RU2822154C2 - Modulation of polymer granules for dna processing - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: group of inventions relates to a polymer granule for conducting a plurality of combined analysis reactions, comprising: a hydrogel polymer precursor; cross-linking agent; and a biomolecule within the polymer granule, wherein the granule is a porous hollow granule, wherein the granule comprises a plurality of polymer layers, wherein each layer is characterized by a certain pore size and pore density, providing diffusion of the reagent through the granule, which retains the biomolecule, also relates to a method for conducting a plurality of successive joint analyses of a biomolecule encapsulated inside a polymer granule, involving: obtaining a polymer granule encapsulating a biomolecule; and sequential bringing a single cell into contact with reagents for multiple sequential combined assays.

EFFECT: group of inventions provides a polymer granule for fast and effective treatment by reducing the loss of biomolecules by retaining them inside the granule, while providing diffusion of one or more reagents through the granule.

32 cl, 29 dwg, 7 ex

Description

TECHNICAL FIELD

[0001] The systems, methods and compositions provided herein relate to polymer beads, methods of encapsulating biomolecules in polymer beads, and methods of using polymer beads to perform assays on encapsulated biomolecules, including, for example, spatial index sequencing and nucleic acid library production.

PREREQUISITES FOR CREATION OF THE INVENTION

[0002] Detection of specific nucleic acid sequences present in a biological sample is used, for example, as a method for identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic abnormalities, identifying genetic changes associated with various diseases such as cancer, studying genetic predisposition to a particular disease or assessing responses to various forms of treatment. Detection of nucleotide sequences in a biological sample requires multiple enzymatic reactions to ultimately determine the nucleotide sequence or generate a nucleic acid library.

[0003] Conducting multiple enzymatic reactions in a single cell does not provide sufficient reliability due to problems associated with isolating and accessing intracellular biomolecules within a single cell across multiple assays. Many cell-based assays fail to isolate intracellular molecules, resulting in loss of biomolecules during the assay.

SUMMARY OF THE INVENTION

[0004] Some embodiments provide a polymer bead for performing multiple co-assay reactions. In some embodiments, the polymer bead contains a hydrogel polymer precursor, a cross-linking agent, and a biomolecule housed within the polymer bead, the bead containing pores that allow diffusion of one or more reactants through the bead while retaining the biomolecule. In some embodiments, the bead is a porous hydrogel bead or a porous hollow bead. In some embodiments, the granule contains multiple layers of polymer, each layer having a specific pore size and pore density. In some embodiments, the pore size is modulated depending on changes in charge, pH, or temperature.

[0005] Some embodiments relate to a method for performing multiple sequential co-assays of a biomolecule encapsulated within a polymer bead. In some embodiments, the method includes producing a polymer bead encapsulating a biomolecule, wherein the polymer bead contains a hydrogel polymer precursor, a cross-linking agent, and a biomolecule housed within the polymer bead, wherein the bead contains pores that allow diffusion of one or more reactants through the bead that retains at the same time a biomolecule. In some embodiments, the method further includes sequentially contacting a single cell with reagents to perform multiple sequential co-assays. In some embodiments, the method further includes modulating the pore size of the polymer bead by adjusting charge, pH, or temperature. In some embodiments, the polymer bead contains multiple polymer layers, and each polymer layer has pores of varying sizes. In some embodiments, the pore size of each polymer layer is modulated in a specific manner by changing charge, pH, or temperature. In some embodiments, the plurality of sequential co-assays include lysis, DNA analysis, RNA analysis, protein analysis, tagmentation, nucleic acid amplification, nucleic acid sequencing, DNA library preparation, analysis of transposase accessible chromatin using sequencing (ATAC-seq), transposition with contiguity preservation (CPT-seq), combinatorial single-cell indexed sequencing (SCI-seq), or single-cell genome amplification, or any combination thereof performed sequentially.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0006] In FIG. 1A is a diagram illustrating an embodiment of spatial indexing of long DNA during flow cell library acquisition and seeding.

[0007] In FIG. 1B is a diagram illustrating spatial indexing using polymer beads encapsulating long DNA molecules. Reagents can be used to spatially generate a library on the surface of a flow cell on polymer beads.

[0008] In FIG. 2 is a flow diagram describing a method for encapsulating long DNA within a polymer bead and producing a library within the polymer bead that can be clustered and sequenced in a flow cell device.

[0009] In FIG. 3 is a diagram illustrating the process of sequencing long DNA encapsulated in polymer beads, including DNA fragments approximately 100 kb in length. (without the multiple displacement amplification (MDA) step (panel (a)) or with the MDA step (panel (b)) before tagmentation) and DNA fragments of about 10-20 kb in length. (panel (c)).

[0010] In FIG. Figure 4 is a graph showing gated reads of long DNA spatial indexing data in a hydrogel from a 100 kb DNA fragment. without MDA.

[0011] In FIG. Figure 5 shows a line graph of linked reads spatially indexing long DNA in a hydrogel of 100 kb DNA fragments. with MDA.

[0012] In FIG. Figure 6 shows a line plot of linked reads spatially indexing long DNA in a hydrogel of 10 kb DNA fragments. with MDA.

[0013] In FIG. 7A and 7B show line plots of spatial reads for long DNA encapsulated within a polymer bead. In FIG. Figure 7A shows spatial reads for cells encapsulated inside a polymer bead, and the inset shows a micrograph of a cell inside a polymer bead. In FIG. Figure 7B shows spatial read data for long DNA fragments encapsulated within a polymer bead, and the inset shows a micrograph of fragments encapsulated within a polymer bead.

[0014] In FIG. Figure 8 is a micrograph showing the identification of various types of microorganisms encapsulated within the polymer bead. Various species of microorganisms are encapsulated inside the polymer bead, and spatial sequencing reads were performed to identify the microbes.

[0015] In FIG. Figure 9 is a graph showing the distribution of barcode reads for long DNA encapsulated within a polymer bead.

[0016] In FIG. 10A is a graph showing short reads and associated reads for one cycle of an E. coli cell encapsulated within a polymer bead. As shown in this figure, linked reads span repeat regions and can improve de novo sequence assembly. In FIG. 10B is a micrograph showing spatially coupled reads and intermediate short reads.

[0017] In FIG. 11A is a diagram illustrating the diffusion of molecules into a polymer bead as a function of pore size and pore density. In FIG. 11B is a diagram illustrating the diffusion of molecules outside a polymer bead.

[0018] In FIG. 12 is a diagram illustrating the retention of nucleic acid libraries within a polymer bead.

[0019] In FIG. 13A and 13B are graphs showing the diffusion of nucleic acid into a polymer bead as a function of nucleic acid size. The graphs show the penetration of short amplicons (220 kb to 1 kb) into the polymer beads, while longer amplicons do not diffuse into the polymer beads.

[0020] In FIG. 14 shows micrographs of porous hollow beads showing PCR amplification within the porous hollow beads for both 200 bp and 5000 bp DNA fragments.

[0021] In FIG. 15 is a diagram showing multilayer polymer beads, each layer having different pore sizes, and the pore sizes of each layer can be independently modulated depending on environmental conditions.

[0022] In FIG. 16A-16C show polymer granules with a stabilized shell with a degradable polyacrylamide core. In FIG. 16A is a schematic diagram of the structure of a polymer bead shelled with N,N'-(1,2-dihydroxyethylene)bisacrylamide (DHEBA) with PEG acrylate and potassium peroxydisulfate (KPS). Inside is a core of filler, such as PEG or polyacrylamide, mixed with a sample (cell or DNA). In FIG. 16B shows the formation of a shell around a granule. In FIG. 16C shows micrographs of DHEBA shells with degradable polyacrylamide cores.

[0023] In FIG. 17A-17C show polymer beads with a stabilized shell with a degradable agarose core. In FIG. 17A is a schematic representation of the structure of a DHEBA shell with an agarose core mixed with a sample (cell or DNA). In FIG. 17B shows micrographs of DHEBA shells with agarose cores. In FIG. 17C shows micrographs of the melting of DHEBA shells with agarose cores under the influence of temperature.

[0024] In FIG. 18A-18C show fluorescence initiated gelation. As shown in FIG. 18A, living cells are contacted with the gelling agent fluorescein isothiocyanate-acrylate polymer (FITC-AETC). Cells coated with FITC-AETC and monomers are irradiated, which results in the formation of gel granules around the cells. In FIG. 18B shows photomicrographs including irradiation microscopy of FITC-AETC-treated cells compared to control cells not treated with FITC-AETC. In FIG. 18C shows micrographs of FITC-initiated gelation around cells under various conditions.

DETAILED DESCRIPTION

[0025] The following detailed description contains references to related drawings that form a part of this document. In graphic materials, similar symbols generally indicate similar components, unless the context otherwise requires. The illustrative embodiments described in the detailed description, drawings and claims are not intended to be limiting. Other embodiments and other changes may be used without departing from the spirit or scope of the claimed subject matter presented herein. It should be understood that the aspects of the present specification generally presented herein and illustrated in the figures can be rearranged, replaced, combined, separated, and constructed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0026] Embodiments relate to compositions, systems, and methods for encapsulating biomolecules within polymer beads and performing one or more assays on the encapsulated biomolecules. The polymer bead retains the biomolecules within the bead but allows smaller molecules, such as reagents, to diffuse into and out of the polymer bead while retaining the biomolecule being analyzed within. As described herein, the polymer bead contains pores, and the size and density of the pores are modulated to control the size or molecules that diffuse into or out of the polymer beads.

[0027] In one embodiment, the polymer bead is a homogeneous porous hydrogel matrix that encapsulates or contains one or more biomolecules. In another embodiment, the polymer bead is a hollow bead with a porous hydrogel shell and a hollow interior with a biomolecule encapsulated within the hollow interior. In some embodiments, a polymer bead (either a homogeneous porous hydrogel matrix or a hollow bead) may include multiple polymer layers, with each polymer layer being a distinct matrix with specific properties, such as pore size. In some embodiments, each polymer layer is controlledly modulated to adjust the pore size in each polymer layer, thereby allowing the user to control the diffusion of molecules into and out of the polymer bead in a controlled, incremental manner. In some embodiments, each polymer layer is controlledly modulated by changing environmental conditions, such as pH, charge, or temperature, wherein such changes in environmental conditions modulate the pore size of the bead or bead shell and thereby allow molecules or reactants to be released from the bead or penetrate into in a controlled and gradual manner.

[0028] Embodiments described herein include reliable and high-throughput systems and methods for conducting sequential reactions with a biomolecule encapsulated within a polymer bead. The methods and systems described herein relate to performing one or more analyzes of an encapsulated biomolecule, including, for example, lysis, DNA analysis, RNA analysis, protein analysis, tagmentation, nucleic acid amplification, nucleic acid sequencing, DNA library preparation, analysis of available for chromatin transposase sequencing (ATAC-seq), contiguity-preserving transposition (CPT-seq), combinatorial single-cell indexed sequencing (SCI-seq), or single-cell genome amplification, or any combination thereof performed sequentially.

[0029] One embodiment is a method of encapsulating a biomolecule within a polymer bead, loading polymer beads encapsulating the biomolecule into a flow cell device, preparing a nucleic acid library, applying the prepared library to the surface of the flow cell device, and clustering and sequencing the deposited library. In some embodiments, the biomolecule is a cell, protein, nucleic acid, DNA, RNA, or any derivative or analog thereof.

[0030] In some embodiments, library production involves tagmentation of DNA contained within a polymer bead. Tagmentation of encapsulated DNA results in the cleavage of longer DNA sequences into shorter tagmentation fragments, which are then used to form DNA clusters on the surface of the flow cell. A cluster is formed from long DNA tagmentation fragments, each of which can be sequenced, for example, using sequencing by synthesis (SBS). A group of clusters of a single long DNA molecule is referred to herein as a “long DNA island.” In some embodiments, a single long DNA molecule or multiple long DNA molecules can be encapsulated in a single polymer bead. Each long DNA molecule forms one long DNA island. Clusters of all long DNA islands within a single polymer bead are referred to herein as a “cluster cloud.” Thus, a cluster cloud represents all the clusters within a single polymer bead and may include multiple long DNA islands (each long DNA island represents one long DNA molecule), and each long DNA island includes multiple clusters.

[0031] The beads may include hydrogel polymers and cross-linking agents that are mixed in the presence of a biomolecule, such as a nucleic acid, such as a long DNA molecule, or a source containing the nucleic acid, subsequently forming polymer beads encapsulating the biomolecule. In some embodiments, the source of the nucleic acid is a cell.

[0032] In some embodiments, the pores of the bead allow diffusion of a molecule that is less than 1000 base pairs, such as a molecule that is less than 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 base pairs or less, or the size within the range defined by any two of the above values, but retains compounds (or prevents the diffusion of compounds) exceeding the above values. Thus, in some embodiments, the polymer beads retain (or prevent diffusion of) compounds that are greater than 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 pairs nucleotides or more, or a value within the range defined by any two of the above values.

[0033] Some embodiments include methods of using biomolecule-encapsulating beads to conduct nucleic acid reactions, including, for example, high-throughput spatial indexing of long DNA molecules. As shown in FIG. 1A, a long DNA molecule library can be easily constructed by clustering and seeding clusters of a single long DNA molecule into a “cluster spot” on a surface, which can then be read and spatially mapped. As used herein, the term “long DNA” can include DNA fragments containing more than 300 base pairs. The term "long DNA fragments" as used herein refers to DNA longer than 1 kb, 2.5 kb, 5 kb. or more, for example 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 kb. or more, including values within the range defined by any two of the above values.

[0034] Some embodiments include methods of using a single bead to fragment a genome sample into a series of long DNA fragments. This single bead can then be mounted at one specific location in a flow cell where long DNA fragments are deposited such that each long DNA fragment is adjacent to another fragment on the surface of the flow cell. The flow cell can then be used in a nucleotide sequencing system, such as the ILLUMINA HISEQ system, to determine the nucleotide sequence of each piece of long DNA. Because the DNA fragments are located adjacent to each other on the surface of the flow cell, the system can use this spatial arrangement data to more efficiently reconstruct the final nucleotide sequence of the original genomic DNA. The system can deposit spatially adjacent reads directly from single cells, long DNA fragments, or chromosomes. In some embodiments, the methods allow the use of a low-input, PCR-free process to produce the library. In some embodiments, the methods can be implemented without the need for molecular barcoding.

[0035] Some embodiments relate to methods for producing a polymer bead encapsulating a biomolecule. In some embodiments, a polymer bead encapsulating long DNA can be used to process a cell genome and prepare a DNA library within the bead. In some embodiments, a polymer bead encapsulating a long DNA fragment encapsulates a single cell and can be used to process the cell's genomic DNA and prepare a library of the total DNA within the bead.

[0036] In some embodiments, the pore size of the polymer bead can be designed to allow diffusion of smaller size enzymes, chemicals, and primers (<50 bp) while retaining larger nucleic acids (>300 bp .) so that long DNA fragments and the resulting DNA library can remain inside the polymer beads during processing. In some embodiments, specific primers can be chemically linked within a polymer bead matrix to hybridize and process specific genomic DNA. The DNA library from a single cell can then be applied to a specific area, such as the surface of a library seeding flow cell. This subsequently leads to the formation of a spatial distribution of “DNA clusters” in the flow cell, which are formed from encapsulated fragments of long DNA, which simplifies read alignment during subsequent processing.

[0037] As used herein, the term “polymer bead” refers to a porous hydrogel bead or a porous hollow bead. In some embodiments, the polymer bead is provided as a porous hydrogel bead. A porous hydrogel granule is a granule that includes a porous matrix with relative uniformity throughout the granule. As described herein, a porous hydrogel bead encapsulates a biomolecule and includes pores that allow diffusion of molecules into or out of the porous hydrogel bead. The pores can also be modulated to change pore size depending on changes in environmental conditions such as pH, temperature, or charge, thereby allowing controlled diffusion of molecules into or out of the porous hydrogel beads. In some embodiments, the porous hydrogel bead may include one or more types of polymers, each polymer having a specific pore size and pore density, and each polymer being independently modulated to allow diffusion of molecules of different sizes depending on changing environmental conditions.

[0038] In some embodiments, the polymer bead is provided in the form of a porous, hollow bead. A porous hollow granule is a granule with a porous polymer shell, but with a hollow interior. As described herein, a porous hollow bead encapsulates a biomolecule within a hollow interior space. The pores of the polymer shell allow the diffusion of molecules into or out of the hollow interior and can be modulated to change pore size depending on changes in environmental conditions such as pH, temperature or charge, thus allowing controlled diffusion of molecules into the hollow interior or out of him. In some embodiments, the porous hollow granule may include a plurality of porous polymer shells, each shell having a specific pore size and pore density, and each shell being independently modulated to allow diffusion of molecules of different sizes depending on changing environmental conditions. For example, as shown in FIG. 15, the porous hollow granule may include a hollow interior (or hollow core) with a plurality of polymer shells. In FIG. 15 shows a porous hollow granule with three porous polymer shells, which are indicated in FIG. 15 as polymer 1, polymer 2 and polymer 3. The molecules are retained within the porous hollow granule, and modulation of the first polymer causes the first molecule to diffuse through the polymer shell. Modulation of the second polymer causes the second molecule to diffuse through the polymer shell. One skilled in the art will appreciate that the example shown in FIG. 15 is illustrative in nature and that a variety of porous polymer shells can be used to control the diffusion of molecules into or out of a porous hollow granule. For example, a porous hollow granule may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more individual polymer shells, each polymer shell having a specific pore size and pore density, and each polymer shell configured to be modulated to control the diffusion of a molecule into or out of the porous hollow granule.

[0039] As used herein, the term "reagent" describes an agent or mixture of two or more agents suitable for reacting, reacting with, diluting, or adding to a sample, and may include agents used in nucleic acid reactions, including eg buffers, chemicals, enzymes, polymerase, primers smaller than 50 bp, template nucleic acids, nucleotides, tags, dyes or nucleases. In some embodiments, the reagent includes lysozyme, proteinase K, random hexamers, polymerase (e.g., F29 DNA polymerase, Taq polymerase, Bsu polymerase), transposase (e.g., Tn5), primers (e.g., P5 and P7 adapter sequences), ligase, catalyzing enzyme, deoxynucleotide triphosphates, buffers or divalent cations. Polymer granules encapsulating biomolecules

[0040] One embodiment includes a bead with a hydrogel polymer and a biomolecule. As used herein, the term “hydrogel” refers to a substance resulting from the cross-linking of an organic polymer (natural or synthetic) through covalent, ionic or hydrogen bonding, creating a three-dimensional open lattice structure that traps water molecules to form a gel. In some embodiments, the hydrogel may be a biocompatible hydrogel. As used herein, the term “biocompatible hydrogel” refers to a gel-forming polymer that is non-toxic to living cells and allows sufficient diffusion of oxygen and nutrients into the entrapped cells to maintain their viability. In some embodiments, the hydrogel polymer includes 60-90% liquid, such as water, and 10-30% polymer. In some embodiments, the water content of the hydrogel is about 70-80%.

[0041] Hydrogels can be prepared by cross-linking hydrophilic biopolymers or synthetic polymers. Thus, in some embodiments, the hydrogel may include a cross-linking agent. As used herein, the term “cross-linking agent” refers to a molecule that can form a three-dimensional network when reacted with appropriate framework monomers. Examples of hydrogel polymers that may include one or more cross-linking agents include, but are not limited to, hyaluronans, chitosans, agar, heparin, sulfate, cellulose, alginates (including alginate sulfate), collagen, dextrans (including dextran sulfate), pectin, carrageenan, polylysine, gelatins (including gelatin type A), agarose, (methyl)acrylate-oligolactide-PEO-oligolactide-(methyl)acrylate, PEO-PPO-PEO copolymers (Pluronics), poly(phosphazene), poly(methacrylates), poly( M-vinylpyrrolidone), copolymers PL(G)A-PEO-PL(G)A, poly(ethyleneimine), polyethylene glycol (PEO)-thiol, PEG-acrylate, maleimide (MAL), PEG/MAL, acrylamide, N,N '-bis(acryloyl)cystamine, PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(M-isopropylacrylamide) (PNIPAAm), poly(lactic) acid ( PLA), copolymer of poly(lactic) and poly(glycolic) acids (PLGA), polycaprolactone (PCL), poly(vinylsulfonic) acid (PVSA), poly(L-aspartic) acid, poly(L-glutamic) acid, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethylene glycol diallyl ether, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylopropane trimethacrylate, ethoxylated trimethylol triacrylate or ethoxylated pentaerythritol tetraacrylate, or combinations thereof. Thus, for example, the combination may include a polymer and a cross-linker such as polyethylene glycol (PEG)-thiol/PEG-acrylate, acrylamide/N,N'-bis(acryloyl)cystamine (BACy), PEG/polypropylene oxide (PPO) or N,N'-(1,2-dihydroxyethylene)bisacrylamide (DHEBA).

[0042] In some embodiments, the cross-linker forms a disulfide bond in the hydrogel polymer, thereby linking the hydrogel polymers. In some embodiments, the hydrogel polymers form a hydrogel matrix or polymer shell with pores. The pores are capable of holding fairly large molecules within the polymer bead, such as long DNA fragments, but allow small components, such as reagents, to pass through the pores, thereby moving in and out of the polymer beads. In some embodiments, the pore size is finely controlled by varying the ratio of polymer concentration to cross-linker concentration. In some embodiments, the polymer to crosslinker ratio is 30:1, 25:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1: 2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20 or 1:30, or the ratio is in range determined by any two of the above relations. In some embodiments, additional functional groups, such as a DNA primer or charged chemical groups, may be grafted onto the polymer matrix to suit the requirements of various applications.

[0043] Moreover, in some embodiments, the pores can be modulated, adjusted, altered, modified, adapted, or optimized in size or density by changing the environmental conditions in which the polymer beads are exposed, including changes in pH, charge, or temperature. Regulation of pores can be accomplished in a controlled manner by introducing incremental changes to the environment, thereby creating conditions for controlled diffusion of molecules.

[0044] As used herein, the term “porosity” means the fractional volume (dimensionless) of a hydrogel that consists of open space, such as pores or other openings. Thus, porosity measures the voids in a material and reflects the proportion of void volume to total volume as a percentage from 0 to 100% (or from 0 to 1). The porosity of the hydrogel may range from 0.5 to 0.99, from about 0.75 to about 0.99, or from about 0.8 to about 0.95.

[0045] Hydrogels can have any pore size. As used herein, the term “pore size” refers to the diameter or effective cross-sectional diameter of the pores. The term "pore size" can also refer to the average diameter or the average effective cross-sectional diameter of the pores as measured by a plurality of pores. The effective diameter of a non-circular cross-section is equal to the diameter of a circular cross-section with the same cross-sectional area as the non-circular cross-section. In some embodiments, the hydrogel may swell when the hydrogel is hydrated. As a result, pore sizes can vary depending on the water content of the hydrogel. In some embodiments, the hydrogel pores may be of sufficient size to retain biomolecules within the polymer bead but allow reactants to pass through, and may be adjusted as described herein to allow molecules to pass through them in a controlled manner.

[0046] In some embodiments, the cross-linking agent is a reversible cross-linking agent. In some embodiments, the reversible cross-linking agent is configured to reversibly cross-link the hydrogel polymer, and such cross-links may be disrupted by the cleavage agent. In some embodiments, the cross-linker may be degraded in the presence of a reducing agent, by elevated temperature, or by exposure to an electric field. In some embodiments, the reversible cross-linking agent may be N,N'-bis(acryloyl)cystamine, a reversible cross-linking agent for polyacrylamide gels in which the disulfide bond can be broken in the presence of a suitable reducing agent. In some embodiments, the interaction of the cross-linker with the reducing agent cleaves the disulfide bonds of the cross-linker, breaking down the polymer beads. The polymer beads degrade and release contents, such as nucleic acids, that were held within them. In some embodiments, the crosslinker is degraded when the temperature is increased to greater than 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100°C. In some embodiments, the cross-linker is degraded when the polymer beads react with a reducing agent. In some embodiments, reducing agents include phosphine compounds, water-soluble phosphines, nitrogen-containing phosphines and salts and derivatives thereof, dithioerythritol (DTE), dithiothreitol (DTT) (cis and trans isomers of 2,3-dihydroxy-1,4-dithiolbutane, respectively) , 2-mercaptoethanol or β-mercaptoethanol (BME), 2-mercaptoethanol or aminoethanethiol, glutathione, thioglycolate or thioglycolic acid, 2,3-dimercaptopropanol, tris(2-carboxyethyl)phosphine (TCEP), tris(hydroxymethyl)phosphine (THP) or P-[tris(hydroxymethyl)phosphine]propionic acid (THPP).

[0047] In some embodiments, increasing the temperature to accelerate diffusion or interaction with the reducing agent causes the cross-linker to degrade, thereby releasing the encapsulated or derived biomolecule from the polymer bead.

[0048] In some embodiments, cross-linking using a cross-linking agent creates pores within the polymer bead. In some embodiments, the pore size of the polymer beads is adjustable and selected to encapsulate biomolecules, such as DNA fragments greater than about 5000 bp, but allow smaller sized particles, such as reagents, or smaller nucleic acids less than about 50 bp. nucleotides, such as primers, pass through the pores, as shown in FIG. 1B. In some embodiments, the reagents include reagents for processing biomolecules or a molecule derived therefrom, such as reagents for isolating nucleic acids from a cell, for amplifying, barcoding or sequencing nucleic acids, or for producing nucleic acid libraries. In some embodiments, the reagents include, for example, lysozyme, proteinase K, random hexamers, polymerase (e.g., F29 DNA polymerase, Taq polymerase, Bsu polymerase), transposase (e.g., Tn5), primers (e.g., P5 adapter sequences and P7), ligase, catalyzing enzyme, deoxynucleotide triphosphates, buffers or divalent cations.

[0049] In some embodiments, long DNA includes genomic DNA, viral nucleic acids, bacterial nucleic acids, or mammalian nucleic acids. In some embodiments, the polymer beads include a source of long DNA, including, for example, a cell. In some embodiments, the cell is a single cell, including a prokaryotic or eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a bacterial cell. Thus, as shown in FIG. 7A and 7B, the method can be implemented on long DNA fragments or on cells, one or both of which are encapsulated within a polymer bead.

[0050] In some embodiments, the polymer bead is a erodible polymer bead with a erodible core encapsulated in a shell. As used herein, the term “erodible” is used in its generally accepted meaning, understood in light of the description, and refers to a granule or portion of a granule that is used to prepare or assemble the granule, but which may be dissolved or removed at a later stage. In some embodiments, the erodible polymer bead includes a polymeric material, such as agarose or polyacrylamide, that is in contact with a sample, such as a cell or DNA, thereby forming a polymer bead. The polymer bead can then be encapsulated in a shell containing another polymer material with different melting characteristics, for example, a shell that includes N,N'-(1,2-dihydroxyethylene)bisacrylamide (DHEBA), acylate-PEG or KPS, or their combination. Once encapsulated, the encapsulated polymer bead can be placed under conditions sufficient to remove the interior of the bead without destroying the shell. Such conditions may include, for example, changes in temperature, pH, or the addition of reducing agents.

[0051] In some additional embodiments, any of the polymer beads described herein may include a fluorescent material associated with the polymeric material. Such fluorescent materials may include, for example, fluorescein isothiocyanate (FITC) or any other fluorescent compound that can be bonded to a polymeric material, such as acrylate polymers, to form a fluorescent polymeric material such as FITC-acrylate polymer (FITC-AETC). The fluorescent material bound to the polymeric material can be brought into contact with the cell, thereby initiating fluorescent gelation to form a fluorescent polymer bead around the cell. In some embodiments, fluorescent images of polymer beads with a cell inside can be obtained without the need for staining. Methods for preparing polymer granules

[0052] Some embodiments provided herein relate to methods for producing beads that encapsulate biomolecules. In some embodiments, the polymer bead is a porous hydrogel bead, as described herein, or a porous hollow bead, as described herein.

[0053] In some embodiments, the polymer pellet is produced from a vortex emulsion. As used herein, the term “vortex emulsion” refers to the vortexing of a hydrogel polymer with long DNA fragments or a source of long DNA fragments in a container such as a tube, vial, or reaction vessel. The components can be mixed, for example, manually or using a mechanical vortex mixer or shaking. In some embodiments, manual mixing results in the formation of polymer beads that encapsulate biomolecules with a size of 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 , 75, 80, 85, 90, 95, 100, 110, 120, 130, 140 or 150 µm in diameter or with a size within the range defined by any two of the above values. In some embodiments, the granule size is not uniform and therefore the granule size includes granules of varying diameters.

[0054] In some embodiments, the granules are produced by forming microfluidic droplets. As shown in FIG. 1B, microfluidic droplet formation involves using a microfluidic device to form a gel emulsion. In some embodiments, the microfluidic device includes microchannels configured to produce a polymer bead of a desired size and configured to encapsulate a selected amount of biomolecule per bead. In some embodiments, the microfluidic device has a height of 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 μm, or a height within a range defined by any two of the above values. In some embodiments, the microfluidic device includes one or more channels. In some embodiments, the microfluidic device includes an aqueous flow channel and an immiscible fluid flow channel. In some embodiments, one or more channels have the same width. In some embodiments, one or more channels have different widths. In some embodiments, the width of one or more channels is 20, 30, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 115, 120, 125, 130, 135, 140, 145 or 150 µm, or the width is within the range determined by any two of the above values. In some embodiments, the aqueous medium channel width is 75 microns. In some embodiments, the immiscible fluid channel width is 78 microns. One skilled in the art will appreciate that the width can be varied to finely adjust the size of the granule. In addition to the size of the microfluidic device and the width of the channels, the flow rate of the aqueous channel and the immiscible fluid channel can also influence the size of the polymer beads.

[0055] In some embodiments, the solution flow rate in the aqueous phase channel is 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 µl/min, or such rate is within the range defined by any two of the above values. In some embodiments, the solution flow rate in the immiscible fluid channel is 20, 30, 50, 80, 100, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, or 400 µl/min, or such rate is within the range defined by any two of the above values. In some embodiments, the aqueous phase solution includes a hydrogel polymer, a cross-linker, and a biomolecule that pass through an aqueous medium channel into an immiscible fluid, such as a carrier oil, at a flow rate below that of the immiscible fluid, thereby forming droplets . In some embodiments, the immiscible fluid is an oil, such as mineral oil, hydrocarbon oil, silicone oil, polydimethylsiloxane oil, tetramethylethylenediamine (TEMED), or mixtures thereof. In some embodiments, hydrogel droplets containing the biomolecule are formed with a uniform size distribution. In some embodiments, the size of the polymer beads is finely controlled by adjusting the size of the microfluidic device, the size of one or more channels, or the flow rate of one or both channels of an aqueous solution or immiscible fluid. In some embodiments, the resulting polymer bead has a diameter in the range of 2 to 150 microns, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140 or 150 microns, or a diameter within the range determined by any two of the above values.

[0056] In some embodiments, the size and uniformity of the polymer bead encapsulating the biomolecule can be further controlled by reacting the hydrogel polymer prior to bead formation with a liquid modifier, such as an alcohol, including isopropyl alcohol.

[0057] In some embodiments, the amount of long DNA fragments encapsulated in the bead can be controlled by diluting or concentrating the long DNA fragments in the input sample. A sample containing long DNA fragments is mixed with a hydrogel polymer, and such hydrogel polymer containing long DNA fragments is emulsified using a vortex mixer or transferred into microfluidic droplets as described herein.

[0058] In some embodiments, polymer beads can be functionalized and used for nucleic acid purification. In some embodiments, the polymer beads may be functionalized with a nucleotide. In some embodiments, the nucleotide is an oligonucleotide or poly-T nucleotide. In some embodiments, the nucleotide is associated with a polymer bead, and the functionalized bead can be used to specifically capture the nucleotide of interest.

[0059] In some embodiments, the polymer bead may be encapsulated by a polymer shell with different characteristics from the polymer bead, such as a different melting point, different pore sizes, or different dissolution characteristics. In some embodiments, the polymer shell may include DHEBA.

Methods for processing biomolecules encapsulated inside polymer granules

[0060] Some embodiments include methods for processing biomolecules within a bead, as shown in FIG. 2, which shows a block diagram of the production and processing of biomolecules inside a polymer granule. In the first step, a DNA sample, for example from genomic data, or a cell is encapsulated inside a polymer bead. In some embodiments, a long DNA fragment is retained within the polymer beads and reagents are able to pass through the pores of the polymer beads. In some embodiments, the reagents may include lysis agents, nucleic acid purification agents, tagmenting agents, PCR agents, or other agents used in the processing of biomolecules or molecules derived therefrom. In this way, the polymer beads form a microenvironment for controlled reactions of long DNA fragments within the polymer beads, providing a barrier to the passage of reagents in and out of the polymer beads while simultaneously retaining long DNA fragments within the beads. Once the DNA is encapsulated in the beads, the next stage of the process begins where the sample can be loaded into a flow cell to form long DNA fragments during the library production process.

[0061] As used herein, the term “tagmentation” refers to the modification of DNA by a transposome complex containing the enzyme transposase in complex with adapters containing the terminal sequence of the transposon. Tagmentation leads to simultaneous DNA fragmentation and ligation of adapters to the 5' ends of both strands of duplex fragments. Following the purification step to remove the transposase enzyme, additional sequences can be added to the ends of the adapted fragments, for example, by PCR, ligation, or any other suitable technique known to those skilled in the art.

[0062] In some embodiments, acquisition of an entire DNA library can be easily accomplished within polymer beads associated with a flow cell by repeatedly changing reagents through a porous hydrogel while retaining the genomic DNA and its library products within the hydrogel matrix. The hydrogel can be resistant to high temperatures up to 95°C for several hours to enable various biochemical reactions.

[0063] In the next step of the process, the polymer bead encapsulating the long DNA fragments from the previous library production is processed to release, purify and isolate the long DNA fragments from the bead. To do this, the polymer bead is brought into contact with, for example, a lysis buffer. As used herein, the term “lysis” means a disruption or alteration of the cell wall or viral particle that facilitates access to or release of cellular RNA or DNA. Neither complete destruction nor rupture of the cell wall is a requirement for lysis. The term "lysis buffer" means a buffer that contains at least one lysis agent. Common enzymatic lysis agents include, but are not limited to, lysozyme, glucolase, zymolose, lyticase, proteinase K, proteinase E, and viral endolysins and exolysins. Thus, for example, lysis of cells in beads can be accomplished by introducing lysis agents such as lysozyme and proteinase K into the polymer beads. The genomic DNA from the cells will then be contained within the beads. In some embodiments, after lysis, the isolated nucleic acid is retained within the polymer bead and can be used during further processing.

[0064] As used herein, the terms “isolated”, “isolate”, “isolating”, “purified”, “purify”, “purification” and their grammatical equivalents as used herein, unless otherwise noted, refer to reducing the amount at least one contaminant (such as a protein and/or nucleotide sequence) in the sample or source (eg, cell) from which the material is isolated. Thus, purification results in “enrichment,” such as an increase in the amount of the desired protein and/or nucleotide sequence in the sample.

[0065] In some embodiments, the encapsulated nucleic acids are sequenced in whole or in part within polymer beads. The encapsulated nucleic acids can be sequenced according to any suitable sequencing technique, such as direct sequencing, including sequencing by synthesis, sequencing by ligation, sequencing by hybridization, sequencing through nanopores, and the like.

[0066] Some embodiments presented herein relate to sequencing by synthesis (SBS), which can be used for long DNA fragments. SBS tracks the extension of a nucleic acid primer along a nucleic acid template (eg, a target nucleic acid or its amplicon) to determine the sequence of nucleotides in the template. The underlying chemical process may be polymerization (eg, catalyzed by the enzyme polymerase). In a particular embodiment of the polymerase-based SBS, fluorescently labeled nucleotides are added to the primer (whereby the primer is extended) in a template-dependent manner such that detection of the order and type of nucleotides added to the primer can be used to determine the sequence of the template.

[0067] For one or more amplified encapsulated nucleic acids, SBS or another detection technique that involves multiple delivery of reagents in a cyclic manner can be used. For example, to start the first round of SBS, one or more labeled nucleotides, DNA polymer azu, etc. can be fed into or passed through a polymer bead containing one or more amplified nucleic acid molecules. It is possible to identify those sites where primer extension leads to the incorporation of a labeled nucleotide. The nucleotides may additionally have a reversible termination property that terminates additional primer extension upon addition of a nucleotide to the primer. For example, a nucleotide analog having a reversible terminator fragment can be added to the primer so that subsequent extension cannot occur until a blocking agent is delivered to remove the fragment. Thus, for reversible termination embodiments, an unblocking reagent may be supplied to the flow cell (before or after detection). Washings can be carried out between the various stages of delivery. The cycle can then be repeated n times to extend the primer by n nucleotides, thereby establishing a sequence of length n. Exemplary SBS procedures, fluidic systems and detection platforms that can be readily adapted for use with amplicons produced by the methods of the present disclosure are provided, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04/018497 ; US Patent No. 7,057,026; WO 91/06678; WO 07/123744; US Patent No. 7,329,492; US Patent No. 7,211,414; US Patent No. 7,315,019; US Patent No. 7,405,281 and US 2008/0108082, each of which is incorporated herein by reference.

[0068] Other sequencing procedures that employ cyclic reactions, such as pyrosequencing, can be used. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as specific nucleotides are incorporated into the growing nucleic acid chain (Ronaghi, et al., Analytical Biochemistry 242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1) , 3-11 (2001); Ronaghi et al. Science 281(5375), 363 (1998); US Patent No. 6,210,891; US Patent No. 6,258,568 and US Patent No. 6,274,320, each of which is incorporated herein by reference). In the pyrosequencing process, the released PPi can be detected when it is immediately converted to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP produced can be detected by photons emitted by luciferase. Thus, the sequencing reaction can be monitored using a luminescence detection system. Pyrosequencing procedures do not require the use of excitation radiation sources used in fluorescence detection systems. Suitable liquid systems, detectors and procedures that can be adapted for the application of pyrosequencing to amplicons obtained in accordance with the present description are provided, for example, in WIPO patent application No. PCT/US11/57111, US 2005/0191698 Al, US patent no. 7,595,883 and U.S. Patent No. 7,244,559, each of which is incorporated herein by reference.

[0069] In some embodiments, methods may be used that include monitoring DNA polymerase activity in real time. For example, nucleotide incorporation can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-containing polymerase and γ-phosphate-labeled nucleotides or through zero-mode waveguides (ZMWs). Methods and reagents for FRET-based sequencing are described, for example, in Levene et al. Science 299, 682-686 (2003); Lundquist et al. Opt. Lett. 33, 1026-1028 (2008); Korlach et al. Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated herein by reference.

[0070] Some embodiments of SBS include detecting a proton released upon insertion of a nucleotide into the extension product. For example, for sequencing based on the detection of released protons, an electrical detector and associated commercially available techniques can be used. Examples of such sequencing systems include pyrosequencing (e.g., the commercially available platform from Roche subsidiary 454 Life Sciences), γ-phosphate-tagged nucleotide sequencing (e.g., the commercially available platform from Pacific Biosciences), and proton-sensing sequencing (eg, a commercially available platform from Ion Torrent, a subsidiary of Life Technologies) or sequencing methods and systems described in US 2009/0026082 A1; US 2009/0127589 A1; US 2010/0137143 A1 or US 2010/0282617 A1, each of which is incorporated herein by reference. The methods described herein for amplifying target nucleic acids using kinetic exclusion can be readily applied to substrates used for proton detection. In particular, the methods described herein can be used to obtain clonal amplicon populations that are used for proton detection.

[0071] Another sequencing technique is nanopore sequencing (see, for example, Deamer et al. Trends Biotechnol. 18, 147-151 (2000); Deamer et al. Acc. Chem. Res. 35:817-825 (2002) ; Li et al. Nat. Mater. 2:611-615 (2003), the descriptions of which are incorporated herein by reference). In some nanopore embodiments, the target nucleic acid or individual nucleotides are removed as the target nucleic acid passes through the nanopore. As a nucleic acid or nucleotide passes through a nanopore, each type of nucleotide can be identified by measuring fluctuations in the electrical conductivity of the pore (US Patent No. 7,001,792; Soni et al. Clin. Chem. 53, 1996-2001 (2007); Healy, Nanomed. 2 , 459-481 (2007); Cockroft et al.

[0072] Examples of on-chip expression methods and genotyping assays that can be used for detection in accordance with the present disclosure are provided in US Pat. No. 7,582,420; 6,890,741; 6,913,884 or 6,355,431 or in publications of US patent applications No. 2005/0053980 A1; 2009/0186349 A1 or US 2005/0181440 A1, each of which is incorporated herein by reference.

[0073] The nucleic acid isolation, amplification, and sequencing methods described herein use various reagents to isolate and produce nucleic acids. Such reagents may include, for example, lysozyme, proteinase K, random hexamers, polymerase (e.g. F29 DNA polymerase, Taq polymerase, Bsu polymerase), transposase (e.g. Tn5), primers (e.g. P5 and P7 adapter sequences ), ligase, catalyzing enzyme, deoxynucleotide triphosphates, buffers or divalent cations. Such reagents pass through the pores of the polymer beads, while the biomolecule or a molecule derived from it is retained within the polymer beads. The advantage of the methods set forth herein is that they provide an encapsulated microenvironment for processing nucleic acids within a polymer bead. This enables single cell processing to quickly and efficiently process the target nucleic acid.

[0074] Adapters may include sequencing primer sites, amplification primer sites, and indices. As used herein, the term "index" may include a nucleotide sequence that can be used as a molecular identifier and/or barcode to tag a nucleic acid and/or to identify the source of a nucleic acid. In some embodiments, the index can be used to identify a single nucleic acid or a subpopulation of nucleic acids. In some embodiments, nucleic acid libraries may be generated within a polymer bead. In some embodiments, a single cell encapsulated within a polymer bead can be used for combinatorial single cell indexing, for example, using a contiguity-preserving transposition (CPTSeq) approach. In some embodiments, DNA from a single cell can be barcoded by encapsulating the single cells after amplifying the WGA with another bead carrying the barcoded transposons, and then dissolving the gel matrix by treating with a reducing agent, for example, to release genomic DNA for barcoding.

[0075] Embodiments of the "spatial indexing" methods and techniques described herein reduce data analysis and simplify the process of obtaining a library from single cells and long DNA molecules. Current single-cell sequencing protocols require efficient physical separation of cells and unique barcoding of each isolated cell, followed by grouping everything together to perform sequencing. Current synthetic long read protocols also require labor-intensive steps of barcoding and grouping together all the barcoded fragments for sequencing, followed by data analysis to delineate the genetic information coming from each barcoded cell. In such processes, loss of material is possible, leading to dropouts in sequences. The embodiments described herein not only shorten the process, but also increase the resolution of single cell data. Moreover, the embodiments proposed herein facilitate the assembly of genomes of new organisms. The embodiments described herein can be used to identify rare genetic variations and co-mutations. In some embodiments, the DNA library contained within the polymer beads prior to release allows the size of the fragments that are released to the surface to be controlled by controlling the release process and the composition of the hydrogel.

[0076] In some embodiments, the surface is a flow cell device. In some embodiments, the flow cell is a specialized flow cell with dimples, grooves, or a pattern. In some embodiments, the flow cell includes a structured surface. In some embodiments, the structured surface includes dimples. In some embodiments, the diameter of the wells is from about 10 μm to about 50 μm, such as 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, or 50 μm in diameter, or in the range , defined by any two of the above values, and wherein the depth of the wells is from about 0.5 μm to about 1 μm, for example 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 µm or 1 µm deep, or in the range determined by any two of the above values. In some embodiments, the wells are made of a hydrophobic material. In some embodiments, the hydrophobic material comprises an amorphous fluoropolymer, such as CYTOP, Fluopel®, or Teflon®.

[0077] In some embodiments, the library can be amplified using primer sites in adapter sequences and sequenced using sequencing primer sites in adapter sequences. In some embodiments, the adapter sequences may include indices to identify the source of the nucleic acids. The efficiency of subsequent amplification steps may be reduced due to the formation of primer dimers. To improve the efficiency of subsequent amplification steps, unligated single-stranded adapters can be removed from the ligation products.

[0078] In some embodiments, a model system can be generated to determine the porosity of polymer beads. As shown in FIG. 11A and 11B, the polymer bead can encapsulate a streptavidin compound (indicated as SA in FIGS. 11A and 11B), retaining the streptavidin compound within the bead. In some embodiments, a biotin compound bound to a specific size amplicon is mixed with a polymer bead (FIG. 11A). A biotin-bound amplicon of sufficient size is able to diffuse into the polymer bead and form a streptavidin conjugate within the bead, thereby retaining the biotin-bound amplicon within the bead. In contrast, a biotin-bound amplicon that is too large will not pass through the polymer bead, and no amplicons will be retained within the bead. Likewise, a polymer bead containing streptavidin conjugated to amplicon-bound biotin can be prepared and reagents can be passed through to perform a reaction such as PCR (FIG. 11B). Fragments of sufficient size diffuse out of the polymer bead, while fragments that are too large do not diffuse through the polymer bead and are retained within the bead.

Preparation of Nucleic Acid Libraries Using Polymer Beads

[0079] Some embodiments of the systems, methods and compositions provided herein include methods in which adapters are ligated to target nucleic acids. Adapters may include sequencing primer binding sites, amplification primer binding sites, and indices. For example, the adapter may include a P5 sequence, a P7 sequence, or a complement thereof. Herein, the P5 sequence contains the sequence identified by SEQ ID NO: 1 (AATGATACGGCGACCACCGA), and the P7 sequence contains the sequence identified by SEQ ID NO: 2 (CAAGCAGAAGACGGCATACGA). In some embodiments, the P5 or P7 sequence may further include a polynucleotide spacer, which may be 1 to 20 nucleotides in length, such as 1 to 15, or 1 to 10 nucleotides, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. In some embodiments, the spacer includes 10 nucleotides. In some embodiments, the spacer is a poly-T spacer, such as a 10T spacer. Spacer nucleotides can be introduced at the 5' ends of polynucleotides, which can be fixed to a suitable support through linkage to the 5' end of the polynucleotide. The coupling may be achieved by a sulfur-containing nucleophile, such as phosphorothioate, at the 5' end of the polynucleotide. In some embodiments, the polynucleotide will include a poly-T spacer and a 5'-phosphorothioate group. Thus, in some embodiments, the P5 sequence is 5'-phosphorothioate-TTTTTTTTTTTTAATATACOOCOASSASSOA-3' (SEQ ID NO: 3), and in some embodiments, the P7 sequence is 5'-phosphorothioate-TTTTTTTTTTTTTTCAAOCAOAAACOOCATACOA-3' (SEQ ID NO : 4)-

[0080] Indexes can be used to identify the source of a nucleic acid molecule. In some embodiments, the adapter may be modified to avoid the formation of concatemers, for example, by introducing blocking groups that prevent elongation of the adapter at one or both ends. Examples of 3' blocking groups include the 3' spacer C3 and the dideoxynucleotide and support anchorage. Examples of 5' blocking groups include dephosphorylated 5' nucleotide and anchorage to a support.

[0081] Adapters include nucleic acids, such as single-stranded nucleic acids. Adapters may include short nucleic acids having a length of less than, greater than, or equal to about 5 nucleotides, 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, or located in the range between any two of the above dimensions. In some embodiments, the adapters are large enough to fit through the pores of the polymer beads. Target nucleic acids include DNA, such as genomic or cDNA; RNA, such as mRNA, sRNA or rRNA; or a hybrid of DNA and RNA. Nucleic acid can be isolated from a single cell encapsulated within a polymer bead. The nucleic acid may contain phosphodiester linkages and may have other types of backbones, including, for example, phosphoramide, phosphorothioate, phosphorodithioate, O-methylphosphoramidite and peptide backbones and nucleic acid linkages. The nucleic acid may contain any combination of deoxyribo- and ribonucleotides and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, isoguanine, and base analogues such as nitropyrrole (including 3-nitropyrrole) and nitroindole (including 5-nitroindole). In some embodiments, the nucleic acid may include at least one universal base. A universal base can base pair with more than one other type of base and can be used, for example, when included in oligonucleotide primers or inserts that are used for random hybridization in complex nucleic acid samples, such as genomic DNA samples. An example of a universal base is inosine, which can pair with adenine, thymine, or cytosine. Other examples include hypoxanthine, 5-nitroindole, acyclic 5-nitroindole, 4-nitropyrazole, 4-nitroimidazole, and 3-nitropyrrole. Universal bases can be used that can be paired with at least two, three, four or more types of bases.

[0082] An example of this method includes dephosphorylation of the 5' ends of target nucleic acids to avoid the formation of concatemers in subsequent ligation steps; ligating the first adapters to the 3' ends of the dephosphorylated target molecules using a ligase, the 3' ends of the first adapters being blocked; re-phosphorylation of the 5' ends of the ligated target molecules; ligating a second adapter to the 5' ends of dephosphorylated target molecules using a single-strand ligase, wherein the 5' ends of the second adapters are not phosphorylated.

[0083] Another example involves partial cleavage of a nucleic acid by a 5' exonuclease to produce a double-stranded nucleic acid with single-stranded 3' overhangs. An adapter containing a 3' blocking group can be ligated to the 3' ends of a double-stranded nucleic acid with 3' overhangs. Double-stranded nucleic acid with 3'-overhangs with ligated adapters can be dehybridized to form single-stranded nucleic acids. An adapter containing a non-phosphorylated 5' end can be ligated to the 5' end of a single-stranded nucleic acid.

[0084] Methods for dephosphorylation of nucleic acids, such as the 5' nucleotide of a nucleic acid, involve contacting the nucleic acid with a phosphatase. Examples of phosphatases include calf intestinal phosphatase, shrimp alkaline phosphatase, Antarctic phosphatase and APEX alkaline phosphatase (Epicentre).

[0085] Methods for ligating nucleic acids include bringing the nucleic acids into contact with a ligase. Examples of ligases include T4 RNA ligase 1, T4 RPHK ligase 2, RtcB ligase, methanobacterial RNA ligase, and TS2126 RNA ligase (CIRCLIGASE).

[0086] Methods for phosphorylating nucleic acids, such as the 5' nucleotide of a nucleic acid, involve contacting the nucleic acid with a kinase. Examples of kinases include T4 polynucleotide kinase.

[0087] Embodiments provided herein relate to the production of nucleic acid libraries within a polymer bead such that the nucleic acid library is produced in a single reaction volume.

[0088] Embodiments of the systems and methods provided herein include kits containing any one or more hydrogel polymers, cross-linkers, or microfluidic devices to produce polymer beads that encapsulate biomolecules, and further include components suitable for processing of biomolecules or molecules derived from them, including reagents for cell lysis and amplification and sequencing of nucleic acids or for obtaining a library of nucleic acids, including lysozyme, proteinase K, random hexamers, polymerase (for example, F29 DNA polymerase, Taq- polymerase, Bsu polymerase), transposase (eg, Tn5), primers (eg, P5 and P7 adapter sequences), ligase, catalyzing enzymes, deoxynucleotide triphosphates, buffer solutions or divalent cations as described herein and which are used in appropriate processing biomolecules or molecules derived from them.

[0089] As shown in FIG. 12, a polymer granule encapsulating the cell is obtained. Polymer granules are exposed to reagents for cell lysis and tagmentation. Treatment of a polymer bead with a detergent such as SDS results in fragmentation and small molecules can diffuse out of the polymer bead. In some embodiments, the ability to retain cellular biomolecules may be limited to a minimum threshold beyond which two-way access of enzymes into cells will be prevented.

[0090] Alternatively, in some embodiments, library production methods are used to increase the physical size of genomic molecules so that they are retained within polymer beads beyond a threshold. Thus, as shown in FIG. 12, during the preparation of a gDNA library, labeled gDNA fragments are held together by Tn5 binding, which prevents diffusion outside the polymer beads. However, after treatment with SDS, Tn5 is released, and the resulting library fragments can diffuse outside the beads if their size is too small. To avoid this, the Tn5 enzyme with sticky transposon ends can be used for tagmentation. Chemical transformations can be performed on the sticky 5' or 3' ends of the transposon to increase the size of library elements or to enable binding to beads or other biomolecules. Various configurations and modifications of transposons can be used to increase the physical size of library fragments.

[0091] For example, a transposon with a 3' overhang can be used to tag DNA. The 3' overhanging end can serve as a substrate for enzymes. Terminal transferase (TdT) can be used to add a specific number of bases in a template-independent manner. For example, TdT can be used to add 100,300 bases to a transposon. If transposon extension with TdT is not sufficient, the resulting extended transposon can be hybridized to oligolinked beads found in polymer beads, a complementary amplicon, or a poly-A-terminal cellular mRNA. After hybridization with the oligonucleotide, an extension reaction can be performed. For example, when annealing a ssDNA plasmid, a rolling circle amplification (RCA) reaction can be performed to increase the size of the ends of the transposon. In an alternative embodiment, the hybridized oligonucleotides can be ligated to the ends of the transposon. Sequential ligation of amplicons, such as cyclic ligation assembly (CLA), can be performed to assemble long DNA fragments from smaller fragments that can diffuse into polymer beads.

[0092] The addition of modified bases to transposons can also be used as targeting molecules to bind additional molecules. The modified bases may be contained in the transposon prior to tagmentation or may be added during an enzymatic reaction after tagmentation. For example, TdT can be used to add a modified core digoxigenin-11-UTP, which can subsequently be bound by an anti-DIG antibody. Other modifications include biotin and 5mC, which can bind to streptavidin and 5mC antibodies, respectively.

[0093] In some embodiments, simultaneous indexing of gDNA and cDNA fragments derived from the same polymer bead and further amplification of library elements can be accomplished through rolling ring amplification.

EXAMPLES

Example 1. Preparation of polymer granules

[0094] The example below presents an embodiment for producing polymer beads encapsulating long DNA fragments using microfluidic droplet forming devices.

[0095] A droplet forming apparatus was used to produce polymer granules. Samples containing long DNA fragments were mixed with the polymer precursor and the mixture was loaded into the sample reservoir in the cartridge. Within 2 minutes, approximately 50,000 polymer beads containing long DNA were formed from each channel (8 channels for 8 independent sample treatments in each cartridge). Polymer beads with long DNA were loaded into a flow cell, where the polymer beads were fixed inside (a channel height of 100 μm and a diameter of polymer beads of 120 μm) for automatic library generation. Enzymes and reagents, including enzymes and reagents for preparing a nucleic acid library, were introduced into a flow cell, contacted with long DNA contained within a polymer bead, and digested long DNA molecules through tagmentation to obtain a DNA library. The library was then seeded onto the surface of a bead flow cell. During the seeding process, the libraries were loaded with oil to fill the voids between the beads, and the flow cell was heated to accelerate the diffusion of the library onto the surface of the flow cell. In the presence of oil, seeding of each labeled library occurred in close proximity to the location of each polymer bead (based on the diameter of the polymer beads being 120 μm, seeding of the library was limited to an area with a diameter of approximately 120 μm).

[0096] The present example shows that long DNA molecules can be loaded and captured by polymer beads (about 120 μm in diameter), and library production is carried out on such long DNA molecules contained within polymer beads. As a result, all DNA libraries from a particular long DNA molecule were stored inside the same polymer beads. The library was then released from the polymer beads onto the surface of the flow cell to seed them as a group on the surface of the flow cell. The clusters released from the long DNA molecule were grouped together as a “cluster spot” on the flow cell. Clusters within a single spot of a single long DNA molecule facilitate genome reconstruction with higher accuracy and fewer scaffold gaps.

Example 2: Spatial indexing of long DNA

[0097] The example below presents an embodiment of gated reads of a 100 kb long DNA fragment encapsulated within a polymer bead with or without MDA.

[0098] Polymer beads were prepared by mixing polymer in the presence of about 100 kb of genomic DNA (Cornell). with the formation of polymer granules using a device for the formation of microdrops. Spatial indexing DNA sequencing was performed by placing formed polymer beads encapsulating DNA fragments into a flow cell device and then bringing the flow cell into contact with the reagents. MDA was not performed. The granules were destroyed and clusters formed in the flow cell device. As shown in FIG. 5, the average clusters per long DNA island were about 33, the average long DNA island size was 64,000 bp, and there were about 405 long DNA islands per bead.

[0099] A second set of polymer beads was prepared by mixing the polymer in the presence of about 100 kb of genomic DNA (Cornell). with the formation of polymer granules using a device for the formation of microdrops. Spatial indexing DNA sequencing was performed by placing formed polymer beads encapsulating DNA fragments into a flow cell device and then bringing the flow cell into contact with the reagents. MDA was performed before tagmentation. The granules were destroyed and clusters formed in the flow cell device. As shown in FIG. 6, the average clusters per long DNA island increased to about 85, the average long DNA island size was 58,000 bp, and there were about 166 long DNA islands per granule.

[0100] A third set of polymer beads was prepared by mixing the polymer in the presence of about 10 kb of genomic DNA (Cornell). with the formation of polymer granules using a device for the formation of microdrops. Spatial indexing DNA sequencing was performed by placing formed polymer beads encapsulating DNA fragments into a flow cell device and then bringing the flow cell into contact with the reagents. MDA was performed before tagmentation. The granules were destroyed and clusters formed in the flow cell device. As shown in FIG. 7, the average clusters per long DNA island were about 57, the average long DNA island size was 10,461 bp, and there were about 85 long DNA islands per bead.

Example 3: Metagenomics of a Complex Mixture of Microbial Species

[0101] The following example presents an embodiment for identifying single-celled microorganisms encapsulated in a hydrogel.

[0102] Polymer beads were prepared as described herein using a microfluidic microdroplet forming apparatus. The polymer material was mixed with a sample containing a number of microorganisms, including L. gasseri, S. aureus, B. cereus, B. vulgatus, A. baumannii, S. agalactiae and P. acnes. The encapsulated cells were then lysed and the library was prepared, after which the polymer beads were destroyed and the libraries were applied to the surface. As shown in FIG. 9, each microorganism could be identified due to its spatial compartmentalization in the flow cell arrangement. Thus, encapsulation and subsequent nucleic acid reactions provide strain-level identification of microbial species in complex mixtures from compartmentalization reads in minimetagenomic analysis.

Example 4: Spatial Indexing in a Flow Cell

[0103] The following example presents an embodiment of spatial indexing in a flow cell.

[0104] The flow cell device was installed and washed with 200 μl of PR2 (inclusion buffer). Treatment beads were also washed with PR2. The diluted hydrogel was prepared in PR2. Increasing dilution leads to an increase in the distance between hydrogels. The hydrogel was introduced into the flow cell, avoiding introducing air bubbles into the flow cell. 200 μL of PR2 was passed through the flow cell to ensure that the beads were still fixed to participate in the process. 100 μl of resuspension buffer (RSB) was passed through the flow cell.

[0105] The tagmentation mixture was prepared by mixing 25 μl of tagmentation reagent, 23 μl of RSB and 2 μl of enzyme. The tagmentation mixture was injected into a narrow channel to remove any possible air bubbles at the inlet. Then the tagmentation mixture was slowly fed to the inlet. The flow cell was sealed and incubated for 10 min at 55°C.

[0106] The stopping buffer mixture was prepared by mixing 25 μl of tagmentation buffer, 25 μl of RSB and 10 μl of stopping buffer. The stopping buffer mixture was slowly introduced into the flow cell without introducing bubbles and incubated at room temperature for 5 minutes. After incubation, 200 μl of PR2 was passed through the device.

[0107] NPM was prepared by mixing 175 μl of RSB and 75 μl of NPM. The NPM mixture was slowly introduced into the flow cell device without introducing air bubbles and incubated for 3 minutes at room temperature. 200 μL of surfactant oil was fed through the flow cell device. The micrographs show that the hydrogels were surrounded by a mixture of NPM and oil. The flow cell was sealed and incubated for 3 min at 72°C to perform the gap filling reaction.

[0108] 20-30 μL of surfactant oil and dithiothreitol (DTT) oil (29/2 ratio) were injected into the flow cell device and the device was sealed. The initial temperature of the release process was 90°C for 3 min, 60°C for 5 min, 40°C for 2 min, and 20°C for 2 min. The flow cell was washed with 400 μL PR2 and 200 μL CLM (digestion mixture). The flow cell was then washed with 400 μL PR2. If it was necessary to inoculate with Phix, a Phix solution with a concentration of 2-3 pM was prepared, the Phix library was fed into the device and incubated at room temperature for 5 minutes. The flow cell was washed with 200 μL PR2. For the first extension, 100–200 μl of AMX was supplied and incubated for 5 min at 50°C. The flow cell was washed with PR2 and 24 or 30 amplification cycles were performed.

Example 5: Simultaneous indexing of gDNA and cDNA fragments

[0109] The following example presents an embodiment for simultaneously indexing gDNA and cDNA fragments from the same polymer bead.

[0110] Polymer beads were prepared as hollow polymer shells with a cell encapsulated inside. About 25 to 50 polymer beads were distributed into the wells of a microtiter plate and treated with cell lysis buffer to disrupt the cell membrane, followed by transposition of gDNA with an indexed Y adapter transposome formed by transposons phosphorylated at the 5' end of both strands. Terminal transferase (TdT) was introduced to add multiple Ts to the 3' end not involved in strand transfer, allowing hybridization to the poly-A tail of the mRNA.

[0111] The gap between the gDNA and the non-transfer strand was formed by a fill-and-ligation reaction, and cDNA synthesis was accomplished using murine leukemia virus reverse transcriptase (MMLV RT). MMLV RT also added a few additional dCMP bases to the 3′ end of the cDNA molecule, which was base-paired with an oligo-G sequence at the 3′ end of the template switch primer (TSP). The TSP was extended after annealing and its sequence was transferred to the 3' end of the newly synthesized cDNA in a template switch reaction. The resulting gDNA-cDNA hybrid contained three different common sequences separating the gDNA and cDNA portions of the hybrid molecule, one at each end and one in the middle of the strand. Such common sequences were used to generate both gDNA and cDNA libraries.

[0112] In a side reaction, elongation of the mRNA and template switching at the 3' end of the transposons were observed, but these transformations did not affect the result. After treatment with RNase H and short washes, the contents of the polymer beads were denatured by heating and a ring ligation reaction was performed. In this reaction, all single-stranded DNA molecules with a phosphorylated 5' end self-ligated into rings that became templates for rolling circle amplification (RCA). In addition to the gDNA-cDNA hybrid, individual tagmented DNAs, as well as cDNA molecules derived from mRNA after annealing to release floating transposomes, were also ring ligated and amplified in an RCA reaction. The long concatemers contained multiple copies of the original molecules, which increased the sensitivity of the assay and were retained within the polymer bead.

[0113] dsDNA approximately <1 kb in length. can pass through polymer beads in case of 15% PEG-MAL hollow polymer bead. Briefly, streptavidin-coated beads (approximately 10 μm in diameter) were encapsulated within polymer beads. Biotinylated amplicons of different sizes were then allowed to diffuse into the polymer beads. The diffusion threshold defining the molecular size cutoff was determined over a variable range of amplicon sizes (eg: 220 bp - 5000 bp) as shown in FIG. 13A-13B.

[0114] Similarly, to test the compatibility of polymerase chain reactions and the ability to retain genomic products, a retention model was developed in which amplicon-bound polymer beads (via biotin-streptavidin conjugation chemistry) were introduced inside the polymer beads. Linear amplification of such amplicons of variable size on beads not only made it possible to confirm the compatibility of polymer beads with PCR, but also determined the possibility of retaining amplified DNA fragments of 200 bp in length. and 5 kb. Products with 200 bp. can diffuse into the surrounding polymer beads, whereas the 5 kb products. are held inside polymer granules (FIG. 14).

[0115] In addition to biomolecular assays that can increase the size of small molecule retention products, polymer beads can be composed of multilayer polymers that can provide controlled diffusion of molecules depending on charge, pH, temperature, or other environmental factors (FIG. 15). Examples of such systems may include materials such as ionic and nonionic polymers, including alginate, polyethylene glycol, N-isopropylacrylamide, N,N'-dimethylacrylamide, or other polymer described herein.

Example 6. Polymer granules with destructible cores

[0116] The following example presents an embodiment for producing polymer beads with a degradable core.

[0117] A core with a degradable polymer with a sample inside (cells or DNA) was obtained. The core included a filler, such as PEG or polyacrylamide, which was mixed with the sample to form polymer beads, as shown in FIG. 16A. The bead was condensed with 50% isopropyl alcohol (IPA) and mixed with DHEBA/acrylamide with KPS/TEMED (FIG. 16B). The result was a polymer bead encapsulated within a DHEBA shell. The DHEBA shell is unstable when exposed to the temperatures used for dehybridization and plating, resulting in the core being released from the DHEBA shell at high temperatures. Additionally, treating the DHEBA polymer bead with a reducing agent such as DTT and swelling in water results in the formation of a DHEBA-acrylamide shell. In FIG. 16C shows micrographs of DHEBA polymer beads corresponding to the schematic diagram shown in FIG. 16V.

[0118] Another embodiment is a DHEBA-shelled polymer bead with an agarose core, as shown in FIG. 17A. The agarose core bead was prepared following a method similar to the polyacrylamide core bead. The cell sample or DNA was mixed with agarose to form a polymer bead following the method described herein. Agarose beads can be functionalized and used to purify a sample, such as DNA. The polymer bead was treated with DHEBA encapsulating the polymer bead within, as shown in FIG. 17V. UV radiation can be used to initiate the formation of a DHEBA shell around the polymer bead. The agarose core was subjected to heat or chemical cleavage to melt the core to form a hollow DHEBA shell, as shown in FIG. 17C.

Example 7. Fluorescent initiation of gelation of polymer beads

[0119] The example below presents an embodiment of producing polymer beads using a fluorescent gel initiation method.

[0120] A sample, such as cells or DNA, is prepared. A polymer containing fluorescein isothiocyanate (FITC)-linked acrylate polymers (FITC-AETC) was added to the sample and coated the sample. FITC-AETC-bound samples and monomers were irradiated, for example, with light, resulting in the formation of FITC-bound cells within the polymer gel, as schematically shown in FIG. 18A. The resulting cells can be visualized without staining due to the interaction of FITC on the cell surface, as shown in FIG. 18B and 18C. No excitation was observed for control samples without FITC-AETC coating. Fluorescein-initiated gelation described herein can be carried out in solution. When carried out in solution, initiation of polymerization occurs in the center of a drop of bulk solution. Triethanolamine (TEA) was added at 210 mM to initiate gelation. 0.05% NaHSO ₃ was also added.

[0121] The embodiments, examples, and figures described herein provide compositions, methods, and systems for containing biomolecules in a physically confined space during the process from lysis to library production. In some embodiments, libraries are provided that are derived from a single long DNA molecule or single cell for release on the surface of a flow cell in a confined space. Once the library of a single DNA molecule or a single cell in separate compartments is released onto the surface of the flow cell, the library from each compartment is seeded in close proximity to each other.

[0122] As used herein, the term “comprising” is synonymous with the terms “including,” “comprising,” or “characterized by,” and is inclusive or non-limiting and does not exclude additional unspecified elements or process steps.

[0123] The description above sets forth several methods and materials of the present invention. The present invention is subject to modifications in methods and materials, as well as changes in manufacturing methods and equipment. Such modifications will become apparent to those skilled in the art upon examination of the present disclosure or upon practice of the invention described herein. Therefore, the present invention is not intended to be limited to the specific embodiments described herein, but is intended to cover all modifications and alternatives within the true scope and spirit of the invention.

[0124] All references cited herein, including, without limitation, published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are therefore a part of this specification. To the extent that publications, patents, or patent applications incorporated by reference are inconsistent with the teachings herein, it is intended that this specification supersedes and/or takes precedence over any such conflicting material.

LIST OF SEQUENCES

<110> Illumina, Inc.

<120> MODULATION OF POLYMER GRANULES FOR DNA PROCESSING

<130> ILLINC.422WO

<150> 62/704,028

<151> 2018-10-26

<160> 4

<170> PatentIn, version 3.5

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 1

aatgatacgg cgaccaccga 20

<210> 2

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 2

caagcagaag acggcatacg a 21

<210> 3

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 3

tttttttttt aatgatacgg cgaccaccga 30

<210> 4

<211> 31

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 4

tttttttttt caagcagaag acggcatacg a 31

Claims (37)

1. Polymer granule for carrying out multiple reactions of joint analysis, containing: hydrogel polymer precursor; cross-linking agent; And a biomolecule inside a polymer granule, wherein the granule is a porous hollow granule, where the granule contains many layers of polymer, with each layer having a certain pore size and pore density, ensuring the diffusion of the reagent through the granule, which retains the biomolecule. 2. Granule according to claim 1, in which the pore size is modulated depending on charge, pH or temperature. 3. A granule according to claim 1, the diameter of which is from about 50 μm to about 150 μm. 4. Granule according to claim 1, in which the hydrogel polymer precursor contains polyethylene glycol (PEG)-thiol/PEG-acrylate, PEG/maleimide (PEG/MAL), acrylamide/N,N'-bis(acryloyl)cystamine (BACy), N,N'-(1,2-dihydroxyethylene)bisacrylamide (DHEBA), PEG/polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) ( PNIPAAm), poly(lactic) acid (PLA), copolymer of poly(lactic) and poly(glycolic) acids (PLGA), polycaprolactone (PCL), poly(vinylsulfonic) acid (PVSA), poly(L-aspartic) acid, poly (L-glutamic) acid, polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose or collagen. 5. The granule of claim 1, wherein the hydrogel polymer precursor contains bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethylene glycol diallyl ether, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylopropane trimethacrylate, ethoxylated trimethylol triacrylate or ethoxylated pentaerythritol tetraacrylate. 6. Granule according to claim 1, in which the biomolecule is a nucleic acid. 7. The bead according to claim 6, wherein the nucleic acid is a long DNA molecule of 50,000 base pairs or more. 8. Granule according to claim 1, in which the reagent contains enzymes, chemicals and primers less than 50 base pairs in size. 9. The granule according to claim 1, in which the reagent contains lysozyme, proteinase K, random hexamers, polymerase, transposase, primers, ligase, catalyzing enzyme, deoxynucleotide triphosphates, buffers or divalent cations. 10. The granule according to claim 9, in which the polymerase is F29 DNA polymerase, Taq polymerase or Bsu polymerase. 11. The granule according to claim 9, wherein the transposase is Tn5. 12. The bead according to claim 9, wherein the primers are P5 adapter sequences or P7 adapter sequences. 13. The granule according to claim 1, further containing a stabilized shell that encapsulates the granule. 14. The granule according to claim 13, wherein the stabilized shell contains N,N'-(1,2-dihydroxyethylene)bisacrylamide (DHEBA), acrylate-PEG and potassium peroxydisulfate (KPS). 15. The granule according to claim 1, further containing a fluorescent compound associated with a hydrogel polymer precursor. 16. The bead according to claim 15, wherein the fluorescent compound is fluorescein isothiocyanate (FITC). 17. A method for performing multiple sequential joint analyzes of a biomolecule encapsulated within a polymer bead, comprising: obtaining a polymer granule encapsulating the biomolecule according to claim 1; And sequentially bringing a single cell into contact with reagents to perform multiple sequential co-assays. 18. The method of claim 17, further comprising modulating the pore size of the polymer bead by adjusting charge, pH or temperature. 19. The method of claim 17, wherein the polymer bead contains multiple layers of polymer, each layer having a specific pore size, and wherein the pore size of each polymer layer is modulated in a specific manner by changes in charge, pH, or temperature. 20. The method of claim 17, wherein the plurality of sequential assays includes lysis, DNA analysis, RNA analysis, protein analysis, tagmentation, nucleic acid amplification, nucleic acid sequencing, DNA library preparation, transposase accessible chromatin analysis using sequencing (ATAC -seq), contiguity-preserving transposition (CPT-seq), combinatorial single-cell indexed sequencing (SCI-seq), or single-cell genome amplification, or any combination thereof performed sequentially. 21. The method according to claim 17, in which a polymer granule encapsulating a biomolecule is seeded onto a solid support. 22. The method of claim 21, wherein the solid support is an etched surface, a well, a flow cell device, a microfluidic channel, a bead, or a column. 23. The method according to claim 17, in which the biomolecule is a nucleic acid. 24. The method of claim 23, wherein the nucleic acid is a long DNA molecule of 50,000 base pairs or more. 25. The method according to claim 17, further comprising performing a nucleic acid amplification reaction on the nucleic acid encapsulated inside the polymer bead before performing the tagmentation reaction. 26. The method of claim 25, wherein the nucleic acid amplification reaction comprises a multiple displacement amplification (MDA) step. 27. The method of claim 26, wherein the tagmentation reaction comprises contacting the biomolecule with a transposase mixture containing adapter sequences and transposomes. 28. The method of claim 20, further comprising seeding the DNA library onto a solid support. 29. The method of claim 28, wherein the seeding comprises cleaving the bead to release the DNA library from the bead. 30. The method of claim 29, wherein the bead is digested by contacting the bead with a digestion mixture or by heating the bead to about 90°C to release the DNA library. 31. The method of claim 30, wherein the digestion mixture contains dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP) or tris(3-hydroxypropyl)phosphine (THP). 32. The method of claim 28, wherein the solid support is a flow cell device.