Methods and systems for sample preparation and analysis

Abstract

The present disclosure provides methods and systems for sample preparation and / or analysis. The sample can be a cell, or can be derived from one or more cells. Sample preparation can include performing one or more reactions on a target. The reactions can be performed in one or more distribution areas. One or more reactions can be performed in one or more continuous operations.

Description

[0001] This application is a divisional application of the Chinese patent application with the application date of October 24, 2018, application number 201880075763.9, and invention name “Method and system for sample preparation and analysis” (the application date of the corresponding PCT application is October 24, 2018, and the application number is PCT / US2018 / 057350).

[0002] Cross-references

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 578,225, filed October 27, 2017, which is incorporated herein by reference in its entirety. Background Art

[0004] Samples can be processed for various purposes, such as identifying the sample type of a portion within a sample. The sample can be a biological sample. Biological samples can be processed for various purposes, such as detecting a disease (e.g., cancer), identifying a specific substance, or analyzing gene expression profiles and gene interactions. There are various methods for processing samples, such as polymerase chain reaction (PCR) and sequencing.

[0005] Biological samples can be processed in various reaction environments, such as partitions. The partitions can be wells or droplets. The biological samples can be processed in droplets or wells in a manner that enables the biological samples to be partitioned and processed independently. For example, such droplets can be fluidically isolated from other droplets, thereby enabling precise control of each environment in the droplet.

[0006] The biological sample in the distribution area can be subjected to various processes, such as chemical processes or physical processes. The sample in the distribution area can be subjected to heating or cooling, or chemical reactions, such as to produce substances that can be qualitatively or quantitatively processed. Summary of the invention

[0007] Biochemical reactions involving sequential operations may not be feasible in microfluidic partition-based systems due to incompatibility of one reagent with another (e.g., one enzyme with another enzyme). Such incompatibility may be caused by inhibition, competing reaction mechanisms, or different reaction conditions, which may also render partition-based merging techniques for sequential introduction of reagents ineffective. The present invention recognizes the need for methods for performing biochemical reactions involving sequential operations in partition-based systems using multiple reagents.

[0008] In some embodiments, disclosed herein is a method for sample preparation or analysis, comprising: (a) providing a plurality of distribution areas, wherein a given distribution area in the plurality of distribution areas comprises a target and a first bead, wherein the first bead comprises one or more reagents for performing one or more reactions on the target, wherein the first bead is destructible; (b) performing one or more reactions on the target using one or more reagents to produce one or more reaction products, wherein after performing the one or more reactions, the given distribution area comprises one or more precursors produced by the destruction of the first bead; and (c) using one or more precursors to produce a second bead, wherein the second bead comprises one or more reaction products. In some embodiments, the method further comprises performing one or more additional reactions on another target to produce one or more additional reaction products. In some embodiments, another target is the target. In some embodiments, another target comprises one or more reaction products. In some embodiments, one or more additional reactions are performed in another distribution area comprising a second bead, wherein the other distribution area is different from the given distribution area. In some embodiments, another distribution area comprises one or more additional reagents for performing one or more additional reactions. In some embodiments, the additional distribution area comprises a third bead, the third bead comprising one or more additional reagents for performing one or more additional reactions. In some embodiments, after performing one or more additional reactions, the additional distribution area comprises one or more precursors produced by the destruction of the second bead. In some embodiments, after performing one or more additional reactions, a given distribution area comprises one or more precursors produced by the destruction of the second bead.

[0009] In some embodiments, the method further comprises producing a third bead using one or more precursors, the third bead comprising one or more other reaction products. In some embodiments, the third bead is a gel bead. In some embodiments, (c) is performed in a given distribution area. In some embodiments, (c) is performed outside a given distribution area. In some embodiments, (c) is performed in another distribution area. In some embodiments, the target is a nucleic acid molecule or is associated with it, wherein one or more reaction products are derived from nucleic acid molecules, and wherein after (c), one or more reaction products or derivatives thereof are sequenced. In some embodiments, a plurality of distribution areas are a plurality of droplets. In some embodiments, a plurality of droplets are produced after contacting a first liquid phase comprising a first bead with a second liquid phase immiscible with the first liquid phase. In some embodiments, a plurality of distribution areas are a plurality of holes.

[0010] In some embodiments, the target is a nucleic acid molecule. In some embodiments, the nucleic acid molecule is a ribonucleic acid molecule. In some embodiments, the nucleic acid molecule is a ribonucleic acid molecule. In some embodiments, the ribonucleic acid molecule is a messenger RNA molecule. In some embodiments, the nucleic acid molecule is a deoxyribonucleic acid molecule. In some embodiments, the target is a protein. In some embodiments, the target is a cell. In some embodiments, one or more reagents include one or more nucleic acid molecules. In some embodiments, one or more nucleic acid molecules include a molecular barcode. In some embodiments, one or more reaction products include a molecular barcode. In some embodiments, one or more nucleic acid molecules are multiple nucleic acid molecules, wherein a given nucleic acid molecule in a plurality of nucleic acid molecules includes a constant barcode sequence between a plurality of nucleic acid molecules. In some embodiments, a given nucleic acid molecule includes another barcode sequence that varies between a plurality of nucleic acid molecules.

[0011] In some embodiments, the first bead is from a plurality of beads, wherein the plurality of beads contain different nucleic acid barcode sequences between the plurality of beads. In some embodiments, one or more reagents include all reagents required to perform one or more reactions. In some embodiments, one or more reactions include reverse transcription reactions. In some embodiments, one or more reactions include nucleic acid modification. In some embodiments, nucleic acid modification is deoxyribonucleic acid modification. In some embodiments, nucleic acid modification is ribonucleic acid modification. In some embodiments, one or more reactions include nucleic acid amplification. In some embodiments, nucleic acid amplification is polymerase chain reaction. In some embodiments, nucleic acid amplification is not polymerase chain reaction. In some embodiments, one or more reactions include cell lysis. In some embodiments, one or more reactions include labeling. In some embodiments, one or more reactions include adding barcode oligonucleotides. In some embodiments, a given distribution area includes cells or gel matrix containing one or more cell components, and wherein the target is from the cell or gel matrix.

[0012] In some embodiments, the first bead is produced by polymerization or cross-linking of one or more precursors. In some embodiments, the first bead is a hydrogel. In some embodiments, one or more precursors include poly-L-lactic acid, poly (ethylene-propylene), poly (ethylene-butylene), polyacrylamide, agarose, alginate, polyvinyl alcohol, PEG, PEG-diacrylate, PEG-acrylate / thiol, PEG-azide / alkyne, other acrylates, chitosan, hyaluronic acid, collagen, fibrin, gelatin, elastin or any combination thereof. In some embodiments, the first bead is a gel bead. In some embodiments, the second bead is a gel bead. In some embodiments, the first bead is destructible after applying a stimulus. In some embodiments, the stimulus is a chemical stimulus, a thermal stimulus, an electromagnetic stimulus or a combination thereof. In some embodiments, a given distribution area includes the stimulus. In some embodiments, (c) includes subjecting one or more precursors to a stimulus to produce a second bead. In some embodiments, the stimulus is thermal energy or electromagnetic energy. In some embodiments, the stimulus is ultraviolet light.

[0013] Other aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, wherein only exemplary embodiments of the present disclosure are shown and described. It should be appreciated that the present disclosure can have other and different embodiments, and that several details thereof can be modified in various aspects, all without departing from the disclosure. Therefore, the drawings and description should be regarded as illustrative rather than restrictive in nature.

[0014] Incorporated by Reference

[0015] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. If 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

[0016] The novel features of the invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the invention may be obtained by reference to the following detailed description and the accompanying drawings (also referred to herein as "Figures and FIG.") which illustrate exemplary embodiments in which the principles of the invention are utilized, wherein:

[0017] Figure 1 An example of a microfluidic channel structure for dispensing single bioparticles is shown.

[0018] Figure 2An example of a microfluidic channel structure for delivering barcode-carrying beads to droplets is shown.

[0019] Figure 3 An example of a microfluidic channel structure for co-dispensing bioparticles and reagents is shown.

[0020] Figure 4 An example of a microfluidic channel structure for controlled dispensing of beads into discrete droplets is shown.

[0021] Figure 5 Shown are examples of microfluidic channel structures for increasing droplet generation throughput.

[0022] Figure 6 Another example of a microfluidic channel structure for increasing droplet generation throughput is shown.

[0023] Figure 7 An exemplary method of sample preparation for the present disclosure is shown.

[0024] Figure 8 Another exemplary method of sample preparation for the present disclosure is shown.

[0025] Fig. 9 An exemplary method for sample preparation of the present disclosure using cell beads is shown.

[0026] Fig.10 Another exemplary method for sample preparation of the present disclosure using cell beads is shown.

[0027] Fig.11 A computer system is shown that has been programmed or otherwise configured to implement the methods provided herein. DETAILED DESCRIPTION

[0028] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Many changes, variations and substitutions may be conceived by those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used.

[0029] Where values ​​are described as a range, it should be understood that such disclosure includes disclosure of all possible sub-ranges within that range, as well as specific values ​​falling within that range, whether or not a specific value or a specific sub-range is explicitly stated.

[0030] As used herein, the term "barcode" generally refers to a label or identifier that conveys or is capable of conveying information about an analyte. A barcode can be part of an analyte. A barcode can be independent of an analyte. In addition to the endogenous characteristics of an analyte (e.g., the size of an analyte or one or more terminal sequences), a barcode can be a label or combination of labels attached to an analyte (e.g., a nucleic acid molecule). A barcode can be unique. A barcode can have a variety of different formats. For example, a barcode can include: a polynucleotide barcode; a random nucleic acid and / or amino acid sequence; and a synthetic nucleic acid and / or amino acid sequence. A barcode can be attached to an analyte in a reversible or irreversible manner. Before, during, and / or after sample sequencing, a barcode can be added to a fragment of, for example, a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample. A barcode can allow real-time identification and / or quantification of individual sequencing reads.

[0031] As used herein, the term "real time" may refer to a response time of less than about 1 second, one tenth of a second, one hundredth of a second, millisecond, or less. The response time may be greater than 1 second. In some cases, real time may refer to simultaneous or substantially simultaneous processing, detection, or identification.

[0032] As used herein, the term "subject" generally refers to an animal, such as a mammal (e.g., a human) or poultry (e.g., a bird), or other organisms (e.g., a plant). The subject may be a vertebrate, a mammal, a rodent (e.g., a mouse), a primate, a monkey, or a human. Animals may include, but are not limited to, farm animals, sports animals, and pets. The subject may be a healthy or asymptomatic individual, an individual suffering from or suspected of suffering from a disease (e.g., cancer) or susceptible to a disease, and / or an individual in need of treatment or suspected of needing treatment. The subject may be a patient.

[0033] As used herein, the term "genome" generally refers to genomic information from a subject, which can be, for example, at least a portion or all of the subject's genetic information. The genome can be encoded in DNA or RNA. The genome can include coding regions (e.g., coding regions for proteins) and non-coding regions. The genome can include the sequence of all chromosomes together in an organism. For example, the human genome generally has a total of 46 chromosomes. The sequence of all these chromosomes together can constitute the human genome.

[0034] The terms "adapter(s), "linker(s)" and "tag(s)" can be used synonymously. The linker or tag can be coupled to the polynucleotide sequence to be "labeled" by any method including ligation, hybridization or other methods.

[0035] As used herein, the term "sequencing" generally refers to methods and techniques for determining the sequence of nucleotide bases in one or more polynucleotides. A polynucleotide can be, for example, a nucleic acid molecule such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (e.g., single-stranded DNA). Sequencing can be performed by a variety of currently available systems, such as, but not limited to, by Pacific Biosciences Oxford or Life Technologies (Ion ) sequencing system. Alternatively or additionally, sequencing can 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 can provide a plurality of raw genetic data corresponding to the genetic information of a subject (e.g., a person), such as generated by the system from a sample provided by the subject. In some instances, such systems provide sequencing reads (also referred to herein as "reads"). The reads can include a string of nucleic acid bases corresponding to the sequence of a nucleic acid molecule that has been sequenced. In some cases, the systems and methods provided herein can be used together with proteomic information.

[0036] As used herein, the term "bead" generally refers to a particle. A bead may be a solid or semi-solid particle. A bead may be a gel bead. A gel bead may include a polymer matrix (e.g., a matrix formed by polymerization or cross-linking). A bead may be formed of a polymer material. A bead may be magnetic or non-magnetic. A bead may be rigid. A bead may be flexible and / or compressible. A bead may be destructible or soluble.

[0037] As used herein, the term "sample" generally refers to a biological sample of a subject. A biological sample may contain any number of macromolecules, such as cellular macromolecules. A biological sample may be a nucleic acid sample or a protein sample. A biological sample may also be a carbohydrate sample or a lipid sample. A biological sample may be derived from another sample. A sample may be a tissue sample, such as a biopsy, a core biopsy, a needle aspirate, or a fine needle aspirate. A sample may be a fluid sample, such as a blood sample, a urine sample, or a saliva sample. A sample may be a skin sample. A sample may be a cheek swab. A sample may be a plasma or serum sample. A sample may be a cell-free or cell free sample. A cell-free sample may include extracellular polynucleotides. Extracellular polynucleotides may be separated from a body sample, and the body sample may be selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal secretions, sputum, feces, and tears.

[0038] As used herein, the term "biological particle" generally refers to a discrete biological system derived from a biological sample. A biological particle can be a virus. A biological particle can be a cell or a cell derivative. A biological particle can be an organelle. A biological particle can be a rare cell from a cell population. A biological particle can be any type of cell, including but not limited to prokaryotic cells, eukaryotic cells, bacteria, fungi, plants, mammalian or other animal cell types, mycoplasmas, normal tissue cells, tumor cells, or any other cell type, whether derived from a unicellular or multicellular organism. A biological particle can be or can include a matrix (e.g., a gel or polymer matrix) containing a cell or one or more components from a cell (e.g., a cell bead), such as DNA, RNA, organelles, proteins, or any combination thereof from a cell. A biological particle can be obtained from a subject's tissue. A biological particle can be a hardened cell. Such a hardened cell may or may not include a cell wall or a cell membrane. A biological particle may include one or more components of a cell, but may not include other components of a cell. An example of such a component is a nucleus or an organelle. A cell can be a living cell. Living cells may be capable of being cultured, for example, when encapsulated in a gel or polymer matrix, or when comprising a gel or polymer matrix.

[0039] As used herein, the term "macromolecular component" generally refers to a macromolecule contained within a bioparticle. A macromolecular component may include a nucleic acid. A macromolecular component may include DNA. A macromolecular component may include RNA. The RNA may be a messenger RNA (mRNA), a ribosomal RNA (rRNA), or a transfer RNA (tRNA). The RNA may be a transcript. A macromolecular component may include a protein. A macromolecular component may include a peptide. A macromolecular component may include a polypeptide.

[0040] As used herein, the term "molecular tag" generally refers to a molecule capable of binding to a macromolecular component. A molecular tag can bind to a macromolecular component with high affinity. A molecular tag can bind to a macromolecular component with high specificity. A molecular tag can comprise a nucleotide sequence. A molecular tag can comprise a nucleic acid sequence. A nucleic acid sequence can be at least a portion or all of a molecular tag. A molecular tag can be a nucleic acid molecule or can be a portion of a nucleic acid molecule. A molecular tag can be an oligonucleotide or a polypeptide. A molecular tag can comprise a DNA aptamer. A molecular tag can be or comprise a primer. A molecular tag can be or comprise a protein. A molecular tag can comprise a polypeptide. A molecular tag can be a barcode.

[0041] Provided herein is a method for sample preparation and / or analysis, which can be performed in a distribution area such as a droplet or a hole, and can include one or more continuous operations. Such continuous operations can be performed in a single distribution area or in multiple distribution areas. Each distribution area can include, for example, a reagent. The reagent can be included in one or more destructible beads. In some cases, one or more precursors can be produced by destructible beads so that the precursor can be used, for example, to produce one or more other beads.

[0042] Sample distribution

[0043] In one aspect, the systems and methods described herein provide for compartmentalization, deposition or distribution of the macromolecular constituent contents of a single bioparticle into discrete compartments or distribution zones (interchangeably referred to herein as distribution zones), wherein each distribution zone maintains its own contents separate from the contents of other distribution zones. A distribution zone may be a droplet in an emulsion. A distribution zone may include one or more other distribution zones.

[0044] The distribution area of ​​the present disclosure may include biological particles and / or their macromolecular components. The distribution area may include one or more gel beads. The distribution area may include one or more cell beads. The distribution area may include a single gel bead, a single cell bead, or a single cell bead and a single gel bead. The cell bead can be, for example, one or more of biological particles and / or their macromolecular components encapsulated in a gel or polymer matrix by polymerization of a droplet containing biological particles and a precursor that can be polymerized or gelled. A unique identifier (such as a barcode) can be injected into the droplet before, after, or simultaneously with the droplet generation, such as by a microcapsule (such as a bead), as further described below. A microfluidic channel network (e.g., on a chip) can be used to generate a distribution area as described herein. Alternative mechanisms can also be used in the distribution of single biological particles, including a porous membrane, through which an aqueous mixture of cells is squeezed into a non-aqueous fluid.

[0045] The distribution area can flow in the fluid stream. The distribution area may include, for example, microvesicles having an external barrier around the center or core of the internal fluid. In some cases, the distribution area may include a porous matrix that can entrain and / or retain material in its matrix. The distribution area may include droplets of an aqueous fluid in a non-aqueous continuous phase (e.g., an oil phase). The distribution area may include droplets of a first phase in a second phase, wherein the first phase and the second phase are immiscible. A variety of different containers are described in, for example, U.S. Patent Application Publication 2014 / 0155295, which is incorporated herein by reference in its entirety for all purposes. An emulsion system for producing stable droplets in a non-aqueous or oily continuous phase is described in, for example, U.S. Patent Application Publication 2010 / 0105112, which is incorporated herein by reference in its entirety for all purposes.

[0046] With regard to the droplets in the emulsion, in a non-limiting example, the distribution of a single biological particle into a discrete distribution area can be achieved by introducing the flow of the biological particles in the aqueous fluid into the flow of the non-aqueous fluid, so that droplets are produced at the confluence of the two streams. By providing an aqueous stream under a certain concentration and / or flow rate of biological particles, the occupancy rate of the resulting distribution area (for example, the number of biological particles in each distribution area) can be controlled. In the case of using a single biological particle distribution area, the relative flow rate of the immiscible fluid can be selected so that, on average, the distribution area can contain less than one biological particle per distribution area to ensure that those distribution areas occupied are mainly occupied individually. In some cases, the distribution area in a plurality of distribution areas can contain at most one biological particle (for example, beads, cells or cell materials). In some embodiments, the relative flow rate of the fluid can be selected so that most distribution areas are occupied, for example, only a small portion of unoccupied distribution areas is allowed. Streams and channel architectures can be controlled to ensure that a given number of individually occupied distribution areas, unoccupied distribution areas less than a certain level, and / or multiple occupied distribution areas less than a certain level.

[0047] Figure 1 An example of a microfluidic channel structure 100 for dispensing a single biological particle is shown. The channel structure 100 may include channel segments 102, 104, 106, and 108 connected at a channel junction 110. In operation, a first aqueous fluid 112 including suspended biological particles (or cells) 114 may be transported along the channel segment 102 into the junction 110, while a second fluid 116 that is immiscible with the aqueous fluid 112 is transported from each of the channel segments 104 and 106 to the junction 110 to produce discrete droplets 118, 120 of the first aqueous fluid 112, which flow into the channel segment 108 and flow away from the junction 110. The channel segment 108 may be fluidically coupled to an outlet reservoir that may store and / or collect the discrete droplets. The discrete droplets produced may include a single biological particle 114 (such as droplet 118). The discrete droplets produced may include more than one individual biological particle 114 (such as droplet 118). Figure 1 14). The discrete droplets may not contain biological particles 114 (e.g., droplet 120). Each discrete dispensing region may maintain its own contents (e.g., individual biological particles 114) separate from the contents of other dispensing regions.

[0048] The second fluid 116 may comprise an oil, such as a fluorinated oil, that includes a fluorosurfactant for stabilizing the resulting droplets (e.g., inhibiting subsequent coalescence of the resulting droplets 118, 120). Examples of particularly useful dispensing fluids and fluorosurfactants are described, for example, in U.S. Patent Application Publication No. 2010 / 0105112, which is incorporated herein by reference in its entirety for all purposes.

[0049] As will be understood, the channel segments described herein can be coupled to any of a variety of different fluid sources or receiving components, including reservoirs, pipelines, manifolds or fluid components of other systems. As will be understood, the microfluidic channel structure 100 may have other geometries. For example, the microfluidic channel structure may have more than one channel junction. For example, the microfluidic channel structure may have 2, 3, 4 or 5 channel segments, each of which carries biological particles, cell beads and / or gel beads that meet at the channel junction). Fluid can be guided to flow along one or more channels or reservoirs by one or more fluid flow units. The fluid flow unit may include a compressor (e.g., providing positive pressure), a pump (e.g., providing negative pressure), an actuator, etc. to control the flow of fluid. Fluid can also or otherwise be controlled by applied pressure difference, centrifugal force, electric pumping, vacuum, capillary or gravity flow, etc.

[0050] The generated droplets may include two subsets of droplets: (1) occupied droplets 118, which contain one or more biological particles 114, and (2) unoccupied droplets 120, which do not contain any biological particles 114. The occupied droplets 118 may include single-occupied droplets (having one biological particle) and multi-occupied droplets (having more than one biological particle). As described elsewhere herein, in some cases, the majority of occupied dispensing areas may include no more than one biological particle per occupied dispensing area, and some of the generated dispensing areas may be unoccupied (by any biological particle). However, in some cases, some of the occupied dispensing areas may include more than one biological particle. In some cases, the dispensing process may be controlled so that less than about 25% of the occupied dispensing areas contain more than one biological particle, and in many cases, less than about 20% of the occupied dispensing areas have more than one biological particle, and in some cases, less than about 10% or even less than about 5% of the occupied dispensing areas include more than one biological particle per dispensing area.

[0051] In some cases, it may be desirable to minimize the generation of an excessive number of empty allocation areas, for example to reduce costs and / or improve efficiency. Although such minimization can be achieved by providing a sufficient number of bioparticles (e.g., bioparticles 114) at the allocation junction 110, for example to ensure that at least one bioparticle is enclosed in the allocation area, the Poisson distribution can be expected to increase the number of allocation areas containing multiple bioparticles. Therefore, in the case of obtaining a single occupied allocation area, up to 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 allocation area can be unoccupied.

[0052] In some cases, the flow of one or more bioparticles (e.g., in channel segment 102) or the flow of other fluids (e.g., in channel segments 104, 106) directed to the distribution junction can be controlled so that in many cases, no more than about 50% of the generated distribution area, no more than about 25% of the generated distribution area, or no more than about 10% of the generated distribution area is unoccupied. These flows can be controlled so as to present a non-Poisson distribution of a single occupied distribution area while providing a lower level of unoccupied distribution area. The above range of unoccupied distribution areas can be achieved while still providing any of the above single occupancy rates. For example, in many cases, using the systems and methods described herein can produce a resulting distribution area with a multiple occupancy 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 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 unoccupied distribution area.

[0053] As will be appreciated, the above occupancy also applies to distribution areas comprising bioparticles and additional reagents, including but not limited to microcapsules carrying barcoded nucleic acid molecules (e.g., oligonucleotides) (see Figure 2 Description). Occupancy of the distribution area (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the distribution area) can include microcapsules (e.g., beads) and bioparticles containing barcoded nucleic acid molecules.

[0054] In another aspect, in addition to droplet-based distribution or as an alternative to droplet-based distribution, the bioparticles can be encapsulated in microcapsules, which include a shell, layer or porous matrix in which one or more individual bioparticles or a small amount of bioparticles are entrained. The microcapsules may include other agents. Encapsulation of bioparticles can be performed by a variety of methods. Such methods combine an aqueous fluid containing bioparticles with a polymer precursor material that can form a gel or other solid or semi-solid matrix after a specific stimulus is applied to the polymer precursor. Such stimulation can include, for example, thermal stimulation (heating or cooling), light stimulation (e.g., by photocuring), chemical stimulation (e.g., by crosslinking, polymerization initiation of precursors (e.g., by added initiators)), etc., and / or combinations of the above.

[0055] The preparation of microcapsules containing biological particles can be performed by a variety of methods. For example, an air knife droplet or an aerosol generator can be used to dispense droplets of a precursor fluid into a gelling solution to form microcapsules containing individual biological particles or a small number of biological particles. Similarly, a membrane-based encapsulation system can be used to produce microcapsules containing encapsulated biological particles as described herein. The microfluidic system of the present disclosure, such as Figure 1As shown, it can be easily used to encapsulate cells as described herein. In particular, and with reference to Figure 1 , an aqueous fluid 112 containing (i) biological particles 114 and (ii) polymer precursor material (not shown) flows into a channel junction 110, where the aqueous fluid 112 is distributed into droplets 118, 120 by the flow of a non-aqueous fluid 116. In terms of the encapsulation method, the non-aqueous fluid 116 may also include an initiator (not shown) to cause polymerization and / or cross-linking of the polymer precursor to form microcapsules containing the entrained biological particles. Examples of polymer precursor / initiator pairs include those described in U.S. Patent Application Publication No. 2014 / 0378345, which is incorporated herein by reference in its entirety for all purposes.

[0056] For example, where the polymer precursor material comprises a linear polymer material (e.g., linear polyacrylamide, PEG, or other linear polymer material), the activator may comprise a crosslinker or a chemical that activates the crosslinker in the formed droplets. Similarly, for polymer precursors comprising polymerizable monomers, the activator may comprise a polymerization initiator. For example, in some cases, when the polymer precursor comprises a mixture of acrylamide monomers and N,N'-bis-(acryloyl)cystamine (BAC) comonomers, an agent such as tetraethylmethanediamine (TEMED) may be provided within the second fluid stream 116 in channel segments 104 and 106, which may initiate copolymerization of acrylamide and BAC into a crosslinked polymer network or hydrogel.

[0057] After the second fluid stream 116 contacts the first fluid stream 112 at the junction 110, during droplet formation, TEMED can diffuse from the second fluid 116 into the aqueous fluid 112 containing 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 in the form of solid or semi-solid beads or particles encapsulating the cells 114. Although polyacrylamide encapsulation is described, other "activatable" encapsulation compositions can also be used in the context of the methods and compositions described herein. For example, alginate droplets are formed and then exposed to divalent metal ions (e.g., Ca 2+ ions), can be used as an encapsulation method using the method. Similarly, agarose droplets can also be converted into capsules by temperature-based gelation (e.g., after cooling, etc.).

[0058] In some cases, the encapsulated bioparticles can be selectively released from the microcapsules, for example, over time or after application of a specific stimulus that degrades the microcapsules sufficiently to allow the bioparticles (e.g., cells) or other contents thereof to be released from the microcapsules, for example, into a distribution region (e.g., a droplet). For example, in the case of the polyacrylamide polymers described above, degradation of the microcapsules can be accomplished by introducing an appropriate reducing agent such as DTT to cleave the disulfide bonds of the cross-linked polymer matrix. See, for example, U.S. Patent Application Publication No. 2014 / 0378345, which is incorporated herein by reference in its entirety for all purposes.

[0059] Bioparticles can be subjected to other conditions sufficient to polymerize or gel precursors. Conditions sufficient to polymerize or gel precursors may include exposure to heating, cooling, electromagnetic radiation and / or light. Conditions sufficient to polymerize or gel precursors may include any conditions sufficient to polymerize or gel precursors. After polymerization or gelation, polymers or gels can be formed around bioparticles. Polymers or gels can diffusely penetrate into chemical or biochemical reagents. Polymers or gels cannot diffusely penetrate into the macromolecular components of bioparticles. In this way, polymers or gels can allow bioparticles to undergo chemical or biochemical operations while spatially limiting macromolecular components to the droplet region defined by polymers or gels. Polymers or gels may include one or more of disulfide cross-linked polyacrylamide, agarose, alginate, polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate, PEG-thiol, PEG-azide, PEG-alkyne, other acrylates, chitosan, hyaluronic acid, collagen, fibrin, gelatin or elastin. Polymers or gels may include any other polymers or gels. The polymer or gel may be a hydrogel. The hydrogel may be a reversible hydrogel. Methods for producing reversible hydrogels are provided in, for example, EP 2343342, U.S. Pat. No. 5,883,211, Aliyar et al. 2005, Wang et al., 2015, and Rosales et al., 2016, each of which is incorporated herein by reference in its entirety for all purposes.

[0060] The polymer or gel can be functionalized to bind target analytes, such as nucleic acids, proteins, carbohydrates, lipids or other analytes. The polymer or gel can be polymerized or gelled by a passive mechanism. The polymer or gel can be stable under alkaline conditions or at elevated temperatures. The polymer or gel can have mechanical properties similar to those of beads. For example, the polymer or gel can have a size similar to that of beads. The polymer or gel can have a mechanical strength (such as tensile strength) similar to that of beads. The density of the polymer or gel can be lower than that of oil. The density of the polymer or gel can be roughly similar to that of a buffer. The polymer or gel can have an adjustable pore size. The pore size can be selected to, for example, retain denatured nucleic acids. The pore size can be selected to maintain diffusion permeability to exogenous chemicals such as sodium hydroxide (NaOH) and / or endogenous chemicals such as inhibitors. The polymer or gel can be biocompatible. The polymer or gel can maintain or enhance cell viability. The polymer or gel can be biochemically compatible. The polymer or gel can be polymerized and / or depolymerized in a thermal, chemical, enzymatic and / or photo manner.

[0061] The polymer may comprise poly(acrylamide-co-acrylic acid) cross-linked with disulfide bonds. The preparation of the polymer may comprise a two-step reaction. In a first activation step, the poly(acrylamide-co-acrylic acid) may be exposed to an acylating agent to convert the carboxylic acid into an ester. For example, the poly(acrylamide-co-acrylic acid) may be exposed to 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride (DMTMM). The polyacrylamide-co-acrylic acid may be exposed to other salts of 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine. In a second cross-linking step, the ester formed in the first step may be exposed to a disulfide cross-linking agent. For example, the ester may be exposed to cystamine (2,2'-disulfide bis(ethylamine)). After these two steps, the bioparticles may be surrounded by polyacrylamide chains linked together by disulfide bridges. In this way, the bioparticles may be encapsulated within or comprise a gel or matrix (e.g., a polymer matrix) to form "cell beads". Cell beads can contain biological particles (e.g., cells) or macromolecular components of biological particles (e.g., RNA, DNA, proteins, etc.). Cell beads can include a single cell or multiple cells, or a derivative of a single cell or multiple cells. For example, after lysing and washing the cells, inhibitory components can be washed away from the cell lysate, and the macromolecular components can be combined into cell beads. The systems and methods disclosed herein are applicable to cell beads (and / or droplets or other distribution areas) containing biological particles and cell beads (and / or droplets or other distribution areas) containing macromolecular components of biological particles.

[0062] Compared with the biological particles distributed based on droplets, encapsulated biological particles can provide some potential advantages of easier storage and more portable. In addition, in some cases, it is desirable to allow the biological particles to be incubated for a selected period of time before analysis, for example, in order to characterize the changes of such biological particles over time in the presence or absence of different stimuli. In such cases, encapsulation can allow longer incubations than distribution in emulsion droplets, but in some cases, the biological particles distributed by droplets can also be incubated for different time periods, for example, at least 10 seconds, at least 30 seconds, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 5 hours, or at least 10 hours or longer. The encapsulation of biological particles can constitute the distribution of biological particles, and other reagents are distributed therein. Alternatively or additionally, the encapsulated biological particles can be easily deposited in other distribution areas (e.g., droplets) as described herein.

[0063] Beads

[0064] The distribution area may include one or more unique identifiers, such as barcodes. The barcode may be delivered in advance, subsequently or simultaneously to a distribution area that maintains compartmentalization or distribution of bioparticles. For example, the barcode may be injected into the droplet before, after or simultaneously with droplet generation. The barcode may be delivered to a specific distribution area allowing the characteristics of individual bioparticles to be attributed to a specific distribution area later. The barcode may be delivered to the distribution area by any suitable mechanism, such as on a nucleic acid molecule (e.g., oligonucleotide). The barcoded nucleic acid molecule may be delivered to the distribution area by microcapsules. In some cases, the barcoded nucleic acid molecule may initially associate with the microcapsule, and then release from the microcapsule after applying a stimulus that dissociates the nucleic acid molecule or releases from the microcapsule. In some cases, the microcapsule may include beads. Beads are described in further detail below.

[0065] Figure 2An example of a microfluidic channel structure 200 for delivering beads carrying barcodes to droplets is shown. The channel structure 200 may include channel segments 201, 202, 204, 206, and 208 connected at a channel junction 210. In operation, the channel segment 201 may transport an aqueous fluid 212 containing a plurality of beads 214 (e.g., having nucleic acid molecules, oligonucleotides, molecular tags) along the channel segment 201 to the junction 210. The plurality of beads 214 may be derived from a suspension of beads. For example, the channel segment 201 may be connected to a reservoir containing an aqueous suspension of beads 214. The channel segment 202 may transport an aqueous fluid 212 containing a plurality of biological particles 216 along the channel segment 202 to the junction 210. The plurality of biological particles 216 may be derived from a suspension of biological particles. For example, the channel segment 202 may be connected to a reservoir containing an aqueous suspension of biological particles 216. In some cases, the aqueous fluid 212 in 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 (e.g., oil) that is immiscible with the aqueous fluid 212 can be delivered to the junction 210 from each of the channel segments 204 and 206. After the aqueous fluid 212 from each of the channel segments 201 and 202 meets the second fluid 218 from each of the channel segments 204 and 206 at the channel junction 210, the aqueous fluid 212 can be distributed in the second fluid 218 in the form of discrete droplets 220 and flow out of the junction 210 along the channel segment 208. The channel segment 208 can deliver the discrete droplets to an outlet reservoir fluidly coupled to the channel segment 208, where they are collected.

[0066] Alternatively, channel segments 201 and 202 may meet at another junction upstream of junction 210. At such a junction, the beads and bioparticles may form a mixture that is directed along another channel to junction 210 to produce droplets 220. The mixture may provide beads and bioparticles in an alternating manner, such that, for example, a droplet contains a single bead and a single bioparticle.

[0067] Beads, bioparticles and droplets can flow along the channel in a substantially conventional flow pattern (e.g., at a conventional flow rate). Such conventional flow patterns may allow droplets to include a single bead and a single bioparticle. Such conventional flow patterns may allow droplets to have an occupancy rate greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% (e.g., droplets with beads and bioparticles). Such conventional flow patterns and devices that can be used to provide such conventional flow patterns are provided in, for example, U.S. Patent Publication 2015 / 0292988, which is incorporated herein by reference in its entirety.

[0068] The second fluid 218 may include an oil, such as a fluorinated oil, which includes a fluorinated surfactant for stabilizing the resulting droplets (eg, inhibiting subsequent coalescence of the resulting droplets 220).

[0069] The generated discrete droplets may include individual bioparticles 216. The generated discrete droplets may include beads 214 carrying barcodes or other reagents. The generated discrete droplets may include individual bioparticles and beads carrying barcodes, such as droplet 220. In some cases, the discrete droplets may include more than one individual bioparticle or no bioparticles. In some cases, the discrete droplets may include more than one bead or no beads. The discrete droplets may be unoccupied (e.g., no beads, no bioparticles).

[0070] Advantageously, dispensing discrete droplets of bioparticles and barcoded beads may effectively allow attribution of barcodes to macromolecular components of the bioparticles within the dispensing region.The contents of a dispensing region may be kept discrete from the contents of other dispensing regions.

[0071] As will be understood, the channel segments described herein can be coupled to any of a variety of different fluid sources or receiving components, including reservoirs, pipelines, manifolds or fluid components of other systems. As will be understood, the microfluidic channel structure 200 can have other geometric shapes. For example, the microfluidic channel structure can have more than one channel junction. For example, the microfluidic channel structure can have 2,3,4 or 5 channel segments, each of which carries the beads that meet at the channel junction. Fluid can be guided to flow along one or more channels or reservoirs by one or more fluid flow units. The fluid flow unit may include a compressor (e.g., providing positive pressure), a pump (e.g., providing negative pressure), an actuator, etc. to control the flow of fluid. Fluid can also or otherwise be controlled by applied pressure difference, centrifugal force, electric pumping, vacuum, capillary or gravity flow, etc.

[0072] The beads can be porous, non-porous, solid, semi-solid, semi-fluid, fluid and / or combinations thereof. In some cases, the beads can be soluble, destructible and / or degradable. In some cases, the beads can be non-degradable. In some cases, the beads can be gel beads. The gel beads can be hydrogel beads. The gel beads can be formed by molecular precursors, such as polymers or monomeric substances. The molecular precursors can be derived from the destruction of the gel beads. The semi-solid beads can be liposome beads. The solid beads can contain metals, including iron oxide, gold and silver. In some cases, the beads can be silica beads. In some cases, the beads can be rigid. In other cases, the beads can be flexible and / or compressible.

[0073] The beads may have any suitable shape. Examples of bead shapes include, but are not limited to, spherical, non-spherical, elliptical, oblong, amorphous, circular, cylindrical, and variations thereof.

[0074] The beads may be of uniform or non-uniform size. In some cases, the diameter of the beads may be at least about 1 micrometer (μm), 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1 mm or more. In some cases, the diameter of the beads may be less than about 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, 1 mm or less. In some cases, the diameter of the beads may be in the range of about 40-75 μm, 30-75 μm, 20-75 μm, 40-85 μm, 40-95 μm, 20-100 μm, 10-100 μm, 1-100 μm, 20-250 μm, or 20-500 μm.

[0075] In certain aspects, the beads can be provided as a population of beads or a plurality of beads having a relatively monodisperse size distribution. Where it is desired to provide a relatively consistent amount of reagent within a dispensing region, maintaining relatively consistent bead characteristics (such as size) can contribute to overall consistency. In particular, the beads described herein can have a size distribution having a coefficient of variation of 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.

[0076] The beads may comprise natural and / or synthetic materials. For example, the beads may comprise natural polymers, synthetic polymers, or 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, silk, polyhydroxyalkanoates, chitosan, dextran, collagen, carrageenan, psyllium ovata, gum arabic, agar, gelatin, shellac, karaya gum, xanthan gum, corn gum, guar gum, karaya gum, agarose, alginic acid, alginate, or its natural polymers. Examples of synthetic polymers include acrylics, nylons, silicones, spandex, viscose rayon, polycarboxylic acids, polyvinyl acetate, polyacrylamide, polyacrylates, polyethylene glycol, polyurethane, poly(ethylene-propylene), poly(ethylene-butylene), polylactic acid, silica, polystyrene, polyacrylonitrile, polybutadiene, polycarbonate, polyethylene, polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethylene oxide), poly(ethylene terephthalate), polyethylene, polyisobutylene, poly(methyl methacrylate), poly(formaldehyde), polyoxymethylene, polypropylene, polystyrene, poly(tetrafluoroethylene), poly(vinyl acetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidene chloride), poly(vinylidene fluoride), poly(vinyl fluoride), and / or combinations thereof (e.g., copolymers). Beads may also be formed from materials other than polymers, including lipids, micelles, ceramics, glass ceramics, material composites, metals, other inorganic materials, and the like.

[0077] In some cases, beads may contain molecular precursors (e.g., monomers or polymers), which can form polymer networks by polymerization of molecular precursors. In some cases, precursors can be already polymerized substances, which can be further polymerized by, for example, chemical crosslinking. In some cases, precursors can include one or more of acrylamide or methacrylamide monomers, oligomers or polymers. In some cases, beads may include prepolymers, which are oligomers that can be further polymerized. For example, polyurethane beads can be prepared using prepolymers. In some cases, beads may contain a single polymer that can be further polymerized together. In some cases, beads can be generated by polymerization of different precursors so that they include mixed polymers, copolymers and / or block copolymers. In some cases, beads can include covalent bonds or ionic bonds between polymer precursors (e.g., monomers, oligomers, linear polymers), nucleic acid molecules (e.g., oligonucleotides), primers and other entities. In some cases, covalent bonds can be carbon-carbon bonds or thioether bonds.

[0078] The crosslinking may be permanent or reversible, depending on the specific crosslinking agent used. Reversible crosslinking may allow the polymer to linearize or dissociate under appropriate conditions. In some cases, reversible crosslinking may also allow reversible attachment of materials bound to the bead surface. In some cases, the crosslinking agent may form disulfide bonds. In some cases, the chemical crosslinking agent that forms disulfide bonds may be cystamine or modified cystamine.

[0079] In some cases, disulfide bonds can be formed between molecular precursor units (e.g., monomers, oligomers, or linear polymers) or precursors incorporated into the beads and nucleic acid molecules (e.g., oligonucleotides). For example, cystamine (including modified cystamine) is an organic reagent containing disulfide bonds that can be used as a cross-linking agent between individual monomers or polymer precursors of the beads. Polyacrylamide can be polymerized in the presence of cystamine or a substance containing cystamine (e.g., modified cystamine) to generate polyacrylamide gel beads containing disulfide bonds (e.g., chemically degradable beads containing a chemically reducible cross-linking agent). Disulfide bonds can cause the beads to be degraded (or dissolved) after the beads are exposed to a reducing agent.

[0080] In some cases, chitosan (a linear polysaccharide polymer) can be cross-linked with glutaraldehyde via hydrophilic chains to form beads. Cross-linking of chitosan polymers can be achieved by chemical reactions initiated by heat, pressure, pH changes, and / or radiation.

[0081] In some cases, the beads may include an acrydite portion, which can be used in some aspects to attach one or more nucleic acid molecules (e.g., barcode sequences, barcoded nucleic acid molecules, barcoded oligonucleotides, primers, or other oligonucleotides) to the beads. In some cases, the acrylamide phosphoramidite portion can refer to an acrylamide phosphoramidite analog generated by the reaction of acrylamide phosphoramidite with one or more substances, such as acrylamide phosphoramidite with other monomers and crosslinkers during a polymerization reaction. The acrydite portion can be modified to form a chemical bond with a substance to be attached, such as a nucleic acid molecule (e.g., a barcode sequence, a barcoded nucleic acid molecule, a barcoded oligonucleotide, a primer, or other oligonucleotide). The acrylamide phosphoramidite portion can be modified with a thiol group capable of forming a disulfide bond, or can be modified with a group that already contains a disulfide bond. A thiol or a disulfide (via disulfide exchange) can be used as an anchor for the substance to be attached, or another portion of the acrylamide phosphoramidite portion can be used for attachment. In some cases, the attachment may be reversible, such that when the disulfide bond is broken (eg, in the presence of a reducing agent), the attached substance is released from the bead. In other cases, the acrydite moiety may contain reactive hydroxyl groups that may be used for attachment.

[0082] Functionalization of beads for attachment of nucleic acid molecules (e.g., oligonucleotides) can be achieved by a variety of different methods, including activation of chemical groups within the polymer, incorporation of active or activatable functional groups into the polymer structure, or attachment at the prepolymer or monomer stage during bead production.

[0083] For example, a precursor (e.g., monomer, cross-linker) that is polymerized to form a bead may include an acrylamide phosphoramidite moiety such that when the bead is generated, the bead also includes the acrylamide phosphoramidite moiety. The acrydite moiety may be attached to a nucleic acid molecule (e.g., an oligonucleotide), which may include a priming sequence (e.g., a primer for amplifying a target nucleic acid, a random primer, a primer sequence for messenger RNA) and / or one or more barcode sequences. The one or more barcode sequences may include a sequence that is the same for all nucleic acid molecules coupled to a given bead and / or a sequence that is different for all nucleic acid molecules coupled to a given bead. Nucleic acid molecules may be incorporated into beads.

[0084] In some cases, the nucleic acid molecule may comprise a functional sequence, such as for attachment to a sequencing flow cell, such as for In some cases, the nucleic acid molecule may include a barcode sequence. In some cases, the primer may also include a unique molecular identifier (UMI). In some cases, the primer may include an R1 primer sequence for Illumina sequencing. In some cases, the primer may include an R2 primer sequence for Illumina sequencing. Examples of such nucleic acid molecules (e.g., oligonucleotides, polynucleotides, etc.) and uses thereof that can be used with the compositions, devices, methods and systems of the present disclosure are provided in U.S. Patent Publications 2014 / 0378345 and 2015 / 0376609, each of which is incorporated herein by reference in its entirety.

[0085] In some cases, precursors containing functional groups that are reactive or can be activated so as to become reactive can be polymerized with other precursors to generate gel beads containing activated or activatable functional groups. The functional groups can then be used to attach additional substances (e.g., disulfide linkers, primers, other oligonucleotides, etc.) to the gel beads. For example, some precursors containing carboxylic acid (COOH) groups can be copolymerized with other precursors to form gel beads that also contain COOH functional groups. In some cases, acrylic acid (a substance containing free COOH groups), acrylamide, and bis(acryloyl)cystamine can be copolymerized together to generate gel beads containing free COOH groups. The COOH groups of the gel beads can be activated (e.g., by 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)) to make them reactive (e.g., reactive with amine functional groups, where EDC / NHS or DMTMM is used for activation). The activated COOH groups can then be reacted with an appropriate substance (e.g., a substance containing an amine functional group, where the carboxylic acid group is activated to be reactive with the amine functional group) that contains a moiety attached to the beads.

[0086] Beads containing disulfide bonds in the polymer network can be functionalized with additional substances by reducing some of the disulfide bonds to free thiols. Disulfide bonds can be reduced by the action of, for example, a reducing agent (e.g., DTT, TCEP, etc.) to generate free thiol groups without dissolving the beads. The free thiols of the beads can then react with the free thiols of the substance or a substance containing another disulfide bond (e.g., by thiol-disulfide exchange) so that the substance can be connected to the beads (e.g., through the generated disulfide bonds). In some cases, the free thiols of the beads can react with any other suitable groups. For example, the free thiols of the beads can react with substances containing acrylamide phosphoramidite moieties. The free thiol groups of the beads can react with acrylamide phosphoramidite by Michael addition chemistry so that the substance containing acrylamide phosphoramidite is connected to the beads. In some cases, uncontrolled reactions can be prevented by including a thiol capping agent such as N-ethylmaleimide or iodoacetate.

[0087] The activation of disulfide bonds within the beads can be controlled so that only a small number of disulfide bonds are activated. For example, this can be controlled by controlling the concentration of the reducing agent used to generate free thiol groups and / or the concentration of the reagent used to form disulfide bonds in the bead polymerization. In some cases, a low concentration (e.g., a reducing agent molecule: gel bead ratio 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 1:1,000,000,000, 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) of reducing agent can be used for reduction. Controlling the number of disulfide bonds reduced to free thiols can be used to ensure the structural integrity of the beads during functionalization. In some cases, optically active agents (eg, fluorescent dyes) can be coupled to beads via free thiol groups of the beads and used to quantify the amount of free thiols present in the beads and / or to track the beads.

[0088] In some cases, it may be advantageous to add each part to the gel beads after the gel beads are formed. For example, adding oligonucleotides (for example, barcoded oligonucleotides) after the gel beads are formed can avoid the loss of material during the chain transfer termination period that may occur during the polymerization. In addition, smaller precursors (for example, monomers or cross-linking agents that do not include side chain groups and connecting parts) can be used for polymerization, and chain end growth can be hindered to a minimum due to the viscosity effect. In some cases, the functionalization after the gel beads are synthesized can minimize the exposure of the material to be loaded (for example, oligonucleotide) in the case of potential destroyers (for example, free radicals) and / or chemical environments. In some cases, the generated gel can have an upper critical solution temperature (UCST), which can allow temperature-driven bead swelling and collapse. During the subsequent functionalization of the oligonucleotide beads, this functional group can help oligonucleotides (for example, primers) to penetrate into the beads. Functionalization after production can also be used to control the load ratio of material in the beads, so that the variability of the load ratio is minimized. Substance loading can also be performed in a batch process, so that multiple beads can be functionalized with substances in a single batch.

[0089] The beads injected or otherwise introduced into the dispensing region may include a releasable, cleavable or reversibly attached barcode. The beads injected or otherwise introduced into the dispensing region may include an activatable barcode. The beads injected or otherwise introduced into the dispensing region may be degradable, destructible or soluble beads.

[0090] The barcode can be releasably, cleavably or reversibly attached to the beads, so that the barcode can be released or releasable by cracking the connection between the barcode molecule and the beads, or released by the degradation of the beads themselves below, thereby allowing the barcode to be approached by other reagents or can be approached by other reagents, or both. In a non-limiting example, it can be achieved by reducing disulfide bonds, using restriction enzymes, photoactivated cleavage or by other types of stimulation (e.g., chemical, heat, pH, enzyme, etc.) and / or reaction to crack, such as described elsewhere herein. Releasable barcodes are sometimes referred to as activatable because they can be used for reactions once released. Therefore, for example, an activatable barcode can be activated by releasing the barcode from the beads (or other suitable types of distribution areas described herein). Other activatable configurations are also contemplated in the context of the described methods and systems.

[0091] In addition to or as an alternative to a cleavable bond between a bead and an associated molecule, such as a nucleic acid molecule containing a barcode (e.g., a barcoded oligonucleotide), the bead can be degradable, destructible, or soluble either spontaneously or upon exposure to one or more stimuli (e.g., a temperature change, a pH change, exposure to a particular chemical or phase, exposure to light, a reducing agent, etc.). In some cases, the bead can be soluble such that a material component of the bead dissolves upon exposure to a particular chemical or environmental change (e.g., a changing temperature or pH change). In some cases, the gel bead can degrade or dissolve at elevated temperatures and / or alkaline conditions. In some cases, the bead can be thermally degradable such that the bead degrades when the bead is exposed to an appropriate temperature change (e.g., heating). Degradation or dissolution of a bead bound to a substance (e.g., a nucleic acid molecule, such as a barcoded oligonucleotide) can result in the release of the substance from the bead.

[0092] It can be understood from the above disclosure that the degradation of beads can refer to the dissociation of bound or entrained substances from the beads with and without degradation of the structure of the physical beads themselves. For example, the degradation of beads can involve the cleavage of cleavable bonds by one or more substances and / or methods described elsewhere herein. In another example, entrained substances can be released from the beads by, for example, an osmotic pressure difference caused by a change in the chemical environment. For example, the change in the pore size of the beads caused by the osmotic pressure difference can generally occur without degradation of the structure of the beads themselves. In some cases, the increase in pore size caused by the osmotic swelling of the beads can allow the release of entrained substances within the beads. In other cases, due to pore size shrinkage, the osmotic shrinkage of the beads may allow the beads to better retain entrained substances.

[0093] Degradable beads can be introduced into a distribution zone (e.g., a droplet or well in an emulsion) such that the beads degrade within the distribution zone and any associated substances (e.g., oligonucleotides) are released within the droplets when an appropriate stimulus is applied. Free substances (e.g., oligonucleotides, nucleic acid molecules) can interact with other reagents contained in the distribution zone. For example, polyacrylamide beads containing cystamine and connected to a barcode sequence via a disulfide bond can be combined with a reducing agent in a droplet of an oil-in-water emulsion. Within the droplet, the reducing agent can destroy various disulfide bonds, causing the beads to degrade and release the barcode sequence into the aqueous internal environment of the droplet. In another example, heating a droplet containing a barcode sequence bound to the beads in an alkaline solution can also cause the beads to degrade and release the attached barcode sequence into the aqueous internal environment of the droplet.

[0094] Any suitable number of molecular tag molecules (e.g., primers, barcoded oligonucleotides) can be associated with beads such that upon release from the beads, the molecular tag molecules (e.g., primers, e.g., barcoded oligonucleotides) are present in the distribution area at a predefined concentration. Such predefined concentrations can be selected to facilitate certain reactions (e.g., amplification) for generating sequencing libraries within the distribution area. In some cases, the predefined concentration of primers can be limited by the process of generating beads carrying nucleic acid molecules (e.g., oligonucleotides).

[0095] In some cases, beads can be non-covalently loaded with one or more reagents. Beads can be non-covalently loaded by, for example, subjecting beads to conditions sufficient to swell the beads, allowing reagents to have enough time to diffuse into the inside of the beads, and subjecting beads to conditions sufficient to deswell the beads. For example, the swelling of beads can be achieved by placing beads in a thermodynamically favorable solvent, subjecting beads to higher or lower temperatures, subjecting beads to higher or lower ion concentrations, and / or subjecting beads to an electric field. The swelling of beads can be achieved by various swelling methods. The deswelling of beads can be achieved by, for example, transferring beads to a thermodynamically unfavorable solvent, subjecting beads to lower or higher temperatures, subjecting beads to lower or higher ion concentrations, and / or removing electric fields. The deswelling of beads can be achieved by various deswelling methods. Transferring beads may cause the holes in the beads to shrink. Then shrinkage may hinder the reagent in the beads from diffusing out from the inside of the beads. The hindrance may be due to the spatial interaction between the reagent and the inside of the beads. Transfer can be completed in a microfluidic manner. For example, the transfer can be achieved by moving the beads from one co-flowing solvent stream to a different co-flowing solvent stream.The swelling properties and / or pore size of the beads can be adjusted by varying the polymer composition of the beads.

[0096] In some cases, the acrydite portion attached to the precursor, another substance attached to the precursor, or the precursor itself may contain an unstable bond, such as a chemical, heat or light sensitive bond, such as a disulfide bond, a UV sensitive bond, etc. Once the acrylamide phosphoramidite portion or other portion containing an unstable bond is incorporated into the bead, the bead may also contain an unstable bond. For example, an unstable bond can be used to reversibly link (e.g., covalently link) a substance (e.g., a barcode, a primer, etc.) to the bead. In some cases, for example, when the oligonucleotide hybridizes with a complementary sequence attached to the bead, the thermally unstable bond may include an attachment based on nucleic acid hybridization, such that thermal melting of the hybrid releases the oligonucleotide (e.g., a sequence containing a barcode) from the bead or microcapsule.

[0097] Adding multiple types of unstable bonds to gel beads can lead to the generation of beads that can respond to different stimuli. Each type of unstable bond may be sensitive to associated stimuli (e.g., chemical stimuli, light, temperature, enzymes, etc.), so that the release of substances attached to the beads by each unstable bond can be controlled by applying appropriate stimuli. Such functional groups can be used to release substances from gel beads in a controlled manner. In some cases, another substance containing an unstable bond can be connected to the gel beads by, for example, an activated functional group of the gel beads as described above after the gel beads are formed. As should be understood, the barcodes releasably, cleavably or reversibly attached to the beads described herein include barcodes that are released or releasable by the cleavage of the linkage between the barcode molecule and the beads, or barcodes that are released by the degradation of the beads themselves below, allowing the barcodes to be approached by other reagents or accessible by other reagents, or both.

[0098] As described herein, releasable barcodes may sometimes be referred to as activatable because they are available for reaction once released. Thus, for example, an activatable barcode may be activated by releasing the barcode from a bead (or other suitable type of dispensing area described herein). Other activatable configurations are also contemplated in the context of the described methods and systems.

[0099] In addition to thermally cleavable bonds, disulfide bonds, and UV-sensitive bonds, other non-limiting examples of labile bonds that can be coupled to a precursor or bead include an ester bond (e.g., cleavable with acid, base, or hydroxylamine), a vicinal diol bond (e.g., cleavable by sodium periodate), a Diels-Alder bond (e.g., cleavable by heat), a sulfone bond (e.g., cleavable by base), a silyl ether bond (e.g., cleavable by acid), a glycosidic bond (e.g., cleavable by amylase), a peptide bond (e.g., cleavable by a protease), or a phosphodiester bond (e.g., cleavable by a nuclease (e.g., DNase)). The bond can be cleaved by other nucleic acid molecule targeting enzymes, such as restriction enzymes (e.g., restriction endonucleases), as further described below.

[0100] During bead generation (e.g., during precursor polymerization), substances can be encapsulated in the beads. Such substances may or may not participate in polymerization. Such substances can enter the polymerization reaction mixture so that the resulting beads contain the substances after the beads are formed. In some cases, such substances can be added to the gel beads after formation. Such substances can include, for example, nucleic acid molecules (e.g., oligonucleotides), reagents for nucleic acid amplification reactions (e.g., primers, polymerases, dNTPs, cofactors (e.g., ionic cofactors), buffers) including those described herein, reagents for enzymatic reactions (e.g., enzymes, cofactors, substrates, buffers), reagents for nucleic acid modification reactions (e.g., polymerization, ligation or digestion) and / or reagents for template preparation (e.g., labeling) for one or more sequencing platforms (e.g., for of ). Such substances may include one or more enzymes described herein, including but not limited to polymerases, reverse transcriptases, restriction enzymes (e.g., endonucleases), transposases, ligases, proteinases K, DNA enzymes, etc. Such substances may include one or more reagents described elsewhere herein (e.g., cleavage agents, inhibitors, inactivators, chelators, stimulation). The capture of such substances may be controlled by the density of the polymer network generated during the polymerization of the precursor, the control of the ionic charge in the gel beads (e.g., by ionic substances connected to polymeric substances), or by the release of other substances. The encapsulated substances may be released from the beads after the beads are degraded and / or by applying a stimulus that can release the substances from the beads. Alternatively or additionally, the substances may be distributed in the distribution area (e.g., droplets) during or after the distribution area is formed. Such substances may include but are not limited to the above-mentioned substances that may also be encapsulated in the beads.

[0101] Degradable beads may contain one or more substances with labile bonds, such that when the beads / substances are exposed to an appropriate stimulus, the bonds break and the beads degrade. The labile bonds may be chemical bonds (e.g., covalent bonds, ionic bonds) or may be another type of physical interaction (e.g., van der Waals interactions, dipole-dipole interactions, etc.). In some cases, the cross-linking agent used to generate the beads may contain labile bonds. Upon exposure to appropriate conditions, the labile bonds may break and the beads degrade. For example, when polyacrylamide gel beads containing a cystamine cross-linking agent are exposed to a reducing agent, the disulfide bonds of the cystamine may break and the beads degrade.

[0102] Compared to non-degradable beads, when appropriate stimuli are applied to the beads, degradable beads can be used to release attached substances (e.g., nucleic acid molecules, barcode sequences, primers, etc.) from the beads faster. For example, for substances bound to the inner surface of porous beads or in the case of encapsulated substances, the substance may have a larger mobility after the beads are degraded and may be approached by other substances in the solution. In some cases, the substance may also be attached to the degradable beads by a degradable linker (e.g., a disulfide linker). The degradable linker may respond to the same stimulus as the degradable beads, or the two degradable substances may respond to different stimuli. For example, a barcode sequence may be attached to polyacrylamide beads containing cystamine by a disulfide bond. After the barcoded beads are exposed to a reducing agent, the beads degrade and the barcode sequence is released after the disulfide bond between the barcode sequence and the beads is broken and the disulfide bond of cystamine in the beads is broken.

[0103] It will be appreciated from the above disclosure that, although referred to as degradation of the beads, in many cases as described above, the degradation may refer to the dissociation of bound or entrained material from the beads with and without degradation of the structure of the physical bead itself. For example, entrained material may be released from the beads by, for example, an osmotic pressure differential caused by a change in the chemical environment. For example, changes in the pore size of the beads caused by an osmotic pressure differential may generally occur without degradation of the structure of the beads themselves. In some cases, the increase in pore size caused by the osmotic swelling of the beads may allow the release of entrained material within the beads. In other cases, the osmotic shrinkage of the beads may allow the beads to better retain entrained material due to shrinkage of the pore size.

[0104] In the case of providing degradable beads, it may be beneficial to avoid exposing such beads to one or more stimuli that cause such degradation before a given time, so as to avoid premature bead degradation and problems caused by such degradation, including, for example, poor flow characteristics and aggregation. For example, in the case where the beads include reducible cross-linking groups such as disulfide groups, it is desirable to avoid contacting such beads with reducing agents such as DTT or other disulfide cleavage agents. In such cases, the treatment of the beads described herein will not provide a reducing agent, such as DTT, in some cases. Because reducing agents are generally provided in commercial enzyme preparations, it may be necessary to provide an enzyme preparation that does not contain a reducing agent (or does not contain DTT) when treating the beads described herein. Examples of such enzymes include, for example, polymerase preparations, reverse transcriptase preparations, ligase preparations, and many other enzyme preparations that can be used to treat the beads described herein. The term "reducing agent-free" or "DTT-free" preparation may refer to a preparation having a lower range of less than about 1 / 10, less than about 1 / 50, or even less than about 1 / 100 for such materials used to degrade these beads. For example, for DTT, a formulation without a reducing agent may 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 may be undetectable.

[0105] A number of chemical triggers may be used to trigger degradation of the beads. Examples of these chemical changes may include, but are not limited to, pH-mediated changes in the integrity of components within the beads, degradation of bead components by cleavage of cross-links, and depolymerization of bead components.

[0106] In some embodiments, beads can be formed by materials comprising degradable chemical crosslinkers (e.g., BAC or cystamine). The degradation of such degradable crosslinkers can be accomplished by many mechanisms. In some instances, beads can be contacted with chemical degradation agents that can induce oxidation, reduction, or other chemical changes. For example, chemical degradation agents can be reducing agents, such as dithiothreitol (DTT). Other examples of reducing agents can include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), tris(2-carboxyethyl)phosphine (TCEP), or a combination thereof. Reducing agents can degrade the disulfide bonds formed between the gel precursors that form beads, and thus degrade beads. In other cases, changes in the pH of the solution (e.g., an increase in pH) can trigger the degradation of beads. In other cases, exposure to an aqueous solution (e.g., water) can trigger hydrolytic degradation, and thus trigger the degradation of beads.

[0107] After applying a thermal stimulus, the beads can also be induced to release their contents. Changes in temperature can result in various changes in the beads. For example, heat can cause solid beads to liquefy. Changes in heat can result in the melting of beads, causing a portion of the beads to degrade. In other cases, heat can increase the internal pressure of the bead components, causing the beads to rupture or explode. Heat can also act on thermosensitive polymers used as materials for building beads.

[0108] Any suitable reagent can degrade the beads. In some embodiments, changes in temperature or pH can be used to degrade thermosensitive or pH-sensitive bonds in the beads. In some embodiments, chemical degradation agents can be used to degrade chemical bonds in the beads by oxidation, reduction or other chemical changes. For example, the chemical degradation agent can be a reducing agent, such as DTT, wherein DTT can degrade the disulfide bonds formed between the cross-linking agent and the gel precursor, thereby degrading the beads. In some embodiments, a reducing agent can be added to degrade the beads, and the reducing agent can release or not release the beads. Examples of reducing agents can include dithiothreitol (DTT), β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), tris (2-carboxyethyl) phosphine (TCEP) or a combination thereof. The reducing agent can be present at a concentration of about 0.1mM, 0.5mM, 1mM, 5mM, 10mM. The reducing agent may be present at a concentration of at least about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, or greater than 10 mM. The reducing agent may be present at a concentration of up to about 10 mM, 5 mM, 1 mM, 0.5 mM, 0.1 mM, or less.

[0109] Any suitable number of molecular tag molecules (e.g., primers, barcoded oligonucleotides) can be associated with beads so that after release from the beads, the molecular tag molecules (e.g., primers, e.g., barcoded oligonucleotides) are present in the distribution area at a predefined concentration. Such predefined concentrations can be selected to facilitate certain reactions (e.g., amplification) for generating sequencing libraries within the distribution area. In some cases, the predefined concentration of primers can be limited by the process of generating beads carrying oligonucleotides.

[0110] Although the above description has been made with respect to providing substantially individually occupied allocation areas Figure 1 and Figure 2, but in some cases, it may be desirable to provide, for example, multiple occupied distribution areas containing two, three, four or more cells and / or microcapsules (e.g., beads) including barcoded nucleic acid molecules (e.g., oligonucleotides) within a single distribution area. Thus, as described above, the flow characteristics of the fluid containing the biological particles and / or beads and the distribution fluid can be controlled to provide such multiple occupied distribution areas. In particular, the flow parameters can be controlled to provide a given occupancy of a distribution area greater than about 50%, greater than about 75%, and in some cases greater than about 80%, 90%, 95%, or more.

[0111] In some cases, additional microcapsules may be used to deliver additional reagents to the distribution area. In such cases, it may be advantageous to introduce different beads from different bead sources (e.g., containing different association reagents) into such a common channel or droplet generation junction (e.g., junction 210) through different channel inlets. In such cases, the flow and frequency of different beads entering the channel or junction can be controlled to provide a certain ratio of microcapsules from each source while ensuring a given pairing or combination of such beads with a given number of bioparticles into the distribution area (e.g., one bioparticle and one bead per distribution area).

[0112] The dispensing zones described herein may comprise small volumes, e.g., less than about 10 microliters (μL), 5 μL, 1 μL, 900 picoliters (pL), 800 pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 20 pL, 10 pL, 1 pL, 500 nanoliters (nL), 100 nL, 50 nL, or less.

[0113] For example, in the case of a droplet-based dispensing zone, the droplets may have a total volume of less than about 1000 pL, 900 pL, 800 pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 20 pL, 10 pL, 1 pL or less. In the case of co-dispensing with microcapsules, it should be understood that the volume of sample fluid in the dispensing zone (e.g., including co-dispensed biological particles and / or beads) may be less than about 90%, 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 volumes.

[0114] As described elsewhere herein, material distribution can produce distribution area group or multiple distribution areas.In such cases, any suitable number of distribution areas can be produced or otherwise provided.For example, at least about 1,000 distribution areas, at least about 5,000 distribution areas, at least about 10,000 distribution areas, at least about 50,000 distribution areas, at least about 100,000 distribution areas, at least about 500,000 distribution areas, at least about 1,000,000 distribution areas, at least about 5,000,000 distribution areas, at least about 10,000,000 distribution areas, at least about 50,000,000 distribution areas, at least about 100,000,000 distribution areas, at least about 50,000,000 distribution areas, at least about 100,000,000 distribution areas, at least about 500,000,000 distribution areas, at least about 1,000,000,000 distribution areas or more distribution areas can be produced or otherwise provided. Furthermore, the plurality of allocations may include unoccupied allocations (eg, empty allocations) and occupied allocations.

[0115] Reagents

[0116] According to certain aspects, the biological particles can be dispensed together with a lysis reagent to release the contents of the biological particles within the dispensing region. In such cases, the lysis reagent can be contacted with the biological particle suspension at the same time or just before the biological particles are introduced into the dispensing junction / droplet generation region (e.g., junction 210), for example, through another channel or multiple channels upstream of the channel junction. According to other aspects, additionally or alternatively, the biological particles can be dispensed together with other reagents, as will be further described below.

[0117] Figure 3 An example of a microfluidic channel structure 300 for co-dispensing biological particles and reagents is shown. Channel structure 300 may include channel segments 301, 302, 304, 306, and 308. Channel segments 301 and 302 are connected at a first channel junction 309. Channel segments 302, 304, 306, and 308 are connected at a second channel junction 310.

[0118] In an exemplary operation, channel segment 301 can transport aqueous fluid 312 including a plurality of biological particles 314 along channel segment 301 into second junction 310. Alternatively or additionally, channel segment 301 can transport beads (eg, gel beads). The beads can contain barcode molecules.

[0119] For example, channel segment 301 can be connected to a reservoir of an aqueous suspension including biological particles 314. Upstream of and just before reaching the second confluence point 310, channel segment 301 can meet channel segment 302 at a first confluence point 309. Channel segment 302 can transport a plurality of reagents 315 (e.g., lysing agents) suspended in aqueous fluid 312 along channel segment 302 into the first confluence point 309. For example, channel segment 302 can be connected to a reservoir containing reagents 315. After the first confluence point 309, the aqueous fluid 312 in channel segment 301 can carry the biological particles 314 and the reagents 315 to the second confluence point 310. In some cases, the aqueous fluid 312 in channel segment 301 can include one or more reagents, which can be the same or different reagents as reagent 315. A second fluid 316 (e.g., oil) that is immiscible with the aqueous fluid 312 may be delivered from each of the channel segments 304 and 306 to the second junction 310. After the aqueous fluid 312 from the channel segment 301 meets the second fluid 316 from each of the channel segments 304 and 306 at the second channel junction 310, the aqueous fluid 312 may be distributed in the second fluid 316 as discrete droplets 318 and flow out of the second junction 310 along the 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 are collected.

[0120] The second fluid 316 may include an oil, such as a fluorinated oil, which includes a fluorosurfactant for stabilizing the resulting droplets (eg, inhibiting subsequent coalescence of the resulting droplets 318).

[0121] The generated discrete droplets can include individual biological particles 314 and / or one or more reagents 315. In some cases, the generated discrete droplets can include beads (not shown) carrying barcodes, such as by other microfluidic structures described elsewhere herein. In some cases, the discrete droplets may be unoccupied (e.g., no reagents, no biological particles).

[0122] Advantageously, when the lysis reagent and the biological particle are co-dispensed, the lysis reagent may promote the release of the contents of the biological particle within the dispensing zone. The released contents in the dispensing zone may remain discrete from the contents of other dispensing zones.

[0123] As will be understood, the channel section described herein can be connected to any one of a variety of different fluid sources or receiving components, including reservoirs, pipelines, manifolds or fluid components of other systems. As will be understood, microfluidic channel structure 300 can have other geometric shapes. For example, the microfluidic channel structure can have more than two channel junctions. For example, the microfluidic channel structure can have 2,3,4,5 or more channel sections, and each channel section carries the same or different types of beads, reagents and / or bioparticles that meet at the channel junction. The fluid flow in each channel section can be controlled to control the distribution of different elements to the droplet. Fluid can be guided to flow along one or more channels or reservoirs by one or more fluid flow units. The fluid flow unit may include a compressor (for example, positive pressure is provided), a pump (for example, negative pressure is provided), an actuator, etc. to control the flow of fluid. Fluid can also or otherwise be controlled by the pressure difference, centrifugal force, electric pumping, vacuum, capillary or gravity flow, etc. applied.

[0124] The example of lysing agent comprises bioactive reagent, for example, lysing enzyme for cracking different cell types (for example Gram-positive or negative bacteria, plant, yeast, mammal etc.), for example lysozyme, colorless peptidase, lysostaphin, labiase, kitalase, lysing enzyme and can be from various other lysing enzymes that for example Sigma-Aldrich company (St Louis, MO) buys and other commercially available lysing enzymes.Other lysing agents can be additionally or alternatively distributed with bioparticles to cause the bioparticle contents to be released into the distribution area.For example, in some cases, the lysing solution based on surfactant can be used for lysing cells, but these may not be suitable for the system based on emulsion, and the surfactant can disturb the stable emulsion in the system based on emulsion.In some cases, the lysing solution can include nonionic surfactant, for example as TritonX-100 and Tween 20.In some cases, the lysing solution can include ionic surfactant, for example as sodium lauryl sarcosine and sodium lauryl sulfate (SDS). In some cases, electroporation, thermal, acoustic, or mechanical cell disruption may also be used, for example, non-emulsion based dispensing regions such as encapsulation of bioparticles may supplement or replace droplet dispensing regions where any pore size of the encapsulant is small enough to retain nucleic acid fragments of a given size after cell disruption.

[0125] In addition to the lysing agent co-assigned with the above-mentioned bioparticles, other reagents can also be co-assigned with the bioparticles, including for example DNA enzyme and RNA enzyme inactivators or inhibitors (such as proteinase K), chelating agents (such as EDTA) and other reagents for removing or otherwise reducing the negative activity of different cell lysate components to nucleic acid subsequent treatment or the impact. In addition, with regard to the encapsulated bioparticles, the bioparticles can be exposed to appropriate stimulation to release the bioparticles or its content from the co-assigned microcapsules. For example, in some cases, chemical stimulation can be co-assigned together with the encapsulated bioparticles, to allow the degradation of microcapsules and the release of cells or their content to a larger distribution area. In some cases, the stimulation can be identical with the stimulation described elsewhere herein for releasing nucleic acid molecules (such as oligonucleotides) from their respective microcapsules (such as beads). In alternative aspects, this can be different and non-overlapping stimulations to allow the encapsulated bioparticles to be released into the distribution area at different times from the nucleic acid molecules released into the same distribution area.

[0126] Additional reagents can also be co-assigned with the bioparticles, such as endonucleases, DNA polymerases, and dNTPs for amplifying the nucleic acid fragments of the bioparticles and attaching barcode molecular tags to the amplified fragments. Other enzymes can be co-assigned, including but not limited to polymerases, transposases, ligases, proteinases K, DNA enzymes, etc. Additional reagents can also include reverse transcriptases, including enzymes with terminal transferase activity, primers and oligonucleotides, and conversion oligonucleotides (also referred to herein as "conversion oligos" or "template conversion oligonucleotides") that can be used for template conversion. In some cases, template conversion can be used to increase the length of cDNA. In some cases, template conversion can be used to attach a predefined nucleic acid sequence to cDNA. In the example of template conversion, cDNA can be generated from reverse transcription of a template (e.g., cellular mRNA), wherein a reverse transcriptase with terminal transferase activity can add additional nucleotides, such as polyC, to cDNA in a template-independent manner. Conversion oligos can include sequences complementary to additional nucleotides (e.g., polyG). Additional nucleotides on the cDNA (e.g., polyC) can hybridize with additional nucleotides on the conversion oligo (e.g., polyG), whereby the conversion oligo can be used as a template by a reverse transcriptase to further extend the cDNA. The template conversion oligonucleotide may comprise a hybridization region and a template region. The hybridization region may comprise any sequence capable of hybridizing with the target. In some cases, as previously described, the hybridization region comprises a series of G bases to complement the protruding C bases at the 3' end of the 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 than 5 G bases. The template sequence may comprise any sequence 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. The conversion oligo may comprise deoxyribonucleic acid; ribonucleic acid; modified nucleic acids including 2-aminopurine, 2,6-diaminopurine (2-amino-dA), reverse dT, 5-methyl dC, 2'-deoxyinosine, Super T (5-hydroxybutyne-2'-deoxyuridine), Super G (8-aza-7-deazaguanosine), locked nucleic acid (LNA), unlocked nucleic acid (UNA, such as UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2' fluoro bases (e.g., fluoro-C, fluoro-U, fluoro-A and fluoro-G), or any combination.

[0127] In some cases, the length of the conversion oligo can 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, 1 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or 250 nucleotides or longer.

[0128] In some cases, the length of the conversion oligo can be at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 ,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137 7, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194 , 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or 250 nucleotides.

[0129] Once the contents of the cells are released into their respective distribution zones, the macromolecular components contained therein (e.g., macromolecular components of biological particles, such as RNA, DNA or proteins) can be further processed in the distribution zones. According to the methods and systems described herein, the macromolecular component contents of a single biological particle can have a unique identifier so that when characterizing those macromolecular components, they can be attributed to the same one or more biological particles. The ability to attribute features to a separate biological particle or biological particle group is provided by specifically assigning a unique identifier to a separate biological particle or biological particle group. A unique identifier (e.g., in the form of a nucleic acid barcode) can be specified or associated with each biological particle or biological particle group so that the macromolecular components of the biological particle (and causing its features) are labeled or marked with a unique identifier. These unique identifiers can then be used to attribute the components and features of the biological particle to a separate biological particle or biological particle group.

[0130] In some aspects, this is performed by co-assigning a single bioparticle or group of bioparticles with a unique identifier, e.g., as described above (see Figure 2 ). In some aspects, the unique identifier is provided in the form of a nucleic acid molecule (e.g., an oligonucleotide) comprising a nucleic acid barcode sequence that can be attached to or otherwise associated with the nucleic acid content of an individual biological particle, or to other components of a biological particle, particularly fragments of those nucleic acids. The nucleic acid molecules are allocated so that the nucleic acid barcode sequences contained therein are identical between nucleic acid molecules in a given allocation, but between different allocations, the nucleic acid molecules can and do have different barcode sequences, or at least represent a large number of different barcode sequences in all allocations in a given analysis. In some aspects, only one nucleic acid barcode sequence can be associated with a given allocation, but in some cases, two or more different barcode sequences can be present.

[0131] Nucleic acid barcode sequences can include about 6 to about 20 or more nucleotides within the sequence of a nucleic acid molecule (e.g., an oligonucleotide). In some cases, the length of the barcode sequence can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of the barcode sequence can be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, the length of the barcode sequence can be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter. These nucleotides can be completely continuous, i.e., in a single segment of adjacent nucleotides, or they can be divided into two or more separate subsequences separated by 1 or more nucleotides. In some cases, the length of the separated barcode subsequences can be about 4 to about 16 nucleotides. In some cases, the barcode subsequences can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequences can be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcode subsequences can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[0132] The co-distributed nucleic acid molecules may also contain other functional sequences that can be used to process nucleic acids from the co-distributed biological particles. These sequences include, for example, targeted or random / universal amplification primer sequences for amplifying genomic DNA from individual biological particles within the distribution zone, while attaching associated barcode sequences, sequencing primers or primer recognition sites, hybridization or detection sequences, such as for identifying sequence presence or for pulling down barcoded nucleic acids, or any of many other potential functional sequences. Other mechanisms for co-distributing oligonucleotides may also be used, including, for example, coalescence of two or more droplets, one of which contains an oligonucleotide, or microdistribution of oligonucleotides into a distribution zone, such as droplets within a microfluidic system.

[0133] In one example, microcapsules (e.g., beads) are provided, each of which includes a large number of the above-described barcoded nucleic acid molecules (e.g., barcoded oligonucleotides) releasably attached to the beads, wherein all nucleic acid molecules attached to a particular bead will include the same nucleic acid barcode sequence, but a large number of different barcode sequences are represented in the population of beads used. In some embodiments, hydrogel beads (e.g., comprising a polyacrylamide polymer matrix) are used as solid supports and delivery vehicles for nucleic acid molecules into a distribution area because they are able to carry a large number of nucleic acid molecules and can be configured to release those nucleic acid molecules after exposure to a specific stimulus, as described elsewhere herein. In some cases, the population of beads provides a diverse library of barcode sequences including 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 have a large number of attached nucleic acid (e.g., oligonucleotide) molecules. In particular, the number of molecules of nucleic acid molecules comprising a barcode sequence on a single bead can be at least about 1,000 nucleic acid molecules, at least about 5,000 nucleic acid molecules, at least about 10,000 nucleic acid molecules, at least about 50,000 nucleic acid molecules, at least about 100,000 nucleic acid molecules, at least about 500,000 nucleic acids, at least about 1,000,000 nucleic acid molecules, at least about 5,000,000 nucleic acid molecules, at least about 10,000,000 nucleic acid molecules, at least about 50,000,000 nucleic acid molecules, at least about 100,000,000 nucleic acid molecules, at least about 250,000,000 nucleic acid molecules, and in some cases at least about 1 billion nucleic acid molecules, or more. The nucleic acid molecules of a given bead can include the same (or shared) barcode sequence, different barcode sequences, or a combination of both. The nucleic acid molecules of a given bead can include a plurality of nucleic acid molecule collections. Nucleic acid molecules of a given collection can include the same barcode sequence. The same barcode sequence can be different from the barcode sequence of nucleic acid molecules of another collection.

[0134] In addition, when the bead population is partitioned, the resulting population of partitioned areas can also include a variety of barcode libraries, which include at least about 1,000 different barcode sequences, at least about 5,000 different barcode sequences, at least about 10,000 different barcode sequences, at least about 50,000 different barcode sequences, at least about 100,000 different barcode sequences, at least about 1,000,000 different barcode sequences, at least about 5,000,000 different barcode sequences, or at least about 10,000,000 different barcode sequences. In addition, each allocation area 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.

[0135] In some cases, it may be desirable to incorporate multiple different barcodes into a given dispensing area, or to attach to a single or multiple beads within a dispensing area. For example, in some cases, a mixed but known set of barcode sequences can provide greater assurance of identification in subsequent processing, for example, by providing a stronger address or barcode attribute to a given dispensing area as a duplicate or independent confirmation of output from a given dispensing area.

[0136] Nucleic acid molecules (e.g., oligonucleotides) can be released from beads after a specific stimulus is applied to the beads. In some cases, the stimulus can be a light stimulus, such as by cleaving a photolabile bond to release the nucleic acid molecule. In other cases, a thermal stimulus can be used, wherein an increase in the temperature of the bead environment will result in bond cleavage or other release of the nucleic acid molecule from the bead. In other cases, a chemical stimulus can be used that cleaves the bond of the nucleic acid molecule to the bead, or otherwise causes the nucleic acid molecule to be released from the bead. In one case, such a composition includes a polyacrylamide matrix for encapsulating bioparticles as described above, and can be degraded by exposure to a reducing agent such as DTT to release the attached nucleic acid molecule.

[0137] In some aspects, systems and methods for controlled dispensing are provided. Droplet size can be controlled by adjusting certain geometric features in a channel architecture (e.g., a microfluidic channel architecture). For example, the expansion angle, width, and / or length of the channel can be adjusted to control droplet size.

[0138] Figure 4An example of a microfluidic channel structure for controlled distribution of beads into discrete droplets is shown. Channel structure 400 may include a channel segment 402 connected to a reservoir 404 at a channel junction 406 (or intersection). Reservoir 404 may be a chamber. As used herein, any reference to a "reservoir" may also refer to a "chamber." In operation, an aqueous fluid 408 including suspended beads 412 may be transported along channel segment 402 to junction 406 to meet a second fluid 410 that is immiscible with the aqueous fluid 408 in reservoir 404 to produce droplets 416, 418 of the aqueous fluid 408 flowing into reservoir 404. At the junction 406 where the aqueous fluid 408 and the second fluid 410 meet, droplets may form based on various factors, such as the fluid dynamics at the junction 406, the flow rates of the two fluids 408, 410, the fluid properties, and certain geometric parameters of the channel structure 400 (e.g., w, h0, α, etc.). Multiple droplets are collected in the reservoir 404 by continuously injecting the aqueous fluid 408 from the channel segment 402 through the junction 406.

[0139] The generated discrete droplets may include a bead (e.g., as in occupied droplet 416). Alternatively, the generated discrete droplets may include more than one bead. Alternatively, the generated discrete droplets may not include any beads (e.g., as in unoccupied droplet 418). In some cases, the generated discrete droplets may contain one or more biological particles, as described elsewhere herein. In some cases, the generated discrete droplets may contain one or more reagents, as described elsewhere herein.

[0140] In some cases, aqueous fluid 408 can have a substantially uniform concentration or frequency of beads 412. Beads 412 can be delivered from separate channels ( Figure 4 The beads 412 are introduced into the channel segment 402 (not shown). The frequency of beads 412 in the channel segment 402 can be controlled by controlling the frequency at which beads 412 are introduced into the channel segment 402 and / or the relative flow rates of fluids in the channel segment 402 and the individual channels. In some cases, beads can be introduced into the channel segment 402 from multiple different channels and the frequency controlled accordingly.

[0141] In some cases, the aqueous fluid 408 in the channel segment 402 may include biological particles (e.g., see Figure 1 and Figure 2402 ). In some cases, the aqueous fluid 408 may have a substantially uniform concentration or frequency of biological particles. As with beads, biological particles may be introduced into the channel segment 402 from a separate channel. The frequency or concentration of biological particles in the aqueous fluid 408 in the channel segment 402 may be controlled by controlling the frequency at which 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 channels. In some cases, biological particles may be introduced into the channel segment 402 from a plurality of different channels and the frequencies may be controlled accordingly. In some cases, a first separate channel may introduce beads and a second separate channel may introduce biological particles into the channel segment 402. The first separate channel where the beads are introduced may be upstream or downstream of the second separate channel where the biological particles are introduced.

[0142] The second fluid 410 may comprise an oil, such as a fluorinated oil, which includes a fluorinated surfactant for stabilizing the resulting droplets (eg, inhibiting subsequent coalescence of the resulting droplets).

[0143] In some cases, the second fluid 410 may not be subjected to and / or directed to any flow into or out of the reservoir 404. For example, the second fluid 410 may be substantially stationary in the reservoir 404. In some cases, the second fluid 410 may flow within the reservoir 404, but not into or out of the reservoir 404, such as by applying pressure to the reservoir 404 and / or being affected by the incoming flow of the aqueous fluid 408 at the confluence point 406. Alternatively, the second fluid 410 may be subjected to and / or directed to flow into or out of the reservoir 404. For example, the reservoir 404 may be a channel that directs the second fluid 410 from upstream to downstream, thereby transporting the generated droplets.

[0144] The channel structure 400 at or near the confluence point 406 may have certain geometric features that at least partially determine the size of the droplets formed by the channel structure 400. The channel segment 402 may have a height h0 and a width w at or near the confluence point 406. For example, the channel segment 402 may include a rectangular cross-section that leads to a reservoir 404 having a wider cross-section (e.g., in terms of width or diameter). Alternatively, the cross-section of the channel segment 402 may be other shapes, such as circular, trapezoidal, polygonal, or any other shape. The top and bottom walls of the reservoir 404 at or near the confluence point 406 may be inclined at a divergence angle α. The divergence angle α allows the tongue (the portion of the aqueous fluid 408 that leaves the channel segment 402 at the confluence point 406 and enters the reservoir 404 before the droplet is formed) to increase in depth and helps reduce the curvature of the droplet formed in the middle. The droplet size may decrease as the divergence angle increases. The resulting droplet radius R can be predicted by the following equation for the above geometric parameters h0, w and α d :

[0145]

[0146] For example, for a channel structure of w = 21 μm, h = 21 μm, and α = 3°, the predicted droplet size is 121 μm. In another example, for a channel structure of w = 25 μm, h = 25 μm, and α = 5°, the predicted droplet size is 123 μm. In another example, for a channel structure of w = 28 μm, h = 28 μm, and α = 7°, the predicted droplet size is 124 μm.

[0147] In some cases, the expansion angle α can range from about 0.5° to about 4°, about 0.1° to about 10°, or about 0° to about 90°. For example, the expansion angle can be at least about 0.01°, 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 6°, 7°, 8°, 9°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 85° or more. In some cases, the expansion angle can be at most about 89°, 88°, 87°, 86°, 85°, 84°, 83°, 82°, 81°, 80°, 75°, 70°, 65°, 60°, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01°, or less. In some cases, the width w can be in the range of about 100 microns (μm) to about 500 μm. In some cases, the width w can be in the range of about 10 μm to about 200 μm. Alternatively, the width can be less than about 10 μm. Alternatively, the width can be greater than about 500 μm. In some cases, the flow rate of aqueous fluid 408 entering junction 406 may be between about 0.04 microliters (μL) / minute (min) and about 40 μL / min. In some cases, the flow rate of aqueous fluid 408 entering junction 406 may be between about 0.01 microliters (μL) / minute (min) and about 100 μL / min. Alternatively, the flow rate of aqueous fluid 408 entering junction 406 may be less than about 0.01 μL / min. Alternatively, the flow rate of aqueous fluid 408 entering junction 406 may be greater than about 40 μL / min, such as 45 μL / min, 50 μL / min, 55 μL / min, 60 μL / min, 65 μL / min, 70 μL / min, 75 μL / min, 80 μL / min, 85 μL / min, 90 μL / min, 95 μL / min, 100 μL / min, 110 μL / min, 120 μL / min, 130 μL / min, 140 μL / min, 150 μL / min, or more. At lower flow rates, such as a flow rate of about less than or equal to 10 microliters / minute, the droplet radius may not depend on the flow rate of aqueous fluid 408 entering junction 406.

[0148] In some cases, at least about 50% of the generated droplets may have a consistent size. In some cases, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the generated droplets may have a consistent size. Alternatively, less than about 50% of the generated droplets may have a consistent size.

[0149] The flux of droplet generation can be increased by increasing the generation points, such as increasing the number of junctions (e.g., junction 406) between a channel segment containing an aqueous fluid 408 (e.g., channel segment 402) and a reservoir 404. Alternatively or additionally, the flux of droplet generation can be increased by increasing the flow rate of the aqueous fluid 408 in the channel segment 402.

[0150] Figure 5 An example of a microfluidic channel structure for increasing droplet generation throughput is shown. The microfluidic channel structure 500 may include 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 may include a plurality of channel junctions 506 between the plurality of channel segments 502 and the reservoir 504. Each channel junction may be a droplet generation point. Figure 4 The channel segment 402 of the channel structure 400 in FIG. 4 and any description of its components may correspond to a given channel segment of the plurality of channel segments 502 in the channel structure 500 and any description of its corresponding components. The reservoir 404 from the channel structure 400 and any description of its components may correspond to the reservoir 504 from the channel structure 500 and any description of its corresponding components.

[0151] Each channel segment in the plurality of channel segments 502 may include an aqueous fluid 508 including suspended beads 512. The reservoir 504 may include a second fluid 510 that is immiscible with the aqueous fluid 508. In some cases, the second fluid 510 may not be subjected to and / or directed to any flow into or out of the reservoir 504. For example, the second fluid 510 may be substantially stationary in the reservoir 504. In some cases, the second fluid 510 may flow within the reservoir 504 but not into or out of the reservoir 504, such as by applying pressure to the reservoir 504 and / or being affected by an incoming flow of the aqueous fluid 508 at a confluence point. Alternatively, the second fluid 510 may be subjected to and / or directed to flow into or out of the reservoir 504. For example, the reservoir 504 may be a channel that directs the second fluid 510 from upstream to downstream, thereby transporting the generated droplets.

[0152] In operation, an aqueous fluid 508 including suspended beads 512 can be transported along multiple channel segments 502 to multiple confluence points 506 to meet a second fluid 510 in a reservoir 504 to produce droplets 516, 518. From each channel segment, at each corresponding confluence point with the reservoir 504, droplets can be formed. At the confluence point where the aqueous fluid 508 and the second fluid 510 meet, droplets can be formed based on various factors, such as fluid dynamics at the confluence point, the flow rates of the two fluids 508, 510, fluid properties, and certain geometric parameters of the channel structure 500 (e.g., w, h0, α, etc.), as described elsewhere herein. Multiple droplets can be collected in the reservoir 504 by continuously injecting the aqueous fluid 508 from the multiple channel segments 502 through the multiple confluence points 506. Through the parallel channel configuration of the channel structure 500, the throughput can be significantly increased. For example, a channel structure having five inlet channel sections including aqueous fluid 508 can produce droplets with a frequency five times that of a channel structure having an inlet channel section, provided that the fluid flow rate in the channel section is substantially the same. The fluid flow rate in different inlet channel sections can be substantially the same or different. The channel structure can have parallel channel sections according to the actual and allowable size of the reservoir. For example, the channel structure can have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 500, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 5000 or more parallel or substantially parallel channel sections.

[0153] For each channel segment in the plurality of channel segments 502, the geometric parameters w, h0 and α may be consistent or inconsistent. 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 heights at or near its respective channel junction with the reservoir 504. In another example, the reservoir 504 may have the same or different expansion angles at different channel junctions with the plurality of channel segments 502. When the geometric parameters are consistent, advantageously, the droplet size may be controlled to be consistent even if the flux is increased. In some cases, when it is desired to have different droplet size distributions, the geometric parameters of the plurality of channel segments 502 may be changed accordingly.

[0154] In some cases, at least about 50% of the generated droplets may have a consistent size. In some cases, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the generated droplets may have a consistent size. Alternatively, less than about 50% of the generated droplets may have a consistent size.

[0155] Figure 6 Another example of a microfluidic channel structure for increasing droplet generation flux is shown. The microfluidic channel structure 600 may include 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 may include a plurality of channel junctions 606 between the plurality of channel segments 602 and the reservoir 604. Each channel junction may be a droplet generation point. Figure 2 The channel segment 402 of the channel structure 400 in FIG. 4 and any description of its components may correspond to a given channel segment of the plurality of channel segments 602 in the channel structure 600 and any description of its corresponding components. The reservoir 404 from the channel structure 400 and any description of its components may correspond to the reservoir 604 from the channel structure 600 and any description of its corresponding components.

[0156] Each channel segment in the plurality of channel segments 602 may include an aqueous fluid 608 including suspended beads 612. The reservoir 604 may include a second fluid 610 that is immiscible with the aqueous fluid 608. In some cases, the second fluid 610 may not be subjected to and / or directed to any flow into or out of the reservoir 604. For example, the second fluid 610 may be substantially stationary in the reservoir 604. In some cases, the second fluid 610 may flow within the reservoir 604 but not into or out of the reservoir 604, such as by applying pressure to the reservoir 604 and / or being affected by an incoming flow of the aqueous fluid 608 at a confluence point. Alternatively, the second fluid 610 may be subjected to and / or directed to flow into or out of the reservoir 604. For example, the reservoir 604 may be a channel that directs the second fluid 610 from upstream to downstream, thereby transporting the generated droplets.

[0157] In operation, an aqueous fluid 608 including suspended beads 612 can be transported along the plurality of channel segments 602 to the plurality of confluence points 606 to meet the second fluid 610 in the reservoir 604 to produce a plurality of droplets 616. From each channel segment, at each respective confluence point with the reservoir 604, a droplet can be formed. At the confluence point where the aqueous fluid 608 and the second fluid 610 meet, droplets can be formed based on a variety of factors, such as the hydrodynamic forces at the confluence point, the flow rates of the two fluids 608, 610, the fluid properties, and certain geometric parameters of the channel structure 600 (e.g., the width and height of the channel segment 602, the divergence angle of the reservoir 604, etc.), 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 confluence points 606. Through the substantially parallel channel configuration of the channel structure 600, the throughput can be significantly increased. The channel structure can have as many substantially parallel channel sections as are practical and the size of the reservoir allows. For example, the channel structure can have at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 5000 or more parallel or substantially parallel channel sections. A plurality of channel sections can be substantially evenly spaced apart, for example, around the edge or periphery of the reservoir. Alternatively, the spacing of a plurality of channel sections can be uneven.

[0158] The reservoir 604 may have a divergence angle α ( Figure 6 602). Each channel segment in the plurality of channel segments 602 may have a width w and a height h0 at or near a channel junction. For each channel segment in the plurality of channel segments 602, the geometric parameters w, h0, and α may be consistent or inconsistent. 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 heights at or near its respective channel junction with the reservoir 604.

[0159] The reservoir 604 can have the same or different expansion angles at different channel junctions with the plurality of channel segments 602. For example, a circular reservoir (such as Figure 6606) can have a conical, dome-shaped, or hemispherical top plate (e.g., top wall) to provide the same or substantially the same expansion angle for each channel segment 602 at or near the multiple channel confluence point 606. When the geometric parameters are consistent, advantageously, the resulting droplet size can be controlled to be consistent even if the flux is increased. In some cases, when it is desired to have different droplet size distributions, the geometric parameters of the multiple channel segments 602 can be changed accordingly.

[0160] In some cases, at least about 50% of the droplets produced may have a consistent size. In some cases, at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the droplets produced may have a consistent size. Alternatively, less than about 50% of the droplets produced may have a consistent size. The beads and / or bioparticles injected into the droplets may have a consistent or inconsistent size.

[0161] For example, the channel network as described above or elsewhere herein can be fluidically coupled to suitable fluid components.For example, the inlet channel section is fluidically coupled to the appropriate source of the material that they will be delivered to the channel junction.These sources can include any of a variety of different fluid components, from the simple reservoir defined in the main structure of the microfluidic device or connected thereto, to the fluid conduit of the delivery fluid from the source, manifold, fluid flow unit (for example, actuator, pump, compressor) etc. outside the device.Similarly, the outlet channel section (for example, channel section 208, reservoir 604 etc.) can be fluidically coupled to the receiving container or conduit of the distribution cell for subsequent processing.Similarly, this can be the reservoir defined in the main body of the microfluidic device, or it can be the fluid conduit for distributing cells to be delivered to subsequent process operations, instrument or parts.

[0162] The methods and systems described herein can be used to greatly improve the efficiency of single-cell applications and / or other applications that receive droplet-based inputs. For example, after sorting cells of occupied cells and / or cells of appropriate size, subsequent operations that can be performed can include generating amplification products, purification (e.g., by solid phase reversible immobilization (SPRI)), further processing (e.g., shearing, connection of functional sequences and subsequent amplification (e.g., by PCR)). These operations can occur in vivo (e.g., outside the distribution area). In the case where the distribution area is a droplet in an emulsion, the emulsion can be destroyed and the contents of the droplet can be collected for additional operations. Other reagents that can be co-distributed together with the beads carrying the barcode can include oligonucleotides that block ribosomal RNA (rRNA) and nucleases that digest genomic DNA in cells. Alternatively, rRNA removers can be applied during additional processing operations. The configuration of the construct generated by this method can help minimize (or avoid) the sequencing of poly-T sequences during sequencing and / or sequence the 5' end of the polynucleotide sequence. Amplification products (e.g., the first amplification product and / or the second amplification product) can be sequenced for sequence analysis. In some cases, amplification can be performed using the Partial Hairpin Amplification for Sequencing (PHASE) method.

[0163] A variety of applications require the assessment and quantification of the presence of different bioparticle or organism types within a bioparticle population, including, for example, microbial flora analysis and characterization, environmental testing, food safety testing, epidemiological analysis, e.g., tracking contamination, etc.

[0164] Sample preparation and / or analysis

[0165] In another aspect, the present disclosure provides a method for sample preparation and / or analysis. Sample preparation may include one or more continuous operations. Each operation may include, for example, one or more reactions to a sample. The sample may be a biological particle (e.g., a cell). In some cases, the sample is derived from a biological particle and may be, for example, a macromolecular component, including but not limited to nucleic acids, proteins, peptides, or polypeptides. The sample may be a target. Alternatively, a sample-derived target may be used. In some aspects, the method of the present disclosure includes generating a plurality of distribution areas, each of which may include a target and a bead. The bead may be a gel bead. In some cases, a plurality of distribution areas may be a plurality of droplets, and may be produced by the method disclosed herein. Alternatively, a plurality of distribution areas may be a plurality of holes. The target may be a target of a reaction, such as a chemical or biochemical reaction. The target may be a biological particle. Alternatively or additionally, the target may be a macromolecular component derived from a biological particle (e.g., a cell). The target may be derived from any part of a cell, including, for example, a cell surface, a cell membrane, a cell wall, a cytosol, or an organelle (e.g., a nucleus, an endoplasmic reticulum, a mitochondria, etc.). In some cases, the target may be associated with a biological particle. The target can be associated by, for example, binding to a bioparticle or otherwise interacting. Examples of targets associated with bioparticles include, but are not limited to, antibodies, ligands, and other bioparticles (e.g., viruses, bacterial cells, etc.). In some cases, the target can be from a cell or gel matrix (e.g., a cell bead) comprising one or more cell components. Before reacting the target, a cell bead can be produced as described herein. For example, a cell bead can be produced, and the cells in the cell bead are lysed to produce a cell component comprising the target. Reversible hydrogels as described herein can be used to produce cells so that the cell bead is destructible (i.e., a destructible cell bead) after applying a stimulus. Multiple distribution areas comprising one or more cell beads can be produced, and each cell bead comprises a target. In some cases, the target is a nucleic acid molecule. The nucleic acid molecule can be, for example, a deoxyribonucleic acid molecule or a ribonucleic acid molecule. The ribonucleic acid molecule can be a messenger RNA, a transfer RNA, or any other RNA. In other examples, the target is a polypeptide, such as a protein.

[0166] The distribution area may also include reagents suitable for performing one or more reactions on the target molecule. In some instances, the reagents are contained within and / or attached to the beads. In some aspects, the reagents can be molecular tag molecules (e.g., primers, barcoded oligonucleotides), which in some instances can include molecular barcodes. Alternatively or in addition, the reagents can be various reagents suitable for performing biological or chemical reactions, and can include, for example, nucleic acid molecules, reagents for nucleic acid amplification reactions (e.g., primers, polymerases, dNTPs, cofactors (e.g., ionic cofactors), buffers), including those described herein, reagents for enzymatic reactions (e.g., enzymes, cofactors, substrates, buffers), reagents for nucleic acid modification reactions such as polymerization, ligation or digestion, reagents for cell lysis (e.g., lysing agents), reagents for labeling, reagents for reverse transcription, and / or for one or more sequencing platforms (e.g., for of ) of a template. A distribution area may contain all reagents required to perform a given reaction.

[0167] In some respects, the present disclosure provides a method for carrying out one or more reactions to produce a reaction product to a target in a distribution area. The reaction can be any type of chemical and / or biochemical reaction. The example of reaction includes but is not limited to the cracking of biological particles (such as cells), nucleic acid amplification, nucleic acid modification (for example, nucleic acid insertion, deletion, mutation, etc.), nucleic acid separation (for example, separation from biological particles), addition of oligonucleotides (for example, barcoded oligonucleotides), reverse transcription and transposition (for example, labeling). In some cases, the number of reactions carried out in the distribution area can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50 reactions, or more. In some cases, the number of reactions carried out in the distribution area can be at most 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1.

[0168] In some cases, the reaction can produce one or more reaction products. In some cases, the reaction can produce at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500 or 1000 reaction products, or more. The reaction product can be any component produced after the reaction, such as, for example, a cellular component (e.g., nucleic acid, protein, lipid, etc.), a modified nucleic acid molecule, a barcoded nucleic acid molecule, and / or a nucleic acid fragment.

[0169] In some cases, after reacting in the distribution area, the distribution area includes precursors from destructible beads, such as after the destructible beads are destroyed. Destructible beads can be, for example, gel beads. Gel beads can be or include hydrogels (i.e., can be hydrogel beads). Hydrogels can be reversible hydrogels. Reversible hydrogels can be produced after, for example, cross-linking polymer chains. Methods for producing reversible hydrogels are provided in, for example, EP 2343342, U.S. Patent No. 5,883,211, Aliyar et al. 2005, Wang et al., 2015 and Rosales et al., 2016, each of which is incorporated herein by reference in its entirety for all purposes. Gel beads can be destroyed by exposure to one or more stimuli (e.g., chemical stimulation, thermal stimulation, electromagnetic stimulation, pH changes, exposure to reducing agents, etc.). Precursors can be produced, for example, by the destruction of destructible beads (e.g., gel beads). Precursors may include, for example, poly-L-lactic acid, poly(ethylene-propylene), poly(ethylene-butylene), polyacrylamide, agarose, alginate, polyvinyl alcohol, PEG, PEG-diacrylate, PEG-acrylate / thiol, PEG-azide / alkyne, other acrylates, chitosan, hyaluronic acid, collagen, fibrin, gelatin, elastin, or a combination thereof. In some aspects of the present disclosure, one or more precursors produced from destructible beads may be used to produce a second bead. A second bead may be produced in the same distribution area. Alternatively, a second bead may be produced in different distribution areas. Precursors may polymerize or gel to produce a second bead. Precursors may polymerize or gel by exposure to one or more stimuli (e.g., chemical stimulation, thermal stimulation, electromagnetic stimulation, pH changes, exposure to reducing agents, etc.). In some instances, a second bead may include one or more reaction products produced by a reaction performed in a distribution area. Alternatively or additionally, a second bead may include all components from a distribution area.

[0170] The present disclosure provides a method for performing one or more second reactions on a second target to produce a second reaction product. The second reaction may be performed after the second bead is produced. The second reaction may be a reaction of the same type as one or more previous reactions, or may be a reaction of a different type. In some instances, the second target may be a reaction product from one or more previous reactions. Alternatively, the second target may be a new target. The second reaction may be performed in the same distribution area as one or more previous reactions, or may be performed in a second distribution area.

[0171] For example, the first distribution area can be destroyed (e.g., destroying the emulsion), and then a second distribution area is formed by any method disclosed herein (including, for example, droplet formation). The second distribution area may include one or more reagents for performing a second reaction. As described herein, the second distribution area may include one or more second beads, which are produced by a precursor produced from a destructible gel bead. The second bead may include a second target (e.g., a reaction product from a previous reaction). In some examples, the second distribution area may include one or more other beads, which may include one or more reagents for performing a second reaction.

[0172] The reagent can be a molecular tag molecule (e.g., a primer, a barcoded oligonucleotide), which in some instances can include a molecular barcode. Alternatively or additionally, the reagent can be a variety of reagents suitable for performing biological or chemical reactions, and can include, for example, nucleic acid molecules, reagents for nucleic acid amplification reactions (e.g., primers, polymerases, dNTPs, cofactors (e.g., ionic cofactors), buffers), including those described herein, reagents for enzymatic reactions (e.g., enzymes, cofactors, substrates, buffers), reagents for nucleic acid modification reactions such as polymerization, ligation or digestion, reagents for cell lysis (e.g., lysing agents), reagents for labeling, reagents for reverse transcription, and / or reagents for template preparation for one or more sequencing platforms (e.g., transposases for labeling). The second reaction can be any type of chemical and / or biochemical reaction. Examples of such reactions include, but are not limited to, lysis of biological particles (e.g., cells), nucleic acid amplification, nucleic acid modification (e.g., nucleic acid insertion, deletion, mutation, etc.), nucleic acid isolation (e.g., isolation from biological particles), addition of oligonucleotides (e.g., barcoded oligonucleotides), reverse transcription, and labeling.

[0173] In some cases, the number of additional reactions performed can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more reactions. In some cases, the number of additional reactions can be at most 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1.

[0174] The method of the present disclosure can be performed with one or more continuous operations. For example, a first reaction can be performed in a first distribution area, and after the first reaction, a second reaction can be performed in a second distribution area. Each continuous operation may include a single reaction, or may include more than one reaction. One or more reactions in each continuous operation may be reactions of the same type, or may be reactions of different types. Each continuous operation may also include producing a precursor by destructible beads (e.g., gel beads), and after one or more reactions, producing other beads comprising one or more reaction products by the precursor. The method of the present disclosure may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 or more operations. The method of the present disclosure may include at most 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 operation.

[0175] The use of continuous operations may be useful in situations where, for example, reagents or conditions for a given reaction are incompatible or otherwise detrimental to another reaction. For example, reagents or conditions for cell lysis (e.g., at high temperatures) may inactivate enzymes necessary to perform another reaction (e.g., such as reverse transcription). The use of continuous operations as described herein may be useful, for example, to lyse cells at a sufficiently high temperature and then reverse transcribe nucleic acids of biological particles at a lower temperature.

[0176] One or more reaction products or derivatives thereof produced by the methods described herein may be further analyzed. Further analysis may include, for example, sequencing one or more reaction products.

[0177] refer to Figure 7 , showing an exemplary method for sample preparation. Droplet 710 contains reagents ( Figure 7 ), a destructible bead 701 containing at least a subset of reagents for performing a biochemical reaction, and a target nucleic acid 702. The biochemical reaction is performed in the droplet to produce a reaction product 703, and the destructible bead 701 is destroyed, resulting in the formation of a precursor ( Figure 7 The second bead 704 containing the components in the droplet is generated from the precursor. The second bead 704 is distributed on a liquid containing reagents ( Figure 7 The biochemical reaction is performed in the second droplet using the reagents contained in the droplet to produce additional reaction products ( Figure 7 not shown).

[0178] refer to Figure 8 , showing an exemplary method for sample preparation. Droplet 810 contains reagents ( Figure 8 ), a destructible bead 801 containing at least a subset of reagents for performing a biochemical reaction, and a target cell 802. The biochemical reaction is performed in the droplet to produce a reaction product 803, and the destructible bead 801 is destroyed, resulting in the formation of a precursor ( Figure 8 The second bead 804 containing the components in the droplet is generated from the precursor. The second bead 804 is distributed on a liquid containing reagents ( Figure 8 The biochemical reaction is performed in the second droplet using the reagents contained in the droplet to produce additional reaction products ( Figure 8 not shown).

[0179] refer to Fig. 9 , showing an exemplary method for sample preparation using destructible cell beads. Droplet 901 contains cells 902 and destructible beads 903 containing barcode molecules. Destruction of destructible beads 903 results in the precursors ( Fig. 9 The precursor is used to generate a destructible cell bead 904 containing a cell 902 and a barcode molecule. The destructible cell bead 904 is subjected to conditions sufficient to lyse the cell 902, thereby producing a cell component 905 (e.g., a DNA molecule, a RNA molecule, a protein, etc.). The destructible cell bead 904 is distributed in a container containing a reagent ( Fig. 9 The destruction of the destructible cell beads 904 results in the precursor ( Fig. 9 The barcode molecules are used to generate barcoded cell components ( Fig. 9 not shown).

[0180] refer to Fig.10 , showing an exemplary method for sample preparation using destructible cell beads. A first destructible bead 1001 comprises one or more first molecular barcodes and one or more molecules (e.g., small molecules, peptides, proteins, etc.) sufficient to stimulate cells. Destructible beads 1001 are dispensed with cells 1003 into droplets 1002 comprising cell growth medium. Destruction of destructible beads 1001 results in the precursor ( Fig.10The cell 1003 is incubated in the droplet 1002 for a time sufficient to allow the molecule to induce stimulation of the cell. A destructible cell bead 1004 containing the cell 1003 is generated from the precursor. The destructible cell bead 1004 is subjected to conditions sufficient to lyse the cell 1003, thereby producing a cell component 1005 (e.g., a DNA molecule, a RNA molecule, a protein, etc.). The destructible cell bead 1004 is distributed in a liquid containing a second destructible bead 1007, one or more second molecular barcodes, and a reagent for barcoding the cell component ( Fig.10 The destruction of the destructible cell beads 1004 and the second destructible beads 1007 results in the precursor ( Fig.10 One or more first molecular barcodes and one or more second molecular barcodes are used to generate barcoded cell components ( Fig.10 The identity of the first molecular barcode can be used to identify the nature of the stimulus applied to the cell prior to cell lysis.

[0181] Computer Systems

[0182] The present disclosure provides a computer system programmed to implement the method of the present disclosure. Fig.11 A computer system 1101 is shown that is programmed or otherwise configured to control a microfluidic system (e.g., fluid flow), sorting occupied droplets from unoccupied droplets, polymerizing droplets, destroying destructible beads to produce precursors, producing beads from precursors, etc. The computer system 1101 can adjust various aspects of the present disclosure, such as, for example, fluid flow rates in one or more channels in a microfluidic structure, destruction conditions, polymerization or gelation conditions, etc. The computer system 1101 can be an electronic device of a user or a computer system remotely located relative to an electronic device. The electronic device can be a mobile electronic device.

[0183] The computer system 1101 includes a central processing unit (CPU, also referred to herein as a "processor" and "computer processor") 1105, which may be a single core or multi-core processor, or multiple processors for parallel processing. The computer system 1101 also includes a memory or storage unit 1110 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 1115 (e.g., a hard disk), a communication interface 1120 (e.g., a network adapter) for communicating with one or more other systems, and peripherals 1125, such as cache memory, other memory, data storage, and / or an electronic display adapter. The memory 1110, storage unit 1115, interface 1120, and peripherals 1125 communicate with the CPU 1105 via a communication bus (solid line) such as a motherboard. The storage unit 1115 may be a data storage unit (or data repository) for storing data. The computer system 1101 may be operably coupled to a computer network ("network") 1130 by means of the communication interface 1120. The network 1130 may be the Internet, the Internet and / or an extranet, or an intranet and / or an extranet in communication with the Internet. The network 1130 is in some cases a telecommunications and / or data network. The network 1130 may include one or more computer servers that may implement distributed computing, such as cloud computing. The network 1130 may, in some cases, implement a peer-to-peer network with the aid of the computer system 1101, which may enable devices coupled to the computer system 1101 to operate as clients or servers.

[0184] The CPU 1105 may execute a sequence of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a storage unit, such as the memory 1110. The instructions may be directed to the CPU 1105, which may then program or otherwise configure the CPU 1105 to implement the methods of the present disclosure. Examples of operations performed by the CPU 1105 may include fetching, decoding, executing, and writing back.

[0185] CPU 1105 may be part of a circuit, such as an integrated circuit. One or more other components of system 1101 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0186] Storage unit 1115 can store files, such as drivers, libraries, and saved programs. Storage unit 1115 can store user data, such as user preferences and user programs. Computer system 1101 may include one or more additional data storage units in some cases, which are external to computer system 1101, such as located on a remote server that communicates with computer system 1101 via an intranet or the Internet.

[0187] Computer system 1101 may communicate with one or more remote computer systems via network 1130. For example, computer system 1101 may communicate with a remote computer system of a user (e.g., an operator). Examples of remote computer systems include personal computers (e.g., portable PCs), tablet PCs or tablet PCs (e.g., iPad, Galaxy Tab), phones, smartphones (e.g. iPhone, Android-enabled devices, ) or a personal digital assistant. A user can access the computer system 1101 via the network 1130.

[0188] The methods described herein may be implemented via machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1101, such as, for example, the memory 1110 or the electronic storage unit 1115. The machine executable or machine readable code may be provided in the form of software. During use, the code may be executed by the processor 1105. In some cases, the code may be retrieved from the storage unit 1115 and stored on the memory 1110 ready for access by the processor 1105. In some cases, the electronic storage unit 1115 may be eliminated and the machine executable instructions are stored on the memory 1110.

[0189] The code may be pre-compiled and configured for use with a machine having a processor suitable for executing the code, or may be compiled during execution time. The code may be provided in a programming language that may be selected to enable the code to be executed in a pre-compiled or as-compiled manner.

[0190] The systems and methods provided herein, such as aspects of computer system 1101, can be specifically implemented in programming. Various aspects of the technology can be considered as "products" or "articles" that are usually in the form of machine (or processor) executable code and / or associated data, which are carried or specifically implemented in a certain type of machine-readable medium. The machine executable code can be stored in an electronic storage unit, such as a memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. "Storage" type media may include any or all tangible memories of a computer, processor, etc., or its associated modules, such as various semiconductor memories, tape drives, disk drives, etc., which can provide non-temporary storage for software programming at any time. All or part of the software may sometimes be transmitted via the Internet or various other telecommunications networks. Such communications, for example, can enable software to be loaded from one computer or processor to another computer or processor, for example, from a management server or host computer to a computer platform of an application server. Therefore, another type of medium that can carry software elements includes light, electricity, and electromagnetic waves, such as light, electricity, and electromagnetic waves used across physical interfaces between local devices, via wired and optical landline networks, and various air links. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., can also be considered media that carry the software. As used herein, unless limited to non-transitory, tangible "storage" media, terms such as computer or machine "readable media" refer to any medium that participates in providing instructions to a processor for execution.

[0191] Thus, machine-readable media, such as computer executable code, can take many forms, including but not limited to tangible storage media, carrier media, or physical transmission media. Non-volatile memory media include, for example, optical or magnetic disks, such as any storage device in any computer, etc., such as the storage device shown in the accompanying drawings that can be used to implement a database, etc. Volatile memory media include dynamic memory, such as the main memory of such computer platforms. Tangible transmission media include coaxial cables; copper wire and optical fiber, including the wires that make up the bus in a computer system. Carrier transmission media can take the form of electrical or electromagnetic signals, or sound or light waves such as signals generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include, for example: floppy disks, floppy disks, hard disks, magnetic tape, any other magnetic media, CD-ROM, DVD or DVD-ROM, any other optical media, punched cardboard tape, any other physical storage medium with a pattern of holes, RAM, ROM, PROM and EPROM, flash EPROM, any other memory chip or cartridge, a carrier that transports data or instructions, a cable or link that transports such a carrier, or any other medium from which a computer can read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0192] Computer system 1101 may include or communicate with an electronic display 1135 that includes a user interface (UI) 1140 for providing, for example, results of a sequencing analysis. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0193] The methods and systems of the present disclosure may be implemented via one or more algorithms. The algorithms may be implemented via software after being executed by the central processing unit 1105. The algorithms may, for example, perform sequencing.

[0194] The present invention provides embodiments including but not limited to the following:

[0195] 1. A method for sample preparation or analysis, comprising:

[0196] (a) providing a plurality of dispensing areas, wherein a given dispensing area of ​​the plurality of dispensing areas comprises a target and a first bead, the first bead comprising one or more reagents for performing one or more reactions on the target, wherein the first bead is destructible;

[0197] (b) performing the one or more reactions on the target using the one or more reagents to produce one or more reaction products, wherein after performing the one or more reactions, the given distribution area comprises one or more precursors produced by destruction of the first bead; and

[0198] (c) using the one or more precursors to produce a second bead, the second bead comprising the one or more reaction products.

[0199] 2. The method of embodiment 1, further comprising performing one or more additional reactions on additional targets to produce one or more additional reaction products.

[0200] 3. A method as described in embodiment 2, wherein the additional target is the target.

[0201] 4. A method as described in embodiment 2 or 3, wherein the additional target comprises the one or more reaction products.

[0202] 5. The method of any one of embodiments 2-4, wherein the one or more additional reactions are performed in an additional distribution area comprising the second beads, the additional distribution area being different from the given distribution area.

[0203] 6. A method as described in embodiment 5, wherein the additional distribution area contains one or more additional reagents for performing the one or more additional reactions.

[0204] 7. A method as described in embodiment 5 or 6, wherein the additional distribution area comprises a third bead containing one or more additional reagents for performing the one or more additional reactions.

[0205] 8. The method of any one of embodiments 5-7, wherein after performing the one or more additional reactions, the additional distribution area comprises the one or more precursors produced by destruction of the second bead.

[0206] 9. The method of any one of embodiments 2-8, wherein after performing the one or more additional reactions, the given distribution area contains the one or more precursors produced by destruction of the second bead.

[0207] 10. The method of embodiment 9, further comprising using the one or more precursors to produce a third bead, the third bead comprising the one or more additional reaction products.

[0208] 11. The method of embodiment 10, wherein the third beads are gel beads.

[0209] 12. A method as described in any of embodiments 1-11, wherein (c) is performed in the given allocation area.

[0210] 13. A method as described in any of embodiments 1-11, wherein (c) is performed outside the given allocation area.

[0211] 14. A method as described in embodiment 13, wherein (c) is performed in another allocation area.

[0212] 15. A method as described in any of embodiments 1-14, wherein the target is a nucleic acid molecule or is associated with the nucleic acid molecule, wherein the one or more reaction products are derived from the nucleic acid molecule, and wherein after (c), the one or more reaction products or their derivatives are sequenced.

[0213] 16. The method of any one of embodiments 1-15, wherein the plurality of distribution areas are a plurality of droplets.

[0214] 17. The method of embodiment 16, wherein the plurality of droplets are generated after a first liquid phase comprising the first beads and a second liquid phase immiscible with the first liquid phase come into contact.

[0215] 18. The method of any one of embodiments 1-15, wherein the plurality of distribution areas are a plurality of holes.

[0216] 19. The method of any one of embodiments 1-18, wherein the target is a nucleic acid molecule.

[0217] 20. A method as described in embodiment 19, wherein the nucleic acid molecule is a ribonucleic acid molecule.

[0218] 21. A method as described in embodiment 20, wherein the RNA molecule is a messenger RNA molecule.

[0219] 22. A method as described in embodiment 19, wherein the nucleic acid molecule is a deoxyribonucleic acid molecule.

[0220] 23. The method of any one of embodiments 1-22, wherein the target is a protein.

[0221] 24. The method of any one of embodiments 1-22, wherein the target is a cell.

[0222] 25. The method of any one of embodiments 1-24, wherein the one or more reagents comprise one or more nucleic acid molecules.

[0223] 26. A method as described in embodiment 25, wherein the one or more nucleic acid molecules comprise a molecular barcode.

[0224] 27. A method as described in embodiment 26, wherein the one or more reaction products contain the molecular barcode.

[0225] 28. The method of embodiment 25, wherein the one or more nucleic acid molecules are a plurality of nucleic acid molecules, wherein a given nucleic acid molecule in the plurality of nucleic acid molecules comprises a barcode sequence that is constant between the plurality of nucleic acid molecules.

[0226] 29. A method as described in embodiment 28, wherein the given nucleic acid molecule contains an additional barcode sequence that varies between the multiple nucleic acid molecules.

[0227] 30. A method as described in embodiment 25, wherein the first bead is from a plurality of beads, wherein the plurality of beads contain nucleic acid barcode sequences that are different between the plurality of beads.

[0228] 31. The method of any one of embodiments 1-30, wherein the one or more reagents include all reagents required to perform the one or more reactions.

[0229] 32. The method of any one of embodiments 1-31, wherein the one or more reactions comprise a reverse transcription reaction.

[0230] 33. A method as described in any of embodiments 1-32, wherein the one or more reactions include nucleic acid modification.

[0231] 34. A method as described in embodiment 33, wherein the nucleic acid modification is a deoxyribonucleic acid modification.

[0232] 35. A method as described in embodiment 33, wherein the nucleic acid modification is ribonucleic acid modification.

[0233] 36. A method as described in any of embodiments 1-35, wherein the one or more reactions include nucleic acid amplification.

[0234] 37. A method as described in embodiment 36, wherein the nucleic acid amplification is a polymerase chain reaction.

[0235] 38. A method as described in embodiment 36, wherein the nucleic acid amplification is not polymerase chain reaction.

[0236] 39. The method of any one of embodiments 1-38, wherein the one or more reactions comprise cell lysis.

[0237] 40. The method of any one of embodiments 1-39, wherein the one or more reactions comprise labeling.

[0238] 41. The method of any one of embodiments 1-40, wherein the one or more reactions comprise adding a barcode oligonucleotide.

[0239] 42. A method as described in any of embodiments 1-41, wherein the given distribution area comprises cells or a gel matrix containing one or more cellular components, and wherein the target is derived from the cells or gel matrix.

[0240] 43. A method as described in any of embodiments 1-42, wherein the first bead is produced by polymerization or cross-linking of the one or more precursors.

[0241] 44. A method as described in embodiment 43, wherein the first bead is a hydrogel.

[0242] 45. A method as described in embodiment 43, wherein the one or more precursors include poly-L-lactic acid, poly(ethylene-propylene), poly(ethylene-butylene), polyacrylamide, agarose, alginate, polyvinyl alcohol, PEG, PEG-diacrylate, PEG-acrylate / thiol, PEG-azide / alkyne, other acrylates, chitosan, hyaluronic acid, collagen, fibrin, gelatin, elastin, or any combination thereof.

[0243] 46. ​​The method of any one of embodiments 1-45, wherein the first beads are gel beads.

[0244] 47. The method of any one of embodiments 1-46, wherein the second beads are gel beads.

[0245] 48. The method of any one of embodiments 1-47, wherein the first bead is destructible upon application of a stimulus.

[0246] 49. The method of embodiment 48, wherein the stimulation is chemical stimulation, thermal stimulation, electromagnetic stimulation, or a combination thereof.

[0247] 50. The method of embodiment 48 or 49, wherein the given distribution area includes the stimulus.

[0248] 51. A method as described in any of embodiments 1-50, wherein (c) comprises subjecting the one or more precursors to stimulation to produce the second bead.

[0249] 52. A method as described in embodiment 51, wherein the stimulus is thermal energy or electromagnetic energy.

[0250] 53. The method of embodiment 52, wherein the stimulus is ultraviolet light.

[0251] Example

[0252] Example 1. Nucleic acid amplification and addition of barcoded oligonucleotides

[0253] In a first operation, a first aqueous phase containing nucleic acid molecules and a second aqueous phase containing destructible gel beads and reagents for nucleic acid amplification are contacted with a third liquid phase containing an oil that is immiscible with both the first and second phases to form aqueous droplets surrounded by the immiscible oil phase. Figure 6 At least some of the droplets formed contain a single nucleic acid molecule, a single destructible gel bead, and a reagent for nucleic acid amplification. The destructible gel bead is destroyed by increasing the temperature, thereby producing a precursor. Nucleic acid amplification is performed on the nucleic acid molecules within the droplets to produce a reaction product comprising one or more nucleic acids. The precursor is then used to produce a gel bead by lowering the temperature.

[0254] In a second operation, the droplets are destroyed by breaking the emulsion and the resulting gel beads are placed in a fourth aqueous phase. The fourth aqueous phase containing gel beads containing the reaction product and the fifth aqueous phase containing gel beads containing the barcoded oligonucleotide are contacted with a sixth liquid phase containing an oil that is immiscible with both the fourth and fifth phases to form aqueous droplets surrounded by the immiscible oil phase. The gel beads containing the reaction product and the gel beads containing the barcoded oligonucleotide are destroyed by increasing the temperature, and the barcoded oligonucleotide is attached to the reaction product.

[0255] Example 2. Nucleic acid capture and amplification

[0256] In a first operation, a first aqueous phase containing single cells and a second aqueous phase containing destructible gel beads are contacted with a third liquid phase containing an oil that is immiscible with both the first and second phases to form aqueous droplets surrounded by the immiscible oil phase. Figure 6 At least some of the droplets formed contain a single cell, a single destructible gel bead, and a reagent for nucleic acid capture. The destructible gel beads are destroyed by increasing the temperature, thereby producing precursors. The cells within the droplets are lysed, and nucleic acids (e.g., RNA and DNA) are captured. The precursors are then used to produce gel beads containing the captured nucleic acids by lowering the temperature.

[0257] In a second operation, the droplets are destroyed by breaking the emulsion and the resulting gel beads are placed in a fourth aqueous phase. The fourth aqueous phase containing gel beads containing captured nucleic acids and a fifth aqueous phase containing reagents for nucleic acid amplification are contacted with a sixth liquid phase containing an oil that is immiscible with both the fourth and fifth phases to form aqueous droplets surrounded by an immiscible oil phase. The gel beads containing the captured nucleic acids are destroyed by increasing the temperature, and nucleic acid amplification is performed on the captured nucleic acids.

[0258] Example 3. Reverse transcription and nucleic acid amplification

[0259] In a first operation, a first aqueous phase containing RNA molecules and a second aqueous phase containing destructible gel beads and reagents for reverse transcription are contacted with a third liquid phase containing an oil that is immiscible with both the first and second phases to form aqueous droplets surrounded by the immiscible oil phase. Figure 6 At least some of the droplets formed contain a single RNA molecule, a single destructible gel bead, and a reagent for reverse transcription. The destructible gel bead is destroyed by increasing the temperature, thereby producing a precursor. Reverse transcription is performed on the RNA molecule to produce a reaction product containing one or more complementary DNA molecules. The precursor is then used to produce a gel bead containing the reaction product by lowering the temperature.

[0260] In a second operation, the droplets are destroyed by breaking the emulsion and the resulting gel beads are placed in a fourth aqueous phase. The fourth aqueous phase containing the gel beads containing the reaction product and the fifth aqueous phase containing the reagent for nucleic acid amplification are contacted with a sixth liquid phase containing an oil that is immiscible with both the fourth phase and the fifth phase to form aqueous droplets surrounded by the immiscible oil phase. The gel beads containing the reaction product are destroyed by increasing the temperature, and nucleic acid amplification is performed on one or more complementary deoxyribonucleic acid molecules.

[0261] Example 4. Cell lysis and nucleic acid amplification using lysis reagents

[0262] In a first operation, a first aqueous phase containing single cells and a second aqueous phase containing destructible gel beads and a reagent for cell lysis are contacted with a third liquid phase containing an oil that is immiscible with both the first and second phases to form aqueous droplets surrounded by the immiscible oil phase. Figure 6 At least some of the droplets formed contain a single cell, a single destructible gel bead, and a reagent for cell lysis. The destructible gel bead is destroyed by increasing the temperature, thereby producing a precursor. The cell within the droplet is lysed using the lysis reagent, thereby producing a cellular component. The precursor is then used to produce a gel bead containing the captured cellular component by lowering the temperature.

[0263] In a second operation, the droplets are destroyed by breaking the emulsion, the lysis reagent is washed off, and the resulting gel beads are placed in a fourth aqueous phase. The fourth aqueous phase containing the gel beads containing the cellular components and the fifth aqueous phase containing the reagents for nucleic acid amplification are contacted with a sixth liquid phase containing an oil that is immiscible with both the fourth and fifth phases to form aqueous droplets surrounded by the immiscible oil phase. The gel beads containing the cellular components are destroyed by increasing the temperature, and nucleic acid amplification is performed on the captured nucleic acids.

[0264] Example 5. Cell lysis and nucleic acid amplification using heating

[0265] In a first operation, a first aqueous phase containing single cells and a second aqueous phase containing destructible gel beads and a reagent for cell lysis are contacted with a third liquid phase containing an oil that is immiscible with both the first and second phases to form aqueous droplets surrounded by the immiscible oil phase. Figure 6 At least some of the droplets formed contain a single cell, a single destructible gel bead, and a reagent for cell lysis. The destructible gel bead is destroyed by increasing the temperature, thereby producing a precursor. The cells within the droplet are lysed using a lysis reagent and heating, thereby producing a cellular component. The precursor is then used to produce a gel bead containing the captured cellular component by lowering the temperature.

[0266] In a second operation, the droplets are destroyed by breaking the emulsion, the lysis reagent is washed off, and the resulting gel beads are placed in a fourth aqueous phase. The fourth aqueous phase containing the gel beads containing the cellular components and the fifth aqueous phase containing the reagents for nucleic acid amplification are contacted with a sixth liquid phase containing an oil that is immiscible with both the fourth and fifth phases to form aqueous droplets surrounded by the immiscible oil phase. The gel beads containing the cellular components are destroyed by increasing the temperature, and nucleic acid amplification is performed on the captured nucleic acids.

[0267] Example 6. Addition of tagging and barcoding oligonucleotides

[0268] In a first operation, a first aqueous phase containing nucleic acids and a second aqueous phase containing destructible gel beads and reagents for tagmentation are contacted with a third liquid phase containing an oil that is immiscible with both the first and second phases to form aqueous droplets surrounded by the immiscible oil phase. Figure 6 At least some of the droplets formed contain a single nucleic acid, a single destructible gel bead, and a reagent for tagging. The destructible gel bead is destroyed by increasing the temperature, thereby producing a precursor. Tagging is performed on the nucleic acid to produce a reaction product containing one or more modified nucleic acids. Then, by lowering the temperature, the precursor is used to produce a gel bead containing the reaction product acid.

[0269] In a second operation, the droplets are destroyed by breaking the emulsion and the resulting gel beads are placed in a fourth aqueous phase. The fourth aqueous phase containing gel beads containing the reaction product and the fifth aqueous phase containing gel beads containing the barcoded oligonucleotide are contacted with a sixth liquid phase containing an oil that is immiscible with both the fourth and fifth phases to form aqueous droplets surrounded by the immiscible oil phase. The gel beads containing the reaction product and the gel beads containing the barcoded oligonucleotide are destroyed by increasing the temperature, and the barcoded oligonucleotide is attached to the reaction product.

[0270] Example 7. Serial addition of barcoded oligonucleotides

[0271] In a first operation, a first aqueous phase comprising nucleic acids and a second aqueous phase comprising destructible gel beads containing barcoded oligonucleotides are contacted with a third liquid phase comprising an oil that is immiscible with both the first and second phases to form aqueous droplets surrounded by the immiscible oil phase. Figure 6 At least some of the droplets formed contain a single nucleic acid and a single destructible gel bead containing a barcoded oligonucleotide. The destructible gel bead is destroyed by increasing the temperature, thereby producing a precursor. The barcoded oligonucleotide is attached to the nucleic acid to produce a reaction product containing one or more barcoded nucleic acids. The precursor is then used to produce a gel bead containing a reaction product acid by lowering the temperature.

[0272] In a second operation, the droplets are broken by breaking the emulsion and the resulting gel beads are placed in a fourth aqueous phase. The fourth aqueous phase containing gel beads containing the reaction product and the fifth aqueous phase containing gel beads containing the additional barcoded oligonucleotide are contacted with a sixth liquid phase containing an oil that is immiscible with both the third and fourth phases to form aqueous droplets surrounded by the immiscible oil phase. The gel beads containing the reaction product and the gel beads containing the barcoded oligonucleotide are broken by increasing the temperature, and the additional barcoded oligonucleotide is attached to the barcoded nucleic acid.

[0273] Example 8. Cell lysis, reverse transcription, addition of barcoded oligonucleotides and nucleic acid amplification using lysis reagents

[0274] In a first operation, a first aqueous phase comprising single cells and a second aqueous phase comprising reagents for lysis, reagents for reverse transcription, and destructible gel beads comprising barcoded oligonucleotides are contacted with a third liquid phase comprising an oil that is immiscible with both the first and second phases to form aqueous droplets surrounded by the immiscible oil phase. Figure 6 . At least some of the formed droplets contain a single cell, a single destructible gel bead containing a barcoded oligonucleotide, a reagent for lysis, and a reagent for reverse transcription. The destructible gel beads are destroyed by increasing the temperature to produce a precursor. The cells in the droplet are lysed using a lysis reagent to produce a cell component containing RNA molecules and DNA molecules. Reverse transcription is performed on the RNA molecules to produce one or more complementary DNA molecules. The barcoded oligonucleotide is attached to the one or more complementary DNA molecules to produce a reaction product containing one or more barcoded complementary DNA molecules. The reaction product also contains the DNA molecules produced by cell lysis. Then, by lowering the temperature, the precursor is used to produce a gel bead containing the reaction product.

[0275] In a second operation, the droplets are destroyed by breaking the emulsion and the resulting gel beads are placed in a fourth aqueous phase. The fourth aqueous phase containing the gel beads containing the reaction product and the fifth aqueous phase containing the gel beads containing the additional barcoded oligonucleotide are contacted with a sixth liquid phase containing an oil that is immiscible with both the third phase and the fourth phase to form aqueous droplets surrounded by the immiscible oil phase. The gel beads containing the reaction product and the gel beads containing the barcoded oligonucleotide are destroyed by increasing the temperature to produce precursors, and the additional barcoded oligonucleotide is attached to the deoxyribonucleic acid molecule to produce a reaction product containing a barcoded complementary deoxyribonucleic acid molecule and a barcoded deoxyribonucleic acid molecule. Then, by lowering the temperature, the precursor is used to produce gel beads containing the reaction product.

[0276] In a third operation, the droplets are destroyed by breaking the emulsion and the resulting gel beads are placed in a seventh aqueous phase. The seventh aqueous phase containing the gel beads containing the reaction product and the eighth aqueous phase containing the reagent for nucleic acid amplification are contacted with a ninth liquid phase containing an oil that is immiscible with both the third phase and the fourth phase to form aqueous droplets surrounded by the immiscible oil phase. The gel beads containing the reaction product are destroyed by increasing the temperature, and nucleic acid amplification is performed on one or more barcoded complementary deoxyribonucleic acid molecules and barcoded deoxyribonucleic acid molecules.

[0277] The apparatus, systems, compositions and methods disclosed herein can 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) of a single cell. For example, biological particles (e.g., cells or cell beads) are distributed in a distribution area (e.g., droplets), and multiple analytes of the biological particles are processed for subsequent processing. Multiple analytes can be from a single cell. This can enable, for example, simultaneous analysis of the proteome, transcriptome and genome of a cell. As described herein, multiple analytes can be analyzed in a single operation or in a continuous operation.

[0278] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. The present invention is not intended to be limited by the specific embodiments provided in this specification. Although the present invention has been described with reference to the above description, the description and illustration of the embodiments herein are not intended to be interpreted in a limiting sense. Those skilled in the art will now think of many variations, changes and substitutions without departing from the present invention. In addition, it should be understood that all aspects of the present invention are not limited to the specific description, configuration or relative proportion described herein, which depend on a variety of conditions and variables. It should be understood that various alternatives of the embodiments of the present invention described herein can be adopted when practicing the present invention. Therefore, it is expected that the present invention should also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the present invention and thus cover methods and structures within the scope of these claims and their equivalents.

Claims

1. A non-volatile computer-readable medium suitable for use in an electronic device, the non-volatile computer-readable medium comprising instructions which, when executed by the electronic device, cause the electronic device to: (a) generating a plurality of dispensing zones, wherein a given dispensing zone of the plurality of dispensing zones comprises a target and a first bead, the first bead comprising a gel matrix or a polymer matrix and one or more reagents for performing one or more reactions on the target, wherein the first bead is destructible upon application of a stimulus; (b) applying the stimulus, thereby destroying the first bead and releasing one or more precursors and the one or more agents, wherein the precursors include a polymer precursor material capable of forming into the gel matrix or polymer matrix; (c) performing the one or more reactions on the target using the one or more reagents to produce one or more reaction products; and d) initiating the production of a second bead using the one or more precursors, the second bead comprising the one or more reaction products and the gel matrix or polymer matrix.

2. The non-transitory computer readable medium of claim 1, wherein the gel matrix or polymer matrix in the first bead is formed from the precursor.

3. The non-transitory computer readable medium of claim 1, wherein the precursor comprises poly-L-lactic acid, poly(ethylene-propylene), poly(ethylene-butylene), polyacrylamide, acrylamide, agarose, alginate, polyvinyl alcohol, PEG, PEG-diacrylate, PEG-acrylate / thiol, PEG-azide / alkyne, chitosan, hyaluronic acid, collagen, fibrin, gelatin, or elastin.

4. The non-transitory computer readable medium of claim 1, wherein the first bead does not contain the target.

5. A non-volatile computer-readable medium as described in claim 1, wherein the electronic device is configured to perform one or more additional reactions on an additional target to produce one or more additional reaction products, the additional target being different from the target, wherein the one or more additional reactions are performed in an additional distribution area containing the second beads, the additional distribution area being different from the given distribution area.

6. The non-transitory computer readable medium of claim 1, wherein the electronic device is configured to perform one or more additional reactions on the target to produce one or more additional reaction products.

7. The non-transitory computer readable medium of claim 5, wherein the additional targets comprise the one or more reaction products.

8. The non-transitory computer readable medium of claim 5, wherein the additional dispensing area comprises one or more additional reagents for conducting the one or more additional reactions.

9. The non-transitory computer readable medium of claim 5, wherein the additional dispensing area comprises a third bead containing one or more additional reagents for performing the one or more additional reactions.

10. The non-transitory computer readable medium of claim 5, wherein after performing the one or more additional reactions, the additional distribution area comprises the one or more precursors produced by destruction of the second bead.

11. The non-transitory computer readable medium of claim 10, wherein the instructions further cause the electronic device to generate a third bead using the one or more precursors, the third bead comprising the one or more additional reaction products.

12. The non-transitory computer readable medium of claim 11, wherein the third beads are gel beads.

13. The non-transitory computer-readable medium of claim 1, wherein (c) is performed in the given allocation.

14. The non-transitory computer-readable medium of claim 1, wherein (d) is performed outside of the given allocation.

15. The non-transitory computer readable medium of claim 14, wherein (d) is performed in another allocation.

16. The non-transitory computer-readable medium of claim 1, wherein the target is or is associated with a nucleic acid molecule, wherein the one or more reaction products are derived from the nucleic acid molecule, and wherein after (d), the one or more reaction products or derivatives thereof are sequenced.

17. The non-transitory computer readable medium of claim 1, wherein the plurality of dispensing areas is a plurality of droplets.

18. The non-transitory computer readable medium of claim 17, wherein the plurality of droplets are generated upon contacting a first liquid phase comprising the first beads and a second liquid phase immiscible with the first liquid phase.

19. The non-transitory computer readable medium of claim 1, wherein the plurality of allocation areas is a plurality of holes.

20. The non-transitory computer readable medium of claim 1, wherein the target is a nucleic acid molecule.

21. The non-transitory computer readable medium of claim 20, wherein the nucleic acid molecule is a ribonucleic acid molecule.

22. The non-transitory computer readable medium of claim 21, wherein the RNA molecule is a messenger RNA molecule.

23. The non-transitory computer readable medium of claim 20, wherein the nucleic acid molecule is a deoxyribonucleic acid molecule.

24. The non-transitory computer readable medium of claim 1, wherein the target is a protein.

25. The non-transitory computer readable medium of claim 1, wherein the target is a cell.

26. The non-transitory computer readable medium of claim 1, wherein the one or more reagents comprise one or more nucleic acid molecules.

27. The non-transitory computer readable medium of claim 26, wherein the one or more nucleic acid molecules comprise a molecular barcode.

28. The non-transitory computer readable medium of claim 27, wherein the one or more reaction products comprise the molecular barcode.

29. The non-transitory computer-readable medium of claim 26, wherein the one or more nucleic acid molecules is a plurality of nucleic acid molecules, wherein a given nucleic acid molecule in the plurality of nucleic acid molecules comprises a barcode sequence that is constant among the plurality of nucleic acid molecules.

30. The non-transitory computer readable medium of claim 29, wherein the given nucleic acid molecule comprises an additional barcode sequence that varies among the plurality of nucleic acid molecules.

31. The non-transitory computer readable medium of claim 26, wherein the first bead is from a plurality of beads, wherein the plurality of beads comprise a nucleic acid barcode sequence that differs between the plurality of beads.

32. The non-transitory computer readable medium of claim 1, wherein the one or more reagents include all reagents required to perform the one or more reactions.

33. The non-transitory computer readable medium of claim 1, wherein the one or more reactions comprise a reverse transcription reaction.

34. The non-transitory computer readable medium of claim 1, wherein the one or more reactions comprise nucleic acid modification.

35. The non-transitory computer readable medium of claim 34, wherein the nucleic acid modification is a deoxyribonucleic acid modification.

36. The non-transitory computer readable medium of claim 34, wherein the nucleic acid modification is a ribonucleic acid modification.

37. The non-transitory computer readable medium of claim 1, wherein the one or more reactions comprise nucleic acid amplification.

38. The non-transitory computer readable medium of claim 37, wherein the nucleic acid amplification is a polymerase chain reaction.

39. The non-transitory computer readable medium of claim 37, wherein the nucleic acid amplification is not polymerase chain reaction.

40. The non-transitory computer readable medium of claim 1, wherein the one or more reactions comprise cell lysis.

41. The non-transitory computer readable medium of claim 1, wherein the one or more reactions comprise tagmentation.

42. The non-transitory computer readable medium of claim 1, wherein the one or more reactions comprise adding a barcode oligonucleotide.

43. The non-transitory computer readable medium of claim 1, wherein the given distribution area comprises cells or a gel matrix containing one or more cellular components, and wherein the target is derived from the cells or gel matrix.

44. The non-transitory computer readable medium of claim 1, wherein the first bead is produced by polymerization or cross-linking of the one or more precursors.

45. The non-transitory computer readable medium of claim 44, wherein the first bead is a hydrogel.

46. ​​The non-transitory computer readable medium of claim 1, wherein the first beads are gel beads.

47. The non-transitory computer readable medium of claim 1, wherein the second beads are gel beads.

48. The non-transitory computer readable medium of claim 1, wherein the stimulus is a chemical stimulus, a thermal stimulus, an electromagnetic stimulus, or a combination thereof.

49. The non-transitory computer readable medium of claim 3, wherein the given allotment includes the stimulus.

50. The non-transitory computer readable medium of claim 1, wherein (d) comprises subjecting the one or more precursors to a second stimulus to produce the second bead.

51. The non-transitory computer readable medium of claim 50, wherein the second stimulus is thermal energy or electromagnetic energy.

52. The non-transitory computer readable medium of claim 50, wherein the second stimulus is ultraviolet light.

53. A system comprising one or more computer processors, the one or more computer processors being individually or collectively programmed to: (a) generating a plurality of dispensing zones, wherein a given dispensing zone of the plurality of dispensing zones comprises a target and a first bead, the first bead comprising a gel matrix or a polymer matrix and one or more reagents for performing one or more reactions on the target, wherein the first bead is destructible upon application of a stimulus; (b) applying the stimulus, thereby destroying the first bead and releasing one or more precursors and the one or more agents, wherein the precursors include a polymer precursor material capable of forming into the gel matrix or polymer matrix; (c) initiating the one or more reactions on the target using the one or more reagents to produce one or more reaction products; and (d) initiating the production of a second bead using the one or more precursors, the second bead comprising the one or more reaction products and the gel matrix or polymer matrix.

54. The system of claim 53, wherein the gel matrix or polymer matrix is ​​formed from a precursor.

55. The system of claim 54, wherein the precursor comprises poly-L-lactic acid, poly(ethylene-propylene), poly(ethylene-butylene), polyacrylamide, acrylamide, agarose, alginate, polyvinyl alcohol, PEG, PEG-diacrylate, PEG-acrylate / thiol, PEG-azide / alkyne, chitosan, hyaluronic acid, collagen, fibrin, gelatin, or elastin.

56. The system of claim 53, wherein the first bead does not comprise the target.

57. A system as described in claim 53, wherein the one or more computer processors are also programmed, individually or collectively, to perform the following steps: performing one or more additional reactions on additional targets within or outside the given distribution area to produce one or more additional reaction products, wherein the additional targets are different from the target.

58. The system of claim 53, wherein the one or more computer processors are further programmed, individually or collectively, to perform one or more additional reactions on the target to produce one or more additional reaction products.

59. The system of claim 57, wherein the additional targets comprise the one or more reaction products.

60. The system of claim 57, wherein the one or more additional reactions are performed in an additional dispensing zone containing the second beads, the additional dispensing zone being different from the given dispensing zone.

61. The system of claim 60, wherein the additional dispensing area comprises one or more additional reagents for performing the one or more additional reactions.

62. The system of claim 60, wherein the additional distribution area comprises a third bead containing one or more additional reagents for performing the one or more additional reactions.

63. The system of claim 60, wherein after performing the one or more additional reactions, the additional distribution area contains the one or more precursors produced by destruction of the second bead.

64. The system of claim 57, wherein after performing the one or more additional reactions, the given distribution area contains the one or more precursors produced by destruction of the second bead.

65. The system of claim 64, wherein the one or more computer processors are further programmed, individually or collectively, to perform the following step: generating a third bead using the one or more precursors, the third bead comprising the one or more additional reaction products.

66. The system of claim 65, wherein the third beads are gel beads.

67. The system of claim 53, wherein (d) is performed in the given allocation area.

68. The system of claim 53, wherein (d) is performed outside of the given allocation area.

69. The system of claim 68, wherein (d) is performed in another allocation area.

70. The system of claim 53, wherein the target is or is associated with a nucleic acid molecule, wherein the one or more reaction products are derived from the nucleic acid molecule, and wherein after (d), the one or more reaction products or derivatives thereof are sequenced.

71. The system of claim 53, wherein the plurality of distribution areas are a plurality of droplets.

72. The system of claim 71, wherein the plurality of droplets are generated upon contact of a first liquid phase comprising the first beads and a second liquid phase immiscible with the first liquid phase.

73. The system of claim 53, wherein the plurality of distribution areas are a plurality of holes.

74. The system of claim 53, wherein the target is a nucleic acid molecule.

75. The system of claim 74, wherein the nucleic acid molecule is a ribonucleic acid molecule.

76. The system of claim 75, wherein the RNA molecule is a messenger RNA molecule.

77. The system of claim 74, wherein the nucleic acid molecule is a deoxyribonucleic acid molecule.

78. The system of claim 53, wherein the target is a protein.

79. The system of claim 53, wherein the target is a cell.

80. The system of claim 53, wherein the one or more reagents comprise one or more nucleic acid molecules.

81. The system of claim 80, wherein the one or more nucleic acid molecules comprise a molecular barcode.

82. The system of claim 81, wherein the one or more reaction products comprise the molecular barcode.

83. The system of claim 80, wherein the one or more nucleic acid molecules is a plurality of nucleic acid molecules, wherein a given nucleic acid molecule in the plurality of nucleic acid molecules comprises a barcode sequence that is constant among the plurality of nucleic acid molecules.

84. The system of claim 83, wherein the given nucleic acid molecule comprises an additional barcode sequence that varies among the plurality of nucleic acid molecules.

85. The system of claim 80, wherein the first bead is from a plurality of beads, wherein the plurality of beads comprise nucleic acid barcode sequences that differ between the plurality of beads.

86. The system of claim 53, wherein the one or more reagents include all reagents required to perform the one or more reactions.

87. The system of claim 53, wherein the one or more reactions comprise a reverse transcription reaction.

88. The system of claim 53, wherein the one or more reactions comprise nucleic acid modification.

89. The system of claim 88, wherein the nucleic acid modification is a deoxyribonucleic acid modification.

90. The system of claim 88, wherein the nucleic acid modification is a ribonucleic acid modification.

91. The system of claim 53, wherein the one or more reactions comprise nucleic acid amplification.

92. The system of claim 91, wherein the nucleic acid amplification is a polymerase chain reaction.

93. The system of claim 91, wherein the nucleic acid amplification is not polymerase chain reaction.

94. The system of claim 53, wherein the one or more reactions comprise cell lysis.

95. The system of claim 53, wherein the one or more reactions comprise labeling.

96. The system of claim 53, wherein the one or more reactions comprise adding a barcode oligonucleotide.

97. The system of claim 53, wherein the given distribution area comprises cells or a gel matrix containing one or more cellular components, and wherein the target is derived from the cells or gel matrix.

98. The system of claim 53, wherein the first bead is produced by polymerization or cross-linking of the one or more precursors.

99. The system of claim 98, wherein the first bead is a hydrogel.

100. The system of claim 53, wherein the first beads are gel beads.

101. The system of claim 53, wherein the second beads are gel beads.

102. The system of claim 53, wherein the stimulation is chemical stimulation, thermal stimulation, electromagnetic stimulation, or a combination thereof.

103. The system of claim 53, wherein the given distribution area includes the stimulus.

104. The system of claim 53, wherein (c) comprises subjecting the one or more precursors to a stimulus to produce the second bead.

105. The system of claim 104, wherein the stimulus is thermal energy or electromagnetic energy.

106. The system of claim 104, wherein the stimulus is ultraviolet light.

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