RU2793717C2 - Methods of encapsulating single cells, the encapsulated cells and uses thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention in particular relates to a solid support for nucleic acid sequencing and a method for conducting multiple consecutive collaboratives analyses of individual cells in a population of cells using said support. The said solid support includes multiple hollow granules, each containing a polymer shell and a single cell located within the polymer shell. The polymer shell contains pores that allow for diffusion of the reagent through the polymer shell while retaining a single cell. A solid substrate is a flow cell device, a well plate, a microarray, a microfluidic channel, a glass slide, or a structured surface. To implement this method, the cell population is first contacted with a polymer in a release oil to form a mixture (a). Subsequently, the mixture (a) is mixed with cross-linking oil to form a mass of granules, each granule encapsulating a single cell. Further, individual cells are sequentially contacted within the mass of granules with a reagent to perform a plurality of sequential joint analyses of the cell population. The process is completed by separating the granules on the solid support and modifying the separated granules to allow diffusion of the encapsulated material from the separated granules.

EFFECT: invention allows for multiple assays to be performed while maintaining cell integrity.

19 cl, 12 dwg, 1 tbl, 3 ex

Description

FIELD OF TECHNOLOGY

The systems, methods, and compositions provided herein relate to single cell encapsulating hollow beads, methods for making hollow beads, and methods for using hollow beads to perform a variety of collaborative assays on an encapsulated cell, including, for example, spatial index sequencing and generation of a nucleic acid library. acids.

BACKGROUND OF THE INVENTION

Detection of specific nucleic acid sequences present in a biological sample is used, for example, as a method for detecting and classifying microorganisms, a method for diagnosing infectious diseases, for detecting and characterizing genetic abnormalities, detecting genetic changes associated with a malignant tumor, studying the genetic predisposition to a disease, and measuring response to various treatments. A common method for detecting specific nucleic acid sequences in a biological sample is nucleic acid sequencing.

Next generation sequencing is a powerful tool that generates large amounts of genomic data per run. Interpreting and analyzing such a large amount of data can be challenging. Single cell DNA sequencing has emerged as a tool to study genomic heterogeneity. Specifically, a microbiome that carries many repetitive genomic regions can be sequenced by obtaining DNA sequences from only one cell. Conducting multiple enzymatic reactions on a single cell is unreliable due to the problems associated with limiting access to intracellular biomolecules in a single cell during multiple assays. For example, many cell-based assays fail to ensure the safety of intracellular molecules, resulting in the loss of biomolecules during the assay.

SUMMARY OF THE INVENTION

The present invention relates to systems, methods, and compositions for performing multiple collaborative assays on a single cell, wherein the single cell is encapsulated in a hollow bead such that the single cell is localized for multiple collaborative assays, including, for example, lysis, DNA analysis, RNA analysis. , protein analysis, tagging, nucleic acid amplification, nucleic acid sequencing, DNA library generation, transposase-accessible chromatin evaluation analysis using sequencing (ATAC-seq), contiguity-preserving transposition (CPT-seq), combinatorial indexed sequencing of a single cells (SCI-seq), or amplification of the genome of a single cell, or any combination thereof, carried out sequentially on a single cell.

Some embodiments provided herein relate to a hollow bead that encapsulates a single cell. In some embodiments, the implementation of the hollow granule includes a polymer shell and a single cell located inside the polymer shell. In some embodiments, the implementation of the polymer shell contains pores that allow the reactants to diffuse through the polymer shell while maintaining a single cell.

Additional embodiments relate to a method for conducting multiple sequential collaborative assays on a single cell encapsulated in a hollow bead. In some embodiments, the method includes making a hollow bead as described herein, wherein the hollow bead encapsulates a single cell, and sequentially contacting the single cell with reagents to perform multiple sequential collaborative assays.

Some embodiments provided herein relate to a method for encapsulating a single cell within a hollow bead. In some embodiments, the method includes mixing the single cell with a polymer to form a blend, and mixing the blend with a cross-linking oil to form a polymer shell encapsulating the single cell, wherein the polymer shell contains pores that allow reactants to diffuse through the polymer shell while maintaining the single cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an embodiment of a microfluidic droplet generator system that can be used to produce cell-encapsulating hollow beads.

FIG. 2 shows micrographs and a schematic representation of an embodiment of hollow granules that are capable of size expansion in the presence of an aqueous buffer. The micrographs show the granules in various states, including (from left to right): dehydrated granules; the beginning of the contact of the granules with the aqueous phase; granules that begin to swell; and fully swollen granules.

FIG. 3 is a schematic diagram that shows an embodiment for increasing the porosity of the granules by thermal or chemical means. As shown in FIG. 3, increasing the porosity of the granules can increase the flow of particles into or out of the granule, and control of porosity provides control over the particles (including particle size) that flow into or out of the granule.

FIG. 4 is a schematic diagram that shows an embodiment of a deviceless cell encapsulation method by placing polymer monomer units over a single cell.

FIG. 5 is a schematic diagram that shows an embodiment of an encapsulated cell within a polymer formed by deviceless methods, where the polymer shell is attached to the cell by specific interactions with specific binding molecules.

FIG. 6 shows micrographs of beads stained with fluorescent dyes showing cells encapsulated within the beads. Micrographs show (left to right): staining with thiol-specific fluorophores targeting free PEG-maleimide; staining with Texas red fluorophores targeting free -SH groups; and staining of nuclei with Hoechst stain.

FIG. 7 is a schematic diagram that shows an embodiment of a method for performing multiple collaborative assays on a cell-encapsulating hollow bead. FIG. 7 shows an embodiment of tagging to obtain fragments for ATAC-seq, reverse transcription to initiate cDNA synthesis, and PCR reactions to obtain an indexed library.

FIG. 8 is a schematic diagram that shows an embodiment of a method for performing multiple collaborative assays on a cell-encapsulating hollow bead. FIG. 8 shows an embodiment of performing tagging to obtain fragments for ATAC-seq, reverse transcription to initiate cDNA synthesis, and random expansion PCR reactions to sequence full-length RNA to obtain an indexed library.

FIG. 9 is a schematic diagram that shows an embodiment of a method for performing multiple collaborative assays on a cell-encapsulating hollow bead. FIG. 9 shows a three-level combinatorial indexing approach using two rounds of indexed split ligation and one round of indexed PCR.

FIG. 10 is a schematic diagram that shows an embodiment of a method for performing multiple collaborative assays on a cell-encapsulating hollow bead. FIG. 10 shows a set of collaborative assays for combinatorial indexing with indexed transposomes.

FIG. 11 is a schematic diagram that shows an embodiment of a method for performing multiple collaborative assays on a cell-encapsulating hollow bead. FIG. 11 shows multiple collaborative assays for whole genome single cell amplification.

FIG. 12 is a flow diagram that shows an embodiment of a method for producing a whole genome library using a cell-encapsulating hollow bead.

DETAILED DESCRIPTION

In the following detailed description, reference is made to the accompanying drawings, which are part of it. In the drawings, like symbols generally denote like components unless the context otherwise requires. The exemplary embodiments described in the detailed description, drawings, and claims are non-limiting. Other variations may be used and other changes may be made without departing from the spirit or scope of the subject matter presented herein. It is understood that aspects of the present disclosure, as generally described herein and illustrated in the figures, may be arranged, replaced, combined, separated, and configured in a wide variety of different configurations, all of which are explicitly discussed herein.

Embodiments provided herein relate to hollow beads encapsulating a single cell, methods for making hollow beads, and methods for performing multiple collaborative assays on a single cell encapsulated in a hollow bead. Hollow beads may include hydrogel polymers and crosslinkers which are mixed in the presence of the cell and which form hollow beads that encapsulate the cell. The hollow beads may contain pores that allow reactants to diffuse through the hollow bead while retaining the cell within the hollow bead, and thus allow reactions to take place within the hollow bead. In some embodiments, hollow beads allow for co-assays on the same single cell while maintaining cell contiguity. In particular, the methods, systems, and compositions provided herein allow intracellular biomolecules to be localized and accessed within encapsulated cells. Thus, in some embodiments, the hollow beads described herein are referred to herein as adjacency particles. Thus, As used herein, the term "adjacency particle" refers to a hollow granule encapsulating a single cell.

The adjacency particles described herein can be used to compartmentalize a cell for "next generation" methods and to perform multiple analyte assays on a single cell. The adjacency particles and their methods of use described herein enable the efficient analysis of millions of cells individually and thus reduce the cost of obtaining a sample and maintaining sample adjacency. The compositions and methods described herein maintain cellular contiguity without the use of external compartmentalization strategies (microfluidic technique) such as emulsions, immobilization, or other microcompartments.

In some embodiments, adjacency particles as described herein can be used in single cell assays. Assays that can be performed on a single cell may include, for example, cell lysis, DNA analysis, RNA analysis, nucleic acid sequencing, protein analysis, tagging, nucleic acid amplification, DNA library preparation, analysis to assess chromatin available to transposase, with using sequencing (ATAC-seq), contiguity-preserving transposition (CPT-seq), single cell combinatorial indexed sequencing (SCI-seq), or single cell genome amplification, or any combination thereof, performed sequentially.

The use of adjacency particles to perform one or more assays on a single cell can be used simultaneously on multiple adjacency particles to simultaneously perform cooperative assays on a number of single cells, e.g. 10,000 to 1 million single cells, such as 10,000, 50,000 , 100,000, 500,000 or 1 million single cells.

The pore size of the adjacency particle can be designed to allow diffusion of reagents such as enzymes, chemicals, and smaller primers (˂ 50 bp) while maintaining a single cell on its own or while maintaining other larger particles such as larger nucleic acids (˃ 300 bp) within a hollow bead during one or more assays.

As used herein, the term "reagent" describes a substance or mixture of two or more substances suitable for reacting, reacting with a sample, dissolving a sample, or adding to a sample, and may include substances used in the assays described herein, including substances for lysis, nucleic acid analysis, nucleic acid amplification reactions, protein analysis, tagmentation reactions, ATAC-seq, CPT-seq or SCI-seq reactions, or other assays. Thus, reagents may include, for example, buffers, chemicals, enzymes, polymerase, primers smaller than 50 base pairs, template nucleic acids, nucleotides, labels, dyes, or nucleases. In some embodiments, the reagent includes lysozyme, proteinase K, random hexamers, polymerase (e.g., Φ29 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.

Particles to Maintain Adjacency

In some embodiments, the implementation of the hollow granule has a polymer shell, which was obtained from the composition of the hydrogel. As used herein, the term "hydrogel" refers to the substance that is formed when an organic polymer (natural or synthetic) is cross-linked through covalent, ionic, or hydrogen bonds to form 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 polymer that forms a gel that is not toxic to living cells and allows sufficient diffusion of oxygen and nutrients to the trapped cells to ensure viability. In some embodiments, the hydrogel material comprises alginate, acrylamide or polyethylene glycol (PEG), PEG acrylate, PEG amine, PEG carboxylate, PEG dithiol, PEG epoxide, PEG isocyanate, PEG maleimide, polyacrylic acid (PAA ), poly(methyl methacrylate) (PMMA), polystyrene (PS), polystyrene sulfonate (PSS), polyvinylpyrrolidone (PVPON), N,N'-bis(acryloyl)cystamine, polypropylene oxide (PPO), poly(hydroxyethyl methacrylate) (PHEMA ), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), lactic acid glycolic acid (PLGA), polycaprolactone (PCL), poly(vinyl sulfonic acid) (PVSA), poly(L-aspartic acid) ), poly(L-glutamic acid), polylysine, agar, agarose, heparin, alginate sulfate, dextran sulfate, hyaluronic acid, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethylene glycol diallyl ether, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylopropoane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetraacrylate, or combinations or mixtures thereof. In some embodiments, the hydrogel is an alginate, acrylamide, or PEG-based material. In some embodiments, the implementation of the hydrogel is a PEG-based material with a chemical composition based on the reactions of acrylate-dithiol and epoxy-amine. In some embodiments, the hydrogel forms a polymer shell that includes PEG maleimide/dithiol oil, PEG epoxide/amine oil, PEG epoxide/PEG amine, or PEG dithiol/PEG acrylate. In some embodiments, the hydrogel material is selected to avoid the formation of free radicals, which have the potential to damage intracellular biomolecules. In some embodiments, the hydrogel polymer comprises 60-90% liquid, such as water, and 10-30% polymer. In certain embodiments, the implementation of the water content of the hydrogel is approximately 70-80%. As used herein, the term "approximately" or "about" when modifying a numeric value refers to the variations that can occur with the numeric value. For example, variations may occur due to differences in the production of a particular substrate or component. In one embodiment, the term "about" means within 1%, 5%, or up to 10% of the specified numerical value.

As used herein, a polymeric shell is the polymeric surface of a hollow granule having a shell with an interior that encapsulates a single cell. Due to the nature of the hollow beads described herein, adjacency particles can retain genetic material after multiple assays and can release it by physical force, chemical degradation, or by creating an osmotic imbalance depending on the thickness of the polymer shell.

Hydrogels can be made by cross-linking hydrophilic biopolymers or synthetic polymers. Thus, in some embodiments, the implementation of the hydrogel may include a crosslinker. As used herein, the term "crosslinker" refers to a molecule that can form a three-dimensional network when reacted with suitable base monomers. Examples of hydrogel polymers that may contain one or more crosslinkers include, but are not limited to, hyaluronic acids, chitosans, agar, heparin, sulfate, cellulose, alginates (including alginate sulfate), collagen, dextrans (including dextran sulfate), pectin, carrageenan, polylysine, gelatins (including type A gelatin), agarose, (meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate, PEO-PPO-PEO copolymers (Pluronics), poly(phosphazene), poly(methacrylates), poly( N-vinylpyrrolidone), PL(G)A-PEO-PL(G)A copolymers, poly(ethylene imine), polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N,N'-bis(acryloyl)cystamine, PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), lactic and glycolic acid (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diallyl ether diethylene glycol, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylopropoane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetraacrylate, or combinations thereof. Thus, for example, the combination may include a polymer and a crosslinker, such as polyethylene glycol (PEG)-thiol/PEG-acrylate, acrylamide/N, N'-bis(acryloyl)cystamine (BACy), or PEG/polypropylene oxide (PPO). In some embodiments, the implementation of the polymer shell includes polyethylene glycol (PEG) with four functional ends. In some embodiments, the polyethylene glycol (PEG) with four functional ends is selected from the group consisting of PEG acrylate, PEG amine, PEG carboxylate, PEG dithiol, PEG epoxide, PEG isocyanate, and PEG maleimide.

In one embodiment, the crosslinker is an instant crosslinker or a slow crosslinker. An instant crosslinker is a crosslinker that crosslinks instantly in a hydrogel polymer and is also referred to herein as "click chemistry". Instant crosslinkers may include dithiol oil + PEG maleimide or PEG epoxide + amine oil. A slow crosslinker is a crosslinker that crosslinks slowly in a hydrogel polymer and may include PEG-epoxide+PEG-amine or PEG-dithiol+PEG-acrylate. A slow crosslinker may take more than a few hours to crosslink, for example more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours to crosslink. In some embodiments provided herein, adjacency particles are formulated with an instantaneous crosslinker, and thus keep the cell in better condition than a slow crosslinker. Without being bound by any theory, cells may possibly undergo physiological changes through intracellular signaling mechanisms during longer cross-linking.

In some embodiments, the crosslinker forms a disulfide bond in the hydrogel polymer, thereby linking the hydrogel polymers. In some embodiments, the hydrogel polymers form a porous hydrogel matrix (eg, a porous hydrogel matrix). These pores are capable of retaining sufficiently large particles, such as a single cell or nucleic acids isolated from it, within the polymer shell, but allow other materials, such as reagents, to pass through the pores, thus allowing them to pass in and out of the hollow granules. In some embodiments, the pore size of the polymer shell is fine-tuned by varying the ratio of polymer concentration to crosslinker concentration. In some embodiments, the ratio of polymer to crosslinker 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 ratio within the range defined by any two of the above ratios. In some embodiments, additional groups, such as a DNA primer or charged chemical groups, may be ligated to the polymer matrix in order to meet the requirements of various applications.

As used herein, the term "porosity" means the proportion of the volume (without dimensions) of the hydrogel, which consists of open space, such as pores or other openings. Thus, "porosity" measures voids in a material and is the proportion of void volume relative to total volume, as a percentage between 0 and 100% (or between 0 and 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.

The polymer shell can have any pore size that allows sufficient diffusion of the reagents while maintaining a single cell or nucleic acids isolated from it. As used herein, the term "pore size" refers to the diameter or effective cross-sectional diameter of the pores. The term "pore size" may also refer to the average diameter or effective average cross-sectional diameter of the pores, based on the measurement of a plurality of pores. The effective diameter of a non-circular cross-section is equal to the diameter of a circular cross-section having the same cross-sectional area as the non-circular cross-section. In some embodiments, the hydrogel may swell when the hydrogel is hydrated. Pore sizes may vary depending on the water content of the hydrogel. In some embodiments, the pores of the hydrogel may have a pore of sufficient size to retain the encapsulated cell within the hydrogel, but allow reagents to pass through. In some embodiments, the interior of the polymer shell is an aqueous medium. In some embodiments, the implementation of a single cell, located in the polymer shell, does not interact with the polymer shell and/or does not contact the polymer shell. In some embodiments, a polymeric envelope is formed around the cell (as described herein in more detail) and the cell is in contact with the polymeric envelope due to the polymeric envelope being delivered to the cell surface by passive adsorption or in a targeted manner such as attachment to an antibody or other molecule for specific binding.

In some embodiments, the adjacency particles are of a size sufficient to encapsulate a single cell. In some embodiments, the adjacency particle has a diameter of about 20 microns to about 200 microns, such as 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160 , 170, 180, 190, or 200 µm, or a diameter in the range defined by any two of the above. The size of the adjacency particle may vary due to environmental factors. In some embodiments, the adjacency particles expand when separated from the continuous oil phase and immersed in an aqueous phase, as shown in FIG. 2. In some embodiments, expanding the particles to maintain contiguity improves the performance of assays on genetic material within encapsulated cells. In some embodiments, expanding the particles to maintain contiguity creates a larger environment for indexed inserts to be amplified during PCR, which may otherwise be limited in current cell assays.

In some embodiments, the pore size is increased enough to retain the encapsulated cell, but allows the isolated nucleic acids to diffuse through the polymeric envelope, as shown in FIG. 3. In some embodiments, the pore size of the particles to maintain contiguity can be controlled by changing the crosslinking chemistry. The final cross-linked pore size can be further modified by the particle's environment to maintain contiguity, for example by changing the salt concentration, pH, or temperature, thereby allowing immobilized molecules to be released from the particle to maintain contiguity.

In some embodiments, the crosslinker is a reversible crosslinker. In some embodiments, the reversible crosslinker is capable of reversibly crosslinking the hydrogel polymer and is capable of becoming uncrosslinked in the presence of a cleaving agent. In some embodiments, the crosslinker can be cleaved with a reducing agent, with elevated temperature, or with an electric field. In some embodiments, the reversible crosslinker can be N,N'-bis(acryloyl)cystamine, a reversible crosslinker for polyacrylamide gels, where the disulfide bond can be broken in the presence of a suitable reducing agent. As shown in FIG. 3, the porosity of the pellets can be increased by thermal or chemical means, thereby facilitating the contact of the crosslinker with a reducing agent that cleaves the disulfide bonds of the crosslinker, destroying the hollow pellets. The hollow granules break down and release the contents that were held inside, such as nucleic acids. In some embodiments, the crosslinker is cleaved by raising the temperature above 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100°C. In some embodiments, the crosslinker is cleaved by contacting the hollow beads with a reducing agent. In some embodiments, reducing agents include phosphine compounds, water-soluble phosphines, nitrogen-containing phosphines, and their salts and derivatives, dithioerythritol (DTE), dithiothreitol (DTT) (cis and trans isomers, respectively, 2,3-dihydroxy-l, 4-dithiolbutane), 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).

In some embodiments, raising the temperature to increase diffusion or contact with the reducing agent destroys the crosslinker, thereby releasing the encapsulated genetic material from the particle to maintain contiguity.

In some embodiments, cross-linking of the crosslinker establishes pores in the particle to maintain contiguity. In some embodiments, the pore size in the polymer shell is adjustable and designed to encapsulate cells or nucleic acids larger than about 300 bp, but allowing smaller particles such as reagents or smaller nucleic acids smaller than about 50 bp. base pairs, such as primers, pass through the pores. In some embodiments, the reagents include reagents for processing genetic material, such as reagents for isolating nucleic acids from a cell, for amplifying or sequencing nucleic acids, or for generating nucleic acid libraries. In some embodiments, the reagents include, for example, lysozyme, proteinase K, random hexamers, polymerase (e.g., Φ29 DNA polymerase, Taq polymerase, Bsu polymerase), transposase (e.g., Tn5), primers (e.g., P5 and P7), ligase, catalyzing enzyme, deoxynucleotide triphosphates, buffers, or divalent cations.

Examples of cell types that can be used in a particle to maintain adjacency may include, for example, cells isolated from tissue in a biopsy (for example, from tissue with a disease, such as cancer of the colon, breast, prostate, lung, skin, or pathogen-infected tissues, etc.) and normal cells from the same tissue, eg from the same patient; cells grown in tissue culture that are immortal (eg, cells with a proliferative mutation or immortalizing transgene), infected with a pathogen, or treated (eg, with environmental or chemical agents such as peptides, hormones, altered temperature, growth conditions, physical stress , cellular transformation, etc.) and normal cells (eg, cells that are identical to experimental cells, except that they are not immortalized, infected, or treated, etc.); cells isolated from a mammal with a cancer, a disease, an elderly mammal or a sick mammal, and cells from a mammal of the same species, for example, from the same family, but healthy or young; and differentiated cells and undifferentiated cells from the same mammal (eg, one cell is the progenitor of another in a mammal, for example). In one embodiment, cells of different types can be compared, eg, neuronal and non-neural cells, or cells of different status (eg, before and after stimulation on the cells). In another embodiment, the experimental material is cells susceptible to infection by a pathogen such as a virus, eg human immunodeficiency virus (HIV), etc., and the control material is cells resistant to infection by the pathogen. In another embodiment of the invention, a pair of samples is represented by undifferentiated cells, such as stem cells, and differentiated cells. Cells from yeast, plants and animals such as fish, birds, reptiles, amphibians and mammals can be used in the methods of the invention. In certain embodiments, mammalian cells, ie, from mice, rabbits, primates, or humans, or cultured derivatives thereof, can be used.

Methods for obtaining particles to maintain adjacency

Some embodiments provided herein relate to methods for encapsulating a single cell within a polymeric shell to form a particle to maintain contiguity. In some embodiments, a sample is obtained containing cells that need to be encapsulated within a particle to maintain contiguity. In some embodiments, cells may be fixed with a fixative prior to being encapsulated within a particle to maintain contiguity. As used herein, a fixative generally refers to a substance that can fix cells. For example, fixed cells can stabilize protein complexes, nucleic acid complexes, or protein-nucleic acid complexes in the cell. Suitable fixatives and crosslinkers may include alcohol- or aldehyde-based fixatives, formaldehyde, glutaraldehyde, ethanol-based fixatives, methanol-based fixatives, acetone, acetic acid, osmium tetroxide, potassium dichromate, chromic acid, potassium permanganate, mercury compounds, picrates , formalin, paraformaldehyde, amine-reactive NHS ester crosslinkers such as bis[sulfosuccinimidyl] suberate (BS3), 3,3'-dithiobis[sulfosuccinimidyl propionate] (DTSSP), ethylene glycol bis[sulfosuccinimidyl succinate] (sulfo-EGS) , divacinimidyl glutarate (DSG), dithiobis[succinimidyl propionate] (DSP), disuccinimidyl suberate (DSS), ethylene glycol bis[succinimidyl succinate] (EGS), NHS-ester/diazirin crosslinkers such as NHS-diazirin, NHS-LC -diazirin, NHS-SS-diazirin, sulfo-NHS-diazirin, sulfo-NHS-LC-diazirin and sulfo-NHS-SS-diazirin. In some embodiments, the fixed cell maintains the internal state of the cell, thus preventing modification of the cell during encapsulation of the cell within a particle to maintain contiguity.

In some embodiments, the adjacency particle is produced by static methods, such as single cell microplate/microarray methods or microdissection methods, without the need for a microfluidic device. Thus, in some embodiments, the adjacency particles described herein can be produced in a device-less manner. Device-free methods may include initiating polymerization at the cell surface by contacting the cell with a polymer to form a polymeric shell around the cell. Polymerization can be initiated by a chemical reaction of an active group on monomeric units with certain groups of membrane proteins, glycans, or other small molecular weight compounds. The initial stage of monomer polymerization may be followed by one or more rounds of precipitation of monomer units caused by electrostatic or hydrophobic forces. Some of the monomer layers may contain functional groups such as biotin or other ligands that can be used later to specifically capture the encapsulated cells. For example, submerged cells may contain biotin, and coating the submerged cells with streptavidin magnetic beads will make them magnetic, allowing for automated and simplified processing, as shown in FIG. 4. Alternatively, encapsulation of a living or fixed cell can be initiated by light or other physical or chemical means, where the initiator molecules are brought to the cell surface either by passive adsorption or in a targeted manner, such as attaching to an antibody or other molecule for specific binding and promoting localized polymeric amplification. as shown in FIG. 5.

Cells for encapsulation in the targeted surface initiated polymerization method allow the user to specifically select cells of interest and simultaneously prepare cells for subsequent assays, thus combining stream sorting with single cell assays. It is possible to enrich certain cell types using cell separation kits using magnetically labeled beads or custom binding molecules specific to certain cell membrane proteins conjugated to the enrichment surface.

In some embodiments, the adjacency particle is produced by dynamic methods, such as emulsion production by vortexing, microfluidic droplet production, or valve-based microfluidics. As used herein, emulsifying by vortexing refers to vortexing a hydrogel polymer with a cell, including a fixed cell, as described herein, in a container such as a test tube, vial, or reaction vessel. The components can be mixed, for example, by manual or mechanical vortexing or shaking. In some embodiments, manual mixing produces hollow beads that encapsulate genetic material in sizes of 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 microns in diameter, or or in a size range defined by any two of the above. In some embodiments, the size of the granules is non-uniform, and thus the granule size includes granules of different diameters.

In some embodiments, adjacency particles are produced by microfluidic flow methods. Microfluidic flow includes the use of a microfluidic device to assist in the generation of a gel emulsion, as shown in FIG. 1. In some embodiments, the microfluidic device includes microchannels configured to produce a contiguity particle of a desired size and configured to encapsulate one cell per contiguity particle. 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 in the range defined by any two of the above values. In some embodiments, the implementation of the microfluidic device includes one or more channels. In some embodiments, the microfluidic device includes a channel for introducing a cell, including a fixed cell that has been introduced into the polymer, a channel for introducing a crosslinker, and a channel for an immiscible liquid. In some embodiments, the width of one or more channels is the same. In some embodiments, the width of one or more channels is different. 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, 110, 115, 120, 125, 130, 135, 140, 145, or 150. µm, or the width is within the range defined by any two of the above values. The width and height of the channel are not necessarily limited to the values described herein, and one skilled in the art will appreciate that the size of the particle to maintain contiguity will depend in part on the size of the channels of the microfluidic device. Thus, the particle size for maintaining adjacency can be partially tuned by changing the size of the channels. In addition to the size of the microfluidic device and the width of the channels, the flow rate of the channels can also affect the particle size to maintain contiguity, and can also affect the number of cells encapsulated in each particle to maintain contiguity.

In some embodiments, the flow rate of a cell in a polymer, including a fixed cell mixed with a polymer, through a microfluidic channel is 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 µl/min, or a rate within the range defined by any two of the above values. In some embodiments, the crosslinker flow rate in the microfluidic channel is 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 µl/min , or a speed within the range defined by any two of the above values. In some embodiments, the flow rate of the immiscible liquid in the microfluidic 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 speed within the range defined by any two of the above values. In some embodiments, polymer-mixed cells, including fixed polymer-mixed cells, and a crosslinker contact each other in a microfluidic droplet generator prior to immiscible liquid. The adjacency particles begin to form upon contact with the crosslinker, encapsulating the cells within the polymeric envelope. The forming adjacency particles continue to flow through the microfluidic droplet generator into the immiscible liquid, such as release oil and/or crosslinking oil, at a flow rate less than the flow rate of the immiscible liquid, thus forming droplets. In some embodiments, the implementation of the immiscible liquid is introduced in two stages, as shown in FIG. 1, including both release oil and crosslink oil. In some embodiments, the release oil is a mineral oil, a hydrocarbon oil, a silicone oil, a fluorocarbon oil, or a polydimethylsiloxane oil, or mixtures thereof. A release oil, as used herein, is used to avoid cross-linking of the polymer in the water-oil interphase channel.

In some embodiments, the adjacency particles are generated instantaneously by crosslinking with an instantaneous crosslinker. For example, cells encapsulated with polymers such as four-arm PEG maleimide or epoxy using a microfluidic droplet generator can be instantaneously cross-linked using oil-soluble crosslinkers such as mineral oil or fluorocarbon oil like HFE-7500, forming crosslinking oil. In some embodiments, the crosslinking oil comprises toluene, acetone, tetrahydrofuran with dithiol, amine functional groups as in the case of toluene-3,4-dithiol, 2,4 diaminotoluene, hexanedithiol, which readily diffuse into the droplets that form, thus instantly crosslinking particles to maintain adjacency.

In some embodiments, the contiguity particles are formulated in a uniform size distribution. In some embodiments, adjacency particle sizes are fine-tuned by adjusting the size of the microfluidic device, the size of one or more channels, or the flow rate through the microfluidic channels. In some embodiments, the resulting adjacency particle has a diameter in the range of 20 to 200 microns, such as 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 , 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 µm, or a diameter within the range defined by any two of the above values.

In some embodiments, particle size and uniformity to maintain contiguity can be further controlled by contacting hydrogel polymers to form particles with a flow modifier, such as alcohol, including isopropyl alcohol. In the absence of isopropyl alcohol, the adjacency particles have a larger diameter than the adjacency particles formed in the presence of isopropyl alcohol. Isopropyl alcohol affects the fluidity of hydrogel polymers, allowing particle size to be controlled to maintain contiguity.

As will be appreciated by those skilled in the art, the microfluidic device depicted in FIG. 1 is an example of a three-channel microfluidic device, but the microfluidic device can be modified, altered or varied to create adjacency particles of a certain size or to create adjacency particles formed from various hydrogel materials or crosslinkers.

In some embodiments, the adjacency particle, prepared either by vortex emulsion or microfluidic inertial flow emulsion, encapsulates a single cell, including a single fixed cell, as described herein. In some embodiments, the number of cells in the particle can be controlled to maintain contiguity by diluting or concentrating the solution containing the cell in the injected sample. The cell-containing sample is mixed with hydrogel polymers, and the cell-containing hydrogel polymers are emulsified by vortexing or emulsified by microfluidic inertial flow, as described herein.

In some embodiments, the particles are functionalized with a nucleotide to maintain contiguity. In some embodiments, the implementation of the nucleotide is an oligonucleotide or poly T nucleotide. In some embodiments, the implementation of the nucleotide is associated with particles to maintain contiguity, and functionalized particles to maintain the contiguity can be used to target capture of the nucleotide of interest.

In some embodiments, adjacency particles encapsulating a single cell are processed to support multiple collaborative assays on a single adjacency particle, including multiple washes with buffer, multiple reagent changes, and multiple assays based on the assay being performed. Formulated adjacency particles prepared by any of the methods described herein, including surface initiated polymerization methods, vortexing, or microfluidic methods, can be loaded or deposited on a structured flow cell, microarray, well plate, etched surface, microfluidic channel, bead, column, or other surface for conducting multiple collaborative assays on the encapsulated cell.

Methods for performing multiple collaborative assays on cells encapsulated within particles to maintain contiguity

Some embodiments provided herein relate to methods for conducting multiple sequential collaborative assays on a single cell encapsulated within a particle to maintain contiguity. In some embodiments, the method includes preparing a coexistence particle as described herein and sequentially contacting a single cell encapsulated within the contiguity particle with reagents to perform multiple sequential collaborative assays.

In some embodiments, the adjacency particles are prepared as described herein and the adjacency particles are loaded onto a surface, such as a flow cell device, plate well, glass slide, or structured surface. In some embodiments, the surface is a flow cell device and includes an insert with microwells or micropillars in the particle distribution device to maintain adjacency for spatial indexing in the flow cell device. In some embodiments, the adjacency particles are loaded onto the well plate, one adjacency particle per well. The well plate may include, for example, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, a 384-well plate, a 1536-well plate, a 3456-well plate, or a 9600-well plate, or plate with any number of wells in the plate, with a single particle to maintain contiguity, and thus a single cell placed in each well. In some embodiments, the adjacency particles are made permeable with a permeability agent. In some embodiments, the penetration agent includes a mild detergent such as TritonX-100. In some embodiments, the permeation agent increases the permeability of the cell membrane within the particle to maintain contiguity to increase the availability of cellular nucleic acids such as genomic DNA and RNA. In some embodiments, the well plate with a single adjacency particle placed in each well is subjected to a plurality of sequential collaborative assays, including, for example, buffer washing, lysis, DNA analysis, RNA analysis, protein analysis, tagging, nucleic acid amplification, nucleic acid sequencing, DNA library preparation, transposase-accessible chromatin analysis using sequencing (ATAC-seq), contiguity-preserving transposition (CPT-seq), combinatorial indexed single cell sequencing (SCI-seq), or genome amplification single cell, or any combination of them, carried out sequentially.

In some embodiments, the adjacency particle, the encapsulating cell or virus particle, is processed to purify and isolate nucleic acids from the cell. Thus, for example, the adjacency particle is contacted with a lysis buffer. As used herein, "lysis" means disruption or alteration of a cell wall or viral particle to facilitate access to, or release of, cellular RNA or DNA. Neither complete destruction nor destruction of the cell wall is a requirement for lysis. The term "lysing buffer" means a buffer that contains at least one lysing agent. Typical enzymatic lysing 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 contiguity particles can be carried out by introducing lysing agents such as lysozyme and proteinase K into contiguity particles. The gDNA from the cells is now contained within the particles to maintain contiguity. In some cases, after lysis treatment, the isolated nucleic acid remains within the particle to maintain contiguity and can be used for further processing.

"DNA analysis" refers to any method used to amplify, sequence or otherwise analyze the DNA contained in an encapsulated cell. DNA amplification can be performed using PCR or pyrosequencing methods. DNA analysis may also include non-targeted, non-PCR based DNA sequencing techniques (eg metagenomics). As a non-limiting example, DNA analysis may include sequencing the 16S rDNA hypervariable region (ribosomal DNA) and using the sequencing to identify species from the DNA.

"RNA analysis" refers to any method used to amplify, sequence or otherwise analyze the RNA contained in an encapsulated cell. For amplification and sequencing of RNA, you can use the same methods that were used for DNA analysis. RNA, which is less stable than DNA, is the reading of DNA in response to stimuli. Thus, RNA analysis can provide a more accurate picture of the metabolically active members of a community and can be used to provide information about the function of a community of organisms in a sample. "Nucleic acid sequencing" refers to the use of sequencing to determine the order of nucleotides in the sequence of a nucleic acid molecule, such as DNA or RNA.

As used herein, the term "sequencing" refers to a method that establishes the identity of at least 10 consecutive nucleotides (for example, an identity of at least 20, at least 50, at least 100, or, at least 200 or more consecutive nucleotides) in a polynucleotide.

The terms "next-generation sequencing" or "high-throughput sequencing" or "NGS" generally refer to high-throughput sequencing technologies, including, but not limited to, massively parallel recognition sequencing, high-throughput sequencing, ligation-assisted sequencing (e.g., SOLiD sequencing), proton ion semiconductor sequencing, nanosphere DNA sequencing, single molecule sequencing, and nanopore sequencing and can refer to parallelized sequencing by synthesis or sequencing by ligation platforms currently used by Illumina, Life Technologies, or Roche, etc. d. Next generation sequencing methods can also include nanopore sequencing methods or electronic detection based methods such as the Ion Torrent technology commercialized by Life Technologies or the fluorescence based single molecule method commercialized by Pacific Biosciences.

"Protein analysis" refers to the study of proteins in an encapsulated cell, and may include proteomic analysis, determination of post-translational modification of proteins of interest, determination of protein expression levels, or determination of the protein's interaction with other molecules, including other proteins or nucleic acids.

As used herein, the term "tagmentation" refers to the modification of DNA by a transposome complex comprising a transposase enzyme in complex with adapters comprising a transposon terminus sequence. Tagging leads to simultaneous fragmentation of DNA and ligation of adapters to the 5' ends of both strands of duplex fragments. Following a purification step to remove the transposase enzyme, additional sequences may be added to the ends of the adapted fragments, for example by PCR, ligation, or any other suitable method known to those skilled in the art.

The assay for evaluating transposase-accessible chromatin using sequencing (ATAC-seq) refers to a fast and sensitive integrative epigenomic analysis method. ATAC-seq captures regions of open chromatin and reveals the interaction between genomic localization of open chromatin, DNA-binding proteins, single nucleosomes, and higher-order compaction in regulatory regions with nucleotide-level resolution. Classes of DNA binding factor have been found that strictly avoid nucleosomes, may allow the presence of nucleosomes, or tend to overlap with nucleosomes. Using ATAC-seq, serial daily epigenomes of resting human T cells in the proband were measured and evaluated using standard blood tests, demonstrating the ability to read personal epigenomes in clinical time frames for health and disease monitoring. More specifically, the ATAC sequence can be performed by treating chromatin from a single encapsulated cell in particles to maintain contiguity with an insertion enzyme complex to produce labeled genomic DNA fragments. In this step, the chromatin is tagged (eg, fragmented and labeled in the same reaction) using an insertion enzyme such as Tn5 or MuA, which cleaves the genomic DNA in open areas in the chromatin and adds adapters to both ends of the fragments.

In some cases, conditions can be adjusted to obtain the desired level of insertion into the chromatin (eg, an insertion that occurs on average every 50-200 base pairs in open areas). The chromatin used in the method may be obtained by any suitable method. In some embodiments, nuclei can be isolated, lysed, and chromatin can be further purified, such as from the nuclear envelope. In other embodiments, chromatin can be isolated by contacting the isolated nuclei with reaction buffer. In these embodiments, isolated nuclei can be lysed when they are contacted with a reaction buffer (including insertion enzyme complexes and other necessary reagents) that allows the insertion enzyme complexes to access chromatin. In these embodiments, the method may include isolating nuclei from a population of cells and combining the isolated nuclei with transposase and adapters, where the combination results in both lysis of the nuclei to release said chromatin and generation of adapter-tagged genomic DNA fragments. Chromatin does not require crosslinking as in other methods (eg ChIP-SEQ methods).

After the chromatin has been fragmented and labeled to produce labeled genomic DNA fragments, at least some of the adapter labeled fragments are sequenced to create multiple consecutive reads. The fragments may be sequenced in any suitable manner. For example, fragments can be sequenced using the reversible termination method from Illumina, the pyrosequencing method from Roche (454), ligation sequencing from Life Technologies (SOLiD platform), or the Ion Torrent platform from Life Technologies. Examples of such methods are described in the following references: Margulies et al. (Nature 2005 437: 376-80); Ronaghi et al. (Analytical Biochemistry 1996 242: 84-9); Shendure et al. (Science 2005 309: 1728-32); Imelfort et al. (Brief Bioinform. 2009 10:609-18); Fox et al. (Methods Mol Biol. 2009;553:79-108); Appleby et al. (Methods Mol Biol. 2009; 513:19-39) and Morozova et al. (Genomics. 2008 92:255-64), which are incorporated by reference herein for a general description of the methods and specific steps of the methods, including all starting materials, library preparation methods, reagents, and end products for each of the steps. As will be appreciated, forward and reverse sequencing primer sites that are compatible with the chosen next generation sequencing platform can be added to the ends of the fragments during the amplification step. In certain embodiments, fragments can be amplified using PCR primers that hybridize to labels that have been added to the fragments, where the PCR primers have 5'-tails that are compatible with a particular sequencing platform. Methods for conducting ATAC-seq are set forth in PCT Application No. PCT/US2014/038825, which is incorporated herein by reference in its entirety.

As used herein, the term "chromatin" refers to a complex of molecules, including proteins and polynucleotides (eg, DNA, RNA), that are found in the nucleus of a eukaryotic cell. Chromatin is composed in part of histone proteins that form nucleosomes, genomic DNA, and other DNA-binding proteins (eg, transcription factors) that are primarily associated with genomic DNA.

Contiguity-preserving transposition-based sequencing (CPT-seq) refers to a sequencing method while maintaining contiguity information by using a transposase to maintain association of a template nucleic acid with fragments adjacent to a target nucleic acid. For example, CPT can be performed on nucleic acid such as DNA or RNA. The CPT nucleic acid can be captured by hybridization with complementary oligonucleotides having unique indices or barcodes and immobilized on a solid support. In some embodiments, the oligonucleotide immobilized on a solid support may further comprise primer binding sites, unique molecular indices, in addition to barcodes. Advantageously, this use of transposomes to maintain the physical proximity of fragmented nucleic acids increases the likelihood that fragmented nucleic acids from the same parent molecule, such as a chromosome, will receive the same unique barcode and index information from oligonucleotides immobilized on a solid substrate. This will result in an adjacent-linked sequencing library with unique barcodes. An adjacent-linked sequencing library can be sequenced to obtain information about adjacent sequences. The adjacency particles described herein can be contacted with CPT-seq reagents to perform CPT-seq on nucleic acids extracted from an encapsulated cell.

As used herein, the term "adjacency information" refers to a spatial relationship between two or more DNA fragments based on common information. This general aspect of information can refer to adjacent, block, and remote spatial relationships. Information about these relationships, in turn, facilitates the hierarchical assembly or mapping of read sequences derived from DNA fragments. This adjacency information improves the efficiency and accuracy of such assembly or mapping because the traditional assembly or mapping methods used in connection with conventional shotgun sequencing do not take into account the relative genomic origin or coordinates of individual read sequences as they refer to spatial relationships between two or more DNA fragments from which an individual read sequence was obtained.

Thus, in accordance with the embodiments described herein, methods for obtaining adjacency information may be implemented using short range adjacency methods to determine adjacent spatial relationships, medium range adjacency methods to determine block spatial relationships, or long range adjacency methods to determine remote spatial links. These methods improve the accuracy and quality of DNA sequence assembly or mapping, and can be used with any of the sequencing methods described herein.

Adjacency information includes the relative original genomic position or sequence coordinates of individual reads, as they refer to the spatial relationships between two or more DNA fragments from which the individual read sequences were derived. In some embodiments, the adjacency information includes sequence information from non-overlapping read sequences.

In some embodiments, the adjacency information for the target nucleic acid sequence indicates haplotype information. In some embodiments, the adjacency information for the target nucleic acid sequence indicates genomic variants.

Combinatorial indexed single cell sequencing (SCI-seq) is a sequencing technique that simultaneously generates low frequency single cell libraries to detect somatic copy number variants.

Thus, many collaborative assays can be performed on a single cell to analyze the cell or nucleic acids from the cell, including the assays described herein alone or in combination with any other assay.

Indexed adjacency particles can also be directly loaded into flow cells that are held by a holder/microwell device. The indexed libraries are released from the particles to maintain contiguity (chemical/thermal release) and are associated with the flow cell. This allows for a powerful approach to indexing where the primary level of indexing comes from a spatial location and then the next level comes from single-particle indexed libraries to maintain contiguity. Alternatively, particle-derived indexed libraries to maintain contiguity can be co-loaded onto the flow cell.

In some embodiments, the cell encapsulated in the adjacency particle is contacted with one or more nucleic acid processing reagents. In some embodiments, the cell is held within the particle to maintain contiguity, and reactants are able to pass through the pores of the particle to maintain contiguity. In some embodiments, the reagents may include lysing agents, nucleic acid purification agents, DNA amplifying agents, tagging agents, PCR agents, or other agents that are used to process genetic materials. Thus, the adjacency particle provides a microenvironment for controlled cell reactions, including nucleic acids released from, within the adjacency particle by providing a polymer shell barrier that reactants can traverse in and out while retaining the cell within the particle to maintain adjacency. adjacency. In some embodiments, the pores of the polymeric shell are altered to allow the flow of reactants and also the flow of nucleic acids, such as DNA and RNA, isolated from the cell, through the polymeric shell.

In some embodiments, the preparation of a complete DNA library can be seamlessly performed within the particle to maintain contiguity with multiple reagent changes passing through the porous hydrogel while maintaining the genomic DNA and its products as a polymer-encased library. The hydrogel can be resistant to high temperatures up to 95°C for several hours to support various biochemical reactions.

As used herein, the terms "isolated", "isolated", "isolation", "purified", "purified", "purification" and their grammatical equivalents as used herein, unless otherwise indicated, refer to the reduction of at least one contaminant (such as a protein and/or nucleic acid sequence) from the sample or source (eg, a cell) from which the material is isolated. Purification thus results in "enrichment", eg, an increase in the amount of the desired protein and/or nucleic acid sequence in the sample.

After lysis and isolation of nucleic acids, amplification can be carried out, such as multiple displacement amplification (MDA), which is widely used to amplify small amounts of DNA, especially from single cells. In some embodiments, the encapsulated nucleic acids are amplified, acid sequenced, or used to generate nucleic acid libraries. The terms "amplify" or "amplified" when used in relation to a nucleic acid or reaction with a nucleic acid refer to methods for making copies of a particular nucleic acid in vitro , such as a target nucleic acid or a nucleic acid encapsulated in a particle to maintain contiguity, for example, by using an embodiment of the present invention. Numerous methods for amplifying nucleic acids are known in the art, and amplification reactions include polymerase chain reactions, ligase chain reactions, strand displacement amplification reactions, rolling ring amplification reactions, multiple anneal looped amplification cycles (MALBAC), methods transcription-mediated amplification such as NASBA, loop-mediated amplification methods (eg, "LAMP" amplification using loop-forming sequences). The nucleic acid to be amplified can be DNA containing, including DNA or RNA, or consisting of DNA or RNA, or a mixture of DNA and RNA, including modified DNA and/or RNA. Products resulting from the amplification of a nucleic acid molecule or molecules (e.g., "amplification products"), whether the parent nucleic acid is DNA, RNA, or both, can be either DNA or RNA, or a mixture of nucleosides or nucleotides like DNA , and RNA, or they may include nucleosides or nucleotides of modified DNA or RNA. "Copy" does not necessarily mean perfect complementarity or sequence identity with the target sequence. For example, copies may include nucleotide analogs such as deoxyinosine or deoxyuridine, intentional sequence changes (such as sequence changes introduced by a primer containing a sequence that hybridizes but is not complementary to the target sequence, and/or sequence errors that occur during amplification time.

Encapsulated nucleic acids that are isolated within a particle to maintain contiguity can be amplified according to any suitable amplification method known in the art. In some embodiments, the encapsulated nucleic acids are amplified within the particle to maintain contiguity. In some embodiments, the adjacency particle is captured onto a solid support and destroyed, whereby the encapsulated nucleic acids are released onto the solid support and the nucleic acids are amplified on the solid support.

In some embodiments, the encapsulated nucleic acids are amplified within the particle to maintain contiguity. For example, in some embodiments, the primers and amplification enzymes pass through the pores of the particle to maintain contiguity and hybridize to the encapsulated nucleic acids.

It should be understood that any of the amplification methods described herein or generally known in the art can be used with universal or target specific primers to amplify encapsulated nucleic acids. Suitable amplification methods include, but are not limited to, polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA) with transcription, and nucleic acid sequence-based amplification (NASBA) as described in US Pat. No. 8003354 incorporated herein by reference in its entirety. The above amplification methods can be used to amplify one or more nucleic acids of interest. For example, PCR can be used, including multiplex PCR, SDA, TMA, NASBA, and the like. for amplification of encapsulated nucleic acids. In some embodiments, primers directed specifically to the nucleic acid of interest are included in the amplification reaction.

Other suitable nucleic acid amplification methods may include oligonucleotide extension and ligation, rolling ring amplification (RCA) (Lizardi et al., Nat. Genet. 19: 225-232 (1998), incorporated herein by reference) and oligonucleotide ligation assay (OLA) technology (See generally US Pat. /12696 and WO 89/09835, all incorporated by reference). It should be understood that these amplification methods may be designed to amplify encapsulated nucleic acids. For example, in some embodiments, the amplification method may include amplification reactions with ligated probes or ligated oligonucleotides (OLAs), which may contain primers directed specifically at the nucleic acid of interest. In some embodiments, the amplification method may include a primer extension-ligation reaction that contains primers that specifically target the nucleic acid of interest and are capable of passing through the pores of the hydrogel. As a non-limiting example of primers for reaction extension and ligation of primers that can be specifically designed to amplify the nucleic acid of interest, the amplification can include primers used for the GoldenGate assay (Illumina, Inc., San Diego, Calif.), as exemplified by US Patent Nos. Nos. 7582420 and 7611869, each of which is incorporated herein by reference in its entirety. In each of the methods described, the reagents and components involved in the reaction of the nucleic acid are able to pass through the pores of the particle to maintain contiguity, while maintaining the nucleic acid itself within the particle to maintain contiguity.

In some embodiments, encapsulated nucleic acids are amplified using the cluster amplification methods disclosed in US Pat. Included materials in US Pat. Nos. 7,985,565 and 7,115,400 describe nucleic acid amplification methods that allow amplification of amplification products on a solid support to form arrays of clusters or "colonies" of immobilized nucleic acids. Each cluster or colony on such an array is formed from a plurality of identical immobilized polynucleotide strands and a plurality of identical immobilized complementary polynucleotide strands. The generated arrays are generally referred to herein as "cluster arrays". The products of solid phase amplification reactions, like those described in US Pat. -terminus, preferably via covalent attachment. Cluster amplification methods are examples of methods where an immobilized nucleic acid template is used to generate immobilized amplicons. You can also use other suitable methods to obtain immobilized amplicons from immobilized DNA fragments obtained in accordance with the methods described herein. For example, one or more clusters or colonies can be formed by solid phase PCR where one or both primers of each amplification primer pair are immobilized. In some embodiments, the encapsulated nucleic acids are amplified within a particle to maintain contiguity and then deposited onto a plate or solid support in a cluster.

Additional amplification methods include isothermal amplification. Typical isothermal amplification methods that can be used as non-limiting examples include multiple displacement amplification (MDA), for example, Dean et al., Proc. Natl. Acad. sci. US 99: 5261-66 (2002) or isothermal amplification with displacement of the nucleic acid strand, for example, in US patent No. 6214587, each of which is incorporated herein by reference in its entirety. Other non-PCR based methods that can be used in the present invention include, for example, strand displacement amplification (SDA) as described in, for example, Walker et al., Molecular Methods for Virus Detection, Academic Press, Inc., 1995; U.S. Patent Nos. 5,455,166 and 5,130,238, and Walker et al., Nucl. Acids Res. 20: 1691-96 (1992) or hyperbranched chain displacement amplification as described, for example, in Lage et al., Genom Research 13: 294-307 (2003), each of which is incorporated herein by reference in its entirety. Isothermal amplification methods can be used with the Phi 29 strand displacement polymerase or the large Bst fragment of Bst DNA polymerase, 5'->3' exo to amplify genomic DNA with a random primer. The use of these polymerases offers the advantage of high processability and strand displacement activity due to their high processivity. High processivity makes it possible to synthesize fragments of 10–20 kb in size. in length.

As mentioned above, smaller fragments can be prepared under isothermal conditions using a polymerase having low processivity and strand displacement activity, such as Klenow polymerase. Additional descriptions of amplification reactions, conditions, and components are detailed in the disclosure of U.S. Patent No. 7,670,810 incorporated herein by reference in its entirety. contiguity to interact with encapsulated nucleic acids, thus amplifying nucleic acids within particles to maintain contiguity. In some embodiments, random hexamers are annealed to denatured DNA followed by strand substitution synthesis at constant temperature in the presence of the catalyzing enzyme, Phi 29. This results in amplification of the DNA in the particles to maintain contiguity, as evidenced by an increase in fluorescence intensity (DNA stained with SYTOX) after MDA. Independently, Nextera-based tagging after lysis and purification and subsequent amplification of genomic DNA by PCR can also be performed, as indicated by a significant increase in fluorescence intensity in the particles to maintain contiguity after Nextera tagging and PCR. After this preparation of the Nextera library, the adjacency particles can be heated to 80° C. for 3 minutes to release the contents of the adjacency particles, namely the products containing the sequencing-ready library, from the cell.

Another method for amplifying nucleic acids that is suitable for the present invention is labeled PCR, which uses a population of two-domain primers having a constant 5 region followed by a random 3' region, as shown, for example, in Grothues et al. Nucleic Acids Res. 21 (5): 1321-2 (1993), incorporated herein by reference in its entirety. The first rounds of amplification are carried out to provide multiple initiation points on heat denatured DNA based on individual hybridization from a randomly synthesized 3' region. Due to the nature of the 3' region, initiation sites are considered random throughout the genome. After that, unbound primers can be removed and further replication can be done using primers complementary to the 5' constant region.

In some embodiments, the encapsulated nucleic acids are sequenced wholly or partially within particles to maintain contiguity. The encapsulated nucleic acids can be sequenced according to any suitable sequencing method such as direct sequencing, including sequencing by synthesis, sequencing by ligation, sequencing by hybridization, nanopore sequencing, and the like.

One of the sequencing methods is sequencing by synthesis (SBS). In SBS, nucleic acid primer extension is monitored along the nucleic acid template (eg, the target nucleic acid or its amplicon) to determine the nucleotide sequence in the template. The main chemical process may be polymerization (eg, catalyzed by a polymerase enzyme). In a specific embodiment of polymerase-based SBS, fluorescently labeled nucleotides are added to the primer (thereby extending the primer) 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.

One or more amplified encapsulated nucleic acids can be subjected to SBS or other detection method that includes repeated delivery of reagents in cycles. For example, to initiate the first round of SBS, one or more labeled nucleotides, DNA polymerase, etc. can be directed into/through the contiguity-maintaining particle containing one or more amplified nucleic acids. It is possible to detect those sites where primer extension causes incorporation of the labeled nucleotide. Optionally, the nucleotides may further include a reversible termination property that stops further extension of the primer once the nucleotide has been added to the primer. For example, a nucleotide analog with a reversible termination group can be added to the primer such that subsequent extension cannot occur until a deblocking agent is delivered to remove the group. Thus, for embodiments that use reversible termination, it is possible to deliver a deblocking reagent to the flow cell (before or after detection). Washes can be carried out between different stages of delivery. The cycle can then be repeated n times to extend the primer by n nucleotides, thus finding a sequence of length n. Examples of SBS methods, fluid systems and detection platforms that can be easily adapted for use with amplicons produced by the methods of the present invention are described, for example, in Bentley et al., Nature 456: 53-59 (2008), WO 04/ 018497; US patent No. 7057026; W091/06678; WO 07/123744; US patent No. 7329492; US patent No. 7211414; US patent No. 7315019; US patent No. 7405281 and US 2008/0108082, each of which is incorporated herein by reference.

You can use other sequencing methods that use cyclic reactions, such as pyrosequencing. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) when certain nucleotides are incorporated into the resulting nucleic acid strand (Ronaghi, et al., Analytical Biochemistry 242(1), 84-9 (1996); Ronaghi, Genom Res. 11(1)), 3-11 (2001); Ronaghi et al. Science 281 (5375), 363 (1998); US patent No. 6210891; US Pat. No. 6,258,568 and US Pat. No. 6,274,320, each of which is incorporated herein by reference). In pyrosequencing, released PPi can be detected by immediate conversion to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP generated can be detected by photons produced by luciferase. Thus, the sequencing reaction can be monitored using a luminescence detection system. Excitation sources used for fluorescence-based detection systems are not needed for pyrosequencing methods. Suitable fluid systems, detectors and methods that can be adapted to apply pyrosequencing to amplicons produced according to the present invention are described, for example, in WIPO patent application serial number PCT/US11/57111, US 2005/0191698 A1, US patent No. 7595883 and US Pat. No. 7,244,559, each of which is incorporated herein by reference.

In some embodiments, methods may be used that include real-time monitoring of DNA polymerase activity. For example, nucleotide inclusions can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-containing formula and γ-phosphate labeled nucleotides or with zero mode waveguides (ZMW). 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 descriptions of which are incorporated herein by reference.

Some embodiments of SBS involve detecting the proton released when the nucleotide is incorporated into the extension product. For example, sequencing based on the detection of released protons may use an electrical detector and related methods that are commercially available. Examples of such sequencing systems are pyrosequencing (for example, a commercially available platform from 454 Life Sciences, a subsidiary of Roche), sequencing using γ-phosphate-labeled nucleotides (for example, a commercially available platform from Pacific Biosciences), and sequencing using proton detection (for example, a commercially available platform from Ion Torrent, a subsidiary of Life Technologies) or the sequencing methods and systems described in US 2009/0026082 A1; US 2009/0127589 A1; US 2010/0137143A1; 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. More specifically, the methods described herein can be used to generate clonal populations of amplicons that are used to detect protons.

Another sequencing method is nanopore-based 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-based embodiments, the target nucleic acid or individual nucleotides removed from the target nucleic acid pass through the nanopores. When a nucleic acid or nucleotide passes through the nanopore, each type of nucleotide can be identified by measuring the fluctuations in the electrical conductivity of the pore. (U.S. Patent No. 7001792; Soni et al. Clin. Chem. 53, 1996-2001 (2007); Healy, Nanomed. 2, 459-481 (2007); Cockroft et al. J. Am. Chem. Soc. 130, 818-820 (2008), the descriptions of which are incorporated herein by reference).

Exemplary methods of massive analysis for expression and genotyping, which can be used for detection in accordance with the present invention, are described in US patent No. 7582420; 6890741; 6,913,884 or 6,355,431 or US Patent Publication No. 2005/0053980 A1; 2009/0186349 A1 or US 2005/0181440 A1, each of which is incorporated herein by reference.

Nucleic acid isolation, amplification, and sequencing methods as described herein use various reagents to isolate and obtain nucleic acids. Such reagents may include, for example, lysozyme, proteinase K, random hexamers, polymerase (e.g., Φ29 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. These reagents pass through the pores of the particles to maintain contiguity while the genetic material is stored within the particles to maintain contiguity. The advantage of the methods described herein is that they provide an encapsulated microenvironment for processing nucleic acids within a particle to maintain contiguity. This allows a single cell to be processed for fast and efficient processing of the target nucleic acid.

Adapters may include sequencing primer regions, amplification primer regions, and indices. As used herein, an "index" may include a sequence of nucleotides that can be used as a molecular identifier and/or a barcode to label a nucleic acid, and/or to identify the source of the nucleic acid. In some embodiments, an index can be used to identify a single nucleic acid or a subset of nucleic acids. In some embodiments, nucleic acid libraries can be generated in a particle to maintain contiguity. In some embodiments, a single cell encapsulated in a contiguity-maintaining particle can be used to combinatorially index a single cell, for example, using a contiguity-preserving transposition (CPT-seq) technique. In some embodiments, DNA from a single cell can be barcoded by encapsulating the single cell after WGA amplification with another adjacency particle carrying barcoded transposons and dissolving the gel matrix by contacting a reductant, e.g., to release genomic DNA for barcoding.

The embodiments of the "spatial indexing" methods and approaches described herein reduce data analysis and simplify the process of obtaining a library from single cells and long DNA molecules. Existing protocols for single cell sequencing require efficient physical separation of cells and unique barcoding of each isolated cell and combining everything together to make sequencing. Current protocols for synthetic long reads also require cumbersome barcoding steps, and combining all barcoded fragments together for sequencing, and allowing data analysis to distinguish between the genetic information coming from each barcoded cell. During these long processes, there is also a loss of genetic material that causes gaps in the sequence. The embodiments described herein not only shorten the process, but also increase the data resolution for single cells. In addition, the embodiments provided herein simplify the assembly of the genomes of new organisms. The embodiments described herein can be used to detect rare genetic variations and the co-occurrence of mutations. In some embodiments of the DNA library within the particle to maintain contiguity until release, it is possible to control the size of the fragments that are released on the surface by controlling the release process and the composition of the hydrogel.

In some embodiments, the library can be amplified using primer regions in adapter sequences, and sequenced using sequencing primer regions in adapter sequences. In some embodiments, adapter sequences may include indexes to identify the source of nucleic acids. The efficiency of subsequent amplification steps can be reduced by the formation of primer dimers. To increase the efficiency of subsequent amplification steps, unligated single strand adapters can be removed from the ligation products.

Obtaining Nucleic Acid Libraries Using Contiguity Particles

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 co-complementary sequence. As stated herein, the P5 sequence contains the sequence defined by SEQ ID NO: 1 (AATGATACGGGCACCACCGA) and the P7 sequence contains the sequence defined by SEQ ID NO: 2 (CAAGCAGAAGACGGCATACGA). In some embodiments, the P5 or P7 sequence may further include a spacer polynucleotide, which may be 1 to 20, such as 1 to 15, or 1 to 10 nucleotides, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some embodiments, the implementation of the spacer includes 10 nucleotides. In some instances, said spacer is a poly T spacer, such as a 10T spacer. Spacer nucleotides can be included at the 5' ends of the polynucleotides, which can be attached to a suitable support by linkage to the 5' end of the polynucleotides. Attachment can be achieved with a sulfur containing nucleophile such as a thiophosphate present at the 5' end of the polynucleotide. In some embodiments, the polynucleotide will include a poly T spacer and a 5' thiophosphate group. Thus, in some embodiments, the P5 sequence is 5' thiophosphate-TTTTTTTTAATGATACGGCGACCACCGA-3' (SEQ ID NO: 3 and in some embodiments, the P7 sequence is 5' thiophosphate-TTTTTTTT CAAGCAGAAGACGGCATACGA-3' (SEQ ID: 4).

Indices can be useful in identifying the origin of a nucleic acid molecule. In some embodiments, the adapter may be modified to prevent the formation of concatemers, for example, by adding blocking groups that prevent the adapter from elongating at one or both ends. Examples of 3' blocking groups include the C3 3' spacer, dideoxynucleotide, and substrate attachment. Examples of 5' blocking groups include a dephosphorylated 5' nucleotide and attachment to a substrate.

Adapters include nucleic acids such as single stranded nucleic acids. Adapters may include short nucleic acids having a length 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 between any two of the above sizes. In some embodiments, they are of sufficient size to pass through the pores of the particles to maintain contiguity. Target nucleic acids include DNA, such as genomic or cDNA; RNA, such as mRNA, rRNA or rRNA; or a hybrid of DNA and RNA. The nucleic acid can be isolated from a single cell encapsulated in a particle to maintain contiguity. The nucleic acid may contain phosphodiester linkages and may include other types of backbone including, for example, phosphoamide, thiophosphate, phospho-dithioate, O-methylphosphoroamidite, 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, xanthanine, hypoxantanine, isocytosine, isoguanine, and base analogs such as nitropyrrole (including 3-nitropyrrole) and nitroindole (including 5-nitroindole). In some embodiments, the implementation of the nucleic acid may include at least one irregular base. An irregular base can be a base pair with more than one different base type, and this can be useful, 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 an irregular base includes inosine, which can be combined with adenine, thymine, or cytosine. Other examples include hypoxanthine, 5-nitroindole, acyl 5-nitroindole, 4-nitropyrazole, 4-nitroimidazole, and 3-nitropyrrole. You can use irregular bases that can pair with at least two, three, four or more types of bases.

Target nucleic acids may include a sample in which the average size of the nucleic acid in the sample is less than, greater than, or equal to about 2 kb, 1 kb, 500 bp, 400 bp. , 200 bp, 100 bp, 50 bp, or a range between any two of the above sizes. In some embodiments, the average size of a nucleic acid in a sample is less than, greater than, or equal to about 2000 nucleotides, 1000 nucleotides, 500 nucleotides, 400 nucleotides, 200 nucleotides, 100 nucleotides, 50 nucleotides, or a range between any two of the above sizes. In some embodiments, the nucleic acid is of sufficient size that the nucleic acid is trapped within the contiguity particles so that it cannot pass through the pores of the contiguity particles.

An exemplary method includes dephosphorylation of the 5' ends of target nucleic acids to prevent formation of concatemers in subsequent ligation steps; ligating the first adapters to the 3' ends of the dephosphorylated targets using a ligase, with the 3' ends of the first adapters blocked; rephosphorylation of the 5' ends of ligated targets; ligating the second adapter to the 5' ends of the dephosphorylated targets using a single strand ligase, with the 5' ends of the second adapters unphosphorylated.

Another example involves partial cleavage of a nucleic acid with a 5'-exonuclease to form a double stranded nucleic acid with single stranded 3' sticky ends. An adapter containing a 3' blocking group can be ligated to the 3' ends of a double stranded nucleic acid with 3' overhang. Double-stranded nucleic acid 3' sticky ends with ligated adapters can be hybridized to form single-stranded nucleic acids. An adapter containing a non-phosphorylated 5' end may be ligated to the 5' end of a single stranded nucleic acid.

Methods for dephosphorylation of nucleic acids, for example the 5'-nucleotide of a nucleic acid, include contacting the nucleic acid with a phosphatase. Examples of phosphatases are calf intestinal phosphatase, shrimp alkaline phosphatase, Antarctic phosphatase and APEX alkaline phosphatase (Epicentre).

Methods for ligating nucleic acids include contacting the nucleic acids with a ligase. Examples of ligases include T4 RNA ligase 1, T4 RNA ligase 2, RtcB ligase, Methanobacterium RNA ligase, and TS2126 RNA ligase (CIRCLIGASE).

Methods for phosphorylation of nucleic acids, for example the 5'-nucleotide of a nucleic acid, involve contacting the nucleic acid with a kinase. Examples of kinases include T4 polynucleotide kinase.

Embodiments provided herein relate to the production of nucleic acid libraries in a contiguity particle such that the nucleic acid library is produced in one volume of the reaction mixture.

Embodiments of the systems and methods provided herein include kits containing any one or more hydrogel polymers, crosslinkers, or microfluidic devices for preparing adjacency particles that encapsulate a cell, and further include components suitable for processing genetic material. , including agents for cell lysis, and for nucleic acid amplification and sequencing, or for the preparation of nucleic acid libraries, including lysozyme, proteinase K, random hexamers, polymerase (e.g., Φ29 DNA polymerase, Taq polymerase, Bsu polymerase), transposase (eg, Tn5), primers (eg, P5 and P7 adapter sequences), enzyme catalyzing ligase, deoxynucleotide triphosphates, buffers, or divalent cations, as described herein, and which are used to appropriately process the genetic material.

EXAMPLES

Example 1 Getting Particles to Maintain Adjacency

The following example demonstrates an embodiment of producing adjacency particles encapsulating a single cell using a microfluidic droplet generator.

Samples containing cells and stored at -80°C were thawed at room temperature. 100 μl of each sample was transferred to a sterile 1.7 ml tube and the sample was washed once with 1 ml of 0.85% NaCl. The sample was precipitated and the washing solution was removed. The cell pellet was mixed with the hydrogel solution to resuspend the cells in the hydrogel solution.

To obtain adjacency particles with a uniform size distribution, microfluidic droplet generators such as the one shown in FIG. 1. A solution containing a hydrogel polymer and a cell was injected into the first channel of the microfluidic droplet generator. The mineral oil used as the release oil was added to the second channel and the crosslinker was added to the third channel. Upon contact with the crosslinker in the third channel, the hydrogel instantly formed adjacency particles encapsulating a single cell. As shown in FIG. 6, the adjacency particles were stained with a fluorescent dye that targets the -SH groups of the polymer shell of the particles to maintain adjacency. Right panel of FIG. 6 shows single cell staining within the adjacency particle, demonstrating a 60x magnification image of the adjacency particle stained with Hoechst (blue core stain) and AF-647 BSA, which diffuses into the adjacency particle through the pores of the polymer shell. The type of crosslinking oil was chosen to adjust the crosslinking speed, including a slow crosslinker or an instantaneous crosslinker, as shown in Table 1:

Table 1: Chemistry of Crosslinking

Type of particle to maintain adjacency Crosslinkers Stitching time Size homogeneity Cross-linking of the granule Slow crosslinker PEG-epoxide+PEG-amine >12 hours Good Complete PEG-dithiol+PEG-acrylate 4 hours Medium incomplete Instant crosslinker Dithiol oil + PEG-maleimide Instantly Good Complete PEG epoxy+amine oil Instantly Good Complete

Example 2 Collaborative Assays Conducted on Particles to Maintain Adjacency

The following example shows examples of assays performed on the contiguity particles of Example 1, including SCI-seq, ATAC-seq, combinatorial indexing, and whole genome single cell amplification.

The adjacency particles from Example 1 were prepared and applied to a well plate such that one adjacency particle was placed in one well. The cells were lysed by introducing a lysis buffer, then washed, thus isolating the nucleic acids from the cells. Particles to maintain contiguity with lysed cells were then subjected to a series of assays as described below.

As indicated in FIG. 7, indexed transposomes (TSMs) were used to tag genomic DNA to generate fragments for ATAC-seq. After proteinase/SDS treatment, an oligo T with the same index as TSM was added to each well to initiate cDNA synthesis by reverse transcription (RT). The PCR adapter at the other end was introduced by extending the randommer. This gave Index 1. The adjacency particles from each well were pooled together and then split into an indexed PCR plate to obtain Index 2. This two-level indexing can be scaled up to 150,000 (384x384) cells. The final generated library was a mixture of ATAC-seq and RNA-seq, in which gDNA and cDNA from the same cell were grouped at the same index, and the T-UMI oligo pattern served as an internal marker to distinguish the RNA signal from ATAC signal.

In addition, random extension was also used for full-length RNA-seq, as shown in FIG. 8. In this case, the TSM indices differed from the randomizer indices. A one-to-one comparison between these two indexes helped identify reads from a single cell and differentiate between DNA and RNA signals. UMI was also used in this method to improve the accuracy of the read analysis.

Three-level combinatorial indexing analysis was also performed using two rounds of indexed bypass ligation and one round of indexed PCR, as indicated in the diagram in FIG. 9. In this method, TSM and oligo T are versatile, both containing a splint1 fragment that allows for index addition by shunt ligation. Three-level indexing has reached a throughput of up to 1 million cells (96×96×96). Another three-level combinatorial indexing for ATAC-seq involves the use of indexed TSM to increase cell throughput, as indicated in FIG. 10. Instead of using a generic TSM, TSMs contained a unique index on the B7G and A7G side. Two different shunts were used to attach indexed adapters required for indexed PCR. 10, the indexing includes the following components: adapter sequence B15 (SEQ ID NO: 5), N6, and Link1, together forming the sequence B15_N6_Link1 (SEQ ID NO: 11); the sequence Phos_Link2, A7G (SEQ ID NO: 9) and the sequence ME (SEQ ID NO: 7), together forming the sequence Phos_Link2_A7G_ME (SEQ ID NO: 12); adapter sequence A14 (SEQ ID NO: 6), N6 and Link1, together forming the sequence A14_N6_Link1 (SEQ ID NO: 13); and Phos_Link2, B7G (SEQ ID NO: 10) and the ME sequence (SEQ ID NO: 7), together forming the Phos_Link2_B7G_ME sequence (SEQ ID NO: 14). Figure 10 also provides a complementary sequence to ME (SEQ ID NO: 8), a Splint 1 sequence (SEQ ID NO: 15), and a Splint 2 sequence (SEQ ID NO: 16).

Whole genome single cell amplification was also performed using adjacency particles as indicated in FIG. 11. This was done using T7 indexed particle transposition to maintain contiguity in individual wells followed by T7 pooling and extension in in vitro transcriptional linear amplification (IVT). The beads were split again for indexed random extension, pooled and split again for the final indexed PCR.

The adjacency particles showed efficient tagmentation when they were targeted to FAM-tagged transposomes. The nuclei were stained with Hoechst (DNA stain with blue light) and the transferred nuclei were stained with fluorescent green with FAM (fluorescent dye). Lysis of cells with SDS increased the background fluorescence of the adjacency particles, while no signal was observed from adjacency particles without cells. The results demonstrate that a library for ATAC-seq can be produced for cells encapsulated within particles to maintain contiguity with only a small fraction of leakage of short fragments from tagmentation.

Example 3 Preparation of a Library of Nucleic Acids in Particles for Maintaining Adjacency

The following example shows a method for producing a genome-wide library from a cell encapsulated in a contiguity particle.

FIG. 12 depicts a scheme for obtaining a whole genome library on a single cell, encapsulated in a particle to maintain contiguity. As indicated in the diagram of FIG. 12, the adjacency particle was used to create a Nextera genome-wide library. Adjacency particles were prepared as described in Example 1. Adjacency particles (CP) were loaded onto a 45 μm cell sieve and washed several times with PBS or Tris-Cl to remove any unencapsulated cells. One of the benefits of an encapsulated cell in a particle for maintaining contiguity is an improved ability to work with and process them. One easy way to do this is to use centrifuge columns or filter plates. The pore size of the filter may be smaller than the granule diameter. Examples of filter plates include, but are not limited to, 20, 40, and 60 µm MultiScreen-Mesh Millipore filter plates, 8.0 µm MultiScreen-Migration Invasion and Chemotaxis Millipore filter plates, or 96-well filter plates. for water filtration Pall's AcroPrep Advance with pores 30-40 microns. With these filter plates, the encapsulated cell pellets were easily separated from the solution, allowing some buffer changes.

The washed granules were suspended in buffer and removed from the filter. To evaluate the final bead concentration, cell loading efficiency, and ensure that no unencapsulated cells remained, an aliquot of the beads was visualized under a microscope.

Millipore Nylon MultiScreen-Mesh 20 µm filter plates were used to perform Nextera tagging. To limit the adhesion of the granules to the filter, they were pre-wetted with Pluronic F-127. After centrifuging the plate for 30 sec at 500 g, the buffer flowed through the filter, saving the beads. The beads were washed twice with 200 μl of Tris-Cl buffer, then suspended in lysis buffer (0.1% SDS). The beads were incubated in lysis buffer for 1 min before being removed by centrifugation. Two additional washes with 200 μl Tris-Cl were performed to remove residual lysis buffer. The cells were then suspended in 45 μl of 1× tag buffer by pipetting up and down, then transferred to a tube strip. To 45 μl of beads, 5 μl of DNA tagging enzyme (TDE, Illumina Inc.) was added and incubated in a thermal cycler for 1 hour at room temperature, 30 minutes at 55°C. Alternatively, tagging can be performed on a filter plate by suspending the bead-encapsulated cells in the tagging master mix and then incubating in an incubator. After tagging, an aliquot of the beads was stained with Hoechst stain and visualized under a microscope. Imaging confirmed that the DNA remained in the granule.

Tagged beads (25 µl) were PCR amplified using Illumina Nextera PCR MM Master Mix (NPM, Illumina) and PCR primers. 0.1% SDS was added to the PCR master mix to remove DNA bound Tn5. Pre-incubation was performed at 75°C to help remove Tn5 in the presence of SDS. Eleven cycles of PCR were performed to generate the final library. After PCR, libraries were purified using 0.9×SPRI, quantified using dsDNA qubit and/or BioAnalyzer, and sequenced on MiSeq and/or NextSeq.

The method described in this example can be scaled up to perform single cell sequencing using a similar approach to sciSEQ (Vitak et al. Nat Meth. 2017; 14: 302-308). For example, using a 96-well filter plate to run 96 tag-indexing reactions simultaneously can be scaled. After the beads are added to the plate, buffers are added one by one, then removed by centrifugation or vacuum. After tagmentation, the beads are collected from the filter and pooled. The pooled beads are redistributed into a second 96-well PCR plate for multiplex PCR. In this two-level indexing scheme (tag-indexing and PCR), all DNA fragments from a single bead-encapsulated cell receive the same barcode, which can be decoded to reconstruct the cell's genome.

The steps described in this example are amenable to automation on liquid handling platforms by adding a vacuum manifold, such as a Millipore MultiScreen HTS vacuum manifold or a 96-well plate vacuum manifold from Orochem. These vacuum manifolds can be added to many fluid handling platforms including Biomek FX, Microlab Star, Tecan Genesis, etc. Tagging can be automated by transferring the filter plate to the thermoblock. The plate is then returned to the vacuum manifold for post-tagment washes.

The embodiments, examples, and drawings described herein provide compositions, methods, and systems for holding genetic material in a physically enclosed space during the process from lysis to library creation. Some embodiments provide a library derived from a single long DNA molecule or a single cell that is released onto the surface of a flow cell in a confined space. Once a single cell DNA or single cell library 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.

As used herein, the term "comprising" is synonymous with "comprising", "including", or "characterized", and is inclusive or open-ended and does not exclude additional, unremarked elements or method steps.

The above description discloses several methods and materials according to the present invention. This invention allows for changes in methods and materials, as well as changes in manufacturing methods and equipment. Such modifications will become apparent to those skilled in the art from a review of this disclosure or the practice of the invention described herein. Thus, it is to be understood that this invention is not to be limited to the specific embodiments described herein, but that it embraces all modifications and alternatives falling within the true scope and spirit of the invention.

All references cited herein, including but not limited to all published and unpublished applications, patents and literature references, are incorporated herein by reference in their entirety and thus form part of the present specification. To the extent that publications and patents or patent applications incorporated by reference conflict with the invention contained in the specification, the description is intended to supersede and/or take precedence over any such conflicting material.

Claims (27)

1. A solid support for nucleic acid sequencing, comprising a plurality of hollow beads, where each bead contains: polymer shell; And a single cell located inside a polymer shell, where the polymer shell contains pores that provide diffusion of the reagent through the polymer shell while retaining a single cell; where the solid support is a flow cell device, a well plate, a microarray, a microfluidic channel, a glass slide, or a structured surface. 2. The solid support of claim 1, wherein the solid support is a flow cell device including a microwell or micropillar insert in the multibead dispenser to maintain contiguity for spatial indexing in the flow cell device. 3. The solid support of claim 1, wherein the solid support is a well plate including a plurality of wells, wherein each of the plurality of beads is loaded into each well of the plurality of wells. 4. The solid support of claim 3, wherein the well plate comprises from about 12 to 960 wells. 5. The solid support of claim 1, wherein each bead has a diameter of from about 20 microns to about 200 microns. 6. The solid support of claim 1 wherein the polymer shell comprises PEG maleimide/dithiol oil, PEG epoxide/amine oil, PEG epoxide/PEG amine, or PEG dithiol/PEG acrylate. 7. The solid support of claim 1, wherein the polymer shell contains a reversible crosslinker. 8. Solid support according to claim 7, wherein the reversible crosslinker is N,N'-bis(acryloyl)cystamine. 9. A method for conducting multiple sequential collaborative analyzes of individual cells in a population of cells, including: contacting the cell population with a polymer in a release oil to form mixture (a); mixing the mixture (a) with the crosslinking oil to form a plurality of beads, each bead encapsulating a single cell, where each bead contains pores that allow diffusion of the reagent through the bead while retaining the single cell; sequentially contacting individual cells within a plurality of reagent beads to perform a plurality of sequential collaborative cell population assays; separation of granules on a solid support as defined in paragraphs. 1-8; and modifying the separated beads to allow diffusion of the encapsulated material from the separated beads. 10. The method of claim. 9, where the modification of the separated granules includes increasing the temperature of the granules. 11. The method of claim 10 wherein the temperature is raised to more than 50°C. 12. The method of claim 9, wherein the modification of the separated beads comprises breaking the crosslinker of the beads. 13. The method of claim 12 wherein the decomposition comprises bringing the pellets into contact with a reducing agent. 14. The method of claim 13 wherein the reducing agent is dithioerythritol (DTE), dithiothreitol (DTT), 2-mercaptoethanol, aminoethanethiol, glutathione, thioglycolate, 2,3-dimercaptopropanol, tris(2-carboxyethyl)phosphine (TCEP), tris (hydroxymethyl)phosphine (THP), or P-[tris(hydroxymethyl)phosphine] propionic acid (THPP). 15. The method according to claim 9, where the modification of the separated granules includes exposing the granules to an electric field. 16. The method of claim 9, wherein the multiple sequential collaborative assays include lysis, DNA analysis, RNA analysis, protein analysis, tagging, nucleic acid amplification, nucleic acid sequencing, DNA library preparation, transposase accessible chromatin analysis using sequencing (ATAC-seq), adjacency-preserving transposition (CPT-seq), combinatorial indexed single cell sequencing (SCI-seq), or single cell genome amplification, or any combination thereof, performed sequentially. 17. The method of claim 9 wherein each bead has a diameter of from about 20 µm to about 200 µm. 18. The method of claim 9 wherein the release oil comprises a mineral oil or a fluorocarbon oil. 19. The method of claim 9, wherein the mixing comprises placing the single cell, polymer, release oil, and cross-linking oil into a droplet generator, where the droplet generator is optionally a microfluidic chip.

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