NL2024596B1

NL2024596B1 - On-flow cell three-dimensional sequencing matrices

Info

Publication number: NL2024596B1
Application number: NL2024596A
Authority: NL
Inventors: Rosàs-Canyelles Elisabet; Lessard-Viger Mathieu; Kumar Khurana Tarun; Black Hayden; Wu Yir-Shyuan; Zimmerley Max; Ramirez Sean
Original assignee: Illumina Inc
Priority date: 2019-11-27
Filing date: 2019-12-31
Publication date: 2021-08-30

Abstract

A method for sequencing in three-dimensions using an on ﬂow-cell three-dimensional sequencing matrix, comprising loading a polymer precursor solution into a ﬂow cell, wherein the polymer precursor 5 solution includes monomers and oligonucleotides, polymerizing the polymer precursor solution to create a permeable three-dimensional matrix within the ﬂow cell, diffusing a sequencing library into the permeable three-dimensional polymer matrix, wherein the sequencing library includes nucleic acid fragments, diffusing enzymes and reagents into the permeable three-dimensional polymer matrix, hybridizing the nucleic acid fragments to the oligonucleotides in the permeable three-dimensional 10 polymer matrix, clonally amplifying the hybridized nucleic acid fragments to create clusters for sequencing within the permeable three-dimensional polymer matrix; sequencing the clusters within the permeable three-dimensional polymer matrix; and optically imaging the sequenced clusters within three- dimensional matrix in multiple, discrete two-dimensional slices to characterize the sequencing library, wherein the multiple, discrete two-dimensional slices represent the entire three-dimensional internal 15 volume of the ﬂow cell.

Description

ON-FLOW CELL THREE-DIMENSIONAL SEQUENCING MATRICES BACKGROUND

[0001] Next-generation sequencing (NGS) 1s a high-throughput sequencing technology capable of sequencing entire genomes in a rapid and cost-effective manner. NGS typically begins with the creation of a sequencing library that includes genomic DNA that has been randomly fragmented, extracted, and purified. NGS processes such as sequencing-by-synthesis can then be utilized for massively parallel sequencing of the entire genomic library. Current NGS platforms rely on optical interrogation of surface-bound nucleic acid clusters and produce data at a fairly static rate and at somewhat significant cost per genome. Increasing the throughput of nucleic sequencing methods is important for driving the cost of sequencing down and improving overall sequencing accuracy. This desired outcome is achievable by sequencing a greater number of nucleic clusters, which are traditionally bound to the top and bottom surfaces of a sequencing flow cell. Thus, either a larger flow cell surface area or a higher cluster density should be implemented to increase the number of clusters that can be sequenced. However, sequencing flow cells are approaching size and cluster density limits, making significant improvements to throughput increasingly challenging using traditional surface-bound sequencing processes. Accordingly, overcoming these limitations would be beneficial.

SUMMARY

[0002] The following provides a summary of certain examples. This summary is not an extensive overview and is not intended to identify key or critical aspects or elements of the present invention or to delineate its scope. It is to be understood that any respective features/examples of each of the aspects of the disclosure as described herein may be implemented together in any combination to achieve the results as described herein, and that any features/examples from any one or more of these aspects may be implemented together with any of the features of the other aspect(s) as described herein in any combination to achieve the benefits as described herein.

[0003] In accordance with one implementation, a for making an on-flow cell three-dimensional sequencing matrix is disclosed. This method comprises embedding oligonucleotides within a permeable three-dimensional matrix, wherein the oligonucleotides facilitate nucleic acid fragment clonal amplification within the matrix; introducing the oligonucleotide-containing permeable three-dimensional matrix into a flow cell, wherein the flow cell includes at least one channel for receiving the oligonucleotide-containing permeable three-dimensional matrix; and immobilizing the oligonucleotide- containing permeable three-dimensional matrix in the at least one channel. The permeable three- dimensional matrix may be a polymer or a hydrogel; may include hydrogel networks of a predetermined size; may include a matrix of particles having the same size or particles of different sizes; may include columnar posts; may include mesoporous or microporous crystalline materials; and may be patterned by photolithography. The oligonucleotides may be adapted for sequencing-by-synthesis. The sequencing flow has an internal volume and the oligonucleotide-containing permeable three-dimensional matrix occupies the entire internal volume of the flow cell. The method may further comprise imaging the permeable three-dimensional matrix may be imaged in discrete two-dimensional layers using different microscopic systems and methods.

[0004] In accordance with another implementation, a first method for sequencing in three- dimensions using an on flow-cell three-dimensional sequencing matrix is disclosed. This method comprises loading a polymer precursor solution into a sequencing flow cell, wherein the polymer precursor solution includes monomers and oligonucleotides: polymerizing the polymer precursor solution to create a permeable three-dimensional matrix within the flow cell; diffusing a sequencing library into the permeable three-dimensional polymer matrix, wherein the sequencing library includes nucleic acid fragments; diffusing enzymes and reagents into the permeable three-dimensional polymer matrix; hybridizing the nucleic acid fragments to the oligonucleotides in the permeable three-dimensional polymer matrix; clonally amplifying the hybridized nucleic acid fragments to create clusters for sequencing within the permeable three-dimensional polymer matrix; sequencing the clusters within the permeable three-dimensional polymer matrix; and optically imaging the sequenced clusters within three- dimensional matrix in multiple, discrete two-dimensional slices to characterize the sequencing library. wherein the multiple, discrete two-dimensional slices represent the entire three-dimensional internal volume of the flow cell.

[0005] The monomers may include polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N.N'-Bis(acryloyleystamine (BACy). PEG, polypropylene oxide (PPO), polvacrylic acid, poly({hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N- isopropylacrylamide) (PNIPAAm). poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA). polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), polylvsine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen. bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethyleneglycol diallyl ether, ethyleneglycol diacrylate, polymethyleneglycol diacrylate, polyethyleneglycol diacrylate, trimethylopropoane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetracrylate, or combinations thereof. The monomers may include polyethylene glycol (PEG)-thiol/PEG-acrylate; acrylamide/N,N'-bis(acrvloyl)cystaming (BACy); PEG/polypropyvlene oxide (PPO): or combinations thereof.

[0006] The polymer precursor solution may include poly(N-(5-azidoacetamidylpentyl) acrylamide-co-acrylamide) (PAZAM) containing azide moieties and the oligonucleotides may be alkyne- linked oligonucleotides adapted to bind to the azide moieties in the PAZAM. The oligonucleotides may be adapted for sequencing-by-synthesis. The permeable three-dimensional matrix may be a hydrogel; may include hydrogel networks of a predetermined size; may include a matrix of particles of the same size or particles of different sizes and may include columnar posts that are fabricated to include alternating materials in Z-direction. The optical imagining may include the use of a confocal microscope or a light- sheet illumination microscope.

[0007] In accordance with still another implementation, a second method for sequencing in three-dimensions using an on flow-cell three-dimensional sequencing matrix is disclosed. This method comprises loading a polymer precursor solution into a sequencing flow cell, wherein the polymer precursor solution includes monomer, crosslinker, photoinitiator, and oligonucleotides; polymerizing the polymer precursor solution using ultraviolet light to create a permeable three-dimensional matrix within the flow cell; diffusing a sequencing library into the permeable three-dimensional polymer matrix, wherein the sequencing library includes nucleic acid fragments having adapters ligated thereto; diffusing enzymes and reagents into the permeable three-dimensional polymer matrix; hybridizing the nucleic acid fragments to the oligonucleotides in the permeable three-dimensional polymer matrix; clonally amplifying the hybridized nucleic acid fragments to create clusters for sequencing within the permeable three-dimensional polymer matrix; sequencing the clusters within the permeable three-dimensional polymer matrix; and using a confocal microscope or a light-sheet illumination microscope to image the sequenced clusters within three-dimensional matrix in multiple, discrete two-dimensional slices to characterize the sequencing library, wherein the multiple, discrete two-dimensional slices represent the entire three-dimensional internal volume of the flow cell.

[0008] The monomer may include polyethylene glvcol (PEG)-thiol, PEG-acrylate, acrylamide, N.N'-Bis(acryvlovl)cystamine (BACy), PEG, polypropylene oxide (PPO), polyacrylic acid. polythydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N- isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA). poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), polv(L-aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide. diacrylate, diallylamme, triallylamine, divinyl sulfone, diethyleneglycol diallyl ether, ethyleneglycol diacrylate, polymethyleneglycol diacrylate, polyethyleneglycol diacrylate, trimethylopropoane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetracrylate, or combinations thereof. The monomer may include polyethylene glycol (PEG)-thiol/PEG-acrylate; acrylamide/N N'-bis(acryloyl)cyvstamine (BACv); or

PEG/polypropylene oxide (PPO). The photoinitiator may be lithium phenyl-2.4.6- trimethylbenzoylphosphinate (LAP), a diazosulfonate initiator; a monoacylphosphineoxide (MAPO) salt, or a bisacylphosphineoxide (BAPO) salt; or combinations thereof.

[0009] The polymer precursor solution may include poly(N-(5-azidoacetamidylpentyl) acrylamide-co-acrylamide) (PAZAM) containing azide moieties and the sequencing primers may be alkvne-linked oligonucleotides adapted to bind to the azide moieties in the PAZAM. The oligonucleotides may be adapted for sequencing-by-synthesis. The permeable three-dimensional matrix may be a hydrogel; may include hydrogel networks of a predetermined size; may include a matrix having particles of the same size or particles of different sizes; and may include columnar posts that are fabricated to include alternating materials in Z-direction. A kit comprising a flow cell, wherein the flow cell includes at least one channel; and an oligonucleotide-containing permeable three-dimensional matrix, wherein the oligonucleotide-containing permeable three-dimensional matrix is adapted to be introduced into the at least one channel and subsequently immobilized therein may also be provided.

[0010] In implementations of the disclosed methods, the monomer may be the compound of formulal: Ngee we + is h

I wherein each R? is independently hydrogen or (C,.) alkyl.

[0011] In implementations of the disclosed methods that include a crosslinker, the crosslinker may be the compound of formula II: FN 4 JS vx ÀL 5 WH a ) U wherein: cach n is independently an integer from 1-6: and each R'is independently a hydrogen or (C.¢) alkyl. It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein and may be implemented to achieve the benefits as described herein. Additional features and aspects of the disclosed system, devices, and methods will become apparent to those of ordinary skill in the art upon reading and understanding the following detailed description of the example embodiments. As will be appreciated by the skilled artisan, further implementations are possible without departing from the scope and spirit of what is disclosed herein.

5 Accordingly, the drawings and associated descriptions are to be regarded as illustrative and not restrictive in nature.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The details of one or more implementations are set forth in the accompanying drawings and the description below. Other features, aspects, and advantages will become apparent from the description, the drawings, and the claims, in which:

[0014] FIG. 1A is a perspective view of a sequencing flow cell in accordance with one implementation of the disclosed systems and methods:

[0015] FIG. 1B is a top view and close-up top view of the flow cell of FIG. 1A wherein arrays of hydrogel structures have been formed on the flow cell;

[0016] FIG. IC depicts the flow cell of FIG. 1A inserted into a cartridge used in sequencing-by- synthesis processes;

[0017] FIG. 2A depicts a first step in an example of the disclosed systems and methods for forming polymer (e.g., hydrogel) structures on a sequencing flow cell such as the flow cell shown in FIG. 1A, wherein a polymer precursor solution has been introduced into a fluidics channel of the flow cell and a prepatterned photomask has been placed over the channel:

[0018] FIG. 2B depicts a second step m an example of the disclosed systems and methods for forming polymer (e.g., hydrogel) structures on a sequencing flow cell wherein ultraviolet light is directed into the channel of the flow cell though openings in the photomask for polymerizing the contents of the polymer precursor solution;

[0019] FIG. 2C depicts a plurality of hydrogel structures formed inside the channel of a flow cell wherein the hydrogel structures are cylindrical in shape and are attached to upper and lower internal surface of the channel:

[0020] FIG. 2D depicts an example method for cleaving hydrogel structures formed in the channel of a flow cell by introducing oil containing a cleaving agent into the channel of the flow cell;

[0021] FIG. 2E depicts an example method for removing cleaved hydrogel structures from the channel of a flow cell by washing the channel;

[0022] FIG. 3A depicts a first step in another example of the disclosed systems and methods for forming polymer (e.g., hydrogel) structures on a sequencing flow cell wherein a prepatterned photomask is placed on or attached to a flow cell that is then inserted into a cartridge;

[0023] FIG. 3B depicts a second step in another example of the disclosed systems and methods for forming polymer (e.g., hydrogel) structures on a sequencing flow cell wherein a polymer precursor solution containing biological cells is loaded into the flow cell of FIG. 3A and the flow cell is then loaded into a device or instrument using an extendable tray.

[0024] FIG. 3C depicts a third step in another example of the disclosed systems and methods for forming polymer (e.g., hydrogel) structures on a sequencing flow cell wherein the flow cell is exposed to ultraviolet light to form an array of hydrogel structures on the flow cell (which are shown in the bright field micrograph), and wherein the flow cell is then washed to remove unpolymerized material and unloaded from the instrument;

[0025] FIGS. 4A-4B depict a flow cell having an array of individual hydrogel pillars located inside the flow cell that were created using a photolithographic method, wherein the pillars contain P5/P7 primers and support the growth of clusters inside the hydrogel matrix;

[0026] FIG. 5A depicts hydrogel pillars fabricated inside a MiSeq"™ flow cell and FIGS. 5B-5K depict time-series images showing the introduction of a fluorescent dye mto the flow cell, diffusion of the dye into hydrogel pillars, and the washing out of the dye from the hydrogel pillars;

[0027] FIGS. 6-6B depict hydrogel beads doped with polv(N-(5-azidoacetamidylpenty[) acrylamide-co-acrylamide) (PAZAM) containing P5/P7 primers, wherein the beads are initially soaked with a sequencing library and then soaked in ExAmp to generate clusters throughout the three- dimensional volume of each bead:

[0028] FIGS. 7A-7B depict index-free sequencing, wherein each hydrogel bead contains clusters from a sample in which it was incubated, wherein hydrogel beads containing such clusters are loaded on a flow cell and sequenced, and wherein the beads from each sample type can be distinguished from one another other using a variety of means such as, for example, fluorophores embedded in beads that are removed before sequencing;

[0029] FIG. 8A depicts a sequencing flow cell, wherein sequencing is occurring in a two- dimensional network of clusters on a top surface and on a bottom surface, and wherein the top surface and the bottom surface are separated by a known distance (e.g.. 100 um) along the Z-axis;

[0030] FIG. 8B depicts a sequencing flow cell, wherein sequencing is occurring in a three- dimensional network of clusters on a top surface and a bottom surface and in discrete regions that are located between the top surface and the bottom surface. and wherein the top surface and the bottom surface are separated by a distance of 100 um along the Z-axis;

[0031] FIG. 9 depicts an example SPIM setup, wherein excitation is delivered by way of a low- NA objective into a sample, and wherein fluorescence emission is collected by a high-NA emission objective;

[0032] FIG. 10 depicts a large hvdrogel network within a sequencing flow cell:

[0033] FIG. 11 depicts a matrix of large particles, small particles, or a combination of large and small particles within a sequencing flow cell;

[0034] FIG. 12 depicts periodic columnar posts within a sequencing flow cell;

[00353] FIG. 13 depicts mesoporous crystalline materials within a sequencing flow cell:

[0036] FIGS. 14A-14D depict an example implementation of a method for forming a hydrogel within a flow cell by polymerization of PAZAM + di-DBCO-PEG:

[0037] FIG. 15 depicts the copolymerization of acrylamide and acrydite-modified oligos into large polyacrvlamide beads:

[0038] FIG. 16A is a brightfield microscopic image depicting hydrogel beads on a glass slide:

[0039] FIG. 16B is a brightfield microscopic image depicting hydrogel beads packed inside a HiSeq™ flow cell;

[0040] FIG. 17A is a fluorescence microscopic image of standard acrylamide beads after incubation with a dve-labeled complementary strand;

[0041] FIG. 17B is a fluorescence microscopic image of oligo-modified acrylamide beads after incubation with a dye-labeled complementary strand:

[0042] FIG. 18A depicts hydrogel beads in which long DNA fragments have been encapsulated trapped within a flow cell;

[0043] FIG. 18B depicts enzymatic processes for library preparation occurring within the trapped hydrogel beads of FIG. 18A;

[0044] FIG. 18C depicts an amplified library generating clusters of linked reads distributed in three-dimensions within each hydrogel bead:

[0045] FIG. 19A depicts template capture and extension occurring on hydrogel beads bearmg oligonucleotides;

[0046] FIG. 19B depicts clonal amplification of library inserts on the hydrogel beads for creating clusters;

[0047] FIG. 20A depicts clustered beads delivered into a flow cell in a hydrogel precursor solution;

[0048] FIG. 20B depicts immobilization of the clustered beads within a crosslinked hydrogel matrix to preserve the spatial locations of the beads m three dimensions during sequencing and subsequent imaging;

[0049] FIG. 21 depicts dimer particles having different orthogonal linearization chemistries;

[0050] FIG. 22 depicts an example system for synthesizing similar dimer particles:

[0051] FIGS. 23A and 23B depict spatial control of clusters in three dimensions using a three- dimensional matrix of columnar posts having alternating material composition in the Z-direction;

[0052] FIGS. 24A-24D depict a simplified example method for creating a polymer scaffold, wherein an unpolymerized monomer solution is embedded with salt particles having a predetermined size distribution: wherein the salt particles displace the monomer, thereby creating a three-dimensional network within the solution; wherein the monomer solution is polymerized to form a three-dimensional polymer scaffold around the salt particles; and wherein the salt particles are dissolved, resulting in a random, three-dimensional array of pores, which define the scaffold.

[0053] FIG. 25 is a flow chart depicting an example method for making a permeable three- dimensional matrix on a sequencing flow cell;

[0054] FIG. 26 is a flow chart depicting a first example method for nucleic acid library sequencing in three-dimensions; and

[0055] FIG. 27 is a flow chart depicting a second example method for nucleic acid library sequencing in three-dimensions.

DETAILED DESCRIPTION

[0056] Various implementations of the disclosed systems, devices, and methods are useful for creating reversible, permeable three-dimensional polymer (e.g. hydrogel) structures within the fluidics channels on sequencing flow cells. These temporary polymer structures expand available sequencing surfaces from two-dimensions to three-dimensions, thereby providing a massive increase to the throughput of a sequencing flow cell. As used herein, the term "hydrogel" refers to a substance formed when an organic polymer (natural or synthetic) is cross-linked by way of covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure that entraps water molecules to form a gel. In some versions, the hydrogel may be a biocompatible hydrogel, which refers to a polvmer that forms a gel that is not toxic to living cells and allows sufficient diffusion of oxygen and nutrients to entrapped cells to maintain viability. In some versions, the hydrogel polymer includes about 60-90% fluid, such as water, and about 10-30% polymer, wherein in other versions, the water content of hydrogel is about 70-80%.

[0057] An example sequencing flow cell includes a channel comprising a surface across which one or more fluid reagents can be flowed and to which adapted fragments of sequencing libraries can transport and bind. A sequencing flow cell includes a solid support having a surface on which sequencing libraries bind. In some examples, the surface contains a lawn of capture nucleotides that can bind to adapted fragments of a sequencing library. In some examples, the surface is a patterned surface. A "patterned surface" refers to an arrangement (such as an array) of different regions (such as amplification sites) in or on an exposed surface of a solid support. For example, one or more of the regions can be IO features where one or more amplification and/or capture primers are present. The features can be separated by interstitial regions where primers are not present. In some examples, the flow cell device has a channel height of about 50 um, about 60 um, about 70 um, about 80 um, about 90 um, about 100 um, about 110 um, about 120 um, about 130 um, about 140 um, or about 150 um, or an amount within a range defined by any two of the aforementioned values.

[0058] As shown in FIG. 1A, an example sequencing flow cell 100 includes top layer of glass 110 having fluidic holes 112 formed therein; channel defining spacer 120, which includes a plurality of fluidic/sequencing channels 122 formed therein; and bottom laver of glass 130 on which array 150 is formed. Array 150 includes a plurality of individual hydrogel structures 152 formed thereon by the disclosed methods. FIG. 1B depicts assembled flow cell 100 upon which an array 150 of individual three- dimensional hydrogel structures 152 has been fabricated in one of the sequencing channels 122 and FIG. IC depicts flow cell 100 having multiple three-dimensional hydrogel structures 152 formed thereon inserted into sequencing cartridge 160, which is used with a sequencing-by-synthesis apparatus. Three- dimensional hydrogel structures having a specific, predetermined geometry may be formed on the flow cell by: (i) introducing a hydrogel precursor solution into a sequencing channel of the flow cell; (ii) placing a photomask having a specific pattern formed thereon over the sequencing channel on the flow cell, either before or after introducing the hydrogel precursor solution into the flow cell: and (iii) exposing the hvdrogel precursor solution to light at a predetermined wavelength through the photomask, wherein the illumination of the hydrogel precursor solution polymerizes the contents thereof and forms three- dimensional structures on the flow cell that correspond to the pattern on the photomask. Once the hydrogel structures have served their purpose, they may be cleaved from the flow cell and washed away without affecting the overall functionality of the flow cell.

[0059] The hydrogel precursor solution may include monomer solutions that can be photopolymerized by activation of a photoinitiator. An example of one such a system includes at least one type of monomer, a reversible or cleavable crosslinker, and a photoinitiator. In one version, the monomer is acrylamide, the reversible crosslinker is N,N'-Bis(acryloyDcystamme (BAC), and the photoinitiator is lithium phenyl-2 4.6-trimethylbenzoylphosphinate (LAP). which is activated by ultraviolet (UV) light at a predetermined wavelength.

[0060] In other versions, the precursor solution may include polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide. N,N'-bis(acryloyl)cystamine, PEG. polypropylene oxide (PPO), polyacrylic acid, poly(hydroxvethyl methacrylate) (PHEMA), polv(methyl methacrylate) (PMMA), poly(N- isopropylacrylamide) (PNIPAAm), poly(lactie acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), polv(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), polylysme. agar, agarose. alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethvleneglycol diallyl ether, ethyleneglycol diacrylate, polymethyleneglycol diacrylate, polyethyleneglycol diacrylate, trimethylopropoane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetracrylate, or combinations thereof. In other versions, the monomer may include PEG-thiol/PEG-acrvlate, acrylamide/N N'-bis(acrvloyl)cystamine (BACy), or PEG/PPO,

[0061] In implementations of the disclosed methods, the monomer may be the compound of formula I:

SE

I wherein each R? is independently hydrogen or (C,) alkyl.

[0062] In implementations of the disclosed methods that include a crosslinker, the crosslinker may be the compound of formula II:

II wherein: each n is independently an integer from 1-6; and each R! is independently hydrogen or (C1) alkyl.

[0063] A crosslinker is capable of crosslinking the polymer chains within the hydrogel. In some versions, if desired, a crosslinker can be cleaved, thereby unlinking the polymer chains, by the presence of a reducing agent; by elevated temperature; by an electric field; or by exposing the hydrogel structures to a wavelength of light that cleaves a photo-cleavable crosslinker that crosslinks polymer of the hydrogel. In some versions, the reducing agent may include phosphine compounds, water soluble phosphines, nitrogen containing phosphines and salts and derivatives thereof, dithioerythritol (DTE), dithiothreitol (DTT) (cis and trans isomers, respectively, of 2,3-dihydroxy-l4-dithiolbutane), 2-mercaptoethanol or f- mercaptoethanol (BME), 2-mercaptoethanol or aminoethanethiol, glutathione, thioglycolate or thioglycolic acid, 2,3-dimercaptopropanol, tris(2-carboxvethyl)phosphine (TCEP). tris(hydroxymethyl)phosphine (THP), or P-[tristhydroxymethyl)phosphine] propionic acid (THPP). In some versions, the crosslinker is cleaved by increasing the temperature to greater than about 50°C, about 535°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, about 95°C, or about 100°C. In some versions, the reducing agent is activated by ultraviolet light.

[0064] Other suitable photoinitiators include biocompatible photoinitiators for radical polymerization that do not damage nucleic acids such as, for example, a diazosulfonate initiator; monoacylphosphineoxide (MAPO) salts such as. for example, NaUTPO and LiOTPO; and bisacylphosphineoxide (BAPO) salts such as, for example, BAPOIONa and BAPOOOI.

[0065] In some examples, crosslinking the polymer chains of the hydrogel structure forms a hydrogel matrix having pores (i.e. a porous hydrogel matrix). In some versions, the size of the pores in the hydrogel structures are regulatable or tunable and may be formulated to encapsulate sufficiently large genetic material, such as cells or nucleic acids, but to allow smaller materials. such as reagents, or smaller sized nucleic acids, such as primers, to pass through the pores, thereby passing in and out of the hydrogel structures. The hydrogels can have any pore size having a diameter sufficient to allow diffusion of reagents through the structure while retaining the encapsulated nucleic acid molecules. The term "pore size" can also refer to an average diameter or an average effective diameter of a cross-section of the pores, based on the measurements of a plurality of pores. The effective diameter of a cross-section that is not circular equals the diameter of a circular cross-section that has the same cross-sectional area as that of the non-circular cross-section. In some examples, the hydrogel structure can be swollen when the hydrogel is hydrated. The sizes of the pores can then change depending on the water content in the hydrogel of the hydrogel structure. In some examples, the pores have a diameter of from about 10 nm to about 100 nm.

[0066] In some examples, the pore size of the hydrogel structures is tuned bv varying the ratio of the concentration of polymer to the concentration of crosslinker. In some examples, the ratio of polymer to crosslinker 1s about 30:1, about 23:1, about 20:1, about 19:1, about 18:1, about 17:1, about 16:1, about 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about

1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, or about 1:30, or about any one of these rations, or a ratio within a range defined by any two of the aforementioned ratios.

[0067] FIGS. 2A-2E depict example method 200 for fabrication and subsequent removal of three-dimensional hydrogel structures on flow cell 210. Flow cell 210 includes upper internal surface 212 and lower internal surface 214 which together define flow cell channel 216. Pre-patterned photomask 218 has been laminated or otherwise attached to the upper surface of flow cell 210. FIG. 2A depicts introducing hydrogel precursor solution 230 containing: (i) monomer (¢.g., acrylamide), (ii) crosslinker (e.g., BAC), and (iii) photo-initiator (c.g., LAP) into flow cell 210. FIG. 2B depicts exposing hydrogel precursor solution 230 to UV light at a predetermined wavelength through pre-patterned photomask 218, which has multiple apertures 200 formed therein. Exposing hydrogel precursor solution 230 to UV light activates the photoinitiator (LAP), thereby generating radicals that lead to controlled polymerization of the monomer (acrylamide) into hydrogel structures 232 containing disulfide bonds. FIG. 2C depicts the formation of hydrogel features 232, which are anchored to top and bottom surfaces 212 and 214 of sequencing channel(s) 216 of flow cell 210, which is adapted to be inserted into cartridge 260. FIG. 2C includes bright field micrograph 250 showing cylindrical hydrogel structure 232 (100-150 um in diameter) having dense gel walls with a less dense core. FIG. 2D depicts cleaving hydrogel features 232 from flow cell 210 using heat or a combination of heat and chemical cleavage of the crosslinker. For example, incubating hydrogel structures 232 with a reducing agent, such as an oil containing DTT, cleaves the structures by reducing the disulfide bonds in the hydrogel crosslinker to thiols, thereby permitting the hydrogel to be washed out of sequencing flow cell 210 as shown in FIG. 2E. The surfaces of flow cell 210 remain functional after the cleaved hydrogel structures have been washed out of the flow cell, ie, removing the hydrogel structures from flow cell 210 does not affect the functionality of sequencing primers that have been bound to the flow cell prior to fabrication and subsequent removal of the hydrogel gel features.

[0068] Fabrication of hydrogel structures such as those previously described can be accomplished in both a factory environment and in a laboratory environment. However, known hydrogel fabrication techniques typically involve the use of expensive and unwieldy equipment such as, for example, a photomask aligner with a collimated UV light source and a chrome mask. Accordingly, to facilitate the fabrication of hydrogel structures on sequencing flow cells directly bv consumers of sequencing products, a relatively small-scale, low-cost instrument for on-flow cell hydrogel fabrication is provided. By way of example, a generic implementation of this instrument includes: (1) a collimated LED UV light source such as, for example, Thor Labs model M385LP1-C1; (ii) a housing that is adapted to receive a flow cell (and flow cell cartridge) therein and that supports and properly positions the light source relative to the flow cell; (iil) a prepatterned mylar photomask that is adapted to be laminate adhered on the upper surface of a particular flow cell; and (iv) a shielding enclosure for containing the light source and housing. An opening in the shielding enclosure allows the flow cell to be inserted into the housing for UV illumination of the flow cell through the prepatterned photomask. The housing may include a movable or adjustable stage apparatus for replicating patterns along the length and width of a flow cell if the illumination zone of the housing is smaller than the area of on the flow cell that is to be photopatterned. In addition to operating as a wide-field illuminator, different versions of the disclosed instrument also perform various reagent exchanges and provide thermal control for facilitating automated library preparation. As described in greater detail below, certain implementations of the disclosed instrument operate as stand-alone library preparation devices that output a ready to cluster or ready to sequence library.

[0069] FIGS. 3A-3C depict an example implementation of the disclosed system and method for fabricating hydrogel structures on a flow cell, wherein the hydrogel structures contain a sample to be sequenced or otherwise analyzed. In this implementation, the disclosed instrument is automated, and the housing includes a processor that executes various programs residing thereon for illuminating the flow cell and for performing reagent exchange and other functions in an automated manner. As shown m FIG. 3A, a customer (or other user) orders sequencing flow cell 310 on which photomask 318, having a region that includes a customer-specified pattern formed therein, has been laminated to form assembly 320. The patterned region of photomask 318 is placed over and aligned with sequencing channel(s) 312 on flow cell 310. Flow cell 310 is then inserted into an appropriate flow cell cartridge 360. As shown in FIG. 3B, the customer then mixes a sample of interest (e.g., biological cells or genomic DNA) with a hydrogel precursor solution that includes, for example, a monomer, a cross linker, and a photoinitiator, and loads the solution onto flow cell 310. As shown in FIG. 3C, assembly 320 and cartridge 360 are then loaded into housing 370 on which a UV light source has been mounted using moveable tray 372. Based on the layout or geometric pattern of photomask 318, the customer chooses an appropriate illumination program and exposes flow cell 310 to UV light for polymerizing the solution and patterning the desired hydrogel structures on flow cell 310. FIG. 3C includes a bright field micrograph of hydrogel pillars fabricated on a flow cell using the disclosed system and method. Flow cell 310 is then washed to remove any unpolymerized solution and excess sample and photomask 318 can be removed from flow cell 310. Flow cell 310 can then be placed into a sequencer or fluid handler for automated downstream processing such as lysis, tagmentation, bridge amplification, clustering, etc.

[0070] Several alternate implementations are provided regarding the assembly of the photomask and the flow cell. In one implementation, a user first inserts a flow cell into the housing and then inserts the photomask, which is separate from the flow cell (e.g., the photomask is not laminated to the flow cell). Because various photomask patterns and designs are possible, a user may select different photomasks based on required pitch or on specific applications or specific uses for the flow cell. In this and other implementations, the housing of the instrument is adapted to receive a variety of different flow cells including HiSeq™, NextSeq™, NovaSeq™. MiniSeq™, and MiSeq™ flow cells available from lumina. In another implementation, the flow cell is provided pre-assembled with the photomask already applied to the exterior surface of the flow cell. Depending on the resolution required, the photomask can be either printed on the flow cell using screen-printing or laminated to the surface of the flow cell using an opaque adhesive film patterned to create structures on the flow cell. The photomask may be peeled off of the flow cell after it has been used, if desired. In another implementation, the photomask may be fabricated from aluminum or another metal deposited inside a fluidic channel, during a microfabrication process used to create the flow cell. The photomask may then be etched away with a high pH buffer after creation of hydrogel structures on the flow cell is complete. Nucleic Acid Sequencing in Three-Dimensions

[0071] As previously discussed, the operational throughput of current top-performing next- generation sequencing (NGS) platforms is determined by: (i) two-dimensional nucleic cluster density; and (i1) the overall size of the active surface area of the flow cell, both of which have already reached practical limits of manufacturing. Example implementations provide systems and methods for overcoming these limitations by expanding the surface upon which sequencing may occur from two dimensions to three dimensions, thereby providing a massive increase to sequencing flow cell throughput and data generation.

[0072] The increase to sequencing flow cell throughput and data generation is facilitated by filling a flow cell (or other item such as, for example, a capillary tube or a miniaturized cuvette} with materials or three-dimensional structures that occupy the entire volume of the flow cell and which support cluster formation at a desired density throughout the entire flow cell volume. Sequencing-by-synthesis or sequencing by another suitable method is then completed, with cluster identification and base calling occurring by optically interrogating a series of stacked two-dimensional slices throughout the flow cell. Example methods for sequentially imaging individual two dimensional slices throughout the flow cell include: (i) using a confocal microscope capable of focusing on discrete two-dimensional slices of the flow cell and repeatedly measuring the same two-dimensional planes; and (ii) using a light-sheet illumination microscope that can rapidly image three-dimensional volumes. In other implementations, multiphoton fluorescence (such as two-photon excited fluorescence (2PEF)) or another multiphoton imaging technique such as three-photon excitation fluorescence (3PEF) or multi-harmonic generation (MHG) is used for imaging the three-dimensional matrix. Multiphoton imaging is a confocal-like excitation modality with similar sectioning capabilities that involves the use of pulsed lasers, but typically at Near-Infrared (NIR) wavelengths, thereby greatly reducing potential photodamage to the matrix and its contents.

[0073] In example implementations, three-dimensional clusters are created throughout permeable hydrogel matrices. such as those described above, by using click chemistry to attach alkyne- linked capture primers (e.g.. P5 and P7) to an acrylamide hydrogel matrix that includes poly(N-(5- azidoacetamidylpentyl) acrvlamide-co-acrylamide) (PAZAM) containing azide moieties. The azide- alkyne click reaction involves the copper-catalyzed reaction of an azide with and alkyne to form a 5- membered heteroatom ring: a Cu(l)-catalvzed azide-alkyne cycloaddition (CuAAC). The azide-alkyne click reaction may be photoinitiated using Cu (II) and a photoinitiator system such as a Type II photoinitiator system, e.g., camphorquinone.. which can use blue light at 470 nm as an excitation source. A sequencing library containing nucleic acid fragments with adapters ligated to the fragment is then diffused into the hydrogel matrix and is clustered by using cluster amplification, bridge amplification, or another suitable method. In some implementations, the nucleic acid fragments are circularized after the adapters are added to create nucleic acid “nanoballs”. The permeability of the hydrogel allows for enzymes and other reagents to diffuse into the hydrogel and perform nucleic acid amplification. As discussed m greater detail below, the hydrogel matrix can be polymerized in various shapes and geometries, such as an array of pillars, posts, or linear trenches, for facilitating reagent exchange around the hydrogel matrix and rapid diffusion into and out of the hydrogel matrix.

[0074] Sequencing flow cells are provided with two types of oligonucleotides (e.g., P5 and P7), referred to in the alternative as grafting primers, capture primers, surface primers, or sequencing primers, bound to the upper and lower surfaces of the flow cell using hydrogel layers or other attachment methods.

The sequences of these primers are complimentary to library adapters, and the fragments of a DNA library are captured by these oligonucleotides. As used herein, P5 and P7 refer to a universal P5 or P7 sequence or P5 or P7 primer for capture and/or amplification purposes. A P5 sequence comprises a sequence defined by SEQ ID NO: 1 (AATGATACGGCGACCACCGA) and a P7 sequence comprises a sequence defined by SEQ ID NO: 2 (CAAGCAGAAGACGGCATACGA).

[0075] FIGS. 4A and 4B depict flow cell 400, which includes an array 402 of individual hydrogel pillars 404 that are formed inside the flow cell using a photolithographic method such as that described above. The pillars contain P5/P7 primers and support the growth of clusters 406 inside the hydrogel matrix. FIG. 5A depicts hydrogel pillars 504 fabricated inside a MiSeq™ flow cell and FIGS. 5B-5F depict time-series images showing the introduction of a fluorescent dve into the flow cell, diffusion of the dye into hydrogel pillars. and the washing out of the dye from the hydrogel pillars. FIGS. 6-GB depict hydrogel beads 604 doped with PAZAM containing PS/P7 primers. The beads are initially soaked with a sequencing library and then soaked in ExAmp to generate clusters throughout the three- dimensional volume of each bead. FIGS. 7A-7B depict index-free sequencing, wherein each hydrogel bead contains clusters (702, 704, 706, 708) from a sample in which it was incubated. Hydrogel beads containing such clusters are loaded on a flow cell and sequenced. The beads from each sample type can be distinguished from one another other using a variety of means such as, for example, fluorophores embedded in beads that are removed before sequencing.

[0076] FIG. 8A depicts sequencing flow cell 800, wherein sequencing is occurring in a two- dimensional network of clusters on top interior surface 810 and bottom interior surface 820, and wherein top interior surface 810 and bottom interior surface 820 are separated by a known distance along the Z- axis (e.g., 100 um). FIG. 8B depicts sequencing flow cell 800, wherein sequencing is occurring in a three- dimensional network of clusters on top interior surface 810 and bottom interior surface 820 and in regions 812, 814, and 816, which are located between top interior surface 810 and bottom interior surface 820, and wherein top interior surface 810 and bottom interior surface 820 are separated by a known distance along the Z-axis (¢.g.. 100 um). Advantages and benefits of this example implementation over existing NGS systems and methods include: (i) enhancing the throughput of an individual flow cell by > 50 fold; (11) fabrication of high-throughput flow cells that do not involve X, Y translation of the optical system; (iii) more efficient consumption of sequencing reagents based on utilization of the entire flow cell volume, thereby reducing waste and improving the economic aspects of the sequencing process; and (iv) compatibility with most or all existing sequencing platforms. In some implementations, cluster locations are identified during first scans and assigned X,Y.Z coordinates for subsequent scans. Drift in the X and Y dimensions can be accounted for by using a ‘reference cluster map’ that 1s generated during the first scan. Herein, the terms X, Y, and Z, or X-axis, Y-axis, and Z-axis refer to the three-dimensional Cartesian coordinate system.

[0077] Throughput can be calculated for a given platform and flow cell size using the number of clusters per two-dimensional plane and the number of two-dimensional slices that can be imaged within the flow cell. The latter is specific to the optical detection system of a specific platform and depends on the optical section thickness (dz) along the Z dimension, which can be calculated using the following formula: az = 08H X Aem 0) n— Vn? - N42 wherein em is the wavelength of excitation, n is the refractive index of the sample, and NA is the numerical aperture. Calculated dz values obtained for a high and low numerical aperture (NA) platform are compiled in TABLE 1, below. Higher magnification objectives may have higher NA (i.e., wider angle for collection of mformation). which also means better resolution in Z (i.e., smaller dz values). In practice, the maximum resolution in Z is about 2X to 3X poorer than in the xy-dimension. Moreover, shorter wavelengths yield higher resolution.

[0078] TABLE 1. Calculated optical thickness (dz) values for a low and high NA platform. 550 7.7 NextSeq 0.35 1.36 oe © 740 10.3 550 1.6 * seq 075 136 60 17 740 21

[0079] Using the dz values and the flow cell thickness, the number of optical sections (or individual two-dimensional slices) that are accessible for each flow cell and potential data output can be extrapolated (see TABLE 2. below). For example. the HiSeq™ system with an optical section thickness of about 2 pm may allow 50 individual 2D slices to be imaged, whereas the NextSeq™ system, having a larger dz of around 10 um, may allow 10 optical sections. Using this three-dimensional sequencing strategy and assuming a constant cluster density in three-dimensions. the yield can be increased from 120 to 600 Gb for a NextSeq™ flow cell and from 900 to 22500 Gb for a HiSeq'" flow cell. Even greater increases in data density are possible by using thicker flow-cells in order to maximize the space for cluster growth in three dimensions.

[0080] TABLE 2. Calculated data output for a low and high NA platform. NextSeq 10 100 10 60 600

DO HiSeq 2 100 50 450 22500

[0081] Similar to confocal microscopy, selective-plane illumination microscopy (SPIM), also referred to as light-sheet microscopy, is an example optical microscopy method for imaging three- dimensional structures. There are multiple implementations for light-sheet microscopy, all of which employ a dual-objective configuration. The first objective, typically low-cost and low-NA, is used for excitation; and the second higher-NA objective is used for collection of fluorescence emission from the sample of interest (see FIG. 9). In this geometry, the lateral (XY) resolution of the system is determined by the collection optics, while the axial (Z) resolution is determined by the excitation objective and wavelength of excitation. This modality is routinely used to image live biological samples from cells to whole organisms up to several millimeters in size with micron-scale resolution at hundreds of images per second. FIG. 9 depicts an example SPIM setup 900, wherein excitation is delivered using low-NA objective 902 into sample 904, and wherein the fluorescence emission is collected by high-NA emission objective 906. The beam can be shaped with a cylindrical lens to simultaneously excite a full sheet of fluorophores or a single-line beam can be raster-scanned across the focal plane of the emission objective to create a full image. Subsequent translation of the sample by way of mechanical stage permits rapid volumetric imaging at high-resolution.

[0082] As previously stated, the disclosed systems and methods provide materials and structures that occupy the entire volume of the flow cell and which support cluster formation and enable three- dimensional sequencing. Suitable materials: (i) occupy the entire height of the flow cell channel; (ii) permit the incorporation of oligonucleotides through various polymerization strategies or through the presence of useful functional groups (e.g., azides); (iii) have a controllable density of functional groups for controlling cluster density; (iv) support the flow of reagents with minimal diffusion gradients; and (v) support confocal microscopy throughout the depth of the flow cell with minimal scattering. Examples of suitable materials include hydrogel networks of a predetermined size; a matrix of large particles, small particles, or a combination of large and small particles (e.g., particles of the same size or particles of different sizes); periodic columnar posts: and mesoporous crystalline materials. FIG. 10 depicts a large hydrogel network 1000 within a sequencing flow cell: FIG. 11 depicts a matrix of large particles 1100, small particles 1102, or a combination of large particles 1100 and small particles 1102 within a sequencing flow cell; FIG. 12 depicts periodic columnar posts 1200 within a sequencing flow cell; and FIG. 13 depicts mesoporous or microporous crystalline materials 1300 within a sequencing flow cell. In some implementations, a three-dimensional matrix may be created from silk fibroin or polymer fibers such as, for example, cellulose/cellulosics and constructions thereof (e.g.. as paper) upon which clusters may be formed.

[0083] A large hydrogel network such as that shown in FIG. 10 may be constructed bv polymerizing a functional hydrogel within a flow cell, thereby creating a continuous polymer network with evenly distributed functional groups that support cluster formation. In one implementation, PAZAM is reacted with DBCO-functionalized PEG. The density of the network may be controlled by adjusting the concentration of PAZAM and the relative concentrations of PAZAM:DBCO-PEG to optimize the density of functional groups and the diffusion characteristics of the hydrogel. This method uses the soft polymeric network as a three-dimensional scaffold with nucleic acid anchor points that define where clusters are located within the hydrogel matrix. FIGS. 14A-14D depict an example implementation of a method for forming a hydrogel within a flow cell by polymerization of PAZAM + di-DBCO-PEG.

[0084] A three-dimensional matrix of solid or porous particles such as that shown in FIG. 11 provides a robust network where sequencing reactions occur on the surface of the particles or inside the particles and diffusion of reagents occurs in the interstitial areas between particles. Clusters are located on the surface of the particles or throughout the particles. In this implementation, the particles are designed to provide optimal surface areca, modulus, and optical transparency. Examples include porous hydrogel beads (e.g.. acrylamide gels), solid polymer particles (e.g., polystyrene), polymer core-shells, and inorganic materials (e.g., silica particles); all bearing grafting primers on their surfaces and/or throughout their three-dimensional structures.

[0085] With reference to FIG. 15, in an example implementation, oligo-bearing hydrogel beads were fabricated using simple droplet generation combined with copolymerization of acrylamide and acrydite-modified oligos (commercially available from Integrated DNA Technologies, Inc.). FIG. 15 depicts the copolymerization of acrylamide and acrydite-modified oligos into large polyacrylamide beads. Because the hydrogel beads are slightly larger in size (~120 um) than the height between the top surface and bottom surface of a typical flow cell (e.g.. 100 um), hydrogel beads can be tightly packed and trapped inside flow cell lanes without any form of chemical attachment thereto. FIG. 16A is a brightfield microscopic image depicting hydrogel beads on a glass slide; and FIG. 16B is a brightfield microscopic image depicting hydrogel beads packed inside a HiSeq™ flow cell. To demonstrate that reagents can readily diffuse into and out of porous hydrogel beads fabricated by the disclosed methods, a dye-labeled complementary strand was hybridized to the oligo-labeled acrylamide beads for detecting fluorescence throughout the entire hydrogel matrix. The control standard acrylamide beads, which were not grafted with oligos, did not show a fluorescence signal upon incubation with the complementary dye-labeled strand, thereby confirming that the signal detected for the oligo-modified acrylamide beads was driven by hybridization and that oligos could be retained within the beads without the presence of hydrogel- bounded complementary strands. FIG. 17A is a fluorescence microscopic image of standard acrylamide beads after incubation with a dye-labeled complementary strand; and FIG 17B is fluorescence microscopic image of oligo-modified acrylamide beads after incubation with a dye-labeled complementary strand.

[0086] This implementation is easily transformable into a simple method for executing long reads, wherein the hydrogel beads bearing grafting primers encapsulate long DNA library fragments (~100 kb) and act as a reaction vessel for enzymatic processes such as, for example, tagmentation, ligation, and clustering to create unique spatially-isolated clusters of linked reads. Internally tagmented and ligated DNA fragments bind to P5 and P7 primers distributed within the hydrogel beads to allow bridge amplification-assisted cluster formation throughout the entire three-dimensional hydrogel structure, and formation of a unique spatial barcode. FIG. 18A depicts hydrogel beads in which long DNA fragments have been encapsulated trapped within a flow cell; FIG. 18B depicts enzymatic processes for library preparation occurring within the trapped hydrogel beads of FIG. 18A; and FIG. 18C depicts an amplified library generating clusters of linked reads distributed in three-dimensions within each hydrogel bead.

[0087] Certain implementations utilizing three-dimensional matrices of solid or porous particles include particles having complex physical and chemical structures outside of a flow cell that are directed into the flow cell and immobilized therein by crosslinking. Clonal amplification of nucleic acid libraries may occur initially on the beads outside of the flow cell, and the beads are then directed into the flow cell in an aqueous solution containing hydrogel precursors such as those previously described. The hydrogel precursors are then crosslinked using previously described methods to create a scaffold within the flow cell. FIG. 19A depicts template capture and extension occurring on hydrogel beads bearing oligonucleotides; and FIG. 19B depicts clonal amplification of library inserts on the hydrogel beads for creating clusters. FIG. 20A depicts clustered beads delivered into a flow cell in a hydrogel precursor solution: and FIG. 20B depicts immobilization of the clustered beads within a crosslinked hydrogel matrix to preserve the spatial locations of the beads in three dimensions during sequencing and subsequent imaging.

[0088] By using external hvdrogel bead preparation, spatially co-localized regions of orthogonal linearization chemistries can be prepared in solution by utilizing specific bead design. Such beads may then be delivered into the flow cell and used for simultaneous forward/reverse strand sequencing in three dimensions. This is achieved using hydrogel particles possessing spatially segregated oligo primers having unique linearization chemistries. Particle dimers having different surface chemistries have been demonstrated using continuous flow reactions, where pre-particles are synthesized and functionalized before dimerization. Dimer particles may be used for spatially linked forward and reverse reads in three dimensions. FIG. 21 depicts dimer particles having different orthogonal linearization chemistries: and FIG. 22 depicts a prior art system for synthesizing similar dimer particles.

[0089] Some implementations of the disclosed systems and methods provide functional three- dimensional scaffolds by utilizing columnar posts that extend vertically from bottom of a flow cell to the top of the flow cell, such as those depicted in FIG. 12. These columnar posts may be fabricated using top- down microfabrication techniques such as, for example, photolithography; thin film deposition; and selective etching. The surfaces of the posts can be functionalized using previously described methods that include PAZAM. for providing a rigid network of chemically active columns while permitting liquid flow and chemical diffusion to occur throughout interstitial areas between the posts. Certain implementations utilize columns that are fabricated to have alternating material composition in the Z-direction. Such posts may be selectively functionalized on the surface of one of the two materials, thereby permitting control of cluster spatial distribution in the Z-direction for limiting multi-clonality and aiding optical imaging. The manifold of Z-slices for optical interrogation is spatially organized in a systematic fashion in this implementation. FIG. 23A and 23B depict spatial control of clusters in three dimensions using a three- dimensional matrix of columnar posts having alternating material composition in the Z-direction.

[0090] Some implementations utilize microporous crystalline materials for creating on- flow cell scaffolds such as that depicted in FIG. 13. Microporous crystalline materials possess well-defined structures that include pores that are ordered and aligned in one direction. Thus, these materials essentially provide multiplexed fluidic channels, with each pore representing a single fluidic channel. The surfaces of many porous materials can be functionalized; therefore, a microporous material with aligned pores can function as a matrix for three-dimensional sequencing with chemical reactions occurring on the walls of the pores. With reference to FIG. 13, both the direction of fluidic flow and optical imaging occurs in the Z-direction, as viewed down the long axis of the pore. Microporous silicon is one example of a material that can be fabricated to have pre-oriented pores of controlled size. Lateral dimensions and thickness of microporous silicon films are easily controlled by selecting a precursor wafer, and flow cells may be prepared by mounting microporous silicon films onto a separate fluidic cell.

[0091] Some implementations utilize polymer scaffolds for on-flow cell sequencing in three- dimensions. FIGS. 24A-24D depict a simplified example method for creating a polymer scaffold, wherein an unpolymerized monomer solution is embedded with salt particles having a predetermined size distribution. The salt particles displace the monomer, thereby creating a three-dimensional network within the solution. The monomer solution is polymerized to form a three-dimensional polymer scaffold around the salt particles and the salt particles are dissolved, resulting in a random, three-dimensional array of pores, which define the scaffold. Such scaffolds may be activated and coated with a hydrogel such as PAZAM. Although such scaffolds are not necessarily equally spaced, multilayer structures such as those previously described, a suitable imaging strategy would image the entire scaffold, and image processing would then be used to identify different clusters. The salt particles used m this example implementation may be spiked with passivated metal particles. After dissolving the salt particles, these particles would remain in the scaffold in fixed locations. During imaging, these particles can be used to provide points to which clusters may be aligned, thereby essentially acting as fiducials.

[0092] FIG. 25 is a flow chart depicting an example method for making a permeable three- dimensional matrix on a flow cell. Method 2500 comprises embedding oligonucleotides within the permeable three-dimensional matrix at block 2502; and mtroducing the oligonucleotide-containing permeable three-dimensional matrix into a flow cell at block 2504, wherein the flow cell includes at least one channel for receiving the oligonucleotide-containing permeable three-dimensional matrix.

[0093] FIG. 26 is a flow chart depicting a first example method for nucleic acid library sequencing in three-dimensions. Method 2600 comprises loading a polymer precursor solution into a flow cell at block 2602, wherein the polymer precursor solution includes monomers and oligonucleotides; polymerizing the polymer precursor solution to create a permeable three-dimensional matrix within the flow cell at block 2604; diffusing a sequencing library into the permeable three-dimensional polymer matrix at block 2606, wherein the sequencing library includes nucleic acid fragments; diffusing enzymes and reagents into the permeable three-dimensional polymer matrix at block 2608; hybridizing the nucleic acid fragments to the oligonucleotides in the permeable three-dimensional polymer matrix at block 2610; clonally amplifying the hybridized nucleic acid fragments to create clusters for sequencing within the permeable three-dimensional polymer matrix at block 2612; sequencing the clusters within the permeable three-dimensional polymer matrix at block 2614; and optically imaging the sequenced clusters within three-dimensional matrix in multiple, discrete two-dimensional slices to characterize the sequencing library at block 2614, wherein the multiple, discrete two-dimensional slices represent the entire three- dimensional internal volume of the flow cell.

[0094] FIG. 27 is a flow chart depicting a second example method for nucleic acid library sequencing in three-dimensions. Method 2700 comprises loading a polymer precursor solution into a flow cell at block 2702, wherein the polymer precursor solution includes monomer, crosslinker, photoinitiator, and oligonucleotides; polymerizing the polymer precursor solution using ultraviolet light to create a permeable three-dimensional matrix within the flow cell at block 2604: diffusing a sequencing library into the permeable three-dimensional polymer matrix at block 2606, wherein the sequencing library includes nucleic acid fragments; diffusing enzymes and reagents into the permeable three-dimensional polymer matrix at block 2608; hybridizing the nucleic acid fragments to the oligonucleotides in the permeable three-dimensional polymer matrix at block 2610; clonally amplifying the hybridized nucleic acid fragments to create clusters for sequencing within the permeable three-dimensional polymer matrix at block 2612: sequencing the clusters within the permeable three-dimensional polymer matrix at block 2614; and using a confocal microscope or a light-sheet illumination microscope to image the sequenced clusters within three-dimensional matrix in multiple, discrete two-dimensional slices to characterize the sequencing library at block 2616, wherein the multiple, discrete two-dimensional slices represent the entire three-dimensional internal volume of the flow cell.

[0095] The foregoing description is provided to enable a person skilled in the art to practice the various configurations described herein. While the subject technology has been particularly described with reference to the various figures and configurations, it should be understood that these are for illustration purposes only and should not be taken as limiting the scope of the subject technology.

[0096] All literature and similar material cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety. In the event that one or more of the incorporated references and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.

[0097] As used herein, the singular forms "a." “an,” and "the." refer to both the singular as well as plural, unless the context clearly indicates otherwise. The term "comprising” as used herein is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. Although many methods and materials similar or equivalent to those described herein can be used. particular suitable methods and materials are described herein. Unless context indicates otherwise, the recitations of numerical ranges by endpoints include all numbers subsumed within that range. Furthermore, references to “one implementation” are not intended to be interpreted as excluding the existence of additional implementations that also incorporate the recited features. Moreover, unless explicitly stated to the contrary, implementations “comprising” or “having” an element or a plurality of elements having a particular property may include additional elements whether or not they have that property.

[0098] The terms “substantially” and “about” used throughout this specification are used to describe and account for small fluctuations, such as due to variations in processing. For example, they can refer to less than or equal to £5%, such as less than or equal to £2%, such as less than or equal to

+1%, such as less than or equal to £0.5%, such as less than or equal to £0.2%, such as less than or equal to +0. 1%, such as less than or equal to £0.05%, and/or 0%.

[0099] There may be many other ways to implement the subject technology. Various functions and elements described herein may be partitioned differently from those shown without departing from the scope of the subject technology. Various modifications to these implementations may be readily apparent to those skilled in the art, and generic principles defined herein may be applied to other implementations. Thus, many changes and modifications may be made to the subject technology, by one having ordinary skill in the art, without departing from the scope of the subject technology. For instance, different numbers of a given module or unit may be employed, a different type or types of a given module or unit may be employed. a given module or unit may be added. or a given module or unit may be omitted.

[0100] Underlined and/or italicized headings and subheadings are used for convenience only, do not limit the subject technology, and are not referred to in connection with the interpretation of the description of the subject technology. All structural and functional equivalents to the elements of the various implementations described throughout this disclosure that are known or later come to be known to those of ordinary skill in the art are expressly incorporated herein by reference and intended to be encompassed by the subject technology. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the above description.

[0101] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein.

[0102] The disclosure also includes the following clauses: I. A method for making an on-flow cell three-dimensional sequencing matrix, comprising: embedding oligonucleotides within a permeable three-dimensional matrix, wherein the oligonucleotides facilitate nucleic acid fragment clonal amplification within the matrix; introducing the oligonucleotide-containing permeable three-dimensional matrix intoa flow cell, wherein the flow cell includes at least one channel for receiving the oligonucleotide-containing permeable three-dimensional matrix; and immobilizing the oligonucleotide-containing permeable three-dimensional matrix in the at least one channel.

2. The method of clause 1, wherein the permeable three-dimensional matrix includes a polymer.

3. The method of any of clauses 1-2, wherein the permeable three-dimensional matrix is a hydrogel.

4. The method of any of clauses 1-2. wherein the permeable three-dimensional matrix includes hydrogel networks of a predetermined size.

5. The method of clause 1, wherein the permeable three-dimensional matrix includes a matrix of particles of the same size or particles of different sizes.

6. The method of any of clauses 1-3, wherein the permeable three-dimensional matrix includes columnar posts.

7. The method of clause 1, wherein the permeable three-dimensional matrix includes mesoporous crystalline materials.

8. The method of any of clauses 1-3, further comprising patterning, by photolithography, the permeable three-dimensional matrix in the flow cell.

9. The method of any of clauses 1-8, wherein the oligonucleotides are adapted for sequencing- by-synthesis.

10. The method of any of clauses 1-9, wherein the flow cell has an internal volume, and wherein the oligonucleotide-containing permeable three-dimensional matrix occupies the entire internal volume of the flow cell.

11. The method of any of clauses 1-10, further comprising imaging the permeable three- dimensional matrix in discrete two-dimensional layers.

12. A method for sequencing in three-dimensions using an on flow-cell three-dimensional sequencing matrix, comprising: loading a polymer precursor solution into a flow cell, wherein the polymer precursor solution includes monomers and oligonucleotides; polvmerizing the polymer precursor solution to create a permeable three-dimensional ~~ matrix within the flow cell; diffusing a sequencing library into the permeable three-dimensional polymer matrix, wherein the sequencing library includes nucleic acid fragments; diffusing enzymes and reagents into the permeable three-dimensional polymer matrix; hybridizing the nucleic acid fragments to the oligonucleotides in the permeable three- dimensional polymer matrix;

clonally amplifying the hybridized nucleic acid fragments to create clusters for sequencing within the permeable three-dimensional polymer matrix; sequencing the clusters within the permeable three-dimensional polymer matrix; and optically imaging the sequenced clusters within three-dimensional matrix in multiple, discrete two-dimensional slices to characterize the sequencing library, wherein the multiple, discrete two- dimensional slices represent the entire three-dimensional internal volume of the flow cell.

13. The method of clause 12, wherein the monomer is the compound of formula I:

SEN

I wherein each R? is independently hydrogen or (C,.) alkyl.

14. The method of any of clauses 12-13, wherein the monomers include polyethylene elycol (PEG)-thiol, PEG-acrylate, acrylamide, N,N'-Bis(acryloyl)cystamine (BACy), PEG, polypropylene oxide (PPO), polyacrylic acid. poly{hydroxvethyl methacrylate) (PHEMA). poly(methyl methacrylate) (PMMA). poly(N-isopropvlacrylamide) (PNIPAAm). poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polvcaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid). poly(L- glutamic acid), polvlysine, agar. agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine. divinyl sulfone, diethyleneglycol diallyl ether, ethyleneglycol diacrylate, polvmethylene glycol diacrylate, polyethyleneglycol diacrvlate, trimethylopropoane trimethacrylate, ethoxylated timethylol triacrylate, or ethoxylated pentaerythritol tetracrylate, or combinations thereof.

15. The method of any of clauses 12-13, wherein the monomers include polyethylene glycol (PEG)- thiol/PEG-acrylate; acrylamide/N,N'-Bis(acryloyDeystamine (BACy); PEG/polypropylene oxide (PPO); or combinations thereof.

16. The method of any of clauses 12-15, wherein the polymer precursor solution further includes poly{N-(5-azidoacetamidylpentyl) acrylamide-co-acrylamide) (PAZAM) containing azide moieties

17. The method of clause 16, wherein the oligonucleotides are alkyne-linked oligonucleotides adapted to bind to the azide moieties in the PAZAM.

18. The method of any of clauses 12-17, wherein the oligonucleotides are adapted for sequencing-by-synthesis.

19 The method of any of clauses 12-18, wherein the permeable three-dimensional matrix includes a hydrogel.

20. The method of any of clauses 12-19, wherein the permeable three-dimensional matrix includes hydrogel networks of predetermined sizes

21. The method of any of clauses 12-19, wherein the permeable three-dimensional matrix includes a matrix of particles of the same size or particles of different sizes,

22. The method of any of clauses 12-19, wherein the permeable three-dimensional matrix includes columnar posts.

23. The method of clause 22, wherein the columnar posts are fabricated to include alternating materials in Z-direction.

24. The method of any of clauses. 12- 23, wherein the optical imaging includes. the use of a confocal microscope, multiphoton, or a light-sheet illumination microscope.

25. A method for sequencing in three-dimensions using an on flow-cell three-dimensional sequencing matrix, comprising: loading a polymer precursor solution into a flow cell, wherein the polymer precursor solution includes monomer, crosslinker, photoinitiator, and oligonucleotides; polymerizing the polymer precursor solution using ultraviolet light to create a permeable three- dimensional matrix within the flow cell; diffusing a sequencing library into the permeable three-dimensional polymer matrix, wherein the sequencing library includes nucleic acid fragments to which adapters have been added; diffusing enzymes and reagents into the permeable three-dimensional polymer matrix; hybridizing the nucleic acid fragments to the oligonucleotides in the permeable three- dimensional polymer matrix: clonally amplifying the hybridized nucleic acid fragments to create clusters for sequencing within the permeable three-dimensional polymer matrix; sequencing the clusters within the permeable three-dimensional polymer matrix; and using a confocal microscope, multiphoton, or a light-sheet illumination microscope to image the sequenced clusters within three-dimensional matrix in multiple, discrete two- dimensional slices to characterize the sequencing library, wherein the multiple, discrete two-dimensional slices represent the entire three-dimensional internal volume of the flow cell.

26. The method of clause 25, wherein the monomer is the compound of formula I: 0 te W Re

I wherein each R? is independently hydrogen or (C.6) alkyl.

27. The method of any of clauses 25-26, wherein the crosslinker is a compound of formula II:

RY NS 5 TI 2 wherein: each n is independently an integer from 1-6; and each R! is independently hydrogen or (C,.) alkyl.

28. The method of any of clauses 25-27, wherein the photoinitiator is a diazosulfonate initiator; a monoacylphosphineoxide (MAPO) salt; a bisacylphosphineoxide (BAPO) salt: or combinations thereof.

29. The method of any of clauses 25-27 | wherein the monomer includes polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N‚N'-Bis(acryloyl)systamme (BACy), PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methvl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PV SA), poly(L-aspartic acid), poly(L- glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethyleneglycol diallyl ether, ethylene glycol diacrylate, polymethyleneglycol diacrylate, polyethyleneglycol diacrylate, trimethylopropoane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetracrylate, or combinations thereof.

30. The method of any of clauses 25-27, wherein the monomer includes polyethylene glycol (PEG)- thiol/PEG-acrylate; acrylamide/N.N'-Bis(acrylovl)eystamine (BACy); PEG/polypropylene oxide (PPO): or combinations thereof.

31. The method of any of clauses 25-30, wherein the photoinitiator is lithium phenyl- 2,4,6- trimethylbenzoylphosphinate (LAP), a diazosulfonate initiator; a monoacylphosphineoxide (MAPO) salt, or a bisacylphosphineoxide (BAPO) salt.

32. The method of any of clauses 25-31, wherem the polymer precursor solution further includes poly(N-(5-azidoacetamidylpentyl) acrylamide-co-acrylamide) (PAZAM) to which azide moieties have been bound.

33. The method of clause 32, wherein the oligonucleotides are alkyne-linked oligonucleotides adapted to bind to the azide moieties in the PAZAM.

34. The method of any of clauses 25-33, wherein the oligonucleotides are adapted for sequencing-by-synthesis.

35. The method of any of clauses 25-34, wherein the permeable three-dimensional matrix includes a hydrogel.

36. The method of any of clauses 25-35, wherem the permeable three-dimensional matrix includes hydrogel networks of a predetermined size.

37. The method of any of clauses 25-35, wherein the permeable three-dimensional matrix includes a matrix of particles of the same size or particles of different sizes.

38. The method of any of clauses 25-35 wherein the permeable three-dimensional matrix includes columnar posts, and wherein the columnar posts are fabricated to include alternating materials in Z-direction.

39. The method of any of clauses 25-38, wherein the nucleic acid fragments to which adapters have been added are circularized after the adapters are added to create nanoballs.

40. A kit, comprising: a flow cell, wherein the flow cell includes at least one channel; and an oligonucleotide-containing permeable three-dimensional matrix, wherein the oligonucleotide-containing permeable three-dimensional matrix is adapted to be introduced into the at least one channel and subsequently immobilized therein.

20200124 Sequence listing.txt

SEQUENCE LISTING <110> Illumina, Inc, San Diego, California, US <120> On-flow cell three dimensional sequencing matrices <130> P168531NL00/37/JED <140> NL2024596 <141> 2019-12-31 <160> 2 <170> BiSSAP 1.3.6 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide P5 <400> 1 aatgatacgg cgaccaccga 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide P7 <400> 2 caagcagaag acggcatacg a 21 Page 1

Claims

CONCLUSIONS

A method for preparing a flow-through cell three-dimensional sequence matrix, comprising: - housing oligonucleotides in a permeable three-dimensional matrix, wherein the oligonucleotides facilitate cloned amplification of nucleic acid fragment within the matrix; - introducing the oligonucleotide-containing permeable three-dimensional matrix into a flow cell, the flow cell comprising at least one channel for receiving the oligonucleotide-containing permeable three-dimensional matrix; and - immobilizing the oligonucleotide-containing permeable three-dimensional matrix in the at least one channel.

The method of claim 1, wherein the permeable three-dimensional matrix comprises a polymer.

A method according to any one of claims 1 to 2, wherein the permeable three-dimensional matrix is a hydrogel.

A method according to any one of claims 1 to 2, wherein the permeable three-dimensional matrix comprises hydrogel networks of a predetermined size.

The method of claim 1, wherein the permeable three-dimensional matrix comprises a matrix of particles of the same size or of different sizes.

A method according to any one of claims 1 - 3, wherein the permeable three-dimensional matrix comprises columnar uprights.

The method of claim 1, wherein the permeable three-dimensional matrix comprises mesoporous crystalline materials.

A method according to any one of claims 1 to 3, additionally comprising patterning, by photolithography, the permeable three-dimensional matrix in the flow cell.

The method of any one of claims 1-8, wherein the oligonucleotides are adapted for sequencing by synthesis.

The method of any one of claims 1-9, wherein the flow cell has an internal volume, and wherein the oligonucleotide-containing permeable three-dimensional matrix occupies the entire internal volume of the flow cell.

A method according to any one of claims 1 to 10, additionally comprising imaging the permeable three-dimensional matrix 1n separate two-dimensional layers.

A method of sequencing in three dimensions using a flow cell three-dimensional sequencing matrix, comprising: loading a polymer precursor solution into a flow cell, the polymer precursor solution comprising a monomer and oligonucleotides; polymerizing the polymer precursor solution to form a permeable three-dimensional matrix in the flow cell; distributing a sequencing library in the permeable three-dimensional polymeric matrix, wherein the sequencing library comprises nucleic acid fragments; distributing enzymes and reagents in the permeable three-dimensional polymeric matrix; hybridizing the nucleic acid fragments to the oligonucleotides in the permeable three-dimensional polymeric matrix; cloning amplifying the hybridized nucleic acid fragments to form clusters for sequencing within the permeable three-dimensional polymeric matrix; sequencing the clusters within the permeable three-dimensional polymeric matrix; and optically imaging the sequenced within the three-dimensional array in multiple, separate two-dimensional slices to characterize the sequencing library, wherein the multiple, individual two-dimensional slices represent the entire three-dimensional internal volume of the flow cell.

A method according to claim 12, wherein the monomer is the substance of the formula I: . Ng y : 82 Re

I where each R? independently hydrogen or (C4) alkyl.

A method according to any one of claims 12 to 13, wherein the monomers are polyethylene glycol (PEG) thiol, PEG-acrylate, acrylamide, N,N'-bis(acryloyl)eystamine (BACy), PEG, propylene glycol (PPO), polyacrylic acid , poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), polylactic acid (PLA), poly(lactic-co-glycolic acid), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethylene glycol diallyl ether, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylolpropane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetraacrylate, or combinations thereof.

A method according to any one of claims 12 to 13, wherein the monomers are polyethylene glycol (PEG) thiol / PEG acrylate, acrylamide / N, N°-bis(acryloyl)cystamine (BACy), PEG / propylene glycol (PPO), or include combinations thereof.

The method of any one of claims 12 to 15, wherein the polymer precursor solution additionally comprises poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM) containing azide moieties.

The method of claim 16, wherein the oligonucleotides are alkyne-linked oligonucleotides arranged to bind to the azide residues in the PAZAM.

The method of any one of claims 12 to 17, wherein the oligonucleotides are arranged for sequencing by synthesis.

A method according to any one of claims 12-18, wherein the permeable three-dimensional matrix comprises a hydrogel.

A method according to any one of claims 12-19, wherein the permeable three-dimensional matrix comprises hydrogel networks of predetermined dimensions.

A method according to any one of claims 12-19, wherein the permeable three-dimensional matrix comprises a matrix of particles of the same size or of different sizes.

A method according to any one of claims 1-19, wherein the permeable three-dimensional matrix comprises columnar uprights.

A method according to claim 22, wherein the columnar uprights are made to comprise alternating materials in the Z direction.

A method according to any one of claims 12 to 23, wherein the optical imaging comprises the use of a confocal microscope, multiphoton, or a light-sheet illumination microscope.

A method of sequencing in three dimensions using a flow cell three-dimensional sequencing matrix, comprising: charging a polymer precursor solution into a flow cell, the polymer precursor solution comprising a monomer, crosslinker, photoinitiator, and oligonucleotides; polymerizing the polymer precursor solution using ultraviolet light to form a permeable three-dimensional matrix in the flow cell;

distributing a sequencing library in the permeable three-dimensional polymeric matrix, the sequencing library comprising nucleic acid fragments to which adapters have been added; distributing enzymes and reagents in the permeable three-dimensional polymeric matrix; hybridizing the nucleic acid fragments to the oligonucleotides in the permeable three-dimensional polymeric matrix; cloning amplifying the hybridized nucleic acid fragments to form clusters for sequencing within the permeable three-dimensional polymeric matrix; sequencing the clusters within the permeable three-dimensional polymeric matrix; and using a confocal microscope, multiphoton, or a light-sheet illumination microscope to image the sequenced within the three-dimensional array in multiple, separate two-dimensional slices to characterize the sequencing library, wherein the multiple, individual two-dimensional slices represent the entire three-dimensional internal volume of the flow cell.

A method according to claim 25, wherein the monomer is the substance of the formula I:

I where each R? independently hydrogen or (C 1 ) alkyl.

A method according to any one of claims 25 to 126, wherein the crosslinker is a substance of the formula II: FN 8 ij} Sg AA

I where: each n is independently an integer of | — 6 is; and every R! independently hydrogen or (C 1-6 ) alkyl.

A method according to any one of claims 25 to 27, wherein the photoinitiator is a diazosulfonate initiator; a monoacylphosphine oxide (MAPO) salt; a bisacylphosphine oxide (BAPO) salt; or combinations thereof.

A method according to any one of claims 25 to 27, wherein the monomer is polyethylene glycol (PEG) thiol, PEG-acrylate, acrylamide, N,N'-bis(acryloyl)cystamine (BACy), PEG, propylene glycol (PPO), polyacrylic acid , poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), polylactic acid (PLA), poly(lactic-co-glycolic acid), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethylene glycol diallyl ether, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylolpropane trimethacrylate, ethoxylated trimethylol triacrylate, ethoxylated pentaerythritol tetraacrylate, or combinations thereof.

The method of any one of claims 25 to 27, wherein the monomer is polyethylene glycol (PEG) thiol/PEG-acrylate, acrylamide/N, N'-bis(acryloyl)cystamime (BACy), PEG/propylene glycol (PPO), or combinations thereof.

The method of any one of claims 25 to 30, wherein the photoinitiator is lithium phenyl 2,4,6-trimethylbenzoylphosphinate (LAP), a diazosulfonate initiator; a monoacylphosphine oxide (MAPO) salt; or a bisacylphosphine oxide (BAPO) salt 1s.

The method of any one of claims 25 to 31, wherein the polymer precursor solution additionally comprises poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM) ame containing azide moieties attached.

The method of claim 32, wherein the oligonucleotides are alkyne-linked oligonucleotides arranged to bind to the azide residues in the PAZAM.

The method of any one of claims 25 to 33, wherein the oligonucleotides are arranged for sequencing by synthesis.

The method of any one of claims 25 to 34, wherein the permeable three-dimensional matrix comprises a hydrogel.

A method according to any one of claims 25 to 35, wherein the permeable three-dimensional matrix comprises hydrogel networks of predetermined dimensions.

A method according to any one of claims 25 to 35, wherein the permeable three-dimensional matrix comprises a matrix of particles of the same size or of different sizes.

A method according to any one of claims 25 to 35, wherein the permeable three-dimensional array comprises columnar sentry posts, and wherein the columnar sentry posts are constructed to include alternating materials in Z direction.

The method of any one of claims 25 to 38, wherein the nucleic acid fragments to which the adapters have been added are circular after the adapters have been added to form nanospheres.

40. An equipment comprising: a flow-through cell, the flow-through cell comprising at least one channel; and an oligonucleotide-containing permeable three-dimensional array, wherein the oligonucleotide-containing permeable three-dimensional array is adapted to be introduced into the at least one channel and subsequently immobilized therein.