RNA), may associate with the cell bead matrix (e.g., via the capture nucleic acid molecules, which may comprise poly-T sequences that are incorporated in the cell bead matrix, e.g., via acrydite-modification), forming associated nucleic acid molecules  1006 . Panel B shows a first partition (droplet)  1008  comprising the cell bead  1002  and a gel bead  1010  comprising a first set of barcode nucleic acid molecules (e.g., barcode RNA molecules)  1016 , each comprising a first barcode sequence. The first partition comprises conditions sufficient to permit the first set of barcode nucleic acid molecules (e.g., barcode RNA molecules)  1016  to be released from the gel bead  1012 . The first partition may also comprise conditions sufficient to permit lysis of the cell  1004  and release of intracellular contents from the cell bead  1010 , such as the first type of nucleic acid molecule (e.g., RNA, mRNA hybridized with the capture poly-T nucleic acid sequence, etc.). The first type of nucleic acid molecules (e.g., RNA) may also associate with the barcode nucleic acid molecules from the gel bead  1012  and form a first set of barcoded nucleic acid molecules  1014  (e.g., barcoded RNA molecules). The released first set of barcode nucleic acid molecules  1016  may also enter the cell bead  1002  and associate with a second type of capture molecule, which may be configured to couple to nucleic acid molecules, thereby forming a second set of barcoded nucleic acid molecules  1018 . The cell bead  1010  comprising the second set of barcoded nucleic acid molecules associated with the cell bead  1014  may be collected. Also, the first set of barcoded nucleic acid molecules (e.g., barcoded RNA molecules)  1014  may be collected and may be sequenced. Panel C shows a second partition (droplet)  1020  comprising a cell bead (e.g.,  1010  collected from the first partition) and a second gel bead  1024  comprising a second set of barcode nucleic acid molecules (e.g., barcode DNA molecules)  1026 , each of which comprise a second barcode sequence. The second partition comprises conditions sufficient to permit the second set of barcode nucleic acid molecules  1026  to be released from the gel bead  1024 . The second partition may also comprise conditions sufficient to release a second type of nucleic acid molecule (e.g., DNA)  1022  from the cell bead. The released second set of barcode nucleic acid molecules  1026  may associate with the released second type of nucleic acid molecule (e.g., DNA)  1022  from the cell bead, forming a third set of barcoded nucleic acid molecules. The released second set of barcode nucleic acid molecules  1026  may also associate with the released, second set of barcoded nucleic acid molecules  1018 , forming a composite barcoded molecule (e.g., barcoded RNA-DNA) comprising the first barcode sequence and the second barcode sequence. 
       FIG.  11    illustrates another example of a method  1100  for barcoding nucleic acid molecules. As shown in panel A, a cell bead  1102  may comprise a single cell  1104 . Capture nucleic acid molecules (not shown) may be bound to the cell bead  1104 . In some cases, the cell bead  1102  comprises an interpenetrating network (IPN) of two or more polymer meshes. Each polymer mesh may be dissociable via different mechanisms. In one example, one polymer network of the IPN may comprise disulfide crosslinks or bonds that may be dissociated when reducing agents (e.g., DTT) are introduced. A second polymer network of the IPN may comprise dihydroxybutane crosslinks or bonds that are dissociable when oxidizing agents (e.g., sodium periodate) are introduced. The pore size of the IPN may be sufficiently small to prevent passage of intracellular contents (e.g., proteins, RNA). In some cases, the cell  1104  is lysed in the cell bead. Panel B shows a first partition (droplet)  1108  comprising the cell bead  1102  and a gel bead  1112  comprising a first set of barcode nucleic acid molecules (e.g., barcode RNA)  1116 , each of which comprises a first barcode sequence. The first partition (droplet) may comprise conditions sufficient to permit the first barcode nucleic acid molecules (e.g., barcode RNA)  1116  to be released from the gel bead  1112  and also for dissociation of one or more of the polymer meshes in the IPN. The dissociation of one or more of the polymer meshes in the IPN may increase the pore size of the cell bead  1102  such that some components of the intracellular contents (e.g., a first type of nucleic acid molecule, e.g., RNA) can exit the cell bead into the first partition. 
     The first partition may also comprise conditions sufficient to permit lysis of the cell  1104  and release of intracellular contents from the cell bead  1110 , such as the first type of nucleic acid molecule (e.g., RNA). The first type of nucleic acid molecules (e.g., RNA) may associate with the first set of barcode nucleic acid molecules  1116  from the gel bead  1112  and form a first set of barcoded nucleic acid molecules  1114 . The released first set of barcode nucleic acid molecules  1116  may also enter the cell bead  1110  and associate with capture nucleic acid molecules in the cell bead, forming a second set of barcoded nucleic acid molecules  1118 . The cell bead  1110  comprising the second set of barcoded nucleic acid molecules  1118  may be collected. Also, the first set of barcoded nucleic acid molecules (e.g., barcoded RNA molecules)  1114  may be collected and may be sequenced. Panel C shows a second partition (droplet)  1120  comprising a cell bead (e.g.,  1102  collected from the first partition) and a second gel bead  1124  comprising a second set of barcode nucleic acid molecules (e.g., barcode DNA molecules)  1126 , each of which comprises a second barcode sequence. The second partition comprises conditions sufficient to permit the second set of barcode nucleic acid molecules  1126  to be released from the gel bead  1124 . The second partition may also comprise conditions to dissociate the second polymer network to release a second type of nucleic acid molecule (e.g., DNA)  1122  from the cell bead. The released second set of barcode nucleic acid molecules  1126  may associate with the released second type of nucleic acid molecule (e.g., DNA)  1122  from the cell bead, forming a third set of barcoded nucleic acid molecule. The released second set of barcode nucleic acid molecules  1126  may also associate with the released, first set of barcoded nucleic acid molecules  1118 , forming a composite barcoded molecule comprising the first barcode sequence and the second barcode sequence. 
     The barcoded nucleic acid molecules may be subjected to further processing and analysis, e.g., sequencing. Sequencing may be used to generate sequencing reads, which can be used to associate barcode sequences with a partition or cell bead. For example, the first barcode nucleic acid molecule may comprise a first barcode sequence. As described herein, the first barcode nucleic acid molecule may associate with (i) the first type of nucleic acid molecule (e.g., RNA) to form a first barcoded nucleic acid molecule comprising the first barcode sequence and (ii) the cell bead (e.g., via a capture sequence) to form a second barcoded nucleic acid molecule, also comprising the first barcode sequence. The first barcoded nucleic acid molecule may be subjected to sequencing. The cell bead, comprising the second barcoded nucleic acid molecule, may be partitioned with a second barcode nucleic acid molecule comprising a second barcode sequence, which may be a different sequence than the first barcode sequence. The second barcode nucleic acid molecule may associate with (i) the second type of nucleic acid molecule (e.g., DNA) to form a third barcoded nucleic acid molecule and (ii) the second barcoded nucleic acid molecule from the cell bead to generate the composite barcode molecule, comprising the first barcode sequence and the second barcode sequence. From sequencing all the barcoded products, the origin of the products may be determined. For example, the first barcode sequence may be used to determine that the first type of nucleic acid molecule (e.g., RNA) arose from a specified cell bead, which cell bead comprises the composite barcode comprising the first (and the second) barcode sequence. The second barcode sequence may be used to determine that the second type of nucleic acid molecule (e.g., DNA) arose from the same specified cell bead, as the cell bead comprises the composite barcode comprising the second (and the first) barcode sequence. Thus, both types of nucleic acid molecules may be associated with a single cell. 
     In some embodiments, a cell bead can comprise one or more polymer networks. These polymer networks may form one or more interpenetrating networks (IPNs). An IPN may comprise two or more polymer networks and/or meshes which are at least partially interlaced but not covalently bonded to one another. In some embodiments, the IPNs are fully-interpenetrating, semi-interpenetrating, or pseudo-interpenetrating. An IPN may comprise one or more polymers, including, in non-limiting examples, polyacrylamide, alginate, poly-lactic acid (PLA), poly-ethylene glycol(PEG), PEG-diol, PEG-diacrylate (PEGDA), and other synthetic and/or natural polymers, as described elsewhere herein. In some cases, one or both polymer networks may be crosslinked using crosslinks that may be dissolved and/or dissociated. In non-limiting examples, one of the polymer networks may be cross-linked with disulfide bonds while a second polymer network may be crosslinked with dihydroxybutane bonds. In some cases, reduction (e.g., with DTT) may disrupt and/or dissolve the disulfide bonds, while a separate oxidation step may be used to disrupt and/or dissolve the dihydroxybutane bonds. See, e.g., U.S. Pat. Pub. 2018/0216162 and U.S. Pat. Pub. 2019/0100632, which are incorporated by reference in their entirety, for exemplary cell bead polymers, polymer functionalization, and strategies for generating cell beads. 
     The IPN may have various tunable properties, e.g., physical, mechanical, chemical, biological, and biochemical. Non-limiting examples of said properties include: pore size, stiffness, ligand density, charge, hydrophobicity, etc. In some cases, one or more properties of IPNs may be tuned by association or dissociation of one or more of the polymer networks. 
     In some cases, the IPN can comprise two layers of polymer networks that may be configured to dissociate by two different dissolution mechanisms. The dissolution mechanisms may be configured such that one dissolution mechanism may dissolve and/or dissociate one of the two polymer networks but not the other. For example, a second dissolution mechanism may be configured to dissolve the second polymer network, but not the first, and vice-versa. The separate dissolution mechanisms may be used to selectively release different types of nucleic acid molecules. 
     In some cases, the IPN may be used to selectively entrap biomolecules (e.g., proteins, lipids, nucleic acids, polysaccharides). In one non-limiting example, the pore size of the IPN may be tuned to inhibit the entrance and exit of biomolecules, such as RNA and DNA. In some cases, the dissociation of one or more of the polymer meshes may release the one or more types of entrapped biomolecules. The biomolecules may be different. 
     In some cases, two or more polymer meshes can be incorporated into the IPN, and the IPN may be tuned for selective release of two biomolecule types. In one non-limiting example, an IPN may comprise two polymer meshes that are dissociable via two different mechanisms. A cell bead may comprise a cell encapsulated in said IPN. During cell lysis, the IPN may prevent biomolecules of interest (e.g., RNA, proteins) from exiting the cell bead. During co-partitioning of the cell bead with a gel bead, a first dissociation mechanism may be introduced that dissociates one of the polymer meshes (e.g., reduction of disulfide bonds or oxidation of dihydroxybutane bonds). This dissociation mechanism may increase the pore size of the IPN, thereby allowing biomolecules of interest (e.g., RNA) to exit out of the cell bead while retaining larger biomolecules of interest (e.g., DNA). In some cases, the dissolution may lead to release of biomolecules of interest without increasing the bead pore size. A second dissociation mechanism may be introduced upon a second co-partitioning that may allow a second type of nucleic acid (e.g., DNA) to be released. In some cases the second dissociation mechanism dissociates the cell bead, thus allowing for larger biomolecules (e.g., DNA) to exit the cell bead into the partition. 
     Systems and Methods for Sample Compartmentalization 
     In an aspect, the systems and methods described herein provide for the compartmentalization, depositing, or partitioning of one or more particles (e.g., biological particles, macromolecular constituents of biological particles, beads, reagents, etc.) into discrete compartments or partitions (referred to interchangeably herein as partitions), where each partition maintains separation of its own contents from the contents of other partitions. The partition can be a droplet in an emulsion. A partition may comprise one or more other partitions. 
     A partition may include one or more particles. A partition may include one or more types of particles. For example, a partition of the present disclosure may comprise one or more biological particles and/or macromolecular constituents thereof. A partition may comprise one or more gel beads. A partition may comprise one or more cell beads. A partition may include a single gel bead, a single cell bead, or both a single cell bead and single gel bead. A partition may include one or more reagents. Alternatively, a partition may be unoccupied. For example, a partition may not comprise a bead. A cell bead can be a biological particle and/or one or more of its macromolecular constituents encased inside of a gel or polymer matrix, such as via polymerization of a droplet containing the biological particle and precursors capable of being polymerized or gelled. Unique identifiers, such as barcodes, may be injected into the droplets previous to, subsequent to, or concurrently with droplet generation, such as via a microcapsule (e.g., bead), as described elsewhere herein. Microfluidic channel networks (e.g., on a chip) can be utilized to generate partitions as described herein. Alternative mechanisms may also be employed in the partitioning of individual biological particles, including porous membranes through which aqueous mixtures of cells are extruded into non-aqueous fluids. 
     The partitions can be flowed within fluid streams. The partitions may comprise, for example, micro-vesicles that have an outer barrier surrounding an inner fluid center or core. In some cases, the partitions may comprise a porous matrix that is capable of entraining and/or retaining materials within its matrix. The partitions can be droplets of a first phase within a second phase, wherein the first and second phases are immiscible. For example, the partitions can be droplets of aqueous fluid within a non-aqueous continuous phase (e.g., oil phase). In another example, the partitions can be droplets of a non-aqueous fluid within an aqueous phase. In some examples, the partitions may be provided in a water-in-oil emulsion or oil-in-water emulsion. A variety of different vessels are described in, for example, U.S. Pat. Application Publication No. 2014/0155295, which is entirely incorporated herein by reference for all purposes. Emulsion systems for creating stable droplets in non-aqueous or oil continuous phases are described in, for example, U.S. Pat. Application Publication No. 2010/0105112, which is entirely incorporated herein by reference for all purposes. 
     In the case of droplets in an emulsion, allocating individual particles to discrete partitions may in one non-limiting example be accomplished by introducing a flowing stream of particles in an aqueous fluid into a flowing stream of a non-aqueous fluid, such that droplets are generated at the junction of the two streams. Fluid properties (e.g., fluid flow rates, fluid viscosities, etc.), particle properties (e.g., volume fraction, particle size, particle concentration, etc.), microfluidic architectures (e.g., channel geometry, etc.), and other parameters may be adjusted to control the occupancy of the resulting partitions (e.g., number of biological particles per partition, number of beads per partition, etc.). For example, partition occupancy can be controlled by providing the aqueous stream at a certain concentration and/or flow rate of particles. To generate single biological particle partitions, the relative flow rates of the immiscible fluids can be selected such that, on average, the partitions may contain less than one biological particle per partition in order to ensure that those partitions that are occupied are primarily singly occupied. In some cases, partitions among a plurality of partitions may contain at most one biological particle (e.g., bead, DNA, cell or cellular material). In some embodiments, the various parameters (e.g., fluid properties, particle properties, microfluidic architectures, etc.) may be selected or adjusted such that a majority of partitions are occupied, for example, allowing for only a small percentage of unoccupied partitions. The flows and channel architectures can be controlled as to ensure a given number of singly occupied partitions, less than a certain level of unoccupied partitions and/or less than a certain level of multiply occupied partitions. 
       FIG.  1    shows an example of a microfluidic channel structure  100  for partitioning individual biological particles. The channel structure  100  can include channel segments  102 ,  104 ,  106  and  108  communicating at a channel junction  110 . In operation, a first aqueous fluid  112  that includes suspended biological particles (or cells)  114  may be transported along channel segment  102  into junction  110 , while a second fluid  116  that is immiscible with the aqueous fluid  112  is delivered to the junction  110  from each of channel segments  104  and  106  to create discrete droplets  118 ,  120  of the first aqueous fluid  112  flowing into channel segment  108 , and flowing away from junction  110 . The channel segment  108  may be fluidically coupled to an outlet reservoir where the discrete droplets can be stored and/or harvested. A discrete droplet generated may include an individual biological particle  114  (such as droplets  118 ). A discrete droplet generated may include more than one individual biological particle  114  (not shown in  FIG.  1   ). A discrete droplet may contain no biological particle  114  (such as droplet  120 ). Each discrete partition may maintain separation of its own contents (e.g., individual biological particle  114 ) from the contents of other partitions. 
     The second fluid  116  can comprise an oil, such as a fluorinated oil, that includes a fluorosurfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets  118 ,  120 . Examples of particularly useful partitioning fluids and fluorosurfactants are described, for example, in U.S. Pat. Application Publication No. 2010/0105112, which is entirely incorporated herein by reference for all purposes. 
     As will be appreciated, the channel segments described herein may be coupled to any of a variety of different fluid sources or receiving components, including reservoirs, tubing, manifolds, or fluidic components of other systems. As will be appreciated, the microfluidic channel structure  100  may have other geometries. For example, a microfluidic channel structure can have more than one channel junction. For example, a microfluidic channel structure can have 2, 3, 4, or 5 channel segments each carrying particles (e.g., biological particles, cell beads, and/or gel beads) that meet at a channel junction. Fluid may be directed to flow along one or more channels or reservoirs via one or more fluid flow units. A fluid flow unit can comprise compressors (e.g., providing positive pressure), pumps (e.g., providing negative pressure), actuators, and the like to control flow of the fluid. Fluid may also or otherwise be controlled via applied pressure differentials, centrifugal force, electrokinetic pumping, vacuum, capillary or gravity flow, or the like. 
     The generated droplets may comprise two subsets of droplets: (1) occupied droplets  118 , containing one or more biological particles  114 , and (2) unoccupied droplets  120 , not containing any biological particles  114 . Occupied droplets  118  may comprise singly occupied droplets (having one biological particle) and multiply occupied droplets (having more than one biological particle). As described elsewhere herein, in some cases, the majority of occupied partitions can include no more than one biological particle per occupied partition and some of the generated partitions can be unoccupied (of any biological particle). In some cases, though, some of the occupied partitions may include more than one biological particle. In some cases, the partitioning process may be controlled such that fewer than about 25% of the occupied partitions contain more than one biological particle, and in many cases, fewer than about 20% of the occupied partitions have more than one biological particle, while in some cases, fewer than about 10% or even fewer than about 5% of the occupied partitions include more than one biological particle per partition. 
     In some cases, it may be desirable to minimize the creation of excessive numbers of empty partitions, such as to reduce costs and/or increase efficiency. While this minimization may be achieved by providing a sufficient number of biological particles (e.g., biological particles  114 ) at the partitioning junction  110 , such as to ensure that at least one biological particle is encapsulated in a partition, the Poissonian distribution may expectedly increase the number of partitions that include multiple biological particles. As such, where singly occupied partitions are to be obtained, at most about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less of the generated partitions can be unoccupied. 
     In some cases, the flow of one or more of the biological particles (e.g., in channel segment  102 ), or other fluids directed into the partitioning junction (e.g., in channel segments  104 ,  106 ) can be controlled such that, in many cases, no more than about 50% of the generated partitions, no more than about 25% of the generated partitions, or no more than about 10% of the generated partitions are unoccupied. These flows can be controlled so as to present a non-Poissonian distribution of single-occupied partitions while providing lower levels of unoccupied partitions. The above noted ranges of unoccupied partitions can be achieved while still providing any of the single occupancy rates described above. For example, in many cases, the use of the systems and methods described herein can create resulting partitions that have multiple occupancy rates of less than about 25%, less than about 20%, less than about 15%, less than about 10%, and in many cases, less than about 5%, while having unoccupied partitions of less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less. 
     As will be appreciated, the above-described occupancy rates are also applicable to partitions that include both biological particles and additional reagents, including, but not limited to, microcapsules or beads (e.g., gel beads) carrying barcoded nucleic acid molecules (e.g., oligonucleotides) (described in relation to  FIG.  2   ). The occupied partitions (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the occupied partitions) can include both a microcapsule (e.g., bead) comprising barcoded nucleic acid molecules and a biological particle. 
     In another aspect, in addition to or as an alternative to droplet based partitioning, biological particles may be encapsulated within a microcapsule that comprises an outer shell, layer or porous matrix in which is entrained one or more individual biological particles or small groups of biological particles. The microcapsule may include other reagents. Encapsulation of biological particles may be performed by a variety of processes. Such processes may combine an aqueous fluid containing the biological particles with a polymeric precursor material that may be capable of being formed into a gel or other solid or semi-solid matrix upon application of a particular stimulus to the polymer precursor. Such stimuli can include, for example, thermal stimuli (e.g., either heating or cooling), photo-stimuli (e.g., through photo-curing), chemical stimuli (e.g., through crosslinking, polymerization initiation of the precursor (e.g., through added initiators)), mechanical stimuli, or a combination thereof. 
     Preparation of microcapsules comprising biological particles may be performed by a variety of methods. For example, air knife droplet or aerosol generators may be used to dispense droplets of precursor fluids into gelling solutions in order to form microcapsules that include individual biological particles or small groups of biological particles. Likewise, membrane based encapsulation systems may be used to generate microcapsules comprising encapsulated biological particles as described herein. Microfluidic systems of the present disclosure, such as that shown in  FIG.  1   , may be readily used in encapsulating cells as described herein. In particular, and with reference to  FIG.  1   , the aqueous fluid  112  comprising (i) the biological particles  114  and (ii) the polymer precursor material (not shown) is flowed into channel junction  110 , where it is partitioned into droplets  118 ,  120  through the flow of non-aqueous fluid  116 . In the case of encapsulation methods, non-aqueous fluid  116  may also include an initiator (not shown) to cause polymerization and/or crosslinking of the polymer precursor to form the microcapsule that includes the entrained biological particles. Examples of polymer precursor/initiator pairs include those described in U.S. Pat. Application Publication No. 2014/0378345, which is entirely incorporated herein by reference for all purposes. 
     For example, in the case where the polymer precursor material comprises a linear polymer material, such as a linear polyacrylamide, PEG, or other linear polymeric material, the activation agent may comprise a cross-linking agent, or a chemical that activates a cross-linking agent within the formed droplets. Likewise, for polymer precursors that comprise polymerizable monomers, the activation agent may comprise a polymerization initiator. For example, in certain cases, where the polymer precursor comprises a mixture of acrylamide monomer with a N,N′-bis-(acryloyl)cystamine (BAC) comonomer, agents such as ammonium persulfate (APS) and tetraethylmethylenediamine (TEMED) and may be provided within the second fluid streams  116  in channel segments  104  and  106 , which can initiate and catalyze the copolymerization of the acrylamide and BAC into a cross-linked polymer network, or hydrogel. Other non-limiting examples of initiators include azide-based reagents (e.g., VA-086) and lithium phenyl-trimethylbenzoylphosphinate. 
     Upon contact of the second fluid stream  116  with the first fluid stream  112  at junction  110 , during formation of droplets, the TEMED may diffuse from the second fluid  116  into the aqueous fluid  112  comprising the linear polyacrylamide, which will activate the crosslinking of the polyacrylamide within the droplets  118 ,  120 , resulting in the formation of gel (e.g., hydrogel) microcapsules, as solid or semi-solid beads or particles entraining the cells  114 . Although described in terms of polyacrylamide encapsulation, other ‘activatable’ encapsulation compositions may also be employed in the context of the methods and compositions described herein. For example, formation of alginate droplets followed by exposure to divalent metal ions (e.g., Ca 2+  ions), can be used as an encapsulation process using the described processes. Likewise, agarose droplets may also be transformed into capsules through temperature based gelling (e.g., upon cooling, etc.). In another example, addition of a complementary nucleic acid (e.g., DNA) may be used to crosslink or un-crosslink nucleic acid molecules that are conjugated to a polymer network. 
     In some cases, encapsulated biological particles can be selectively releasable from the microcapsule, such as through passage of time or upon application of a particular stimulus, that degrades the microcapsule sufficiently to allow the biological particles (e.g., cell), or its other contents to be released from the microcapsule, such as into a partition (e.g., droplet). For example, in the case of the polyacrylamide polymer described above, degradation of the microcapsule may be accomplished through the introduction of an appropriate reducing agent, such as DTT or the like, to cleave disulfide bonds that cross-link the polymer matrix. See, for example, U.S. Pat. Application Publication No. 2014/0378345, which is entirely incorporated herein by reference for all purposes. 
     The biological particle can be subjected to other conditions sufficient to polymerize or gel the precursors. The conditions sufficient to polymerize or gel the precursors may comprise exposure to heating, cooling, electromagnetic radiation, and/or light. The conditions sufficient to polymerize or gel the precursors may comprise any conditions sufficient to polymerize or gel the precursors. Following polymerization or gelling, a polymer or gel may be formed around the biological particle. The polymer or gel may be diffusively permeable to chemical or biochemical reagents. The polymer or gel may be diffusively impermeable to macromolecular constituents of the biological particle. In this manner, the polymer or gel may act to allow the biological particle to be subjected to chemical or biochemical operations while spatially confining the macromolecular constituents to a region of the droplet defined by the polymer or gel. The polymer or gel may include one or more of disulfide cross-linked polyacrylamide, agarose, alginate, polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate, PEG-thiol, PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronic acid, collagen, fibrin, gelatin, or elastin. The polymer or gel may comprise any other polymer or gel. 
     The polymer or gel may be functionalized to bind to targeted analytes, such as nucleic acids, proteins, carbohydrates, lipids or other analytes. The polymer or gel may be polymerized or gelled via a passive mechanism. The polymer or gel may be stable in alkaline or acidic conditions or at elevated temperature. The polymer or gel may have mechanical properties similar to the mechanical properties of the bead. For instance, the polymer or gel may be of a similar size to the bead. The polymer or gel may have a mechanical strength (e.g., tensile strength, compressive strength, stiffness, toughness, etc.) similar to that of the bead. The polymer or gel may be of a lower density than an oil. The polymer or gel may be of a density that is roughly similar to that of a buffer. The polymer or gel may have a tunable pore size. The pore size may be chosen to, for instance, retain denatured nucleic acids. The pore size may be chosen to maintain diffusive permeability to exogenous chemicals such as sodium hydroxide (NaOH) and/or endogenous chemicals such as inhibitors. The polymer or gel may be biocompatible. The polymer or gel may maintain or enhance cell viability. The polymer or gel may be biochemically compatible. The polymer or gel may be polymerized and/or depolymerized thermally, chemically, enzymatically, and/or optically. 
     The polymer may comprise poly(acrylamide-co-acrylic acid) crosslinked with disulfide linkages. The preparation of the polymer may comprise a two-step reaction. In the first activation step, poly(acrylamide-co-acrylic acid) may be exposed to an acylating agent to convert carboxylic acids to esters. For instance, the poly(acrylamide-co-acrylic acid) may be exposed to 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM). The polyacrylamide-co-acrylic acid may be exposed to other salts of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium. In the second cross-linking step, the ester formed in the first step may be exposed to a disulfide crosslinking agent. For instance, the ester may be exposed to cystamine (2,2′-dithiobis(ethylamine)). Following the two steps, the biological particle may be surrounded by polyacrylamide strands linked together by disulfide bridges. In this manner, the biological particle may be encased inside of or comprise a gel or matrix (e.g., polymer matrix) to form a “cell bead.” A cell bead can contain biological particles (e.g., a cell) or macromolecular constituents (e.g., RNA, DNA, proteins, etc.) of biological particles, as described elsewhere herein. 
     Encapsulated biological particles can provide certain potential advantages of being more storable and more portable than droplet-based partitioned biological particles. Furthermore, in some cases, it may be desirable to allow biological particles to incubate for a select period of time before analysis, such as in order to characterize changes in such biological particles over time, either in the presence or absence of different stimuli. In such cases, encapsulation may allow for longer incubation than partitioning in emulsion droplets, although in some cases, droplet partitioned biological particles may also be incubated for different periods of time, e.g., at least 10 seconds, at least 30 seconds, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours, or at least 10 hours or more. The encapsulation of biological particles may constitute the partitioning of the biological particles into which other reagents are co-partitioned. Alternatively or in addition, encapsulated biological particles may be readily deposited into other partitions (e.g., droplets) as described above. 
     Beads 
     A partition may comprise one or more unique identifiers, such as barcodes. Barcodes may be previously, subsequently or concurrently delivered to the partitions that hold the compartmentalized or partitioned biological particle. For example, barcodes may be injected into droplets previous to, subsequent to, or concurrently with droplet generation. The delivery of the barcodes to a particular partition allows for the later attribution of the characteristics of the individual biological particle to the particular partition. Barcodes may be delivered, for example on a nucleic acid molecule (e.g., an oligonucleotide), to a partition via any suitable mechanism. Barcoded nucleic acid molecules can be delivered to a partition via a microcapsule. A microcapsule, in some instances, can comprise a bead. Beads are described in further detail below. 
     In some cases, barcoded nucleic acid molecules can be initially associated with the microcapsule and then released from the microcapsule. Release of the barcoded nucleic acid molecules can be passive (e.g., by diffusion out of the microcapsule). In addition or alternatively, release from the microcapsule can be upon application of a stimulus which allows the barcoded nucleic acid nucleic acid molecules to dissociate or to be released from the microcapsule. Such stimulus may disrupt the microcapsule, an interaction that couples the barcoded nucleic acid molecules to or within the microcapsule, or both. Such stimulus can include, for example, a thermal stimulus, photo-stimulus, chemical stimulus (e.g., change in pH or use of a reducing agent(s)), a mechanical stimulus, a radiation stimulus; a biological stimulus (e.g., enzyme), or any combination thereof. 
       FIG.  2    shows an example of a microfluidic channel structure  200  for delivering barcode carrying beads to droplets. The channel structure  200  can include channel segments  201 ,  202 ,  204 ,  206  and  208  communicating at a channel junction  210 . In operation, the channel segment  201  may transport an aqueous fluid  212  that includes a plurality of beads  214  (e.g., with nucleic acid molecules, oligonucleotides, molecular tags) along the channel segment  201  into junction  210 . The plurality of beads  214  may be sourced from a suspension of beads. For example, the channel segment  201  may be connected to a reservoir comprising an aqueous suspension of beads  214 . The channel segment  202  may transport the aqueous fluid  212  that includes a plurality of biological particles  216  along the channel segment  202  into junction  210 . The plurality of biological particles  216  may be sourced from a suspension of biological particles. For example, the channel segment  202  may be connected to a reservoir comprising an aqueous suspension of biological particles  216 . In some instances, the aqueous fluid  212  in either the first channel segment  201  or the second channel segment  202 , or in both segments, can include one or more reagents, as further described below. A second fluid  218  that is immiscible with the aqueous fluid  212  (e.g., oil) can be delivered to the junction  210  from each of channel segments  204  and  206 . Upon meeting of the aqueous fluid  212  from each of channel segments  201  and  202  and the second fluid  218  from each of channel segments  204  and  206  at the channel junction  210 , the aqueous fluid  212  can be partitioned as discrete droplets  220  in the second fluid  218  and flow away from the junction  210  along channel segment  208 . The channel segment  208  may deliver the discrete droplets to an outlet reservoir fluidly coupled to the channel segment  208 , where they may be harvested. 
     As an alternative, the channel segments  201  and  202  may meet at another junction upstream of the junction  210 . At such junction, beads and biological particles may form a mixture that is directed along another channel to the junction  210  to yield droplets  220 . The mixture may provide the beads and biological particles in an alternating fashion, such that, for example, a droplet comprises a single bead and a single biological particle. 
     Beads, biological particles and droplets may flow along channels at substantially regular flow profiles (e.g., at regular flow rates). Such regular flow profiles may permit a droplet to include a single bead and a single biological particle. Such regular flow profiles may permit the droplets to have an occupancy (e.g., droplets having beads and biological particles) greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. Such regular flow profiles and devices that may be used to provide such regular flow profiles are provided in, for example, U.S. Pat. Publication No. 2015/0292988, which is entirely incorporated herein by reference. 
     The second fluid  218  can comprise an oil, such as a fluorinated oil, that includes a fluorosurfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets  220 . Other surfactants such as Span80, Triton X-100, SDS, perfluorooctanol (PFO), perfluoropolyethers, etc. may also be employed to prevent coalescence of droplets. 
     A discrete droplet that is generated may include an individual biological particle  216 . A discrete droplet that is generated may include a barcode or other reagent carrying bead  214 . A discrete droplet generated may include both an individual biological particle and a barcode carrying bead, such as droplets  220 . In some instances, a discrete droplet may include more than one individual biological particle or no biological particle. In some instances, a discrete droplet may include more than one bead or no bead. A discrete droplet may be unoccupied (e.g., no beads, no biological particles). 
     Beneficially, a discrete droplet partitioning a biological particle and a barcode carrying bead may effectively allow the attribution of the barcode to macromolecular constituents of the biological particle within the partition. The contents of a partition may remain discrete from the contents of other partitions. 
     As will be appreciated, the channel segments described herein may be coupled to any of a variety of different fluid sources or receiving components, including reservoirs, tubing, manifolds, or fluidic components of other systems. As will be appreciated, the microfluidic channel structure  200  may have other geometries. For example, a microfluidic channel structure can have more than one channel junctions. For example, a microfluidic channel structure can have 2, 3, 4, or 5 channel segments each carrying beads that meet at a channel junction. Fluid may be directed flow along one or more channels or reservoirs via one or more fluid flow units. A fluid flow unit can comprise compressors (e.g., providing positive pressure), pumps (e.g., providing negative pressure), actuators, and the like to control flow of the fluid. Fluid may also or otherwise be controlled via applied pressure differentials, centrifugal force, electrokinetic pumping, vacuum, capillary or gravity flow, or the like. 
     A bead may be porous, non-porous, solid, semi-solid, semi-fluidic, fluidic, and/or a combination thereof. In some instances, a bead may be dissolvable, disruptable, and/or degradable. In some cases, a bead may not be degradable. In some cases, the bead may be a gel bead. A gel bead may be a hydrogel bead. A gel bead may be formed from molecular precursors, such as a polymeric or monomeric species. A semi-solid bead may be a liposomal bead. Solid beads may comprise metals including iron oxide, gold, and silver. In some cases, the bead may be a silica bead. In some cases, the bead can be rigid. In other cases, the bead may be flexible and/or compressible. 
     A bead may be of any suitable shape. Examples of bead shapes include, but are not limited to, spherical, non-spherical, oval, oblong, amorphous, circular, cylindrical, and variations thereof. 
     Beads may be of uniform size or heterogeneous size. In some cases, the diameter of a bead 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 greater. In some cases, a bead may have a diameter of 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, a bead may have a diameter 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. 
     In certain aspects, beads can be provided as a population or plurality of beads having a relatively monodisperse size distribution. Where it may be desirable to provide relatively consistent amounts of reagents within partitions, maintaining relatively consistent bead characteristics, such as size, can contribute to the overall consistency. In particular, the beads described herein may have size distributions that have a coefficient of variation in their cross-sectional dimensions of less than 50%, less than 40%, less than 30%, less than 20%, and in some cases less than 15%, less than 10%, less than 5%, or less. 
     A bead may comprise natural and/or synthetic materials. For example, a bead can comprise a natural polymer, a synthetic polymer or both natural 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, silks, polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan, ispaghula, acacia, agar, gelatin, shellac, sterculia gum, xanthan gum, Corn sugar gum, guar gum, gum karaya, agarose, alginic acid, alginate, or natural polymers thereof. Examples of synthetic polymers include acrylics, nylons, silicones, spandex, viscose rayon, polycarboxylic acids, polyvinyl acetate, polyacrylamide, polyacrylate, polyethylene glycol, 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), polyformaldehyde, polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinyl acetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidene dichloride), poly(vinylidene difluoride), poly(vinyl fluoride) and/or combinations (e.g., co-polymers) thereof. Beads may also be formed from materials other than polymers, including lipids, micelles, liposomes, ceramics, glass-ceramics, material composites, metals, other inorganic materials, and others. 
     In some instances, the bead may contain molecular precursors (e.g., monomers or polymers), which may form a polymer network via polymerization of the molecular precursors. In some cases, a precursor may be an already polymerized species capable of undergoing further polymerization via, for example, a chemical cross-linkage. In some cases, a precursor can comprise one or more of an acrylamide or a methacrylamide monomer, oligomer, or polymer. In some cases, the bead may comprise prepolymers, which are oligomers capable of further polymerization. For example, polyurethane beads may be prepared using prepolymers. In some cases, the bead may contain individual polymers that may be further polymerized together. In some cases, beads may be generated via polymerization of different precursors, such that they comprise mixed polymers, co-polymers, and/or block co-polymers. In some cases, the bead may comprise covalent or ionic bonds between polymeric precursors (e.g., monomers, oligomers, linear polymers), nucleic acid molecules (e.g., oligonucleotides), primers, and other entities. In some cases, the covalent bonds can be carbon-carbon bonds, thioether bonds, or carbon-heteroatom bonds. 
     Cross-linking may be permanent or reversible, depending upon the particular cross-linker used. Reversible cross-linking may allow for the polymer to linearize or dissociate under appropriate conditions. In some cases, reversible cross-linking may also allow for reversible attachment of a material bound to the surface of a bead. In some cases, a cross-linker may form disulfide linkages. In some cases, the chemical cross-linker forming disulfide linkages may be cystamine or a modified cystamine. 
     In some cases, disulfide linkages can be formed between molecular precursor units (e.g., monomers, oligomers, or linear polymers) or precursors incorporated into a bead and nucleic acid molecules (e.g., oligonucleotides). Cystamine (including modified cystamines), for example, is an organic agent comprising a disulfide bond that may be used as a crosslinker agent between individual monomeric or polymeric precursors of a bead. Polyacrylamide may be polymerized in the presence of cystamine or a species comprising cystamine (e.g., a modified cystamine) to generate polyacrylamide gel beads comprising disulfide linkages (e.g., chemically degradable beads comprising chemically-reducible cross-linkers). The disulfide linkages may permit the bead to be degraded (or dissolved) upon exposure of the bead to a reducing agent. 
     In some cases, chitosan, a linear polysaccharide polymer, may be crosslinked with glutaraldehyde via hydrophilic chains to form a bead. Crosslinking of chitosan polymers may be achieved by chemical reactions that are initiated by heat, pressure, change in pH, and/or radiation. 
     In some cases, a bead may comprise an acrydite moiety, which in certain aspects may be used to attach one or more nucleic acid molecules (e.g., barcode sequence, barcoded nucleic acid molecule, barcoded oligonucleotide, primer, or other oligonucleotide) to the bead. In some cases, an acrydite moiety can refer to an acrydite analogue generated from the reaction of acrydite with one or more species, such as, the reaction of acrydite with other monomers and cross-linkers during a polymerization reaction. Acrydite moieties may be modified to form chemical bonds with a species to be attached, such as a nucleic acid molecule (e.g., barcode sequence, barcoded nucleic acid molecule, barcoded oligonucleotide, primer, or other oligonucleotide). Acrydite moieties may be modified with thiol groups capable of forming a disulfide bond or may be modified with groups already comprising a disulfide bond. The thiol or disulfide (via disulfide exchange) may be used as an anchor point for a species to be attached or another part of the acrydite moiety may be used for attachment. In some cases, attachment can be reversible, such that when the disulfide bond is broken (e.g., in the presence of a reducing agent), the attached species is released from the bead. In other cases, an acrydite moiety can comprise a reactive hydroxyl group that may be used for attachment. 
     Functionalization of beads for attachment of nucleic acid molecules (e.g., oligonucleotides) may be achieved through a wide range of different approaches, including activation of chemical groups within a polymer, incorporation of active or activatable functional groups in the polymer structure, or attachment at the pre-polymer or monomer stage in bead production. 
     For example, precursors (e.g., monomers, cross-linkers) that are polymerized to form a bead may comprise acrydite moieties, such that when a bead is generated, the bead also comprises acrydite moieties. The acrydite moieties can be attached to a nucleic acid molecule (e.g., oligonucleotide), which may include a priming sequence (e.g., a primer for amplifying target nucleic acids, random primer, primer sequence for mRNA) and/or one or more barcode sequences. The one or more barcode sequences may include sequences that are the same for all nucleic acid molecules coupled to a given bead and/or sequences that are different across all nucleic acid molecules coupled to the given bead. The nucleic acid molecule may be incorporated into the bead. 
     In some cases, the nucleic acid molecule can comprise a functional sequence, for example, for attachment to a sequencing flow cell, such as, for example, a P5 sequence for Illumina® sequencing. In some cases, the nucleic acid molecule or derivative thereof (e.g., oligonucleotide or polynucleotide generated from the nucleic acid molecule) can comprise another functional sequence, such as, for example, a P7 sequence for attachment to a sequencing flow cell for Illumina sequencing. In some cases, the nucleic acid molecule can comprise a barcode sequence. In some cases, the primer can further comprise a unique molecular identifier (UMI). In some cases, the primer can comprise an R1 primer sequence for Illumina sequencing. In some cases, the primer can comprise an R2 primer sequence for Illumina sequencing. Examples of such nucleic acid molecules (e.g., oligonucleotides, polynucleotides, etc.) and uses thereof, as may be used with compositions, devices, methods and systems of the present disclosure, are provided in U.S. Pat. Pub. Nos. 2014/0378345 and 2015/0376609, each of which is entirely incorporated herein by reference. 
       FIG.  8    illustrates an example of a barcode carrying bead. A nucleic acid molecule  802 , such as an oligonucleotide, can be coupled to a bead  804  by a releasable linkage  806 , such as, for example, a disulfide linker. The same bead  804  may be coupled (e.g., via releasable linkage) to one or more other nucleic acid molecules  818 ,  820 . The nucleic acid molecule  802  may be or comprise a barcode. As noted elsewhere herein, the structure of the barcode may comprise a number of sequence elements. The nucleic acid molecule  802  may comprise a functional sequence  808  that may be used in subsequent processing. For example, the functional sequence  808  may include one or more of a sequencer specific flow cell attachment sequence (e.g., a P5 sequence for Illumina® sequencing systems) and a sequencing primer sequence (e.g., a R1 primer for Illumina® sequencing systems). The nucleic acid molecule  802  may comprise a barcode sequence  810  for use in barcoding the sample (e.g., DNA, RNA, protein, etc.). In some cases, the barcode sequence  810  can be bead-specific such that the barcode sequence  810  is common to all nucleic acid molecules (e.g., including nucleic acid molecule  802 ) coupled to the same bead  804 . Alternatively or in addition, the barcode sequence  810  can be partition-specific such that the barcode sequence  810  is common to all nucleic acid molecules coupled to one or more beads that are partitioned into the same partition. The nucleic acid molecule  802  may comprise a specific priming sequence  812 , such as an mRNA specific priming sequence (e.g., poly-T sequence), a targeted priming sequence, and/or a random priming sequence. The nucleic acid molecule  802  may comprise an anchoring sequence  814  to ensure that the specific priming sequence  812  hybridizes at the sequence end (e.g., of the mRNA). For example, the anchoring sequence  814  can include a random short sequence of nucleotides, such as a 1-mer, 2-mer, 3-mer or longer sequence, which can ensure that a poly-T segment is more likely to hybridize at the sequence end of the poly-A tail of the mRNA. 
     The nucleic acid molecule  802  may comprise a unique molecular identifying sequence  816  (e.g., unique molecular identifier (UMI)). In some cases, the unique molecular identifying sequence  816  may comprise from about 5 to about 8 nucleotides. Alternatively, the unique molecular identifying sequence  816  may compress less than about 5 or more than about 8 nucleotides. The unique molecular identifying sequence  816  may be a unique sequence that varies across 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 identifying sequence  816  may be a random sequence (e.g., such as a random N-mer sequence). For example, the UMI may provide a unique identifier of the starting mRNA molecule that was captured, in order to allow quantitation of the number of original expressed RNA. As will be appreciated, although  FIG.  8    shows three nucleic acid molecules  802 ,  818 ,  820  coupled to the surface of the bead  804 , an individual bead may be coupled to any number of individual nucleic acid molecules, for example, from one to tens to hundreds of thousands or even millions of individual nucleic acid molecules. The respective barcodes for the individual nucleic acid molecules can comprise both common sequence segments or relatively 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. 
     In operation, a biological particle (e.g., cell, DNA, RNA, etc.) can be co-partitioned along with a barcode bearing bead  804 . The barcoded nucleic acid molecules  802 ,  818 ,  820  can be released from the bead  804  in the partition. By way of example, in the context of analyzing sample RNA, the poly-T segment (e.g.,  812 ) of one of the released nucleic acid molecules (e.g.,  802 ) can hybridize to the poly-A tail of a mRNA molecule. Reverse transcription may result in a cDNA transcript of the mRNA, but which transcript includes each of the sequence segments  808 ,  810 ,  816  of the nucleic acid molecule  802 . Because the nucleic acid molecule  802  comprises an anchoring sequence  814 , it will more likely hybridize to and prime reverse transcription at the sequence end of the poly-A tail of the mRNA. Within any given partition, all of the cDNA transcripts of the individual mRNA molecules may include a common barcode sequence segment  810 . However, the transcripts made from the different mRNA molecules within a given partition may vary at the unique molecular identifying sequence  812  segment (e.g., UMI segment). Beneficially, even following any subsequent amplification of the contents of a given partition, the number of different UMIs can be indicative of the quantity of mRNA originating from a given partition, and thus from the biological particle (e.g., cell). As noted above, the transcripts can be amplified, cleaned up and sequenced to identify the sequence of the cDNA transcript of the mRNA, as well as to sequence the barcode segment and the UMI segment. While a poly-T primer sequence is described, other targeted or random priming sequences may also be used in priming the reverse transcription reaction. Likewise, although described as releasing the barcoded oligonucleotides into the partition, in some cases, the nucleic acid molecules bound to the bead (e.g., gel bead) may be used to hybridize and capture the mRNA on the solid phase of the bead, for example, in order to facilitate the separation of the RNA from other cell contents. 
     In some cases, precursors comprising a functional group that is reactive or capable of being activated such that it becomes reactive can be polymerized with other precursors to generate gel beads comprising the activated or activatable functional group. The functional group may then be used to attach additional species (e.g., disulfide linkers, primers, other oligonucleotides, etc.) to the gel beads. For example, some precursors comprising a carboxylic acid (COOH) group can copolymerize with other precursors to form a gel bead that also comprises a COOH functional group. In some cases, acrylic acid (a species comprising free COOH groups), acrylamide, and bis(acryloyl)cystamine can be co-polymerized together to generate a gel bead comprising free COOH groups. The COOH groups of the gel bead 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-methylmorpholinium chloride (DMTMM)) such that they are reactive (e.g., reactive to amine functional groups where EDC/NHS or DMTMM are used for activation). The activated COOH groups can then react with an appropriate species (e.g., a species comprising an amine functional group where the carboxylic acid groups are activated to be reactive with an amine functional group) comprising a moiety to be linked to the bead. 
     Beads comprising disulfide linkages in their polymeric network may be functionalized with additional species via reduction of some of the disulfide linkages to free thiols. The disulfide linkages may be reduced via, for example, the action of a reducing agent (e.g., DTT, TCEP, etc.) to generate free thiol groups, without dissolution of the bead. Free thiols of the beads can then react with free thiols of a species or a species comprising another disulfide bond (e.g., via thiol-disulfide exchange) such that the species can be linked to the beads (e.g., via a generated disulfide bond). In some cases, free thiols of the beads may react with any other suitable group. For example, free thiols of the beads may react with species comprising an acrydite moiety. The free thiol groups of the beads can react with the acrydite via Michael addition chemistry, such that the species comprising the acrydite is linked to the bead. In some cases, uncontrolled reactions can be prevented by inclusion of a thiol capping agent such as N-ethylmalieamide or iodoacetate. 
     Activation of disulfide linkages within a bead can be controlled such that only a small number of disulfide linkages are activated. Control may be exerted, for example, by controlling the concentration of a reducing agent used to generate free thiol groups and/or concentration of reagents used to form disulfide bonds in bead polymerization. In some cases, a low concentration (e.g., molecules of reducing agent:gel bead ratios of 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, less than or equal to about 1:100,000, less than or equal to about 1:10,000) of reducing agent may be used for reduction. Controlling the number of disulfide linkages that are reduced to free thiols may be useful in ensuring bead structural integrity during functionalization. In some cases, optically-active agents, such as fluorescent dyes, may be coupled to beads via free thiol groups of the beads and used to quantify the number of free thiols present in a bead and/or track a bead. 
     In some cases, addition of moieties to a gel bead after gel bead formation may be advantageous. For example, addition of an oligonucleotide (e.g., barcoded oligonucleotide) after gel bead formation may avoid loss of the species during chain transfer termination that can occur during polymerization. Moreover, smaller precursors (e.g., monomers or cross linkers that do not comprise side chain groups and linked moieties) may be used for polymerization and can be minimally hindered from growing chain ends due to viscous effects. In some cases, functionalization after gel bead synthesis can minimize exposure of species (e.g., oligonucleotides) to be loaded with potentially damaging agents (e.g., free radicals) and/or chemical environments. In some cases, the generated gel may possess an upper critical solution temperature (UCST) that can permit temperature driven swelling and collapse of a bead. Such functionality may aid in oligonucleotide (e.g., a primer) infiltration into the bead during subsequent functionalization of the bead with the oligonucleotide. Post-production functionalization may also be useful in controlling loading ratios of species in beads, such that, for example, the variability in loading ratio is minimized. Species loading may also be performed in a batch process such that a plurality of beads can be functionalized with the species in a single batch. 
     A bead injected or otherwise introduced into a partition may comprise releasably, cleavably, or reversibly attached barcodes. A bead injected or otherwise introduced into a partition may comprise activatable barcodes. A bead injected or otherwise introduced into a partition may be degradable, disruptable, or dissolvable beads. 
     Barcodes can be releasably, cleavably or reversibly attached to the beads such that barcodes can be released or be releasable through cleavage of a linkage between the barcode molecule and the bead, or released through degradation of the underlying bead itself, allowing the barcodes to be accessed or be accessible by other reagents, or both. In non-limiting examples, cleavage may be achieved through reduction of di-sulfide bonds, use of restriction enzymes, photo-activated cleavage, or cleavage via other types of stimuli (e.g., chemical, thermal, pH, enzymatic, etc.) and/or reactions, such as described elsewhere herein. Releasable barcodes may sometimes be referred to as being activatable, in that they are available for reaction once released. 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 envisioned in the context of the described methods and systems. 
     In addition to, or as an alternative to the cleavable linkages between the beads and the associated molecules, such as barcode containing nucleic acid molecules (e.g., barcoded oligonucleotides), the beads may be degradable, disruptable, or dissolvable spontaneously or upon exposure to one or more stimuli (e.g., temperature changes, pH changes, exposure to particular chemical species or phase, exposure to light, reducing agent, etc.). In some cases, a bead may be dissolvable, such that material components of the beads are solubilized when exposed to a particular chemical species or an environmental change, such as a change temperature or a change in pH. In some cases, a gel bead can be degraded or dissolved at elevated temperature and/or in alkaline conditions. In some cases, a bead may be thermally degradable such that when the bead is exposed to an appropriate change in temperature (e.g., heat), the bead degrades. Degradation or dissolution of a bead bound to a species (e.g., a nucleic acid molecule, e.g., barcoded oligonucleotide) may result in release of the species from the bead. 
     As will be appreciated from the above disclosure, the degradation of a bead may refer to the dissociation of a bound or entrained species from a bead, both with and without structurally degrading the physical bead itself. For example, the degradation of the bead may involve cleavage of a cleavable linkage via one or more species and/or methods described elsewhere herein. In another example, entrained species may be released from beads through osmotic pressure differences due to, for example, changing chemical environments. By way of example, alteration of bead pore sizes due to osmotic pressure differences can generally occur without structural degradation of the bead itself. In some cases, an increase in pore size due to osmotic swelling of a bead can permit the release of entrained species within the bead. In other cases, osmotic shrinking of a bead may cause a bead to better retain an entrained species due to pore size contraction. 
     A degradable bead may be introduced into a partition, such as a droplet of an emulsion or a well, such that the bead degrades within the partition and any associated species (e.g., oligonucleotides) are released within the droplet when the appropriate stimulus is applied. The free species (e.g., oligonucleotides, nucleic acid molecules) may interact with other reagents contained in the partition. For example, a polyacrylamide bead comprising cystamine and linked, via a disulfide bond, to a barcode sequence, may be combined with a reducing agent within a droplet of a water-in-oil emulsion. Within the droplet, the reducing agent can break the various disulfide bonds, resulting in bead degradation and release of the barcode sequence into the aqueous, inner environment of the droplet. In another example, heating of a droplet comprising a bead-bound barcode sequence in basic solution may also result in bead degradation and release of the attached barcode sequence into the aqueous, inner environment of the droplet. 
     Any suitable number of molecular tag molecules (e.g., primer, barcoded oligonucleotide) can be associated with a bead such that, upon release from the bead, the molecular tag molecules (e.g., primer, e.g., barcoded oligonucleotide) are present in the partition at a pre-defined concentration. Such pre-defined concentration may be selected to facilitate certain reactions for generating a sequencing library, e.g., amplification, within the partition. In some cases, the pre-defined concentration of the primer can be limited by the process of producing nucleic acid molecule (e.g., oligonucleotide) bearing beads. 
     In some cases, beads can be non-covalently loaded with one or more reagents. The beads can be non-covalently loaded by, for instance, subjecting the beads to conditions sufficient to swell the beads, allowing sufficient time for the reagents to diffuse into the interiors of the beads, and subjecting the beads to conditions sufficient to de-swell the beads. The swelling of the beads may be accomplished, for instance, by placing the beads in a thermodynamically favorable solvent, subjecting the beads to a higher or lower temperature, subjecting the beads to a higher or lower ion concentration, and/or subjecting the beads to an electric field. The swelling of the beads may be accomplished by various swelling methods. The de-swelling of the beads may be accomplished, for instance, by transferring the beads in a thermodynamically unfavorable solvent, subjecting the beads to lower or high temperatures, subjecting the beads to a lower or higher ion concentration, and/or removing an electric field. The de-swelling of the beads may be accomplished by various de-swelling methods. Transferring the beads may cause pores in the bead to shrink. The shrinking may then hinder reagents within the beads from diffusing out of the interiors of the beads. The hindrance may be due to steric interactions between the reagents and the interiors of the beads. The transfer may be accomplished microfluidically. For instance, the transfer may be achieved by moving the beads from one co-flowing solvent stream to a different co-flowing solvent stream. The swellability and/or pore size of the beads may be adjusted by changing the polymer composition of the bead. 
     In some cases, an acrydite moiety linked to a precursor, another species linked to a precursor, or a precursor itself can comprise a labile bond, such as chemically, thermally, or photosensitive bond e.g., disulfide bond, UV-sensitive bond, or the like. Once acrydite moieties or other moieties comprising a labile bond are incorporated into a bead, the bead may also comprise the labile bond. The labile bond may be, for example, useful in reversibly linking (e.g., covalently linking) species (e.g., barcodes, primers, etc.) to a bead. In some cases, a thermally labile bond may include a nucleic acid hybridization based attachment, e.g., where an oligonucleotide is hybridized to a complementary sequence that is attached to the bead, such that thermal melting of the hybrid releases the oligonucleotide, e.g., a barcode containing sequence, from the bead or microcapsule. 
     The addition of multiple types of labile bonds to a gel bead may result in the generation of a bead capable of responding to varied stimuli. Each type of labile bond may be sensitive to an associated stimulus (e.g., chemical stimulus, light, temperature, enzymatic, etc.) such that release of species attached to a bead via each labile bond may be controlled by the application of the appropriate stimulus. Such functionality may be useful in controlled release of species from a gel bead. In some cases, another species comprising a labile bond may be linked to a gel bead after gel bead formation via, for example, an activated functional group of the gel bead as described above. As will be appreciated, barcodes that are releasably, cleavably or reversibly attached to the beads described herein include barcodes that are released or releasable through cleavage of a linkage between the barcode molecule and the bead, or that are released through degradation of the underlying bead itself, allowing the barcodes to be accessed or accessible by other reagents, or both. 
     In addition to thermally cleavable bonds, disulfide bonds and UV sensitive bonds, other non-limiting examples of labile bonds that may be coupled to a precursor or bead include an ester linkage (e.g., cleavable with an acid, a base, or hydroxylamine), a vicinal diol linkage (e.g., cleavable via sodium periodate), a Diels-Alder linkage (e.g., cleavable via heat), a sulfone linkage (e.g., cleavable via a base), a silyl ether linkage (e.g., cleavable via an acid), a glycosidic linkage (e.g., cleavable via an amylase), a peptide linkage (e.g., cleavable via a protease), or a phosphodiester linkage (e.g., cleavable via a nuclease (e.g., DNAase)). A bond may be cleavable via other nucleic acid molecule targeting enzymes, such as restriction enzymes (e.g., restriction endonucleases), as described further below. 
     Species may be encapsulated in beads during bead generation (e.g., during polymerization of precursors). Such species may or may not participate in polymerization. Such species may be entered into polymerization reaction mixtures such that generated beads comprise the species upon bead formation. In some cases, such species may be added to the gel beads after formation. Such species may include, for example, nucleic acid molecules (e.g., oligonucleotides), reagents for a nucleic acid amplification reaction (e.g., primers, polymerases, dNTPs, co-factors (e.g., ionic co-factors), buffers) including those described herein, reagents for enzymatic reactions (e.g., enzymes, co-factors, substrates, buffers), reagents for nucleic acid modification reactions such as polymerization, ligation, or digestion, and/or reagents for template preparation (e.g., tagmentation) for one or more sequencing platforms (e.g., Nextera® for Illumina®). Such species may include one or more enzymes described herein, including without limitation, polymerase, reverse transcriptase, restriction enzymes (e.g., endonuclease), transposase, ligase, proteinase K, DNAse, etc. Such species may include one or more reagents described elsewhere herein (e.g., lysis agents, inhibitors, inactivating agents, chelating agents, stimulus). Trapping of such species may be controlled by the polymer network density generated during polymerization of precursors, control of ionic charge within the gel bead (e.g., via ionic species linked to polymerized species), or by the release of other species. Encapsulated species may be released from a bead upon bead degradation and/or by application of a stimulus capable of releasing the species from the bead. Alternatively or in addition, species may be partitioned in a partition (e.g., droplet) during or subsequent to partition formation. Such species may include, without limitation, the abovementioned species that may also be encapsulated in a bead. 
     A degradable bead may comprise one or more species with a labile bond such that, when the bead/species is exposed to the appropriate stimuli, the bond is broken and the bead degrades. The labile bond may be a chemical bond (e.g., covalent bond, ionic bond) or may be another type of physical interaction (e.g., van der Waals interactions, dipole-dipole interactions, etc.). In some cases, a crosslinker used to generate a bead may comprise a labile bond. Upon exposure to the appropriate conditions, the labile bond can be broken and the bead degraded. For example, upon exposure of a polyacrylamide gel bead comprising cystamine crosslinkers to a reducing agent, the disulfide bonds of the cystamine can be broken and the bead degraded. 
     A degradable bead may be useful in more quickly releasing an attached species (e.g., a nucleic acid molecule, a barcode sequence, a primer, etc) from the bead when the appropriate stimulus is applied to the bead as compared to a bead that does not degrade. For example, for a species bound to an inner surface of a porous bead or in the case of an encapsulated species, the species may have greater mobility and accessibility to other species in solution upon degradation of the bead. In some cases, a species may also be attached to a degradable bead via a degradable linker (e.g., disulfide linker). The degradable linker may respond to the same stimuli as the degradable bead or the two degradable species may respond to different stimuli. For example, a barcode sequence may be attached, via a disulfide bond, to a polyacrylamide bead comprising cystamine. Upon exposure of the barcoded-bead to a reducing agent, the bead degrades and the barcode sequence is released upon breakage of both the disulfide linkage between the barcode sequence and the bead and the disulfide linkages of the cystamine in the bead. 
     Where degradable beads are provided, it may be beneficial to avoid exposing such beads to the stimulus or stimuli that cause such degradation prior to a given time, in order to, for example, avoid premature bead degradation and issues that arise from such degradation, including for example poor flow characteristics and aggregation. By way of example, where beads comprise reducible cross-linking groups, such as disulfide groups, it will be desirable to avoid contacting such beads with reducing agents, e.g., DTT or other disulfide cleaving reagents. In such cases, treatment to the beads described herein will, in some cases be provided free of reducing agents, such as DTT. Because reducing agents are often provided in commercial enzyme preparations, it may be desirable to provide reducing agent free (or DTT free) enzyme preparations in treating the beads described herein. Examples of such enzymes include, e.g., polymerase enzyme preparations, reverse transcriptase enzyme preparations, ligase enzyme preparations, as well as many other enzyme preparations that may be used to treat the beads described herein. The terms “reducing agent free” or “DTT free” preparations can refer to a preparation having less than about ⅒th, less than about 1/50th, or even less than about 1/100th of the lower ranges for such materials used in degrading the beads. For example, for DTT, the reducing agent free preparation 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 can be undetectable. 
     Numerous chemical triggers may be used to trigger the degradation of beads. Examples of these chemical changes may include, but are not limited to pH-mediated changes to the integrity of a component within the bead, degradation of a component of a bead via cleavage of cross-linked bonds, and depolymerization of a component of a bead. 
     In some embodiments, a bead may be formed from materials that comprise degradable chemical crosslinkers, such as BAC or cystamine. Degradation of such degradable crosslinkers may be accomplished through a number of mechanisms. In some examples, a bead may be contacted with a chemical degrading agent that may induce oxidation, reduction or other chemical changes. For example, a chemical degrading agent may be a reducing agent, such as dithiothreitol (DTT). Additional examples of reducing agents may include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinations thereof. A reducing agent may degrade the disulfide bonds formed between gel precursors forming the bead, and thus, degrade the bead. In other cases, a change in pH of a solution, such as an increase in pH, may trigger degradation of a bead. In other cases, exposure to an aqueous solution, such as water, may trigger hydrolytic degradation, and thus degradation of the bead. In some cases, any combination of stimuli may trigger degradation of a bead. For example, a change in pH may enable a chemical agent (e.g., DTT) to become an effective reducing agent. 
     Beads may also be induced to release their contents upon the application of a thermal stimulus. A change in temperature can cause a variety of changes to a bead. For example, heat can cause a solid bead to liquefy. A change in heat may cause melting of a bead such that a portion of the bead degrades. In other cases, heat may increase the internal pressure of the bead components such that the bead ruptures or explodes. Heat may also act upon heat-sensitive polymers used as materials to construct beads. 
     Any suitable agent may degrade beads. In some embodiments, changes in temperature or pH may be used to degrade thermo-sensitive or pH-sensitive bonds within beads. In some embodiments, chemical degrading agents may be used to degrade chemical bonds within beads by oxidation, reduction or other chemical changes. For example, a chemical degrading agent may be a reducing agent, such as DTT, wherein DTT may degrade the disulfide bonds formed between a crosslinker and gel precursors, thus degrading the bead. In some embodiments, a reducing agent may be added to degrade the bead, which may or may not cause the bead to release its contents. Examples of reducing agents may include dithiothreitol (DTT), β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinations thereof. The reducing agent may be present at a concentration of about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM. 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 greater than 10 mM. The reducing agent may be present at concentration of at most about 10 mM, 5 mM, 1 mM, 0.5 mM, 0.1 mM, or less. 
     Although  FIG.  1    and  FIG.  2    have been described in terms of providing substantially singly occupied partitions, above, in certain cases, it may be desirable to provide multiply occupied partitions, e.g., containing two, three, four or more cells and/or microcapsules (e.g., beads) comprising barcoded nucleic acid molecules (e.g., oligonucleotides) within a single partition. Accordingly, as noted above, the flow characteristics of the biological particle and/or bead containing fluids and partitioning fluids may be controlled to provide for such multiply occupied partitions. In particular, the flow parameters may be controlled to provide a given occupancy rate at greater than about 50% of the partitions, greater than about 75%, and in some cases greater than about 80%, 90%, 95%, or higher. 
     In some cases, additional microcapsules can be used to deliver additional reagents to a partition. In such cases, it may be advantageous to introduce different beads into a common channel or droplet generation junction, from different bead sources (e.g., containing different associated reagents) through different channel inlets into such common channel or droplet generation junction (e.g., junction  210 ). In such cases, the flow and frequency of the different beads into the channel or junction may be controlled to provide for a certain ratio of microcapsules from each source, while ensuring a given pairing or combination of such beads into a partition with a given number of biological particles (e.g., one biological particle and one bead per partition). 
     The partitions described herein may comprise small volumes, for example, less than about 10 microliters (µL), 5 µL, 1 µL, 500 nanoliters (nL), 100 nL, 50 nL, 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, or less. 
     For example, in the case of droplet based partitions, the droplets may have overall volumes that are 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. Where co-partitioned with microcapsules, it will be appreciated that the sample fluid volume, e.g., including co-partitioned biological particles and/or beads, within the partitions may be less than about 90% of the above described volumes, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% of the above described volumes. 
     As is described elsewhere herein, partitioning species may generate a population or plurality of partitions. In such cases, any suitable number of partitions can be generated or otherwise provided. For example, at least about 1,000 partitions, at least about 5,000 partitions, at least about 10,000 partitions, at least about 50,000 partitions, at least about 100,000 partitions, at least about 500,000 partitions, at least about 1,000 ,000 partitions, at least about 5,000 ,000 partitions at least about 10,000 ,000 partitions, at least about 50,000 ,000 partitions, at least about 100,000 ,000 partitions, at least about 500,000 ,000 partitions, at least about 1,000 ,000,000 partitions, or more partitions can be generated or otherwise provided. Moreover, the plurality of partitions may comprise both unoccupied partitions (e.g., empty partitions) and occupied partitions. 
     Reagents 
     In accordance with certain aspects, biological particles may be partitioned along with lysis reagents in order to release the contents of the biological particles within the partition. In such cases, the lysis agents can be contacted with the biological particle suspension concurrently with, or immediately prior to, the introduction of the biological particles into the partitioning junction/droplet generation zone (e.g., junction  210 ), such as through an additional channel or channels upstream of the channel junction. In accordance with other aspects, additionally or alternatively, biological particles may be partitioned along with other reagents, as will be described further below. 
       FIG.  3    shows an example of a microfluidic channel structure  300  for co-partitioning biological particles and reagents. The channel structure  300  can include channel segments  301 ,  302 ,  304 ,  306  and  308 . Channel segments  301  and  302  communicate at a first channel junction  309 . Channel segments  302 ,  304 ,  306 , and  308  communicate at a second channel junction  310 . 
     In an example operation, the channel segment  301  may transport an aqueous fluid  312  that includes a plurality of biological particles  314  along the channel segment  301  into the second junction  310 . As an alternative or in addition to, channel segment  301  may transport beads (e.g., gel beads). The beads may comprise barcode molecules. 
     For example, the channel segment  301  may be connected to a reservoir comprising an aqueous suspension of biological particles  314 . Upstream of, and immediately prior to reaching, the second junction  310 , the channel segment  301  may meet the channel segment  302  at the first junction  309 . The channel segment  302  may transport a plurality of reagents  315  (e.g., lysis agents) suspended in the aqueous fluid  312  along the channel segment  302  into the first junction  309 . For example, the channel segment  302  may be connected to a reservoir comprising the reagents  315 . After the first junction  309 , the aqueous fluid  312  in the channel segment  301  can carry both the biological particles  314  and the reagents  315  towards the second junction  310 . In some instances, the aqueous fluid  312  in the channel segment  301  can include one or more reagents, which can be the same or different reagents as the reagents  315 . A second fluid  316  that is immiscible with the aqueous fluid  312  (e.g., oil) can be delivered to the second junction  310  from each of channel segments  304  and  306 . Upon meeting of the aqueous fluid  312  from the channel segment  301  and the second fluid  316  from each of channel segments  304  and  306  at the second channel junction  310 , the aqueous fluid  312  can be partitioned as discrete droplets  318  in the second fluid  316  and flow away from the second junction  310  along channel segment  308 . The channel segment  308  may deliver the discrete droplets  318  to an outlet reservoir fluidly coupled to the channel segment  308 , where they may be harvested. 
     The second fluid  316  can comprise an oil, such as a fluorinated oil, that includes a fluorosurfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets  318 . 
     A discrete droplet generated may include an individual biological particle  314  and/or one or more reagents  315 . In some instances, a discrete droplet generated may include a barcode carrying bead (not shown), such as via other microfluidics structures described elsewhere herein. In some instances, a discrete droplet may be unoccupied (e.g., no reagents, no biological particles). 
     Beneficially, when lysis reagents and biological particles are co-partitioned, the lysis reagents can facilitate the release of the contents of the biological particles within the partition. The contents released in a partition may remain discrete from the contents of other partitions. 
     As will be appreciated, the channel segments described herein may be coupled to any of a variety of different fluid sources or receiving components, including reservoirs, tubing, manifolds, or fluidic components of other systems. As will be appreciated, the microfluidic channel structure  300  may have other geometries. For example, a microfluidic channel structure can have more than two channel junctions. For example, a microfluidic channel structure can have 2, 3, 4, 5 channel segments or more each carrying the same or different types of beads, reagents, and/or biological particles that meet at a channel junction. Fluid flow in each channel segment may be controlled to control the partitioning of the different elements into droplets. Fluid may be directed flow along one or more channels or reservoirs via one or more fluid flow units. A fluid flow unit can comprise compressors (e.g., providing positive pressure), pumps (e.g., providing negative pressure), actuators, and the like to control flow of the fluid. Fluid may also or otherwise be controlled via applied pressure differentials, centrifugal force, magnetic force, electrokinetic pumping, vacuum, capillary or gravity flow, or the like. 
     Examples of lysis agents include bioactive reagents, such as lysis enzymes that are used for lysis of different cell types, e.g., gram-positive or gram-negative bacteria, plants, yeast, mammalian, etc., such as lysozymes, achromopeptidase, lysostaphin, labiase, kitalase, lyticase, and a variety of other lysis enzymes available from, e.g., Sigma-Aldrich, Inc. (St Louis, MO), as well as other commercially available lysis enzymes. Other lysis agents may additionally or alternatively be co-partitioned with the biological particles to cause the release of the biological particle’s contents into the partitions. For example, in some cases, surfactant-based lysis solutions may be used to lyse cells, although these may be less desirable for emulsion-based systems where the surfactants can interfere with stable emulsions. In some cases, lysis solutions may include non-ionic surfactants such as, for example, TritonX-100, CHAPS, and Tween 20. In some cases, lysis solutions may include ionic surfactants such as, for example, sarcosyl and sodium dodecyl sulfate (SDS). In some cases, lysis may be achieved through osmotic pressure, e.g., using a hypotonic lysis buffer. Electroporation, thermal, acoustic or mechanical cellular disruption may also be used in certain cases, e.g., non-emulsion based partitioning such as encapsulation of biological particles that may be in addition to or in place of droplet partitioning, where any pore size of the encapsulate is sufficiently small to retain nucleic acid fragments of a given size, following cellular disruption. 
     Alternatively or in addition to the lysis agents co-partitioned with the biological particles described above, other reagents can also be co-partitioned with the biological particles, including, for example, DNase and RNase, inactivating agents or inhibitors, such as proteinase K and/or other protease inhibitors, phosphatase inhibitors, chelating agents, such as EDTA, and other reagents employed in removing or otherwise reducing negative activity or impact of different cell lysate components on subsequent processing of nucleic acids. In addition, in the case of encapsulated biological particles, the biological particles may be exposed to an appropriate stimulus to release the biological particles or their contents from a co-partitioned microcapsule. For example, in some cases, a chemical stimulus may be co-partitioned along with an encapsulated biological particle to allow for the degradation of the microcapsule and release of the cell or its contents into the larger partition. In some cases, this stimulus may be the same as the stimulus described elsewhere herein for release of nucleic acid molecules (e.g., oligonucleotides) from their respective microcapsule (e.g., bead). In alternative aspects, this may be a different and non-overlapping stimulus, in order to allow an encapsulated biological particle to be released into a partition at a different time from the release of nucleic acid molecules into the same partition. 
     Additional reagents may also be co-partitioned with the biological particles, such as endonucleases to fragment a biological particle’s DNA, DNA polymerase enzymes and dNTPs used to amplify the biological particle’s nucleic acid fragments and to attach the barcode molecular tags to the amplified fragments. Other enzymes may be co-partitioned, including without limitation, polymerase, transposase, ligase, proteinase K, DNAse, etc. Additional reagents may also include reverse transcriptase enzymes, including enzymes with terminal transferase activity, primers and oligonucleotides, and switch oligonucleotides (also referred to herein as “switch oligos” or “template switching oligonucleotides”) which can be used for template switching. In some cases, template switching can be used to increase the length of a cDNA. In some cases, template switching can be used to append a predefined nucleic acid sequence to the cDNA. In an example of template switching, cDNA can be generated from reverse transcription of a template, e.g., cellular mRNA, where a reverse transcriptase with terminal transferase activity can add additional nucleotides, e.g., polyC, to the cDNA in a template independent manner. Switch oligos can include sequences complementary to the additional nucleotides, e.g., polyG. The additional nucleotides (e.g., polyC) on the cDNA can hybridize to the additional nucleotides (e.g., polyG) on the switch oligo, whereby the switch oligo can be used by the reverse transcriptase as template to further extend the cDNA. Template switching oligonucleotides may comprise a hybridization region and a template region. The hybridization region can comprise any sequence capable of hybridizing to the target. In some cases, as previously described, the hybridization region comprises a series of G bases to complement the overhanging C bases at the 3′ end of a cDNA molecule. The series of G bases may comprise 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G bases or more than5 G bases. The template sequence can comprise any sequence to be incorporated into the cDNA. In some cases, the template region comprises at least 1 (e.g., at least 2, 3, 4, 5 or more) tag sequences and/or functional sequences. Switch oligos may comprise deoxyribonucleic acids; ribonucleic acids; modified nucleic acids including 2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA), inverted dT, 5-Methyl dC, 2′-deoxyInosine, Super T (5-hydroxybutynl-2′-deoxyuridine), Super G (8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlocked nucleic acids (UNAs, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2′ Fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and Fluoro G), or any combination. 
     In some cases, the length of a switch oligo may be at least 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, 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 or longer. 
     In some cases, the length of a switch oligo may 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, 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. 
     Once the contents of the cells are released into their respective partitions, the macromolecular components (e.g., macromolecular constituents of biological particles, such as RNA, DNA, or proteins) contained therein may be further processed within the partitions. In accordance with the methods and systems described herein, the macromolecular component contents of individual biological particles can be provided with unique identifiers such that, upon characterization of those macromolecular components they may be attributed as having been derived from the same biological particle or particles. The ability to attribute characteristics to individual biological particles or groups of biological particles is provided by the assignment of unique identifiers specifically to an individual biological particle or groups of biological particles. Unique identifiers, e.g., in the form of nucleic acid barcodes can be assigned or associated with individual biological particles or populations of biological particles, in order to tag or label the biological particle’s macromolecular components (and as a result, its characteristics) with the unique identifiers. These unique identifiers can then be used to attribute the biological particle’s components and characteristics to an individual biological particle or group of biological particles. 
     In some aspects, this is performed by co-partitioning the individual biological particle or groups of biological particles with the unique identifiers, such as described above (with reference to  FIG.  2   ). In some aspects, the unique identifiers are provided in the form of nucleic acid molecules (e.g., oligonucleotides) that comprise nucleic acid barcode sequences that may be attached to or otherwise associated with the nucleic acid contents of individual biological particle, or to other components of the biological particle, and particularly to fragments of those nucleic acids. The nucleic acid molecules are partitioned such that as between nucleic acid molecules in a given partition, the nucleic acid barcode sequences contained therein are the same, but as between different partitions, the nucleic acid molecule can, and do have differing barcode sequences, or at least represent a large number of different barcode sequences across all of the partitions in a given analysis. In some aspects, only one nucleic acid barcode sequence can be associated with a given partition, although in some cases, two or more different barcode sequences may be present. 
     The nucleic acid barcode sequences can include from about 6 to about 20 or more nucleotides within the sequence of the nucleic acid molecules (e.g., oligonucleotides). The nucleic acid barcode sequences can include from about 6 to about 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides. In some cases, the length of a barcode sequence may be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of a barcode sequence may be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of a barcode sequence may be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter. These nucleotides may be completely contiguous, i.e., in a single stretch of adjacent nucleotides, or they may be separated into two or more separate subsequences that are separated by 1 or more nucleotides. In some cases, separated barcode subsequences can be from about 4 to about 16 nucleotides in length. In some cases, the barcode subsequence may be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequence may be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequence may be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter. 
     The co-partitioned nucleic acid molecules can also comprise other functional sequences useful in the processing of the nucleic acids from the co-partitioned biological particles. These sequences include, e.g., targeted or random/universal amplification primer sequences for amplifying the genomic DNA from the individual biological particles within the partitions while attaching the associated barcode sequences, sequencing primers or primer recognition sites, hybridization or probing sequences, e.g., for identification of presence of the sequences or for pulling down and/or collecting barcoded nucleic acids, or any of a number of other potential functional sequences. Other mechanisms of co-partitioning oligonucleotides may also be employed, including, e.g., coalescence of two or more droplets, where one droplet contains oligonucleotides, or microdispensing of oligonucleotides into partitions, e.g., droplets within microfluidic systems. 
     In an example, microcapsules, such as beads, are provided that each include large numbers of the above described barcoded nucleic acid molecules (e.g., barcoded oligonucleotides) releasably attached to the beads, where all of the nucleic acid molecules attached to a particular bead will include the same nucleic acid barcode sequence, but where a large number of diverse barcode sequences are represented across the population of beads used. In some embodiments, hydrogel beads, e.g., comprising polyacrylamide polymer matrices, are used as a solid support and delivery vehicle for the nucleic acid molecules into the partitions, as they are capable of carrying large numbers of nucleic acid molecules, and may be configured to release those nucleic acid molecules upon exposure to a particular stimulus, as described elsewhere herein. In some cases, the population of beads provides a diverse barcode sequence library that includes at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000 ,000 different barcode sequences, at least about 5,000 ,000 different barcode sequences, or at least about 10,000 ,000 different barcode sequences, or more. Additionally, each bead can be provided with large numbers of nucleic acid (e.g., oligonucleotide) molecules attached. In particular, the number of molecules of nucleic acid molecules including the barcode sequence on an individual bead can be at least about 1,000 nucleic acid molecules, at least about5,000 nucleic acid molecules, at least about 10,000 nucleic acid molecules, at least about 50,000 nucleic acid molecules, at least about 100,000 nucleic acid molecules, at least about 500,000 nucleic acids, at least about 1,000 ,000 nucleic acid molecules, at least about 5,000 ,000 nucleic acid molecules, at least about 10,000 ,000 nucleic acid molecules, at least about 50,000 ,000 nucleic acid molecules, at least about 100,000 ,000 nucleic acid molecules, at least about 250,000 ,000 nucleic acid molecules and in some cases at least about 1 billion nucleic acid molecules, or more. Nucleic acid molecules of a given bead can include identical (or common) barcode sequences, different barcode sequences, or a combination of both. Nucleic acid molecules of a given bead can include multiple sets of nucleic acid molecules. Nucleic acid molecules of a given set can include identical barcode sequences. The identical barcode sequences can be different from barcode sequences of nucleic acid molecules of another set. 
     Moreover, when the population of beads is partitioned, the resulting population of partitions can also include a diverse barcode library that includes at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000 ,000 different barcode sequences, at least about 5,000 ,000 different barcode sequences, or at least about 10,000 ,000 different barcode sequences. Additionally, each partition of the population can include at least about 1,000 nucleic acid molecules, at least about 5,000 nucleic acid molecules, at least about 10,000 nucleic acid molecules, at least about 50,000 nucleic acid molecules, at least about 100,000 nucleic acid molecules, at least about 500,000 nucleic acids, at least about 1,000 ,000 nucleic acid molecules, at least about 5,000 ,000 nucleic acid molecules, at least about 10,000 ,000 nucleic acid molecules, at least about 50,000 ,000 nucleic acid molecules, at least about 100,000 ,000 nucleic acid molecules, at least about 250,000 ,000 nucleic acid molecules and in some cases at least about 1 billion nucleic acid molecules. 
     In some cases, it may be desirable to incorporate multiple different barcodes within a given partition, either attached to a single or multiple beads within the partition. For example, in some cases, a mixed, but known set of barcode sequences may provide greater assurance of identification in the subsequent processing, e.g., by providing a stronger address or attribution of the barcodes to a given partition, as a duplicate or independent confirmation of the output from a given partition. 
     The nucleic acid molecules (e.g., oligonucleotides) are releasable from the beads upon the application of a particular stimulus to the beads. In some cases, the stimulus may be a photo-stimulus, e.g., through cleavage of a photo-labile linkage that releases the nucleic acid molecules. In other cases, a thermal stimulus may be used, where elevation of the temperature of the beads environment will result in cleavage of a linkage or other release of the nucleic acid molecules from the beads. In still other cases, a chemical stimulus can be used that cleaves a linkage of the nucleic acid molecules to the beads, or otherwise results in release of the nucleic acid molecules from the beads. In one case, such compositions include the polyacrylamide matrices described above for encapsulation of biological particles, and may be degraded for release of the attached nucleic acid molecules through exposure to a reducing agent, such as DTT. 
     In some aspects, provided are systems and methods for controlled partitioning. Droplet size may be controlled by adjusting certain geometric features in channel architecture (e.g., microfluidics channel architecture). For example, an expansion angle, width, and/or length of a channel may be adjusted to control droplet size. 
       FIG.  4    shows an example of a microfluidic channel structure for the controlled partitioning of beads into discrete droplets. A channel structure  400  can include a channel segment  402  communicating at a channel junction  406  (or intersection) with a reservoir  404 . The reservoir  404  can be a chamber. Any reference to “reservoir,” as used herein, can also refer to a “chamber.” In operation, an aqueous fluid  408  that includes suspended beads  412  may be transported along the channel segment  402  into the junction  406  to meet a second fluid  410  that is immiscible with the aqueous fluid  408  in the reservoir  404  to create droplets  416 ,  418  of the aqueous fluid  408  flowing into the reservoir  404 . At the junction  406  where the aqueous fluid  408  and the second fluid  410  meet, droplets can form based on factors such as the hydrodynamic forces at the junction  406 , flow rates of the two fluids  408 ,  410 , fluid properties, and certain geometric parameters (e.g., w, h 0 , α, etc.) of the channel structure  400 . A plurality of droplets can be collected in the reservoir  404  by continuously injecting the aqueous fluid  408  from the channel segment  402  through the junction  406 . 
     A discrete droplet generated may include a bead (e.g., as in occupied droplets  416 ). Alternatively, a discrete droplet generated may include more than one bead. Alternatively, a discrete droplet generated may not include any beads (e.g., as in unoccupied droplet  418 ). In some instances, a discrete droplet generated may contain one or more biological particles, as described elsewhere herein. In some instances, a discrete droplet generated may comprise one or more reagents, as described elsewhere herein. 
     In some instances, the aqueous fluid  408  can have a substantially uniform concentration or frequency of beads  412 . The beads  412  can be introduced into the channel segment  402  from a separate channel (not shown in  FIG.  4   ). The frequency of beads  412  in the channel segment  402  may be controlled by controlling the frequency in which the beads  412  are introduced into the channel segment  402  and/or the relative flow rates of the fluids in the channel segment  402  and the separate channel. In some instances, the beads can be introduced into the channel segment  402  from a plurality of different channels, and the frequency controlled accordingly. 
     In some instances, the aqueous fluid  408  in the channel segment  402  can comprise biological particles (e.g., described with reference to  FIGS.  1  and  2   ). In some instances, the aqueous fluid  408  can have a substantially uniform concentration or frequency of biological particles. As with the beads, the biological particles can be introduced into the channel segment  402  from a separate channel. The frequency or concentration of the biological particles in the aqueous fluid  408  in the channel segment  402  may be controlled by controlling the frequency in which the biological particles are introduced into the channel segment  402  and/or the relative flow rates of the fluids in the channel segment  402  and the separate channel. In some instances, the biological particles can be introduced into the channel segment  402  from a plurality of different channels, and the frequency controlled accordingly. In some instances, a first separate channel can introduce beads and a second separate channel can introduce biological particles into the channel segment  402 . The first separate channel introducing the beads may be upstream or downstream of the second separate channel introducing the biological particles. 
     The second fluid  410  can comprise an oil, such as a fluorinated oil, that includes a fluorosurfactant for stabilizing the resulting droplets, for example, inhibiting subsequent coalescence of the resulting droplets. 
     In some instances, the second fluid  410  may not be subjected to and/or directed to any flow in or out of the reservoir  404 . For example, the second fluid  410  may be substantially stationary in the reservoir  404 . In some instances, the second fluid  410  may be subjected to flow within the reservoir  404 , but not in or out of the reservoir  404 , such as via application of pressure to the reservoir  404  and/or as affected by the incoming flow of the aqueous fluid  408  at the junction  406 . Alternatively, the second fluid  410  may be subjected and/or directed to flow in or out of the reservoir  404 . For example, the reservoir  404  can be a channel directing the second fluid  410  from upstream to downstream, transporting the generated droplets. 
     The channel structure  400  at or near the junction  406  may have certain geometric features that at least partly determine the sizes of the droplets formed by the channel structure  400 . The channel segment  402  can have a height, h 0  and width, w, at or near the junction  406 . By way of example, the channel segment  402  can comprise a rectangular cross-section that leads to a reservoir  404  having a wider cross-section (such as in width or diameter). Alternatively, the cross-section of the channel segment  402  can be other shapes, such as a circular shape, trapezoidal shape, polygonal shape, or any other shapes. The top and bottom walls of the reservoir  404  at or near the junction  406  can be inclined at an expansion angle, α. The expansion angle, α, allows the tongue (portion of the aqueous fluid  408  leaving channel segment  402  at junction  406  and entering the reservoir  404  before droplet formation) to increase in depth and facilitate decrease in curvature of the intermediately formed droplet. Droplet size may decrease with increasing expansion angle. The resulting droplet radius, R d , may be predicted by the following equation for the aforementioned geometric parameters of h 0 , w, and α: 
     
       
         
           
             
               R 
               d 
             
             ≈ 
             0.44 
             
               
                 1 
                 + 
                 2.2 
                 
                   
                     tan 
                     α 
                   
                 
                 
                   w 
                   
                     
                       h 
                       0 
                     
                   
                 
               
             
             
               
                 
                   h 
                   0 
                 
               
               
                 
                   
                     tan 
                     α 
                   
                 
               
             
           
         
       
     
     By way of example, for a channel structure with w = 21 µm, h = 21 µm, and α = 3°, the predicted droplet diameter is 121 µm. In another example, for a channel structure with w = 25 µm, h = 25 µm, and α = 5°, the predicted droplet diameter is 123 µm. In another example, for a channel structure with w = 28 µm, h = 28 µm, and α = 7°, the predicted droplet diameter is 124 µm. 
     In some instances, the expansion angle, α, may be between a range of from about 0.5° to about 4°, from about 0.1° to about 10°, or from 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 higher. In some instances, 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 instances, the width, w, can be between a range of from about 100 micrometers (µm) to about 500 µm. In some instances, the width, w, can be between a range of from 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 instances, the flow rate of the aqueous fluid  408  entering the junction  406  can be between about 0.04 microliters (µL)/minute (min) and about 40 µL/min. In some instances, the flow rate of the aqueous fluid  408  entering the junction  406  can be between about 0.01 microliters (µL)/minute (min) and about 100 µL/min. Alternatively, the flow rate of the aqueous fluid  408  entering the junction  406  can be less than about 0.01 µL/min. Alternatively, the flow rate of the aqueous fluid  408  entering the 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 greater. At lower flow rates, such as flow rates of about less than or equal to 10 microliters/minute, the droplet radius may not be dependent on the flow rate of the aqueous fluid  408  entering the junction  406 . 
     In some instances, at least about 50% of the droplets generated can have uniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the droplets generated can have uniform size. Alternatively, less than about 50% of the droplets generated can have uniform size. 
     The throughput of droplet generation can be increased by increasing the points of generation, such as increasing the number of junctions (e.g., junction  406 ) between aqueous fluid  408  channel segments (e.g., channel segment  402 ) and the 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 . 
       FIG.  5    shows an example of a microfluidic channel structure for increased droplet generation throughput. A microfluidic channel structure  500  can comprise a plurality of channel segments  502  and a reservoir  504 . Each of the plurality of channel segments  502  may be in fluid communication with the reservoir  504 . The channel structure  500  can comprise a plurality of channel junctions  506  between the plurality of channel segments  502  and the reservoir  504 . Each channel junction can be a point of droplet generation. The channel segment  402  from the channel structure  400  in  FIG.  4    and any description to the components thereof may correspond to a given channel segment of the plurality of channel segments  502  in channel structure  500  and any description to the corresponding components thereof. The reservoir  404  from the channel structure  400  and any description to the components thereof may correspond to the reservoir  504  from the channel structure  500  and any description to the corresponding components thereof. 
     Each channel segment of the plurality of channel segments  502  may comprise an aqueous fluid  508  that includes suspended beads  512 . The reservoir  504  may comprise a second fluid  510  that is immiscible with the aqueous fluid  508 . In some instances, the second fluid  510  may not be subjected to and/or directed to any flow in or out of the reservoir  504 . For example, the second fluid  510  may be substantially stationary in the reservoir  504 . In some instances, the second fluid  510  may be subjected to flow within the reservoir  504 , but not in or out of the reservoir  504 , such as via application of pressure to the reservoir  504  and/or as affected by the incoming flow of the aqueous fluid  508  at the junctions. Alternatively, the second fluid  510  may be subjected and/or directed to flow in or out of the reservoir  504 . For example, the reservoir  504  can be a channel directing the second fluid  510  from upstream to downstream, transporting the generated droplets. 
     In operation, the aqueous fluid  508  that includes suspended beads  512  may be transported along the plurality of channel segments  502  into the plurality of junctions  506  to meet the second fluid  510  in the reservoir  504  to create droplets  516 ,  518 . A droplet may form from each channel segment at each corresponding junction with the reservoir  504 . At the junction where the aqueous fluid  508  and the second fluid  510  meet, droplets can form based on factors such as the hydrodynamic forces at the junction, flow rates of the two fluids  508 ,  510 , fluid properties, and certain geometric parameters (e.g., w, h 0 , α, etc.) of the channel structure  500 , as described elsewhere herein. A plurality of 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 . Throughput may significantly increase with the parallel channel configuration of channel structure  500 . For example, a channel structure having five inlet channel segments comprising the aqueous fluid  508  may generate droplets five times as frequently than a channel structure having one inlet channel segment, provided that the fluid flow rate in the channel segments are substantially the same. The fluid flow rate in the different inlet channel segments may or may not be substantially the same. A channel structure may have as many parallel channel segments as is practical and allowed for the size of the reservoir. For example, the channel structure may 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 segments. 
     The geometric parameters, w, h 0 , and α, may or may not be uniform for each of the channel segments 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 reservoir  504 . For example, each channel segment may have the same or different height at or near its respective channel junction with the reservoir  504 . In another example, the reservoir  504  may have the same or different expansion angle at the different channel junctions with the plurality of channel segments  502 . When the geometric parameters are uniform, beneficially, droplet size may also be controlled to be uniform even with the increased throughput. In some instances, when it is desirable to have a different distribution of droplet sizes, the geometric parameters for the plurality of channel segments  502  may be varied accordingly. 
     In some instances, at least about 50% of the droplets generated can have uniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the droplets generated can have uniform size. Alternatively, less than about 50% of the droplets generated can have uniform size. 
       FIG.  6    shows another example of a microfluidic channel structure for increased droplet generation throughput. A microfluidic channel structure  600  can comprise a plurality of channel segments  602  arranged generally circularly around the perimeter 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  can comprise a plurality of channel junctions  606  between the plurality of channel segments  602  and the reservoir  604 . Each channel junction can be a point of droplet generation. The channel segment  402  from the channel structure  400  in  FIG.  4    and any description to the components thereof may correspond to a given channel segment of the plurality of channel segments  602  in channel structure  600  and any description to the corresponding components thereof. The reservoir  404  from the channel structure  400  and any description to the components thereof may correspond to the reservoir  604  from the channel structure  600  and any description to the corresponding components thereof. 
     Each channel segment of the plurality of channel segments  602  may comprise an aqueous fluid  608  that includes suspended beads  612 . The reservoir  604  may comprise a second fluid  610  that is immiscible with the aqueous fluid  608 . In some instances, the second fluid  610  may not be subjected to and/or directed to any flow in or out of the reservoir  604 . For example, the second fluid  610  may be substantially stationary in the reservoir  604 . In some instances, the second fluid  610  may be subjected to flow within the reservoir  604 , but not in or out of the reservoir  604 , such as via application of pressure to the reservoir  604  and/or as affected by the incoming flow of the aqueous fluid  608  at the junctions. Alternatively, the second fluid  610  may be subjected and/or directed to flow in or out of the reservoir  604 . For example, the reservoir  604  can be a channel directing the second fluid  610  from upstream to downstream, transporting the generated droplets. 
     In operation, the aqueous fluid  608  that includes suspended beads  612  may be transported along the plurality of channel segments  602  into the plurality of junctions  606  to meet the second fluid  610  in the reservoir  604  to create a plurality of droplets  616 . A droplet may form from each channel segment at each corresponding junction with the reservoir  604 . At the junction where the aqueous fluid  608  and the second fluid  610  meet, droplets can form based on factors such as the hydrodynamic forces at the junction, flow rates of the two fluids  608 ,  610 , fluid properties, and certain geometric parameters (e.g., widths and heights of the channel segments  602 , expansion angle of the reservoir  604 , etc.) of the channel structure  600 , as described elsewhere herein. A 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 . Throughput may significantly increase with the substantially parallel channel configuration of the channel structure  600 . A channel structure may have as many substantially parallel channel segments as is practical and allowed for by the size of the reservoir. For example, the channel structure may 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 channel segments. The plurality of channel segments may be substantially evenly spaced apart, for example, around an edge or perimeter of the reservoir. Alternatively, the spacing of the plurality of channel segments may be uneven. 
     The reservoir  604  may have an expansion angle, α (not shown in  FIG.  6   ) at or near each channel junction. Each channel segment of the plurality of channel segments  602  may have a width, w, and a height, h 0 , at or near the channel junction. The geometric parameters, w, h 0 , and α, may or may not be uniform for each of the channel segments in 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 reservoir  604 . For example, each channel segment may have the same or different height at or near its respective channel junction with the reservoir  604 . 
     The reservoir  604  may have the same or different expansion angle at the different channel junctions with the plurality of channel segments  602 . For example, a circular reservoir (as shown in  FIG.  6   ) may have a conical, dome-like, or hemispherical ceiling (e.g., top wall) to provide the same or substantially same expansion angle for each channel segments  602  at or near the plurality of channel junctions  606 . When the geometric parameters are uniform, beneficially, resulting droplet size may be controlled to be uniform even with the increased throughput. In some instances, when it is desirable to have a different distribution of droplet sizes, the geometric parameters for the plurality of channel segments  602  may be varied accordingly. 
     In some instances, at least about 50% of the droplets generated can have uniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the droplets generated can have uniform size. Alternatively, less than about 50% of the droplets generated can have uniform size. The beads and/or biological particle injected into the droplets may or may not have uniform size. 
       FIG.  7 A  shows a cross-section view of another example of a microfluidic channel structure with a geometric feature for controlled partitioning. A channel structure  700  can include a channel segment  702  communicating at a channel junction  706  (or intersection) with a reservoir  704 . In some instances, the channel structure  700  and one or more of its components can correspond to the channel structure  100  and one or more of its components.  FIG.  7 B  shows a perspective view of the channel structure  700  of  FIG.  7 A . 
     An aqueous fluid  712  comprising a plurality of particles  716  may be transported along the channel segment  702  into the junction  706  to meet a second fluid  714  (e.g., oil, etc.) that is immiscible with the aqueous fluid  712  in the reservoir  704  to create 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 form based on factors such as the hydrodynamic forces at the junction  706 , relative flow rates of the two fluids  712 ,  714 , fluid properties, and certain geometric parameters (e.g., Δh, etc.) of the channel structure  700 . A plurality of droplets can be collected in the reservoir  704  by continuously injecting the aqueous fluid  712  from the channel segment  702  at the junction  706 . 
     A discrete droplet generated may comprise one or more particles of the plurality of particles  716 . As described elsewhere herein, a particle may be any particle, such as a bead, cell bead, gel bead, biological particle, macromolecular constituents of biological particle, or other particles. Alternatively, a discrete droplet generated may not include any particles. 
     In some instances, the aqueous fluid  712  can have a substantially uniform concentration or frequency of particles  716 . As described elsewhere herein (e.g., with reference to  FIG.  4   ), the particles  716  (e.g., beads) can be introduced into the channel segment  702  from a separate channel (not shown in  FIG.  7   ). The frequency of particles  716  in the channel segment  702  may be controlled by controlling the frequency in which the particles  716  are introduced into the channel segment  702  and/or the relative flow rates of the fluids in the channel segment  702  and the separate channel. In some instances, the particles  716  can be introduced into the channel segment  702  from a plurality of different channels, and the frequency controlled accordingly. In some instances, different particles may be introduced via separate channels. For example, a first separate channel can introduce beads and a second separate channel can introduce biological particles into the channel segment  702 . The first separate channel introducing the beads may be upstream or downstream of the second separate channel introducing the biological particles. 
     In some instances, the second fluid  714  may not be subjected to and/or directed to any flow in or out of the reservoir  704 . For example, the second fluid  714  may be substantially stationary in the reservoir  704 . In some instances, the second fluid  714  may be subjected to flow within the reservoir  704 , but not in or out of the reservoir  704 , such as via application of pressure to the reservoir  704  and/or as affected by the incoming flow of the aqueous fluid  712  at the junction  706 . Alternatively, the second fluid  714  may be subjected and/or directed to flow in or out of the reservoir  704 . For example, the reservoir  704  can be a channel directing the second fluid  714  from upstream to downstream, transporting the generated droplets. 
     The channel structure  700  at or near the junction  706  may have certain geometric features that at least partly determine the sizes and/or shapes of the droplets formed by the channel structure  700 . The channel segment  702  can have a first cross-section height, h 1 , and the reservoir  704  can have a second cross-section height, h 2 . The first cross-section height, h 1 , and the second cross-section height, h 2 , may be different, such that at the junction  706 , there is a height difference of Δh. The second cross-section height, h 2 , may be greater than the first cross-section height, h 1 . In some instances, the reservoir may thereafter gradually increase in cross-section height, for example, the more distant it is from the junction  706 . In some instances, the cross-section height of the reservoir may increase in accordance with expansion angle, β, at or near the junction  706 . The height difference, Δh, and/or expansion angle, β, can allow the tongue (portion of the aqueous fluid  712  leaving channel segment  702  at junction  706  and entering the reservoir  704  before droplet formation) to increase in depth and facilitate decrease in curvature of the intermediately formed droplet. For example, droplet size may decrease with increasing height difference and/or increasing expansion angle. 
     The height difference, Δh, can be at least about 1 µm. Alternatively, the height difference can 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 can 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 instances, the expansion angle, β, may be between a range of from about 0.5° to about 4°, from about 0.1° to about 10°, or from 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 higher. In some instances, 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 instances, the flow rate of the aqueous fluid  712  entering the junction  706  can be between about 0.04 microliters (µL)/minute (min) and about 40 µL/min. In some instances, the flow rate of the aqueous fluid  712  entering the junction  706  can be between about 0.01 microliters (µL)/minute (min) and about 100 µL/min. Alternatively, the flow rate of the aqueous fluid  712  entering the junction  706  can be less than about 0.01 µL/min. Alternatively, the flow rate of the aqueous fluid  712  entering the 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 greater. At lower flow rates, such as flow rates of about less than or equal to 10 microliters/minute, the droplet radius may not be dependent on the flow rate of the aqueous fluid  712  entering the junction  706 . The second fluid  714  may be stationary, or substantially stationary, in the reservoir  704 . Alternatively, the second fluid  714  may be flowing, such as at the above flow rates described for the aqueous fluid  712 . 
     In some instances, at least about 50% of the droplets generated can have uniform size. In some instances, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater of the droplets generated can have uniform size. Alternatively, less than about 50% of the droplets generated can have uniform size. 
     While  FIGS.  7 A and  7 B  illustrate the height difference, Δh, being abrupt at the junction  706  (e.g., a step increase), the height difference may increase gradually (e.g., from about 0 µm to a maximum height difference). Alternatively, the height difference may decrease gradually (e.g., taper) from a maximum height difference. A gradual increase or decrease in height difference, as used herein, may refer to a continuous incremental increase or decrease in height difference, wherein an angle between any one differential segment of a height profile and an immediately adjacent differential segment of the height profile is greater than 90°. For example, at the junction  706 , a bottom wall of the channel and a bottom wall of the reservoir can meet at an angle greater than 90°. Alternatively or in addition, a top wall (e.g., ceiling) of the channel and a top wall (e.g., ceiling) of the reservoir can meet an angle greater than 90°. A gradual increase or decrease may be linear or non-linear (e.g., exponential, sinusoidal, etc.). Alternatively or in addition, the height difference may variably increase and/or decrease linearly or non-linearly. While  FIGS.  7 A and  7 B  illustrate the expanding reservoir cross-section height as linear (e.g., constant expansion angle, β), the cross-section height may expand non-linearly. For example, the reservoir may be defined at least partially by a dome-like (e.g., hemispherical) shape having variable expansion angles. The cross-section height may expand in any shape. 
     The channel networks, e.g., as described above or elsewhere herein, can be fluidically coupled to appropriate fluidic components. For example, the inlet channel segments are fluidly coupled to appropriate sources of the materials they are to deliver to a channel junction. These sources may include any of a variety of different fluidic components, from simple reservoirs defined in or connected to a body structure of a microfluidic device, to fluid conduits that deliver fluids from off-device sources, manifolds, fluid flow units (e.g., actuators, pumps, compressors) or the like. Likewise, the outlet channel segment (e.g., channel segment  208 , reservoir  604 , etc.) may be fluidically coupled to a receiving vessel or conduit for the partitioned cells for subsequent processing. Again, this may be a reservoir defined in the body of a microfluidic device, or it may be a fluidic conduit for delivering the partitioned cells to a subsequent process operation, instrument or component. 
     The methods and systems described herein may be used to greatly increase the efficiency of single-cell applications and/or other applications receiving droplet-based input. For example, following the sorting of occupied cells and/or appropriately-sized cells, subsequent operations that can be performed can include generation of amplification products, purification (e.g., via solid phase reversible immobilization (SPRI)), further processing (e.g., shearing, ligation of functional sequences, and subsequent amplification (e.g., via PCR)). These operations may occur in bulk (e.g., outside the partition). In the case where a partition is a droplet in an emulsion, the emulsion can be broken and the contents of the droplet pooled for additional operations. Additional reagents that may be co-partitioned along with the barcode bearing bead may include oligonucleotides to block ribosomal RNA (rRNA) and nucleases to digest genomic DNA from cells. Alternatively, rRNA removal agents may be applied during additional processing operations. The configuration of the constructs generated by such a method can help minimize (or avoid) sequencing of the poly-T sequence during sequencing and/or sequence the 5′ end of a polynucleotide sequence. The amplification products, for example, first amplification products and/or second amplification products, may be subject to sequencing for sequence analysis. In some cases, amplification may be performed using the Partial Hairpin Amplification for Sequencing (PHASE) method. 
     A variety of applications require the evaluation of the presence and quantification of different biological particle or organism types within a population of biological particles, including, for example, microbiome analysis and characterization, environmental testing, food safety testing, epidemiological analysis, e.g., in tracing contamination or the like. 
     Computer Systems 
     The present disclosure provides computer systems that are programmed to implement methods of the disclosure.  FIG.  12    shows a computer system  1201  that is programmed or otherwise configured to control a microfluidics system (e.g., fluid flow) and perform sequencing applications. The computer system  1201  can regulate various aspects of the present disclosure. The computer system  1201  can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device. 
     The computer system  1201  includes a central processing unit (CPU, also “processor” and “computer processor” herein)  1205 , which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system  1201  also includes memory or memory location  1210  (e.g., random-access memory, read-only memory, flash memory), electronic storage unit  1215  (e.g., hard disk), communication interface  1220  (e.g., network adapter) for communicating with one or more other systems, and peripheral devices  1225 , such as cache, other memory, data storage and/or electronic display adapters. The memory  1210 , storage unit  1215 , interface  1220  and peripheral devices  1225  are in communication with the CPU  1205  through a communication bus (solid lines), such as a motherboard. The storage unit  1215  can be a data storage unit (or data repository) for storing data. The computer system  1201  can be operatively coupled to a computer network (“network”)  1230  with the aid of the communication interface  1220 . The network  1230  can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network  1230  in some cases is a telecommunication and/or data network. The network  1230  can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network  1230 , in some cases with the aid of the computer system  1201 , can implement a peer-to-peer network, which may enable devices coupled to the computer system  1201  to behave as a client or a server. 
     The CPU  1205  can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory  1210 . The instructions can be directed to the CPU  1205 , which can subsequently program or otherwise configure the CPU  1205  to implement methods of the present disclosure. Examples of operations performed by the CPU  1205  can include fetch, decode, execute, and writeback. 
     The CPU  1205  can be part of a circuit, such as an integrated circuit. One or more other components of the system  1201  can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC). 
     The storage unit  1215  can store files, such as drivers, libraries and saved programs. The storage unit  1215  can store user data, e.g., user preferences and user programs. The computer system  1201  in some cases can include one or more additional data storage units that are external to the computer system  1201 , such as located on a remote server that is in communication with the computer system  1201  through an intranet or the Internet. 
     The computer system  1201  can communicate with one or more remote computer systems through the network  1230 . For instance, the computer system  1201  can communicate with a remote computer system of a user (e.g., operator). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system  1201  via the network  1230 . 
     Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system  1201 , such as, for example, on the memory  1210  or electronic storage unit  1215 . The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor  1205 . In some cases, the code can be retrieved from the storage unit  1215   and stored on the memory  1210  for ready access by the processor  1205 . In some situations, the electronic storage unit  1215  can be precluded, and machine-executable instructions are stored on memory  1210 . 
     The code can be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion. 
     Aspects of the systems and methods provided herein, such as the computer system  1201 , can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution. 
     Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may 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. 
     The computer system  1201  can include or be in communication with an electronic display  1235  that comprises a user interface (UI)  1240  for providing, for example, results of sequencing analysis, etc. Examples of UIs include, without limitation, a graphical user interface (GUI) and web-based user interface. 
     Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit  1205 . The algorithm can, for example, perform sequencing. 
     Devices, systems, compositions and methods of the present disclosure may be used for various applications, such as, for example, 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 single cell. For example, a biological particle (e.g., a cell or cell bead) is partitioned in a partition (e.g., droplet), and multiple analytes from the biological particle are processed for subsequent processing. The multiple analytes may be from the single cell. This may enable, for example, simultaneous proteomic, transcriptomic and genomic analysis of the cell. 
     While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.