CN113710364A

CN113710364A - Three-dimensional polymer structures on flow-through cells

Info

Publication number: CN113710364A
Application number: CN202080025897.7A
Authority: CN
Inventors: T·K·库拉纳; E·洛萨斯-坎耶勒斯; 吴怡萱; H·布莱克; M·莱萨德-维格; M·齐默利; S·拉米雷斯
Original assignee: Illumina Inc
Current assignee: Illumina Inc
Priority date: 2019-11-27
Filing date: 2020-11-25
Publication date: 2021-11-26
Also published as: CA3134848A1; IL286667A; EP3930888A1; AU2020391457A1

Abstract

A method for preparing a three-dimensional polymer structure on a flow cell includes loading a polymer precursor solution onto the flow cell. The polymer precursor solution includes a monomer, a crosslinking agent, and a photoinitiator. The flow cell includes at least one channel for receiving the polymer precursor solution. The at least one channel has an upper inner surface and a lower inner surface. The method further includes irradiating the polymer precursor solution through a patterned photomask with light of a wavelength sufficient to activate the photoinitiator. Activation of the photoinitiator polymerizes at least some of the polymer precursor solutions below the openings in the patterned photomask and forms a three-dimensional polymer structure extending from the upper interior surface to the lower interior surface of the at least one channel.

Description

Three-dimensional polymer structures on flow-through cells

RELATED APPLICATIONS

Priority is claimed in this application for U.S. provisional patent application No. 62/941,197 entitled "On-Flow Cell Three-Dimensional Polymer Structures" filed On 27/11/2019, the disclosure of which is incorporated herein by reference in its entirety.

This application also claims priority from U.S. provisional patent application No. 62/941,215 entitled "On-Flow Cell Three-Dimensional Sequencing substrates," filed On 2019, 11, month 27, the disclosure of which is incorporated herein by reference in its entirety.

This application also claims priority from U.S. provisional patent application No. 62/941,242 entitled "On-Flow Cell Three-Dimensional Polymer Structures Having functional Surfaces," filed On 27.11.2019, the disclosure of which is incorporated herein by reference in its entirety.

The present application also claims priority from Dutch patent application number N2024527 entitled "On-Flow Cell Three-Dimensional Polymer Structures" filed On 2019, 12, month 20, the disclosure of which is incorporated herein by reference in its entirety.

The present application also claims priority from Dutch patent application No. N2024596 entitled "On-Flow Cell Three-Dimensional Sequencing substrates," filed 2019, 12, 31, the disclosure of which is incorporated herein by reference in its entirety.

The present application also claims priority from Dutch patent application No. N2024528 entitled "On-Flow Cell Three-Dimensional Polymer Structures Having functional Surfaces", filed 2019, 12, month 20, the disclosure of which is incorporated herein by reference in its entirety.

Sequence listing in electronic format

This application is filed in conjunction with a sequence listing in electronic format. The sequence listing is provided in a file named Illumina0737385sequence listing _ st25.txt, created on 20.11 months of 2020 and saved last that day, which is 1 kilobyte in size. The information in electronic format of the sequence listing is incorporated herein by reference in its entirety.

Background

Next generation sequencing ("NGS") is a high throughput sequencing technology that is capable of sequencing entire genomes in a rapid and cost-effective manner. In at least one embodiment, NGS begins with the creation of a sequencing library that includes genomic DNA that has been randomly fragmented, extracted, and purified. The entire genomic library can then be massively sequenced in parallel using NGS methods (such as sequencing-by-synthesis). Single cell sequencing decodes changes in the genome and transcriptome of single cells, helping to elucidate the underlying mechanisms of both health and disease. Many problems with cell-to-cell variations require sequencing of hundreds to thousands of cells. However, high throughput single cell sequencing may be limited by the difficulty in processing hundreds to thousands of single cells while achieving (i) efficient library preparation, (ii) indexing of library molecules, and (iii) minimal loss. Compartmentalization strategies can overcome these challenges by distributing single cells in separate compartments that not only (i) separate cells from each other, but also (ii) allow efficient reagent exchange so that library preparation can occur in parallel across hundreds to thousands of samples and without cross-contamination.

Some NGS platforms may rely on optical interrogation of surface-bound nucleic acid clusters and generate data at a fairly static rate and at a significant cost per genome. Increasing the throughput of nucleic acid sequencing methods can be important to reduce sequencing costs and improve overall sequencing accuracy. This desired result may be able to be achieved by sequencing a greater number of nucleic acid clusters. Thus, in some cases, a larger flow cell surface area or higher cluster density can be implemented to increase the number of clusters that can be sequenced. However, significant improvements in throughput using traditional surface-bound sequencing methods can be increasingly challenging as sequencing flow cells approach the limits of size and cluster density. Therefore, it would be beneficial to overcome these limitations.

In some cases, the large amount of data generated by whole genome sequencing can complicate data processing and analysis. Thus, as a variation, various techniques can be used to enrich multiple portions of a genome to focus on a gene of interest or other particular target of interest. However, some current methods for library preparation and library enrichment may require multiple manual manipulations and reagent transfers, which results in the loss of the targeted library. Therefore, automated systems and methods for mitigating the losses associated with current sequencing library preparation and enrichment methods may be beneficial; and are disclosed herein.

Disclosure of Invention

The following provides an overview of some examples. This summary is not an extensive overview and is intended to neither identify key or critical aspects nor delineate the scope of the disclosed systems, apparatuses, and methods. It should be understood that any respective features/examples of each aspect of the present disclosure as described herein may be implemented together in any combination to achieve a result as described herein, and any features/examples from any one or more of these aspects may be implemented together with any features of the other aspects as described herein in any combination to achieve a beneficial effect as described herein.

One embodiment relates to a method for making a three-dimensional polymeric structure on a flow cell, comprising: loading a polymer precursor solution onto a flow cell, wherein the polymer precursor solution comprises a monomer, a cross-linking agent, and a photoinitiator, and wherein the flow cell comprises at least one channel for receiving the polymer precursor solution, and wherein the at least one channel has an upper interior surface and a lower interior surface; and irradiating the polymer precursor solution through the patterned photomask using light of a wavelength sufficient to activate the photoinitiator, wherein activation of the photoinitiator polymerizes at least some of the polymer precursor solution below the openings in the patterned photomask and forms a three-dimensional polymer structure extending from the upper interior surface to the lower interior surface of the at least one channel.

There are variations on any one or more of the above embodiments, wherein the method further comprises washing unpolymerized polymer precursor solution out of the flow cell.

There are variations on any one or more of the above embodiments wherein the method further comprises cleaving at least some of the three-dimensional polymeric structures from the flow cell using heat, a cleaving chemical, or a combination of heat and a cleaving chemical.

There are variations on any one or more of the above embodiments wherein the flow cell has an oligonucleotide of a predetermined length on both the upper and lower surfaces of the at least one channel, and wherein the oligonucleotide comprises a primer.

There are variations on any one or more of the above embodiments, wherein the polymer is a hydrogel.

There are variations on any one or more of the above embodiments wherein the monomer is a compound of formula I:

wherein each R²Independently is hydrogen or (C)_1-6) An alkyl group.

There are variations on any one or more of the above embodiments wherein the crosslinker is a compound of formula II:

wherein:

each n is independently an integer from 1 to 6; and is

Each R¹Independently is hydrogen or (C)_1-6) An alkyl group.

Variations exist regarding any one or more of the above embodiments, wherein the photoinitiator is a diazonium sulfonate initiator; monoacylphosphine oxide (MAPO) salts; bisacylphosphine oxide (BAPO) salts; or a combination thereof.

There are variations on any one or more of the above embodiments wherein the monomer is acrylamide, the crosslinker is N, N' -bis (acryloyl) cystamine (BACy), and the photoinitiator is lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (LAP).

There are variations on any one or more of the above embodiments wherein the polymer precursor solution comprises polyethylene glycol (PEG) -thiol, PEG-acrylate, acrylamide, N' -bis (acryloyl) cystamine (BACy), PEG, polypropylene oxide (PPO), polyacrylic 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), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronic acid, pectin, dextran sulfate, and mixtures thereof, Carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinylsulfone, diethylene glycol diallyl ether, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylolpropane trimethacrylate, ethoxylated trimethylolpropane triacrylate, ethoxylated pentaerythritol tetraacrylate, or combinations thereof.

There are variations on any one or more of the above embodiments wherein the polymer precursor solution comprises polyethylene glycol (PEG) -thiol/PEG-acrylate; acrylamide/N, N' -bis (acryloyl) cystamine (BACy); PEG/polypropylene oxide (PPO), or combinations thereof.

Variations on any one or more of the above embodiments exist wherein the photomask comprises polyethylene terephthalate, carbon ink, a chemically etched metal film, or combinations thereof.

There are variations on any one or more of the above embodiments wherein the photomask is laminated to an upper exterior surface of the flow cell.

There are variations on any one or more of the above embodiments wherein irradiating the polymer precursor comprises emitting light using an ultraviolet light source.

There are variations to any one or more of the above embodiments wherein the three-dimensional polymeric structure is cylindrical.

Variations exist regarding any one or more of the above embodiments, wherein the three-dimensional polymeric structure is an inverted C-shape.

Another embodiment relates to a method for making a three-dimensional polymeric structure on a flow cell, comprising: loading a polymer precursor solution onto a flow cell, wherein the polymer precursor solution comprises biological cells or biological cell colonies comprising genetic material, monomers, a cross-linking agent and a photoinitiator, and wherein the flow cell comprises at least one channel for receiving the polymer precursor solution, wherein the at least one channel has an upper inner surface and a lower inner surface, and wherein primers are bound to both the upper surface and the lower surface of the at least one channel; and illuminating the polymer precursor solution through a patterned photomask using a light source emitting light of a wavelength that activates the photoinitiator, and wherein activation of the photoinitiator polymerizes at least some of the polymer precursor solution below the openings in the photomask and forms a three-dimensional polymer structure extending from an upper interior surface to a lower interior surface of the at least one channel, and wherein the biological cells or biological cell colonies are compartmentalized in the three-dimensional polymer structure.

There are variations on any one or more of the above embodiments, wherein the method further comprises diffusing an agent into the three-dimensional polymeric structure, wherein the agent comprises a lytic agent that lyses a biological cell and releases genetic material therefrom, and wherein the genetic material comprises a nucleic acid.

There are variations on any one or more of the above embodiments, wherein the method further comprises fragmenting the released nucleic acids and ligating adapters to the ends of the nucleic acid fragments.

There are variations on any one or more of the above embodiments, wherein the method further comprises seeding the nucleic acid fragments on the upper and lower surfaces of at least one sequencing channel by: introducing a diffusion barrier into the at least one channel, heating the flow cell to a temperature that cleaves the polymeric structure and releases the nucleic acid fragments therefrom, hybridizing the nucleic acid fragments to oligonucleotides on upper and lower surfaces of the at least one channel, and washing the cleaved polymeric structure out of the flow cell.

There are variations on any one or more of the above embodiments, wherein the method further comprises clonally amplifying the hybridized nucleic acids using bridge amplification to produce a nucleic acid cluster.

There are variations on any one or more of the above embodiments wherein the polymer is a hydrogel and wherein the diffusion barrier layer comprises a hydrophobic liquid or a viscous aqueous solution, wherein the hydrophobic liquid comprises a mineral, silicone, or perfluorinated oil, or a combination thereof, and wherein the viscous aqueous solution comprises polyethylene glycol (PEG), polyvinylpyrrolidone, pluronic dextran, sucrose, poly (N-isopropylacrylamide), or polyethylene oxide-polypropylene oxide-polyethylene oxide, PEO-PPO-peoyiponite, or a combination thereof.

wherein each R²Independently is hydrogen or (C)_1-6) An alkyl group.

wherein:

each n is independently an integer from 1 to 6; and is

Each R¹Independently is hydrogen or (C)_1-6) An alkyl group.

There are variations on any one or more of the above embodiments wherein the polymer precursor solution comprises polyethylene glycol (PEG) -thiol, PEG-acrylate, acrylamide, N' -bis (acryloyl) cystamine (BACy), PEG, polypropylene oxide (PPO), polyacrylic 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), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronic acid, pectin, dextran sulfate, and mixtures thereof, Carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinylsulfone, diethylene glycol diallyl ether, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylolpropane trimethacrylate, ethoxylated trimethylolpropane triacrylate, or ethoxylated pentaerythritol tetraacrylate, or combinations thereof.

There are variations on any one or more of the above embodiments wherein the polymer precursor solution comprises polyethylene glycol (PEG) -thiol/PEG-acrylate; acrylamide/N, N' -bis (acryloyl) cystamine (BACy); PEG/polypropylene oxide (PPO); or a combination thereof.

There are variations on any one or more of the above embodiments wherein the photomask is a polyethylene terephthalate, a carbon ink, or a chemically etched metal film.

There are variations to any one or more of the above embodiments wherein the light source is an ultraviolet light source.

Variations exist with respect to any one or more of the above embodiments, wherein the biological cell is a mammalian cell.

There are variations on any one or more of the above embodiments, wherein the biological cell is a cell of a bacterium.

Variations exist with respect to any one or more of the above embodiments, wherein the nucleic acid is a deoxyribonucleic acid.

Variations exist with respect to any one or more of the above embodiments, wherein the nucleic acid is a ribonucleic acid.

Another embodiment relates to a method for making a three-dimensional polymeric structure on a flow cell, comprising: loading a hydrogel precursor solution onto a flow cell, wherein the hydrogel precursor solution comprises biological cells or biological cell colonies comprising genetic material, monomers, a cross-linking agent and a photoinitiator, and wherein the flow cell comprises at least one channel for receiving the polymer precursor solution, wherein the at least one channel has an upper inner surface and a lower inner surface, and wherein primers are bound to both the upper inner surface and the lower inner surface of the at least one channel; illuminating the hydrogel precursor solution through a patterned photomask using a light source that emits light at a wavelength that activates the photoinitiator, and wherein activation of the photoinitiator polymerizes at least some of the hydrogel precursor solution below the openings in the photomask and forms a three-dimensional hydrogel structure extending from an upper interior surface to a lower interior surface of the at least one channel, and wherein the biological cells or biological cell colonies are compartmentalized in the three-dimensional hydrogel structure; diffusing a lysing agent into the three-dimensional hydrogel structure, wherein the lysing agent lyses the biological cells and releases genetic material therefrom, and wherein the genetic material comprises nucleic acids; fragmenting the released nucleic acid and ligating adaptors to the ends of the fragments; and seeding the nucleic acid fragments on the upper and lower interior surfaces of the at least one channel by: introducing a diffusion barrier into the at least one channel, wherein the diffusion barrier prevents cross-contamination between hydrogel structures, heating the flow cell to a temperature that cleaves the hydrogel structures and releases the nucleic acid fragments, hybridizing the nucleic acid fragments to primers on upper and lower internal surfaces of the at least one channel, and washing the cleaved hydrogel structures out of the flow cell; and clonally amplifying the hybridized nucleic acid fragments to generate clusters for sequencing.

wherein each R²Independently is hydrogen or (C)_1-6) An alkyl group.

wherein:

each n is independently an integer from 1 to 6; and is

Each R¹Independently is hydrogen or (C)_1-6) An alkyl group.

There are variations on any one or more of the above embodiments, wherein the hydrogel precursor solution comprises polyethylene glycol (PEG) -thiol, PEG-acrylate, acrylamide, N' -bis (acryloyl) cystamine (BACy), PEG, polypropylene oxide (PPO), polyacrylic 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), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronic acid, pectin, dextran sulfate, and mixtures thereof, Carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinylsulfone, diethylene glycol diallyl ether, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylolpropane trimethacrylate, ethoxylated trimethylolpropane triacrylate, or ethoxylated pentaerythritol tetraacrylate, or combinations thereof.

There are variations on any one or more of the above embodiments, wherein the hydrogel precursor solution comprises polyethylene glycol (PEG) -thiol/PEG-acrylate; acrylamide/N, N' -bis (acryloyl) cystamine (BACy); PEG/polypropylene oxide (PPO); or a combination thereof.

There are variations on any one or more of the above embodiments wherein the diffusion barrier layer comprises a hydrophobic liquid or a viscous aqueous solution, wherein the hydrophobic liquid comprises a mineral oil, a silicone oil, or a perfluorinated oil, or combinations thereof, and wherein the viscous aqueous solution comprises polyethylene glycol (PEG), polyvinylpyrrolidone, pluronic dextran, sucrose, poly (N-isopropylacrylamide) or polyethylene oxide-polypropylene oxide-polyethylene oxide, PEO-PPO-peoyiponite, or combinations thereof.

There are variations to any one or more of the above embodiments wherein the photomask is a polyethylene terephthalate, a carbon ink, or a chemically etched metal film, and wherein the photomask is laminated to an upper exterior surface of the flow cell.

There are variations to any one or more of the above embodiments wherein the hydrogel structure is cylindrical.

There are variations on any one or more of the above embodiments wherein the hydrogel structure is inverted C-shaped.

Another embodiment relates to a flow-through cell comprising: a channel, wherein the channel comprises an upper interior surface having a primer coated thereon and a lower interior surface having a primer coated thereon; and a reversible, permeable three-dimensional polymeric structure formed from a polymer precursor solution in the channel, wherein the three-dimensional polymeric structure extends from an upper interior surface of the channel to a lower interior surface of the channel.

There are variations on any one or more of the above embodiments wherein the flow cell further comprises a photomask placed over an exterior surface of the channel.

Variations exist with respect to any one or more of the above embodiments in which the three-dimensional polymeric structure is cylindrical, inverted C-shaped, tubular, or a combination thereof.

There are variations on any one or more of the above embodiments, wherein the three-dimensional polymeric structure comprises a hydrogel.

There are variations on any one or more of the above embodiments in which the flow cell, polymer precursor solution, and photomask are provided in a kit.

Another embodiment relates to a method for preparing a three-dimensional sequencing matrix on a flow cell, comprising: embedding oligonucleotides within a permeable three-dimensional matrix, wherein the oligonucleotides facilitate clonal amplification of nucleic acid fragments within the matrix; introducing the permeable three-dimensional oligonucleotide-containing matrix into a flow cell, wherein the flow cell comprises at least one channel for receiving the permeable three-dimensional oligonucleotide-containing matrix; and the permeable three-dimensional matrix containing oligonucleotides is immobilized in the at least one channel.

There are variations to any one or more of the above embodiments wherein the permeable three-dimensional matrix comprises a polymer.

There are variations on any one or more of the above embodiments wherein the permeable three-dimensional matrix is a hydrogel.

There are variations on any one or more of the above embodiments wherein the permeable three-dimensional matrix comprises a hydrogel network of predetermined dimensions.

There are variations on any one or more of the above embodiments wherein the permeable three-dimensional matrix comprises a matrix of particles of the same size or particles of different sizes.

There are variations on any one or more of the above embodiments wherein the permeable three-dimensional matrix comprises columnar pillars.

Variations exist with respect to any one or more of the above embodiments, wherein the permeable three-dimensional matrix comprises a mesoporous crystalline material.

There are variations on any one or more of the above embodiments, wherein the method further comprises patterning the permeable three-dimensional matrix in the flow cell by photolithography.

There are variations on any one or more of the above embodiments wherein the oligonucleotides are suitable for sequencing-by-synthesis.

There are variations on any one or more of the above embodiments wherein the flow cell has an interior volume, and wherein the permeable three-dimensional matrix containing oligonucleotides occupies the entire interior volume of the flow cell.

There are variations on any one or more of the above embodiments wherein the method further comprises imaging the permeable three-dimensional substrate in discrete two-dimensional layers.

Another embodiment relates to a method of three-dimensional sequencing using a three-dimensional sequencing matrix on a flow cell, comprising: loading a polymer precursor solution into a flow cell, wherein the polymer precursor solution comprises monomers and oligonucleotides; polymerizing the polymer precursor solution to produce a permeable three-dimensional matrix within the flow cell; diffusing a sequencing library into the permeable three-dimensional polymer matrix, wherein the sequencing library comprises nucleic acid fragments; diffusing an enzyme and a reagent 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 produce 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 the three-dimensional matrix in a plurality of discrete two-dimensional slices to characterize the sequencing library, wherein the plurality of discrete two-dimensional slices represent the entire three-dimensional internal volume of the flow cell.

wherein each R²Independently is hydrogen or (C)_1-6) An alkyl group.

Variations on any one or more of the above embodiments exist in which the monomer comprises polyethylene glycol (PEG) -thiol, PEG-acrylate, acrylamide, N' -bis (acryloyl) cystamine (BACy), PEG, polypropylene oxide (PPO), polyacrylic 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), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronic acid, pectin, carrageenan, dextran sulfate, hyaluronic acid, and mixtures thereof, Gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinylsulfone, 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.

Variations exist with respect to any one or more of the above embodiments, wherein the monomer comprises polyethylene glycol (PEG) -thiol/PEG-acrylate; acrylamide/N, N' -bis (acryloyl) cystamine (BACy); PEG/polypropylene oxide (PPO); or a combination thereof.

There are variations on any one or more of the above embodiments wherein the polymer precursor solution further comprises poly (N- (5-azidoacetamidylpentyl) acrylamide-co-acrylamide) (PAZAM) containing an azide moiety.

There are variations on any one or more of the embodiments above wherein the oligonucleotide is an alkyne-linked oligonucleotide adapted to bind to an azide moiety in PAZAM.

There are variations on any one or more of the above embodiments wherein the permeable three-dimensional matrix comprises a hydrogel.

There are variations on any one or more of the above embodiments in which the columnar posts are fabricated to comprise alternating materials in the Z-direction.

There are variations on any one or more of the above embodiments wherein the optical imaging comprises using a confocal microscope, a multi-photon or light sheet illumination microscope.

Another embodiment relates to a method of three-dimensional sequencing using a three-dimensional sequencing matrix on a flow cell, comprising: loading a polymer precursor solution into a flow cell, wherein the polymer precursor solution comprises a monomer, a crosslinker, a photoinitiator, and an oligonucleotide; 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 comprises nucleic acid fragments to which adaptors have been added; diffusing an enzyme and a reagent 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 produce clusters for sequencing within the permeable three-dimensional polymer matrix; sequencing the clusters within the permeable three-dimensional polymer matrix; and imaging the sequenced clusters within the three-dimensional matrix in a plurality of discrete two-dimensional slices using confocal microscopy, multiphoton or light sheet illumination microscopy to characterize the sequencing library, wherein the plurality of discrete two-dimensional slices represent the entire three-dimensional interior volume of the flow-through cell.

wherein each R²Independently is hydrogen or (C)_1-6) An alkyl group.

wherein:

each n is independently an integer from 1 to 6; and is

Each R¹Independently is hydrogen or (C)_1-6) An alkyl group.

Variations exist regarding any one or more of the above embodiments wherein the photoinitiator is lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (LAP), a diazonium sulfonate initiator; monoacylphosphine oxide (MAPO) salts or bisacylphosphine oxide (BAPO) salts.

There are variations on any one or more of the above embodiments wherein the polymer precursor solution further comprises poly (N- (5-azidoacetamidylpentyl) acrylamide-co-acrylamide) (PAZAM) to which an azide moiety has been bound.

There are variations on any one or more of the above embodiments wherein the permeable three-dimensional matrix comprises columnar pillars, and wherein the columnar pillars are fabricated to comprise alternating materials in the Z-direction.

There are variations on any one or more of the above embodiments in which the adapter-added nucleic acid fragments are circularized after addition of the adapter to produce nanospheres.

Another embodiment relates to a kit, comprising: a flow-through cell, wherein the flow-through cell comprises at least one channel; and a permeable three-dimensional matrix containing oligonucleotides, wherein the permeable three-dimensional matrix containing oligonucleotides is adapted to be introduced into the at least one channel and subsequently immobilized therein.

Another embodiment relates to a method for preparing a three-dimensional polymeric structure having a functionalized surface on a flow cell, comprising: loading a polymer precursor solution into a flow cell, wherein the polymer precursor solution comprises a monomer, a crosslinker, a photoinitiator, and a functionalized polymer, and wherein the flow cell comprises at least one channel for receiving the polymer precursor solution, and wherein the at least one channel has an upper interior surface and a lower interior surface; and irradiating the polymer precursor solution through a photomask with light that activates the photoinitiator with a wavelength, wherein the photomask includes a series of openings formed therein, wherein the photomask has been placed over the outer surface of the channel, and wherein activation of the photoinitiator polymerizes at least some of the polymer precursor solution below the openings in the photomask and forms a three-dimensional polymer structure extending from an upper inner surface to a lower inner surface of the at least one channel.

There are variations on any one or more of the above embodiments, wherein the method further comprises reacting a bifunctional linker having a first end and a second end with the functionalized polymer, wherein the first end of the bifunctional linker is chemically or enzymatically attached to the functionalized polymer, and wherein the second end of the bifunctional linker selectively binds a predetermined type of molecule.

There are variations on any one or more of the above embodiments wherein the functionalized polymer is poly (N- (5-azidoacetamidylpentyl) acrylamide-co-acrylamide (PAZAM) containing an azide moiety, and wherein the bifunctional linker is a biotin-PEG-alkyne complex, and the method further comprises reacting the biotin-PEG-alkyne complex with the azide moiety in the PAZAM using an azide-alkyne click reaction.

There are variations on any one or more of the above embodiments wherein the method further comprises binding streptavidin to biotin in the biotin-PEG-alkyne complex.

There are variations on any one or more of the above embodiments, wherein the method further comprises binding a biotinylated capture oligonucleotide to the streptavidin, wherein the biotinylated capture oligonucleotide is specific for a target of interest in a sequencing library.

There are variations on any one or more of the above embodiments wherein the flow cell has an oligonucleotide of a predetermined length and a sequence that binds to both the upper and lower interior surfaces of the at least one channel, and wherein the oligonucleotide comprises a primer suitable for nucleic acid amplification.

There are variations on any one or more of the above embodiments wherein the polymer is a hydrogel.

wherein each R²Independently is hydrogen or (C)_1-6) An alkyl group.

wherein:

each n is independently an integer from 1 to 6; and is

Each R¹Independently is hydrogen or (C)_1-6) An alkyl group.

There are variations on any one or more of the above embodiments wherein the polymer precursor solution comprises polyethylene glycol (PEG) -thiol, PEG-acrylate, acrylamide, N' -bis (acryloyl) cystamine (BACy), PEG, polypropylene oxide (PPO), polyacrylic 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), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronic acid, pectin, dextran sulfate, and mixtures thereof, Carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinylsulfone, diethylene glycol diallyl ether, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylolpropane trimethacrylate, ethoxylated trimethylolpropane triacrylate, or ethoxylated pentaerythritol tetraacrylate, or combinations or mixtures thereof.

There are variations to any one or more of the above embodiments wherein the photomask is polyethylene terephthalate.

There are variations on any one or more of the above embodiments wherein the photomask is laminated to an upper surface of the flow cell.

There are variations on any one or more of the above embodiments wherein irradiating the polymer precursor solution comprises using an ultraviolet light source.

Another embodiment relates to a method for preparing a three-dimensional polymeric structure having a functionalized surface on a flow cell, comprising: loading a hydrogel precursor solution into a flow cell, wherein the hydrogel precursor solution comprises a monomer, a cross-linking agent, a photoinitiator, and a PAZAM containing an azide moiety, and wherein the flow cell comprises at least one channel for receiving the hydrogel precursor solution, and wherein the at least one channel has an upper interior surface and a lower interior surface; placing a photomask over the at least one channel, wherein the photomask comprises a series of openings formed therein; and irradiating the hydrogel precursor solution through the photomask with light of a wavelength that activates the photoinitiator, and wherein activation of the photoinitiator polymerizes at least some of the hydrogel precursor solution below the openings in the photomask and forms a three-dimensional hydrogel structure extending from an upper interior surface to a lower interior surface of the at least one channel; reacting the biotin-PEG-alkyne complex with an azide moiety in the PAZAM in the three-dimensional polymer structure using an azide-alkyne click reaction; binding streptavidin to biotin in the biotin-PEG-alkyne complex; and binding a biotinylated capture oligonucleotide to the streptavidin, wherein the biotinylated capture oligonucleotide is specific for the target molecule of interest in a sequencing library.

wherein each R²Independently is hydrogen or (C)_1-6) An alkyl group.

wherein:

each n is independently an integer from 1 to 6; and is

Each R¹Independently is hydrogen or (C)_1-6) An alkyl group.

Variations exist regarding any one or more of the above embodiments, wherein the photoinitiator is a diazonium sulfonate initiator; monoacylphosphine oxide (MAPO) salts; bisacylphosphine oxide (BAPO) salts; or combinations or mixtures thereof.

There are variations on any one or more of the above embodiments wherein the photomask comprises a polyester film.

Another embodiment relates to a method for preparing a three-dimensional polymeric structure having a functionalized surface on a flow cell, comprising: loading a polymer precursor solution into a flow cell, wherein the polymer precursor solution comprises a monomer, a cross-linking agent, a photoinitiator, and a streptavidin-labeled acrylamide monomer, and wherein the flow cell comprises at least one channel for receiving the polymer precursor solution, and wherein the at least one channel has an upper interior surface and a lower interior surface, and wherein oligonucleotides of a predetermined length are bound to both the upper surface and the lower surface of the at least one channel; placing a photomask over the at least one channel, wherein the photomask comprises a series of openings formed therein; irradiating the polymer precursor solution through the photomask with light of a wavelength that activates the photoinitiator, and wherein activation of the photoinitiator polymerizes at least some of the polymer precursor solution below the openings in the photomask and forms a three-dimensional polymer structure extending from an upper interior surface to a lower interior surface of the at least one channel; selectively binding biotinylated capture oligonucleotides to streptavidin in the three-dimensional polymer structure, wherein the biotinylated capture oligonucleotides are specific for and bind to target molecules of interest in the library; and eluting the bound target molecules and seeding the eluted target molecules on the surface of the flow cell on which the oligonucleotides are bound.

wherein each R²Independently is hydrogen or (C)_1-6) An alkyl group.

wherein:

each n is independently an integer from 1 to 6; and is

Each R¹Independently is hydrogen or (C)_1-6) An alkyl group.

It should be understood 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 systems, devices, and methods will become apparent to those of ordinary skill in the art upon reading and understanding the following detailed description of the exemplary embodiments. As the skilled person will appreciate, further embodiments are possible without departing from the scope and spirit of the disclosure herein. Accordingly, the drawings and associated descriptions are to be regarded as illustrative in nature and not as restrictive.

Drawings

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, wherein:

FIG. 1A is a perspective view of a flow cell in accordance with one embodiment of the disclosed systems and methods;

FIG. 1B is a top view and close-up top view of the flow cell of FIG. 1A, wherein an array of hydrogel structures has been formed on the flow cell;

FIG. 1C depicts the flow cell of FIG. 1A properly inserted into a cassette used in a sequencing-by-synthesis process;

FIG. 2A depicts one example of the presently disclosed system and method for forming a polymer (e.g., hydrogel) structure on a flow cell (such as the flow cell shown in FIG. 1A) in which a polymer precursor solution has been introduced into a fluidic channel of the flow cell and a pre-patterned photomask has been placed over the channel;

FIG. 2B depicts one example of the presently disclosed system and method for forming a polymer (e.g., hydrogel) structure on a flow cell, wherein ultraviolet light is directed through an opening in a photomask into a channel of the flow cell for polymerizing the contents of a polymer precursor solution;

fig. 2C depicts an array of hydrogel structures formed within a channel of a flow-through cell, wherein the hydrogel structures have a cylindrical shape and are attached to upper and lower interior surfaces of the channel;

fig. 2D depicts an exemplary method of cutting a hydrogel structure formed in a channel of a flow cell by introducing oil containing a cutting agent into the channel of the flow cell;

fig. 2E depicts an exemplary method of removing a cut hydrogel structure from a channel of a flow cell by washing the channel;

FIG. 3A depicts a first step in another example of the presently disclosed systems and methods for forming polymer (e.g., hydrogel) structures on a sequencing flow cell, wherein a pre-patterned photomask is placed on or attached to the flow cell and the flow cell is then inserted into a cassette;

FIG. 3B depicts a second step in another example of the presently 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;

fig. 3C depicts a third step in another example of the presently 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 (shown in bright field micrographs) on the flow cell, and wherein the flow cell is subsequently washed to remove unpolymerized material and to detach the flow cell from the instrument;

FIG. 4A depicts one example of the presently disclosed system and method for cell encapsulation and in situ preparation of a sequencing library, in which single cells or cell colonies are mixed with a polymer precursor solution and loaded into a flow cell, and then irradiated with ultraviolet light through a photomask to produce an array of cell-embedded hydrogel structures (e.g., pillars) on the flow cell, shown in bright field micrographs;

FIG. 4B depicts an example of the presently disclosed systems and methods for cell encapsulation and in situ preparation of a sequencing library, wherein a lysing agent and a labeling agent are diffused into the hydrogel structure of FIG. 4A, and wherein the cells are subsequently lysed and labeled within the hydrogel structure;

FIG. 4C depicts one example of the presently disclosed system and method for cell encapsulation and in situ preparation of sequencing libraries, wherein the library of FIG. 4B is seeded onto the top and bottom surfaces of a flow cell by introducing oil into the flow cell and raising the temperature to release library fragments contained in a hydrogel structure, which are then hybridized to surface primers attached to the flow cell surface;

FIG. 4D depicts an example of the presently disclosed systems and methods for cell encapsulation and in situ preparation of a sequencing library, wherein the hybridized library fragments of FIG. 4C are then clonally amplified using a bridge amplification method for generating clusters;

FIG. 5A is a side view of a set of hydrogel structures formed on a flow cell for capturing cells on the flow cell to prepare libraries in situ using an alternative version of the disclosed systems and methods, wherein the hydrogel structures have an inverted C-shaped geometry;

FIG. 5B is several top views of the cell-trapping hydrogel feature of FIG. 5A, showing its inverted C-shaped geometry;

FIG. 5C is a side view of the hydrogel structure of FIG. 5A, showing individual cells captured in each hydrogel structure;

FIG. 5D is a side view of one of the hydrogel structures of FIG. 5C, showing individual cells captured in the hydrogel structure and the direction of the fluid containing cells being directed into and through the flow cell;

FIG. 6 is a flow diagram depicting an exemplary embodiment of a method for producing a reversible, permeable three-dimensional polymeric structure on a flow-through cell;

FIG. 7 is a flow diagram depicting an exemplary embodiment of a method for sequencing library preparations using reversible, permeable three-dimensional polymer structures formed on a flow cell;

FIG. 8 is a flow chart depicting an exemplary embodiment of a method of sequencing library preparations using a reversible, permeable three-dimensional hydrogel structure formed on a flow cell;

figures 9A to 9B depict a flow cell having an array of individual hydrogel columns formed using photolithography located within the flow cell, wherein the columns contain P5/P7 primers and support the growth of clusters within the hydrogel matrix;

FIG. 10A depicts a MiSeq^TMA hydrogel column fabricated within the flow cell, and fig. 10B-10K depict time series images showing fluorescent dye introduced into the flow cell, dye diffusion into the hydrogel column, and dye washout from the hydrogel column;

11A-11B depict hydrogel beads doped with poly (N- (5-azidoacetamidylpentyl) acrylamide-co-acrylamide) (PAZAM) containing P5/P7 primer, wherein the beads are initially soaked with a sequencing library and then soaked in ExAmp to create clusters throughout the three-dimensional volume of each bead;

FIGS. 12A-12B depict index-free sequencing, wherein each hydrogel bead contains clusters from the sample in which the bead is incubated, wherein hydrogel beads containing such clusters are loaded onto a flow cell and sequenced, and wherein beads from each sample type can be distinguished from each other using a variety of means, such as fluorophores embedded in the beads that are removed prior to sequencing;

FIG. 13A depicts a sequencing flow cell, wherein sequencing occurs in a two-dimensional network of clusters on a top surface and a bottom surface, and wherein the top surface and the bottom surface are separated by a known distance (e.g., 100 μm) along the Z-axis;

FIG. 13B depicts a sequencing flow cell, wherein sequencing occurs on a top surface and a bottom surface and in a three-dimensional network of clusters located in discrete regions between the top surface and the bottom surface, and wherein the top surface and the bottom surface are separated by a distance of 100 μm along the Z-axis;

fig. 14 depicts an exemplary SPIM setup, wherein excitation is delivered into the sample through a low NA objective lens, and wherein fluorescence emissions are collected through a high NA emission objective lens;

FIG. 15 depicts a large hydrogel network within a sequencing flow cell;

FIG. 16 depicts a matrix for sequencing large particles, small particles, or a combination of large and small particles within a flow cell;

FIG. 17 depicts periodic columnar pillars within a sequencing flow cell;

FIG. 18 depicts sequencing a mesoporous crystalline material within a flow cell;

fig. 19A-19D depict exemplary embodiments of methods of forming a hydrogel within a flow-through cell by polymerizing PAZAM + di DBCO-PEG;

FIG. 20 depicts the copolymerization of acrylamide and acrylamide-based modified oligonucleotides into large polyacrylamide beads;

fig. 21A is a bright field micrograph depicting hydrogel beads on a slide;

FIG. 21B is a bright field micrograph depicting stacking in HiSeq^TMHydrogel in flow cellBeads;

FIG. 22A is a fluorescence micrograph of standard acrylamide beads after incubation with a dye-labeled complementary strand;

FIG. 22B is a fluorescence micrograph of oligonucleotide-modified acrylamide beads after incubation with a dye-labeled complementary strand;

FIG. 23A depicts a hydrogel bead in which long DNA fragments have been encapsulated to be captured within a flowcell;

FIG. 23B depicts an enzymatic process for library preparation performed within the trapped hydrogel beads of FIG. 23A;

FIG. 23C depicts an amplified library that produces linked read clusters distributed three-dimensionally within each hydrogel bead;

figure 24A depicts template capture and extension occurring on a hydrogel bead carrying oligonucleotides;

FIG. 24B depicts clonal amplification of library inserts on hydrogel beads, which were used to generate clusters;

figure 25A depicts clustered beads delivered into a flow cell in a hydrogel precursor solution;

figure 25B depicts immobilization of clustered beads within a cross-linked hydrogel matrix for maintaining spatial position of the beads in three dimensions during sequencing and subsequent imaging;

figure 26 depicts dimer particles with different orthogonal linearization chemistries;

fig. 27 depicts an exemplary system for synthesizing similar dimeric particles;

FIGS. 28A and 28B depict spatial control of clusters in three dimensions using a three-dimensional matrix having columnar pillars of alternating material composition in the Z-direction;

29A-29D depict a simplified exemplary method for producing a polymer scaffold in which unpolymerized monomer solution is embedded with salt particles having a predetermined size distribution; wherein the salt particles displace the monomers, thereby creating a three-dimensional network within the solution; wherein the monomer solution is polymerized to form a three-dimensional polymer scaffold surrounding the salt particles; and wherein the salt particles dissolve, thereby creating a random three-dimensional array of pores defining a scaffold.

FIG. 30 is a flow chart depicting an exemplary method for preparing a permeable three-dimensional matrix on a sequencing flow cell;

FIG. 31 is a flow chart depicting a first exemplary method for three-dimensional sequencing of a nucleic acid library;

FIG. 32 is a flow chart depicting a second exemplary method for three-dimensional sequencing of a nucleic acid library;

fig. 33 depicts the formation of hydrogel micropillars on a channel within a flow-through cell, wherein individual hydrogel micropillars are visible in a bright field micrograph;

fig. 34A depicts an exemplary method for fabricating a hydrogel microcolumn on a flow cell, wherein a hydrogel precursor solution comprising monomers and a photoinitiator is introduced into the flow cell;

FIG. 34B depicts an exemplary method for fabricating hydrogel micropillars on a flow cell, wherein a pre-patterned photomask is placed on the flow cell of FIG. 34A and irradiated with ultraviolet light;

fig. 34C depicts an exemplary method for fabricating a hydrogel microcolumn on a flow cell, wherein the hydrogel microcolumn is formed on the flow cell of fig. 34A, and wherein the hydrogel microcolumn is attached to an upper surface and a lower surface of one of the channels in the flow cell;

fig. 35A depicts an exemplary method for making a functionalized hydrogel structure on a flow cell, wherein a hydrogel precursor solution containing 10% Polyacrylamide (PA), a crosslinking agent, and 0.25% PAZAM into which an azide moiety has been incorporated is loaded onto the flow cell;

fig. 35B depicts an exemplary method for fabricating a functionalized hydrogel structure on a flow cell, wherein a photomask including a plurality of openings formed therein is placed over the flow cell of fig. 35A, then exposed to ultraviolet light for 10 seconds to copolymerize acrylamide and PAZAM, and an array of azide-functionalized hydrogel micropillars is formed in a narrow channel of the flow cell;

fig. 35C depicts an exemplary method for making a functionalized hydrogel structure on a flow cell, wherein a biotin-PEG-alkyne complex is clicked onto the azide portion of the hydrogel microcolumn of fig. 35B;

fig. 35D depicts an exemplary method for making a functionalized hydrogel structure on a flow cell, wherein streptavidin labeled with fluorescein binds to biotin in the hydrogel microcolumn of fig. 35C;

fig. 35E depicts an exemplary method for making functionalized hydrogel structures on a flow cell, wherein streptavidin is bound to biotinylated capture oligonucleotides to enable immobilization of target sequencing library molecules;

fig. 36A depicts a 4X bright field micrograph of a PA/PAZAM control (no biotin);

FIG. 36B depicts a 4X bright field micrograph of PA/PAZAM plus Blackpool;

FIG. 36C depicts a 4X fluorescence micrograph of the PA/PAZAM control (no biotin) after five minutes of reaction time;

FIG. 36D depicts a 4X fluorescence micrograph of PA/PAZAM plus Blackpool after five minutes reaction time;

FIG. 36E depicts a 4X fluorescence micrograph of the PA/PAZAM control (no biotin) after ten minute reaction time at 40 ℃;

FIG. 36F depicts a 4X fluorescence micrograph of PA/PAZAM plus Blackpool after ten minute reaction time at 40 ℃;

FIG. 37A depicts another exemplary method for making a functionalized hydrogel structure on a flow cell, wherein a hydrogel precursor solution containing 10% Polyacrylamide (PA) and 0.25% streptavidin-labeled acrylamide monomers is loaded onto the flow cell;

fig. 37B depicts another exemplary method for fabricating a functionalized hydrogel structure on a flow cell, wherein a photomask including a plurality of openings formed therein is placed over the flow cell of fig. 37A, then exposed to ultraviolet light for 10 seconds to copolymerize acrylamide-streptavidin labeled acrylamide monomer and form an array of streptavidin functionalized hydrogel micropillars in the narrow channel of the flow cell;

FIG. 37C depicts another exemplary method for fabricating a functionalized hydrogel structure on a flow cell, wherein a biotinylated capture oligonucleotide binds to the streptavidin moiety in the hydrogel microcolumn of FIG. 37B, and wherein the target library molecules hybridize to the biotinylated capture oligonucleotide and become immobilized on the hydrogel microcolumn of FIG. 37B;

FIG. 37D depicts another exemplary method for fabricating a functionalized hydrogel structure on a flow cell, wherein the immobilized target molecule is eluted from the capture oligonucleotide of FIG. 37C and seeded on a wide channel of the flow cell;

FIG. 38A depicts biotinylated P5 primer and P7 primer bound to streptavidin-functionalized hydrogel microcolumns;

FIG. 38B depicts the biotinylated P5 primer and P7 primer of FIG. 38A being incubated with TET-labeled complementary P5 'and P7' oligonucleotides;

FIG. 38C depicts the TET-labeled complementary P5 'and P7' oligonucleotides of FIG. 38B hybridized to biotinylated P5 and P7 primers;

FIG. 39A is a bright field micrograph showing hydrogel micropillars incubated with TET-P5 'and TET-P7' in the absence of biotin-P5 oligonucleotide and biotin-P7 oligonucleotide;

FIG. 39B is a fluorescence micrograph (488nm excitation) showing a hydrogel microcolumn incubated with TET-P5 'and TET-P7' in the absence of biotin-P5 oligonucleotide and biotin-P7 oligonucleotide, in which uniform staining of the P5 primer and P7 primer on the flow cell surface was observed;

FIG. 39C is a bright field micrograph showing hydrogel micropillars incubated with TET-P5 'and TET-P7' after incubation with biotin-P5 oligonucleotide and biotin-P7 oligonucleotide;

FIG. 39D is a fluorescence micrograph (488nm excitation) showing hydrogel micropillars incubated with TET-P5 'and TET-P7' after incubation with biotin-P5 oligonucleotide and biotin-P7 oligonucleotide, where TET staining was observed to localize to the edges of the hydrogel micropillars, indicating that the TET labeled oligonucleotide had hybridized to the streptavidin-conjugated biotinylated P5 primer and P7 primer;

FIG. 40A is a fluorescence micrograph depicting the level of TET-P5 'oligonucleotide/TET-P7' oligonucleotide in the interstitial spaces between hydrogel microcolumns at an incubation time of one minute;

FIG. 40B is a graph depicting the level of TET-P5 'oligonucleotide/TET-P7' oligonucleotide in the interstitial spaces between hydrogel microcolumns at an incubation time of one minute;

FIG. 40C is a fluorescence micrograph depicting the level of TET-P5 'oligonucleotide/TET-P7' oligonucleotide in the interstitial space between hydrogel microcolumns at an incubation time of five minutes;

FIG. 40D is a graph depicting the level of TET-P5 'oligonucleotide/TET-P7' oligonucleotide in the interstitial spaces between hydrogel microcolumns at an incubation time of five minutes;

FIG. 40E is a fluorescence micrograph depicting the level of TET-P5 'oligonucleotide/TET-P7' oligonucleotide in the interstitial space between hydrogel microcolumns at an incubation time of ten minutes;

FIG. 40F is a graph depicting the level of TET-P5 'oligonucleotide/TET-P7' oligonucleotide in the interstitial space between hydrogel microcolumns at an incubation time of ten minutes;

FIG. 41A depicts the hybridization of the P7 'and P5' regions of the sequenced library molecules to biotinylated P5 and P7 oligonucleotides;

figure 41B depicts capture of sequencing library molecules with streptavidin-functionalized hydrogel columns attached to the surface of a flow cell;

figure 41C depicts the vaccination-bound sequencing library molecules by: incubation at 85 ℃ to denature the hybridized biotinylated primer, followed by warming to 20 ℃ to allow the sequencing library molecules to hybridize to the surface primer;

FIG. 42A is a bright field micrograph of an untreated flow cell (control);

FIG. 42B is a fluorescence micrograph (488nm) of a SYTOX stained untreated flow cell (control) with no clusters shown;

FIG. 42C is a bright field micrograph of a flow cell with streptavidin micropillars;

FIG. 42D is a fluorescence micrograph (488nm) of a SYTOX stained flow-through cell with streptavidin mini-columns;

FIG. 42E is a bright field micrograph of the hydrogel micropillars of FIG. 42C;

FIG. 42F is a fluorescence micrograph (488nm) of the SYTOX stained microcolumn of FIG. 42D;

FIG. 43A depicts a flow cell in a cassette in which streptavidin microcolumns have been formed in narrow channels of the flow cell, but not in wide channels of the flow cell;

FIG. 43B is a photomicrograph of the wide channel of the flow cell of FIG. 43A stained with SYTOX dye after 24 cycles of bridge amplification;

FIG. 43C is a photomicrograph of the narrow channel of the flow cell of FIG. 43A stained with SYTOX dye after 24 cycles of bridge amplification;

FIG. 44 is a flow chart depicting a first method for preparing a functionalized three-dimensional polymeric structure on a flow cell;

FIG. 45 is a flow diagram depicting a second method for preparing a functionalized three-dimensional polymeric structure on a flow-through cell; and is

Fig. 46 is a flow diagram depicting a third method for preparing a functionalized three-dimensional polymer structure on a flow cell.

Detailed Description

I. Overview

Embodiments of the disclosed systems and methods can be used to form reversible hydrogel polymer structures on flow cells used as part of the workflow of sequencing-by-synthesis and other sequencing methods. The workflow may include library preparation and sequencing. These hydrogel structures may be particularly useful for addressing the challenges associated with high throughput single cell or single colony sequencing on flow-through cells due to low initial nucleic acid input from single cells and the inability to compartmentalize sequencing libraries on flow-through cells. The disclosed systems and methods enable high throughput single cell or single colony sequencing by providing entrapment or encapsulation of cells and genetic material in a reversible hydrogel structure on a flow cell. These hydrogel structures entrap or compartmentalize individual cells or individual colonies while allowing efficient exchange of reagents for cell lysis and ultimately preparation of sequencing libraries in situ.

Various embodiments of the disclosed systems, devices, and methods can be used to create reversible, permeable three-dimensional polymer (e.g., hydrogel) structures within a fluidic channel on a sequencing flow cell. These temporary polymer structures extend the available sequencing surface from two dimensions to three dimensions, thereby greatly increasing the throughput of sequencing flow cells.

Various embodiments of the disclosed systems, devices, and methods can be used to create reversible three-dimensional polymer (e.g., hydrogel) structures within a fluid channel on a flow-through cell. These structures can be used to introduce temporary functional surfaces in addition to pre-existing sequencing surfaces within a flow cell for a variety of applications including, for example: (i) enriching target DNA; (ii) clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) screening; and (iii) highly multiplexed screening applications using DNA-conjugated antigens.

As used herein, the term "hydrogel" refers to a substance formed when organic polymers (natural or synthetic) are cross-linked by 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, meaning that the gel-forming polymer is non-toxic to living cells and allows sufficient diffusion of oxygen and nutrients to entrapped cells to maintain viability. In some versions, the hydrogel polymer comprises about 60% to 90% fluid (such as water) and about 10% to 30% polymer, wherein in other versions the hydrogel has a water content of about 70% to 80%.

The term "adaptor" as used herein refers to a linear oligonucleotide that can be fused to a nucleic acid molecule, e.g., by ligation or tagging. In some examples, the adaptor is not substantially complementary to the 3 'end or the 5' end of any target sequence present in the sample. In some examples, suitable adaptors are in the range of about 10 to 100 nucleotides, about 12 to 60 nucleotides, or about 15 to 50 nucleotides in length. Generally, the adapters may include any combination of nucleotides and/or nucleic acids. The adapter may comprise one or more cleavable groups at one or more positions. The adapter can further include a sequence complementary to at least a portion of a primer (e.g., a primer comprising a universal nucleotide sequence). The adaptors may also include barcodes (also known as tags or indexes) to aid in downstream error correction, identification or sequencing. As used herein, the term "index" refers to a nucleotide sequence that can be used as a molecular identifier or barcode to tag or identify a nucleic acid as being derived from. The index may be used to identify individual nucleic acids or subpopulations of nucleic acids.

A flow cell herein may refer to a flow cell to be used during a sequencing workflow. For example, flow cells may be used for library preparation, sequencing, or both. In one embodiment, the same flow cell may be used for both library preparation and sequencing. An exemplary flow cell includes a channel that includes a surface through which one or more fluidic reagents can flow and to which the adapted fragments of the sequencing library can be transported and bound. The flow cell includes a solid support having a surface to which a sequencing library is bound. In some examples, the solid surface is covered with a hydrogel layer. In some examples, the surface contains a layer of capture nucleotides lawn (lawn) that can bind to the adaptation fragments of the sequencing library. In some examples, the surface is a patterned surface. When referring to a surface, the term "patterning" may refer to an arrangement (such as an array) of different regions (such as amplification sites) in or on the exposed surface of a solid support. For example, one or more of these regions may be characteristic of the presence of one or more amplification primers and/or capture primers. These features may be separated by interstitial regions where no primers are present. In some examples, the channel height of the flow cell device is about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, about 100 μm, about 110 μm, about 120 μm, about 130 μm, about 140 μm, or about 150 μm, or an amount within a range defined by any two of the foregoing values.

As shown in fig. 1A, an exemplary sequencing flow cell 100 includes a glass top layer 110 having a fluidic aperture 112 formed therein; a channel-defining spacer 120 comprising a plurality of fluidic/sequencing channels 122 formed therein; and a glass substrate 130 on which the array 150 is formed. Array 150 includes a plurality of individual structures 152 formed thereon by the disclosed method. The individual structures 152 may be three-dimensional structures. The structure may comprise a polymer. In one embodiment, the polymer is a hydrogel. It should be noted that while a hydrogel is used to refer to structure 152 in some instances herein, a "hydrogel" is used only as a representative material in this embodiment, and the structure need not comprise a hydrogel, but may comprise any suitable polymeric material. Fig. 1B depicts an assembled flow cell 100 on which an array 150 of individual three-dimensional hydrogel structures 152 have been fabricated in one of the sequencing channels 122, and fig. 1C depicts a flow cell 100 having a plurality of three-dimensional hydrogel structures 152 formed thereon, which is inserted into a sequencing cassette 160 for use with a sequencing-by-synthesis apparatus. A three-dimensional hydrogel structure having a specific predetermined geometry may be formed on a flow cell by: (i) introducing a hydrogel precursor solution into a sequencing channel of a flow cell; (ii) placing a photomask having a specific pattern formed thereon over a sequencing channel on a flow cell before or after introducing a hydrogel precursor solution into the flow cell; and (iii) exposing the hydrogel precursor solution to light of a predetermined wavelength transmitted through the photomask, wherein irradiation of the hydrogel precursor solution polymerizes its contents and forms a three-dimensional structure on the flow cell corresponding to the pattern on the photomask. Once the hydrogel structures have been used for their purpose, they can be cut and washed out of the flow cell without affecting the overall function of the flow cell.

The hydrogel precursor solution may include a monomer solution capable of being photopolymerized by activating a photoinitiator. An example of one such system comprises 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 (acryloyl) cystamine (BAC), and the photoinitiator is lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (LAP), which is activated by Ultraviolet (UV) light of a predetermined wavelength.

In other versions, the precursor solution may comprise polyethylene glycol (PEG) -thiol, PEG-acrylate, acrylamide, N' -bis (acryloyl) cystamine, PEG, polypropylene oxide (PPO), polyacrylic acid, poly (hydroxyethyl methacrylate) (PHEMA), poly (methyl methacrylate) (PMMA), poly (N-isopropylacrylamide) (PNIPAAm), poly (lactic acid) (PLA), poly (lactic-co-glycolic acid) (PLGA), Polycaprolactone (PCL), poly (vinylsulfonic acid) (PVSA), poly (L-aspartic acid), poly (L-glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronic acid, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, chitosan, cellulose, and mixtures thereof, 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. In other versions, the monomer may include PEG-thiol/PEG-acrylate, acrylamide/N, N' -bis (acryloyl) cystamine (BACy), or PEG/PPO.

In some embodiments of the disclosed methods, the monomer can be a compound of formula I:

wherein each R²Independently is hydrogen or (C)_1-6) An alkyl group.

In some embodiments of the disclosed methods that include a crosslinking agent, the crosslinking agent can be a compound of formula II:

wherein:

each n is independently an integer from 1 to 6; and is

Each R¹Independently is hydrogen or (C)_1-6) An alkyl group.

The crosslinking agent is capable of crosslinking polymer chains within the polymer. In one embodiment, the polymer is a hydrogel. In some versions, the cross-linking agent is capable of being cleaved, thereby releasing the polymer chains, for the following reasons: the presence of a reducing agent; an elevated temperature; an electric field; or exposing the hydrogel structure to a wavelength of light that cleaves a photocleavable crosslinker that crosslinks the polymer of the hydrogel. In some versions, the reducing agent may include a phosphine compound, a water-soluble phosphine, a nitrogen-containing phosphine, and salts and derivatives thereof, Dithioerythritol (DTE), Dithiothreitol (DTT) (cis and trans isomers of 2, 3-dihydroxy-1, 4-dithiolbutane, respectively), 2-mercaptoethanol or β -mercaptoethanol (BME), 2-mercaptoethanol or aminoethanethiol, glutathione, thioglycolates or thioglycolic acid, 2, 3-dimercaptopropanol, tris (2-carboxyethyl) phosphine (TCEP), tris (hydroxymethyl) phosphine (THP), or p- [ tris (hydroxymethyl) phosphine ] propionic acid (THPP). In some versions, the cross-linking agent is cleaved by raising the temperature to greater than about 50 ℃, about 55 ℃, about 60 ℃, about 65 ℃, about 70 ℃, about 75 ℃, about 80 ℃, about 85 ℃, about 90 ℃, about 95 ℃, or about 100 ℃. In some versions, the reducing agent is activated by ultraviolet light.

Other suitable photoinitiators include biocompatible photoinitiators for free radical polymerization that do not damage nucleic acids, such as diazosulfonate initiators; monoacylphosphine oxide (MAPO) salts such as Na-TPO and Li-TPO; and bisacylphosphine oxide (BAPO) salts such as BAPO-ONa and BAPO-Oli.

In some examples, crosslinking the polymer chains of the hydrogel structure forms a hydrogel matrix with pores (i.e., a porous hydrogel matrix). In some versions, the size of the pores in the hydrogel structure are adjustable or fine-tuned, and may be formulated to encapsulate sufficiently large genetic material, such as cells or nucleic acids (e.g., greater than about 300 base pairs), but allow smaller materials, such as reagents, or smaller nucleic acids (e.g., less than about 50 base pairs), such as primers, to pass through the pores, and thus into and out of the hydrogel structure. The size of the pores may vary depending on the water content in the hydrogel of the hydrogel structure. In some examples, the pores have a diameter of about 10nm to about 100 nm.

In some examples, the pore size of the hydrogel structure is fine-tuned by changing the ratio of the polymer concentration to the crosslinker concentration. In some examples, the ratio of polymer to crosslinker is about 30:1, about 25:1, 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 1:2, 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 any of these ratios, or a ratio within a range defined by any two of the foregoing ratios.

Fig. 2A-2E depict an exemplary method 200 for fabricating and subsequently removing a three-dimensional hydrogel structure on a flow cell 210. Flow cell 210 includes an upper interior surface 212 and a lower interior surface 214 that together define a flow cell channel 216. A pre-patterned photomask 218 has been laminated or otherwise attached to the upper surface of the flow cell 210. Fig. 2A depicts the introduction of a hydrogel precursor solution 230 into the flow cell 210, the solution comprising: (i) monomers (e.g., acrylamide), (ii) cross-linkers (e.g., BAC), and (iii) photoinitiators (e.g., LAP). Figure 2B depicts exposing the hydrogel precursor solution 230 to ultraviolet light of a predetermined wavelength through a pre-patterned photomask 218 having a plurality of openings 200 formed therein. Exposing the hydrogel precursor solution 230 to ultraviolet light activates the photoinitiator (LAP) generating free radicals that result in the controlled polymerization of the monomer (acrylamide) into the disulfide bond containing hydrogel structure 232. Fig. 2C depicts the formation of hydrogel features 232 that are anchored to the top surface 212 and the bottom surface 214 of the sequencing channel 216 of the flow cell 210 that is adapted to be inserted into the cassette 260. Fig. 2C includes a bright field micrograph 250 showing a cylindrical hydrogel structure 232 (100 um to 150um in diameter) with dense gel walls and a less dense core. Figure 2D depicts cutting of the hydrogel features 232 from the flow cell 210 using heat or a combination of chemical cutting of heat and a crosslinking agent. For example, incubating the hydrogel structure 232 with a reducing agent (such as an oil containing DTT) cleaves the structure by reducing disulfide bonds in the hydrogel crosslinker to thiols, allowing the hydrogel to wash out of the flow cell 210, as shown in fig. 2E. After the cleaved hydrogel structures have been washed out of the flow cell, the surface of the flow cell 210 remains functional, i.e., removal of the hydrogel structures from the flow cell 210 does not affect the functionality of the sequencing primers that have been bound to the flow cell prior to fabrication and subsequent removal of the gel features of the hydrogel.

The manufacture of hydrogel structures, such as those previously described, can be accomplished in both a factory environment and a laboratory environment. However, known hydrogel fabrication techniques typically involve the use of expensive and cumbersome equipment, such as a photomask aligner with a collimated ultraviolet light source and a chrome mask. Accordingly, to facilitate the manufacture of hydrogel structures directly on flow cells by consumers of sequencing products, a relatively small scale, low cost instrument for manufacturing hydrogels on flow cells is provided. By way of example, a general implementation of the apparatus comprises: (i) a collimated LED ultraviolet light source, such as Thor Labs model M385LP 1-C1; (ii) a housing adapted to receive a flow cell (and flow cell cassette) therein and to support and properly position the light source relative to the flow cell; (iii) pre-patterned

A photomask adapted to be laminated and adhered to an upper surface of a particular flow cell; and (iv) a shield for housing the light source and the housing. An opening in the shield allows the flow cell to be inserted into the housing for uv irradiation of the flow cell through the pre-patterned photomask. The housing may comprise a movable or adjustable stage device for replicating the pattern along the length and width of the flow cell if the illumination area of the housing is smaller than the area to be photopatterned on the flow cell. In addition to operating as a wide field illuminator, the different versions of the disclosed instrument also perform various reagent exchanges and provide thermal control to facilitate automated library preparation. As described in more detail below, certain implementations of the disclosed apparatus operate as an independent library preparation device that outputs ready clusters or ready library sequencing. The photomask may include (but is not necessarily limited to)

(polyethylene terephthalate); screen printed light absorbing materials, such as carbon ink; or a chemically etched metal film; such aluminum, chromium, gold or platinum, and other light absorbing materials.

Fig. 3A-3C depict exemplary embodiments of the disclosed systems and methods for manufacturing hydrogel structures on flow-through cells, wherein the hydrogel structures contain a sample to be sequenced or otherwise analyzed. In this embodiment, the disclosed instrument is automated and the housing includes a processor that executes various programs resident thereon for illuminating the flow cell and for performing reagent exchanges and other functions in an automated fashion. As shown in fig. 3A, a customer (or other user) orders a flow cell 310 on which a photomask 318 (having an area including a customer-specified pattern formed therein) has been laminated to form an assembly 320. The patterned region of the photomask 318 is placed over and aligned with the channel 312 on the flow cell 310. The flow cell 310 is then inserted into the appropriate flow cell cassette 360. As shown in fig. 3B, the customer then mixes the sample of interest (e.g., biological cell or genomic DNA) with a hydrogel precursor solution containing, for example, monomers, cross-linking agents, and photoinitiators, and loads the solution onto flow cell 310. As shown in fig. 3C, the assembly 320 and cartridge 360 are then loaded into a housing 370 on which the ultraviolet light source has been mounted using a removable tray 372. Based on the layout or geometry of the photomask 318, the customer selects the appropriate illumination program and exposes the flow cell 310 to ultraviolet light to polymerize the solution and pattern the desired hydrogel structure on the flow cell 310. Fig. 3C includes a bright field micrograph of a hydrogel column 332 fabricated on a flow cell using the disclosed system and method. The flow cell 310 is then washed to remove unpolymerized solution and excess sample, and the photomask 318 may be removed from the flow cell 310. In one embodiment, more than half of each of the unpolymerized solution, excess sample, and photomask is removed. In one embodiment, all of each of the unpolymerized solution, excess sample, and photomask are removed. The flow cell 310 may then be placed into a sequencer or fluidic processor for automated downstream processing, such as lysis, labeling, bridge amplification, clustering, and the like.

Several other embodiments are provided regarding the assembly of a photomask and a flow cell. In one embodiment, the user first inserts the flow cell into the housing and then inserts the photomask separate from the flow cell (e.g., the photomask is not laminated to the flow cell). Because a variety of photomask patterns and designs are possible, a user may select different photomasks based on the desired pitch or based on the particular application or particular use of the flow cell. In this and other embodiments, the housing of the instrument is adapted to receive a plurality of different flow cells, including HiSeq^TM、NextSeq^TM、NovaSeq^TM、MiniSeq^TM、iSeq^TMAnd MiSeq^TMA flow-through cell, or other suitable flow-through cell available from Illumina, inc. In another embodiment, a flow cell is provided that is pre-assembled with a photomask that has been applied to the outer surface of the flow cell. Depending on the resolution, a photomask may be printed on the flow cell using screen printing, orThe photomask was laminated to the surface of the flow cell using an opaque adhesive film, the photomask being patterned to create structures on the flow cell. If desired, the photomask may be stripped from the flow cell after it has been used. In another embodiment, the photomask may be made of aluminum or another metal deposited within the fluid channels during the microfabrication process used to create the flow cell. Then, after the formation of the hydrogel structure on the flow cell is complete, the photomask may be etched away with a high pH buffer.

Cell compartmentalization and in situ sequencing library preparation

The disclosed systems and methods can have the beneficial effects of high throughput single cell or single colony sequencing by providing compartmentalization of biological cells (and genetic material contained therein) on a flow-through cell by encapsulating the single cells or single cell colonies in a reversible hydrogel structure that allows efficient exchange of reagents for cell lysis and sequencing library preparation. The following exemplary embodiments are used to achieve in situ library preparation and spatial indexing of clusters, including biological cell encapsulation, library preparation, library inoculation, and bridge amplification on flow-through cells. The flow cell is provided with two types of oligonucleotides (e.g., P5 and P7), called surface primers or sequencing primers, bound to the upper and lower surfaces of the flow cell. The sequences of these surface primers are complementary to the library adaptors, and fragments of the DNA library are captured by these oligonucleotides. As used herein, P5 and P7 refer to the universal P5 or P7 sequences or P5 or P7 primers used for capture purposes and/or amplification purposes. The P5 sequence includes the sequence defined by SEQ ID NO:1(AATGATACGGCGACCACCGA) and the P7 sequence includes the sequence defined by SEQ ID NO:2 (CAAGCAGAAGACGGCATACGA).

As used herein, "genetic material" refers to cells, microbial populations, or nucleic acids. In some versions, the cell is a single cell, including a prokaryotic cell or a eukaryotic cell. In some versions, the cell is a mammalian cell. In some versions, the cell is a human cell. In some versions, the cell is a bacterial cell. In some versions, the genetic material is a viral particle. In some versions, the nucleic acid is a long DNA molecule, genomic DNA, viral nucleic acid, bacterial nucleic acid, or mammalian nucleic acid. Any genetic material of interest can be encapsulated within the disclosed hydrogel structures.

Genetic material encapsulated with the disclosed hydrogel structures is of a size large enough that it is trapped within the hydrogel structure and cannot pass through the pores of the hydrogel structure. In some examples, the target nucleic acid molecule encapsulated within the hydrogel structure is at least about 100 nucleotides, at least about 150 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 5,000 nucleotides, at least about 10,000 nucleotides, at least about 20,000 nucleotides, at least about 50,000 nucleotides, at least about 100,000 nucleotides, or more nucleotides in length. In several examples, the nucleic acid molecule encapsulated within the hydrogel structure is a genomic DNA fragment having the following length: about 1,000 to about 10,000 nucleotides, about 10,000 to about 20,000 nucleotides, about 10,000 to about 50,000 nucleotides, about 50,000 to about 100,000 nucleotides, or about 300, about 500, about 1000, about 10,000, about 20,000, about 50,000, or about 100,000 nucleotides, or a range between any two of the foregoing dimensions, or a length longer than the foregoing dimensions. In some examples, the encapsulated nucleic acid molecule is at most about 3M bases in length.

Some versions of the disclosed systems and methods involve processing genetic material within a hydrogel structure to generate a sequencing library, which can be defined as a collection of fragments of one or more target nucleic acid molecules or amplicons of these fragments. In some versions, genetic material encapsulated within a hydrogel structure is contacted with one or more reagents for nucleic acid processing. In some versions, the genetic material is retained within the hydrogel structure and the agent passes through the pores of the hydrogel structure. The reagents may include lysis reagents, nucleic acid purification reagents, DNA amplification reagents, tagging reagents, PCR agents, or other reagents for processing genetic material (e.g., lysozyme, proteinase K, random hexamer, polymerase (e.g., Φ 29DNA polymerase, Taq polymerase, Bsu polymerase), transposase (e.g., Tn5), primers (e.g., P5 and P7 adaptor sequences), ligase, catalytic enzymes, deoxynucleotide triphosphates, buffers, or divalent cations Sequencing the encapsulated nucleic acids, such as direct sequencing, including sequencing-by-synthesis, sequencing-by-ligation, sequencing by hybridization, nanopore sequencing, and the like.

For cell encapsulation, as shown in fig. 4A, single cells or single colonies are mixed with a polymer precursor solution comprising a monomer, a cleavable crosslinker, and a photoinitiator. The solution containing the cells is then loaded into the flow-through cell and irradiated with ultraviolet light through a photomask in the manner previously described to produce an array of cell-embedded hydrogel structures (e.g., pillars) on the flow-through cell. Excess precursor solution is washed away to obtain clear interstitial spaces between the hydrogel structures. The combined bright field and fluorescence micrographs show the hydrogel structure with the e.coli (e.coli) cells encapsulated therein, and a bright field image of the hydrogel column formed on the flow cell appears at the bottom of fig. 4A. Alternatively, as shown in fig. 5A-5D, an array of hydrogel structures 532 having cell-trapping features formed therein may first be produced on the flow cell 510, and then single cells or colonies may be flowed over the cell-trapping hydrogel features 532 such that the single cells or colonies are trapped in the hydrogel features. Figure 5A depicts a side view of exemplary cell-trapping hydrogel features 532 attached to the upper surface 512 and lower surface 514 of the channel 516. Figure 5B is a top view of the cell-trapping hydrogel feature of figure 5A. Fig. 5C is a side view depicting an exemplary cell-trapping hydrogel array in which cells 550 have been trapped, and fig. 5D is a top view of one of the cell-trapping hydrogel features of fig. 5C, showing cells 550 trapped therein. As shown in fig. 5D, the hydrogel features 532 can include beveled edges and various channels and passageways formed therein to facilitate fluid flow through and around these features.

In one embodiment, for library preparation, as shown in figure 4B, the cleavage reagent and the labeling reagent are diffused into the hydrogel structure. Fine tuning or otherwise changing the pore size of the hydrogel may allow for optimal buffer exchange and efficient diffusion of reagents into and out of the hydrogel structure. Cells trapped in the hydrogel structure are lysed with an enzymatic lysis buffer or a chemical lysis buffer. The DNA released by lysis of the cells is then tagged. Tagging involves modification of the nucleic acid molecule by a transposome complex to fragment the nucleic acid molecule in a single step and ligating adaptors to the 5 'and 3' ends of these fragments. The tagging reaction can combine random DNA fragmentation and adaptor ligation into a single step to increase the efficiency of the sequencing library preparation process. Once adapters have been ligated to the fragments, additional motifs such as indexes, barcodes, and other kinds of molecular modifications that serve as reference points during amplification, sequencing, and analysis can be added. The index and barcode are unique DNA sequences that are ligated into fragments within the sequencing library for downstream computer sorting and identification. A bright field micrograph showing the hydrogel structure array appears at the bottom of fig. 4B.

As previously described, the adaptors may include sequencing primer sites, amplification primer sites, and indices. For example, the adaptor may include a P5 sequence, a P7 sequence, or the complement of either. As previously described, an "index" may include a nucleotide sequence that may be used as a molecular identifier and/or barcode to tag a nucleic acid and/or identify the source of the nucleic acid. In some versions, the index may be used to identify a single nucleic acid or a subpopulation of nucleic acids.

For library inoculation, as shown in fig. 4C, to inoculate libraries resulting from library preparation onto the top and bottom surfaces of a flow cell while maintaining spatial compartmentalization, a liquid diffusion barrier was introduced into the flow cell. The liquid diffusion barrier may contain a cleaving agent, such as DTT, that degrades the hydrogel structure. The temperature of the flow cell is raised and the hydrogel structure is cleaved to release the library fragments contained therein, which are then hybridized to surface primers attached to the flow cell surface at region 250. In this embodiment, the cleaved hydrogel is removed from the flow cell using a washing step with an aqueous buffer. A bright field micrograph showing the melted hydrogel structure in mineral oil appears at the bottom of fig. 4C.

The hydrogel structures are degraded while surrounded by a liquid diffusion barrier to release the sequencing library from these structures and to seed the sequencing library on a flow cell. A liquid diffusion barrier is loaded onto the flow cell to fill the void volume between and surround the hydrogel structures. Surrounding the captured hydrogel structures with a liquid diffusion barrier inhibits diffusion of the sequencing library out of the structure volume as the structures degrade, thereby reducing (and in some cases even preventing) cross-contamination between hydrogel structures. After structural degradation, the encapsulated sequencing library is transported to the surface of the flow cell where it is captured. Thus, seeding the flow-through cell occurs immediately adjacent to the footprint of each hydrogel structure in the presence of a liquid diffusion barrier. It should be noted that in certain embodiments where discrete compartmentalization of the library fragments generated within the hydrogel structure is desired, a diffusion barrier is used. However, in embodiments where compartmentalization is not required, a diffusion barrier may not be used. Thus, the diffusion barrier layer may be referred to as "optional".

In some examples, the liquid diffusion barrier layer may be a hydrophobic liquid, such as an oil, examples of which include mineral, silicone, or perfluorinated oils, or a combination of two or more thereof. In some examples, the liquid diffusion barrier layer is a viscous aqueous solution, e.g., comprising polyethylene glycol (PEG), polyvinylpyrrolidone, pluronic dextran, sucrose, poly (N-isopropylacrylamide) or polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEOyiaponite, or a combination of two or more thereof Laponite nanoparticle composites. In some examples, the liquid diffusion barrier layer used in the disclosed embodiments is comprised of a combination of any two or more of the liquid diffusion barrier layers discussed above.

The hydrogel structures can be degraded using any suitable method that does not substantially reduce the effectiveness of the liquid diffusion barrier to inhibit the diffusion of the sequencing library beyond the diameter of the hydrogel structure. The hydrogel structure does not need to be completely degraded to release the sequencing library from the hydrogel structure and the sequencing library is seeded on a flow cell. Sufficient degradation includes an increase in porosity of the hydrogel structure to allow diffusion of the encapsulated sequencing library and transport of the sequencing library to the surface of the flow cell.

For bridge amplification, as shown in fig. 4D, the hybridized library fragments are then clonally amplified using a bridge amplification method for generating clusters. During bridge amplification, the polymerase moves along the single-stranded DNA fragments (polynucleotides) bound to the flow cell, thereby producing its complementary polynucleotides. The original polynucleotide is washed away, leaving only the inverted polynucleotide. An adapter sequence (e.g., P5 or P7) is present on top of the inverted polynucleotide. The DNA fragments are bent and attached to oligonucleotides complementary to the top adaptor sequence on the flow cell surface. The polymerase attaches to the reverse polynucleotide and prepares its complementary polynucleotide (which is identical to the original polynucleotide). The now double stranded DNA is denatured so that each polynucleotide can be individually attached to an oligonucleotide sequence anchored to the flow cell. One will be the reverse strand; the other would be the forward chain. The process is then repeated iteratively, and may be repeated at the same timeIn millions of clusters, resulting in clonal amplification of all fragments in a DNA library. After bridge amplification, the resulting clusters 260 are positioned to the top and bottom surfaces of the flow cell where the hydrogel structures have been previously anchored. FIG. 4D bottom fluorescent micrograph showing the fluorescence intensity at the time of detection using SYTOX commercially available from ThermoFisher Scientific^TMThe presence of cluster 260 was sequenced after the dye staining.

Fig. 6 is a flow diagram depicting an exemplary embodiment of a method for producing a three-dimensional polymeric structure on a flow cell. The method 600 includes loading a polymer precursor solution into a flow cell at block 602, wherein the polymer precursor solution comprises a monomer, a crosslinker, and a photoinitiator, wherein the flow cell includes at least one channel for receiving the polymer precursor solution, and wherein the at least one channel has an upper interior surface and a lower interior surface; placing a photomask over the at least one channel, wherein the photomask comprises a series of openings formed therein, at block 604; and at block 606, irradiating the polymer precursor solution through the photomask with light of a wavelength sufficient to activate the photoinitiator. Activation of the photoinitiator polymerizes at least some of the polymer precursor solution below the openings in the photomask and forms a three-dimensional polymer structure extending from the upper interior surface to the lower interior surface of the at least one channel. In other embodiments, the photomask is integral with the flow cell, rather than being placed on or attached to it.

Fig. 7 is a flow diagram depicting another exemplary embodiment of a method for producing a three-dimensional polymeric structure on a flow cell. The method 700 includes loading a polymer precursor solution into a flow cell at block 702, wherein the polymer precursor solution comprises biological cells or biological cell colonies comprising genetic material, monomers, a cross-linking agent, and a photoinitiator, wherein the flow cell comprises at least one channel for receiving the polymer precursor solution, wherein the at least one channel has an upper interior surface and a lower interior surface, and wherein primers are bound to both the upper interior surface and the lower interior surface of the at least one channel; at block 704, placing a photomask over the at least one channel, wherein the photomask includes a series of openings formed therein; and at block 706, irradiating the polymer precursor solution through the photomask using light of a wavelength that activates the photoinitiator, wherein activation of the photoinitiator polymerizes at least some of the polymer precursor solution below the openings in the photomask and forms a three-dimensional polymer structure extending from an upper interior surface to a lower interior surface of the at least one channel; and wherein the biological cells or biological cell colonies are compartmentalized in the three-dimensional polymer structure. In other embodiments, the photomask is integral with the flow cell, rather than being placed on or attached to it.

Fig. 8 is a flow chart depicting yet another exemplary embodiment of a method for producing a three-dimensional hydrogel structure on a flow cell. The method 800 includes loading a hydrogel precursor solution onto a flow cell at block 802, wherein the hydrogel precursor solution comprises biological cells or biological cell colonies comprising genetic material, monomers, a cross-linking agent, and a photoinitiator, wherein the flow cell comprises at least one channel for receiving the polymer precursor solution, wherein the at least one channel has an upper interior surface and a lower interior surface, and wherein primers are bound to both the upper interior surface and the lower interior surface of the at least one channel; at block 804, placing a photomask over the at least one channel, wherein the photomask includes a series of openings formed therein; at block 806, irradiating the hydrogel precursor solution through the photomask with light of a wavelength that activates the photoinitiator, wherein activation of the photoinitiator polymerizes the hydrogel precursor solution below the openings in the photomask and forms a three-dimensional hydrogel structure extending from an upper interior surface to a lower interior surface of the at least one channel, and wherein the biological cells or biological cell colonies are compartmentalized in the three-dimensional hydrogel structure; at block 808, diffusing a lysing agent into the three-dimensional hydrogel structure, wherein the lysing agent lyses the biological cells and releases genetic material therefrom, and wherein the genetic material comprises nucleic acids; at block 810, fragmenting the released nucleic acids and ligating adaptors to ends of the nucleic acid fragments; at block 812, the nucleic acid fragments are seeded on the upper and lower surfaces of the channel by: introducing a diffusion barrier into the at least one channel at 814 to prevent cross-contamination between hydrogel structures, heating the flow cell to a temperature that cleaves the hydrogel structures and releases the nucleic acid fragments at 816, hybridizing the nucleic acid fragments to primers on upper and lower interior surfaces of the at least one channel at 818, and washing the cleaved hydrogel structures out of the flow cell at 820; and clonally amplifying the hybridized nucleic acids at block 822 to generate clusters for sequencing. In other embodiments, the photomask is integral with the flow cell, rather than being placed on or attached to it.

The methods and systems described herein provide certain benefits. Versions of the "spatial indexing" methods and techniques described herein shorten data analysis and simplify the process of preparing libraries from single cells and long DNA molecules. Existing protocols for single cell sequencing involve efficient physical separation of cells, and unique barcode labeling of each separated cell and pooling the cells together for sequencing. Current protocols for synthesizing long reads also involve cumbersome barcode labeling, pooling together each barcode labeled fragment for sequencing, and performing data analysis to distinguish the genetic information derived from each barcode labeled cell. During these long processes there is also a loss of genetic material, which leads to deletions in the nucleotide sequence. The versions described herein not only shorten the overall sequencing process, but also improve the data resolution of single cells.

The following non-limiting working examples are provided to show specific features of certain embodiments, but the scope of the claims should not be limited to those features illustrated.

Example 1: integration from genomic DNA on flow cellsLibrary preparation

This example demonstrates the sequencing of genomic DNA trapped in a hydrogel structure, where the library preparation by sequencing is integrated and performed directly on the flow cell.

A10% T hydrogel precursor solution was prepared from a 40% (w/v) acrylamide/N, N' -bis (acryloyl) cystamine (BACy) (19:1) monomer stock solution (3.8g acrylamide, 0.2g BACy, and 6mL double distilled (dd) H2O) with 1mg/mL LAP photoinitiator and E.coli genomic DNA (0.008 ng/. mu.L). Introduction of the solution into MiSeq^TMIn a flow cell and exposing the flow cell to collimated ultraviolet light (OAI mask aligner, power at about 30 mW/cm) through a chrome mask (HTA Photomasks) patterned with 200 μm circular features²To 40mW/cm²Within) to form a hydrogel structure.

The precursor solution containing excess genomic DNA was washed out with PR-2. The flow cell was incubated with the tagged enzyme solution at 55 ℃ for 15 minutes, followed by washing with PR-2 and incubation with the tagged stop buffer (10 minutes at 37 ℃). The flow cell was then washed with PR-2 and the AMS-1 enzyme was incubated at 50 ℃ for 5 minutes. The library was denatured by washing with 0.1M NaOH, followed by HT-2. The flow cell was incubated with HT-1 for 5 minutes, then loaded with mineral oil plus surfactant and DTT (312.5. mu.L mineral oil + 4.5% Span 80, 0.4% Tween 20, and 0.05% Triton X-100, and 0.5. mu.L 12mg DTT/400. mu.L EtOH). Inoculation was achieved by incubating the flow cell with temperature ramps of 60 ℃, 40 ℃ and 20 ℃.

The flow cell was then washed with HT-1 and the inoculated library was expanded with AMS-1 (maintained at 50 ℃ for 5 minutes). The remaining hydrogel was then thawed with CLM (40 ℃ for 5 minutes) and the flow cell was washed with PR-2. The flow cell was then inserted into a sequencer for bridge amplification (24 cycles) and sequencing. This method demonstrates that genomic DNA can be trapped within a hydrogel structure on a flow cell, and that library preparation and library sequencing can be performed directly on the flow cell.

Example 2: integration of library preparation on flow cell for minigenome sequencing

The following examples demonstrate the direct integration of microbial cell sequencing, from lysis and library preparation of microorganisms encapsulated in hydrogel structures on flow cells, to seeding, clustering and sequencing of library molecules.

A10% T hydrogel solution was prepared from a 40% (w/v) acrylamide/N, N' -bis (acryloyl) cystamine (BACy) (19:1) monomer stock solution (3.8g acrylamide, 0.2g BACy, and 6mL double distilled (dd) H2O) with a mixture of 1mg/mL LAP photoinitiator and 0.01M Tris/HCl and 10 microorganisms (ZYMICS microbiological Community Standard D6300) and introduced into MiSeq^TMIn the flow-through cell. The flow cell was exposed to collimated ultraviolet light (OAI mask aligner, power at about 30 mW/cm) through a chrome mask (HTA Photomasks) patterned with 200 μm circular features (OAI mask aligner)²To 40mW/cm²Within) to form a hydrogel structure.

Excess precursor solution was washed out with PR-2 and the microorganisms were lysed using the charge switch gDNA miniprep kit (Thermo Fisher CS 11301); the first incubation was performed with lysozyme and lysostaphin, followed by a second incubation with proteinase K. The flow cell was washed with PR-2 and the tagged enzyme solution was introduced and incubated at 55 ℃ for 15 minutes, followed by washing with PR-2 and incubation with the tagged stop buffer (10 minutes at 37 ℃). The flow cell was then washed with PR-2 and the AMS-1 enzyme was incubated at 50 ℃ for 5 minutes. The library was denatured by washing with 0.1M NaOH, followed by HT-2. The flow cell was incubated with HT-1 for 5 minutes, then loaded with mineral oil plus surfactant and DTT (312.5. mu.L mineral oil + 4.5% Span 80, 0.4% Tween 20, and 0.05% Triton X-100, and 0.5. mu.L 12mg DTT/400. mu.L EtOH). Inoculation was achieved by incubating the flow cell with temperature ramps of 60 ℃, 40 ℃ and 20 ℃.

The flow cell was then washed with HT-1 and the inoculated library was expanded with AMS-1 (maintained at 50 ℃ for 5 minutes). The remaining hydrogel was then thawed with CLM (40 ℃ for 5 minutes) and the flow cell was washed with PR-2. The flow cell was then inserted into a sequencer for bridge amplification (24 cycles) and sequencing. The method demonstrates that microorganisms can be trapped within hydrogel structures on a flow cell, and that genomic library preparation and library sequencing can be performed directly on the flow cell.

Example 3: integration of library preparation from mammalian cells on flow cells

The following example demonstrates the encapsulation, lysis, library preparation and sequencing of genetic material from mammalian cells on a flow cell.

A10% T hydrogel solution was prepared from a 40% (w/v) acrylamide/N, N' -bis (acryloyl) cystamine (BACy) (19:1) monomer stock solution (3.8g acrylamide, 0.2g BACy, and 6mL PBS) with 1mg/mL LAP photoinitiator and mammalian cells (GM12878 cells). Introduction of the solution into MiSeq^TMIn a flow cell and exposing the flow cell to collimated ultraviolet light (OAI mask aligner at a power of about 30 mW/cm) through a chrome mask (HTA Photomasks) patterned with 200 to 500 μm circular features²To 40mW/cm²Within) to form a hydrogel structure encapsulating the cell. The flow cell was then washed with PBS.

Cells were lysed with the Charge Switch lysis buffer and proteinase K (10 min, 50 ℃). The flow cell was washed with PR-2 and the tagged enzyme solution was added to the flow cell (55 ℃ for 15 minutes), followed by washing with PR-2 and incubation with the tagged stop buffer (37 ℃ for 10 minutes). The flow cell was washed with PR-2 and the AMS-1 enzyme was incubated at 50 ℃ for 5 minutes. The library was then denatured by washing with 0.1M NaOH, followed by HT-2 and incubation with HT-1 for 5 minutes. Flow cells were loaded with mineral oil plus surfactant and DTT (312.5. mu.L mineral oil + 4.5% Span 80, 0.4% Tween 20 and 0.05% Triton X-100 and 0.5. mu.L 12mg DTT/400. mu.L EtOH) and incubated at temperature ramps of 60 ℃, 40 ℃ and 20 ℃.

The flow cell was washed with HT-1, followed by incubation with AMS-1 (50 ℃ for 5 minutes). Any remaining hydrogel was cut with CLM (40 ℃ for 5 minutes) and the flowcell was washed with PR-2. Insert flow cell into MiSeq^TMUsed in the sequencer for bridge amplification (24 cycles) and subsequent sequencing. The method demonstrates hydraulic binding that mammalian cells can be trapped on a flow cellIntraframe, and genomic library preparation and library sequencing can be performed directly on the flow cell.

Example 4: integration of amplicon sequencing from genomic DNA on flow cell

This example demonstrates integrated amplicon sequencing on a flow cell, where genomic DNA is encapsulated in a hydrogel structure for subsequent amplification of the target region, addition of sequencing primers, inoculation, and sequencing.

Encapsulating genomic DNA in a hydrogel structure by: genomic DNA was first mixed with a 10% T hydrogel solution prepared from 40% (w/v) acrylamide/N, N' -bis (acryloyl) cystamine (BACy) (19:1) monomer stock solution (3.8g acrylamide, 0.2g BACy, and 6mL PBS) and 1mg/mL LAP photoinitiator. Introducing the hydrogel precursor solution into MiSeq^TMIn the flow-through cell. The flow cell was exposed to collimated ultraviolet light (OAI mask aligner, power at about 30 mW/cm) through a chrome mask (HTA Photomasks) patterned with 200 μm circular features (OAI mask aligner)²To 40mW/cm²Within) resulting in the formation of a hydrogel column comprising genomic DNA. Excess solution and DNA were washed out with PR-2.

10 μ L of 1 μ M oligonucleotide pair (forward and reverse primer pairs containing target sequence and Illumina adapter sequence overhangs) was mixed with 25 μ L of KAPA HiFi 2X mix (Roche) and 5 μ L of resuspension buffer and introduced into the flow cell. PCR was performed on a thermal cycler using the following procedure: the temperature was maintained at 92 ℃ for 5 minutes for 25 cycles: (i) 30 seconds at 92 ℃, (ii) 30 seconds at 55 ℃ and (iii) 2 minutes at 72 ℃ and 5 minutes at 72 ℃. The flow cell was then washed with PR-2.

Next, 8 cycles of PCR were run using the thermocycler program described in the previous paragraph, this time with 5 μ Ι _

Nextera XT primer

1, 5 μ Ι _ Nextera XT primer 2, 25 μ Ι _ KAPA HiFi 2X mix, 15 μ Ι _ PCR grade water. The flow cell was then washed with PR-2.

The library molecules were denatured by washing with 0.1M NaOH, followed by washing with HT-2 and incubation with HT-1 for 5 minutes. The flow cell was loaded with mineral oil plus surfactant and DTT (312.5. mu.L mineral oil + 4.5% Span 80, 0.4%Tween 20 and 0.05% Triton X-100 and 0.5. mu.L of 12mg DTT/400. mu.L EtOH) and incubated at 60 ℃, 40 ℃ and 20 ℃ temperature ramps. The flow cell was washed with HT-1, followed by incubation with AMS-1 (50 ℃ for 5 minutes). Any remaining hydrogel was cut with CLM (40 ℃ for 5 minutes) and the flowcell was washed with PR-2. The flow cell was then inserted into the MiSeq^TMUsed in the sequencer for bridge amplification (24 cycles) and subsequent sequencing. The method demonstrates that genomic DNA can be encapsulated within a hydrogel structure on a flow cell, and that subsequent target region amplification, sequencing primer addition, inoculation, and sequencing can be performed on the flow cell.

In embodiments disclosed herein, each individual hydrogel structure contains a sequencing library generated from genetic material or nucleic acids contained within the hydrogel structure. Thus, a sequencing library seeded from a single hydrogel structure corresponds to a nucleic acid encapsulated within that hydrogel structure. Because the seeding occurs in the coverage area on the flow cell immediately adjacent to each hydrogel structure, the seeded sequencing library from each structure is spatially separated (or "indexed") on the flow cell based on the location of the structure.

Three-dimensional nucleic acid sequencing

As discussed previously, the operational throughput of the currently most excellent Next Generation Sequencing (NGS) platform is determined by: (i) a two-dimensional nucleic acid cluster density; and (ii) the overall size of the effective surface area of the flow-through cell, both of which have reached practical limits of manufacture. Exemplary embodiments provide systems and methods that overcome these limitations by extending the surface on which sequencing can be performed from two dimensions to three dimensions, thereby providing a substantial increase in sequencing flow cell throughput and data generation.

By filling the flow-through cell (or other item, such as a capillary tube or miniaturized cuvette) with a material or three-dimensional structure that occupies the entire volume of the flow-through cell and supports cluster formation at a desired density throughout the entire flow-through cell volume, it is beneficial to increase flow-through cell sequencing throughput and data generation. Sequencing-by-synthesis or by another suitable method is then done, where cluster identification and base calling are performed by optically interrogating a series of stacked two-dimensional slices throughout the flow cell. An exemplary method for sequentially imaging individual two-dimensional slices throughout a flow cell includes: (i) using a confocal microscope capable of focusing on discrete two-dimensional slices of the flow-through cell and repeatedly measuring the same two-dimensional plane; and (ii) the use of light sheet illumination microscopy, which allows rapid imaging of three-dimensional volumes. In other embodiments, the three-dimensional substrate is imaged using multi-photon fluorescence, such as two-photon excited fluorescence (2PEF), or another multi-photon imaging technique, such as three-photon excited fluorescence (3PEF) or multi-harmonic generation (MHG). Multi-photon imaging is a similarly confocal excitation modality with similar segmentation capabilities that involves the use of pulsed laser light, but typically at Near Infrared (NIR) wavelengths, thereby greatly reducing potential light damage to the stroma and its contents.

In exemplary embodiments, three-dimensional clusters are created throughout a permeable hydrogel matrix, such as those described above, by attaching alkyne-linked capture primers (e.g., P5 and P7) to an acrylamide hydrogel matrix comprising poly (N- (5-azidoacetamidylpentyl) acrylamide-co-acrylamide) (PAZAM) containing an azide moiety using click chemistry. The azide-alkyne click reaction involves a copper-catalyzed reaction of the azide with the alkyne to form a 5-membered heteroatom ring: cu (i) catalyzed azido-alkyne cycloaddition (CuAAC). The azide-alkyne click reaction can be photoinitiated using cu (II) and a photoinitiator system (such as a type II photoinitiator system, e.g., camphorquinone), which can use 470nm blue light as an excitation source. The sequencing library containing the nucleic acid fragments to which the adaptors are ligated is then spread into a hydrogel matrix and clustered by using cluster amplification, bridge amplification or another suitable method. In some embodiments, the nucleic acid fragments are circularized after addition of adapters to produce nucleic acid "nanospheres. The permeability of the hydrogel allows enzymes and other reagents to diffuse into the hydrogel and perform nucleic acid amplification. As discussed in more detail below, the hydrogel matrix can be polymerized into various shapes and geometries, such as an array of pillars, or linear grooves, to facilitate exchange of reagents around and rapid diffusion into and out of the hydrogel matrix.

Sequencing flow cells are provided with two types of oligonucleotides (e.g., P5 and P7), referred to in the alternative as graft primers, capture primers, surface primers, or sequencing primers, that are bound to the upper and lower surfaces of the flow cell using hydrogel layers or other attachment methods. The sequences of these primers are complementary to the library adaptors, and fragments of the DNA library are captured by these oligonucleotides. As used herein, P5 and P7 refer to the universal P5 or P7 sequences or P5 or P7 primers used for capture purposes and/or amplification purposes. The P5 sequence includes the sequence defined by SEQ ID NO:1(AATGATACGGCGACCACCGA) and the P7 sequence includes the sequence defined by SEQ ID NO:2 (CAAGCAGAAGACGGCATACGA).

Fig. 9A and 9B depict a flow cell 1400 that includes an array 1402 of individual hydrogel posts 1404 formed within the flow cell using photolithography, such as the photolithography described above. These columns contained P5/P7 primers and supported the growth of cluster 1406 within the hydrogel matrix. FIG. 10A depicts a MiSeq^TM A hydrogel column 1410 fabricated within the flow cell, and fig. 10B-10F depict time series images showing fluorescent dye introduced into the flow cell, dye diffusion into the hydrogel column, and dye washout from the hydrogel column. Fig. 11-11B depict hydrogel beads 1412 doped with PAZAM containing P5/P7 primers. These beads were initially soaked with the sequencing library and then soaked in the ExAmp to create clusters throughout the three-dimensional volume of each bead. Fig. 12A-12B depict index-less sequencing, in which each hydrogel bead contains a cluster (1420, 1422, 1424, 1426) from the sample in which the bead is incubated. Hydrogel beads containing such clusters were loaded onto flow cells and sequenced. Beads from each sample type can be distinguished from each other using a variety of means, such as fluorophores embedded in the beads that are removed prior to sequencing.

Fig. 13A depicts a sequencing flow cell 1430 in which sequencing occurs in a two-dimensional network of clusters on the top interior surface 1440 and the bottom interior surface 1450, and in which the top interior surface 1440 and the bottom interior surface 1450 are separated by a known distance (e.g., 100 μm) along the Z-axis. Fig. 13B depicts a sequencing flow cell 1430 in which sequencing occurs on the top and bottom

interior surfaces

1440, 1450 and in a three-dimensional network of clusters in

regions

1442, 1444, and 1446 located between the top and bottom

interior surfaces

1440, 1450, and in which the top and bottom

interior surfaces

1440, 1450 are separated by a known distance (e.g., 100 μm) along the Z-axis. Advantages and benefits of the exemplary embodiment over existing NGS systems and methods include: (i) the flux of each flow cell is improved by more than 50 times; (ii) manufacturing a high-throughput flow-through cell that does not involve X, Y translation of the optical system; (iii) based on the utilization of the entire flow cell volume, sequencing reagents are more efficiently consumed, thereby reducing waste and improving the economic aspects of the sequencing process; and (iv) compatibility with most or all existing sequencing platforms. In some embodiments, the cluster location is identified during the first scan and X, Y, Z coordinates are assigned for subsequent scans. By using the "reference cluster map" generated during the first scan, the offset in the X and Y dimensions can be accounted for. Herein, the terms X, Y and Z, or X, Y, and Z axes, refer to a three-dimensional Cartesian coordinate system.

The number of clusters per two-dimensional plane and the number of two-dimensional slices that can be imaged within the flowcell can be used to calculate the flux for a given platform and flowcell size. The latter number is specific to the optical detection system of a particular platform and depends on the optical cross-sectional thickness (dz) along the Z dimension, which can be calculated using the following equation:

wherein λ_emFor the excitation wavelength, n is the refractive index of the sample, and NA is the numerical aperture. The calculated dz values obtained for the high Numerical Aperture (NA) plateau and the low NA plateau are summarized in table 1 below. Higher magnification objectives may have a higher NA (i.e., wider angles for collecting information), which also means better resolution in the Z direction (i.e., smaller dz values). In practice, the maximum resolution in the Z direction is about 1/3 to the latter, compared to the difference in the xy plane1/2. In addition, shorter wavelengths produce higher resolution.

Table 1: calculated optical thickness (dz) values for low and high NA plateaus。

Using the dz values and flow cell thickness, the number of optical sections (or individual two-dimensional slices) that can be accessed per flow cell and the potential data output can be extrapolated (see table 2 below). For example, HiSeq having an optical cross-sectional thickness of about 2 μm^TMThe system may allow 50 individual 2D slices to be imaged with a NextSeq with a large dz of about 10 μm^TMThe system may allow 10 optical cross-sections. Using this three-dimensional sequencing strategy and assuming a constant cluster density in three dimensions, NextSeq^TMThe flow cell yield can be increased from 120Gb to 600Gb, and HiSeq^TMThe throughput of the flow cell can be increased from 1460Gb to 22500 Gb. By using a thicker flow cell, the data density can be increased even more in order to maximize the cluster growth space in three dimensions.

Table 2: data output for low NA platform and high NA platform calculations。

Similar to confocal microscopy, Selective Planar Illumination Microscopy (SPIM), also known as light sheet microscopy, is an exemplary optical microscopy for imaging three-dimensional structures. There are various embodiments of light sheet microscopy, all of which employ a dual objective configuration. A first objective (typically low cost and low NA) is used for excitation; and a second higher NA objective lens is used to collect fluorescence emissions from the sample of interest (see fig. 14). 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 the excitation wavelength. This mode is commonly used to image living biological samples from cells to intact organisms up to several millimeters in size at speeds of hundreds of images per second with micron resolution. Fig. 14 depicts an exemplary SPIM setup 1460 in which excitation is delivered into a sample 1464 using a low NA objective lens 1462, and in which fluorescence emissions are collected by a high NA emission objective lens 1466. The beam may be shaped with a cylindrical lens to excite the entire fluorophore simultaneously, or the single beam may be raster scanned across the entire focal plane of the emission objective to create a complete image. Subsequent translation of the sample by the mechanical stage allows rapid volume imaging at high resolution.

As previously mentioned, the disclosed systems and methods provide materials and structures that occupy the entire volume of a flow cell and support cluster formation and enable three-dimensional sequencing. Suitable materials are: (i) occupies the entire height of the flow cell channel; (ii) allows incorporation of oligonucleotides by various polymerization strategies or by the presence of available functional groups (e.g., azides); (iii) functional groups having a controllable density for controlling cluster density; (iv) supporting the flow of reagents with a minimal diffusion gradient; and (v) support confocal microscopy with minimal scattering over the entire depth of the flow cell. Examples of suitable materials include hydrogel networks of predetermined dimensions; a matrix of large particles, small particles, or a combination of large and small particles (e.g., same size particles or different size particles); periodic columnar pillars; and a mesoporous crystalline material. FIG. 15 depicts a large hydrogel network 1000 within a sequencing flow cell; FIG. 16 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. 17 depicts periodic columnar pillars 1200 within a sequencing flow cell; and fig. 18 depicts a mesoporous or microporous crystalline material 1300 within a sequencing flow cell. In some embodiments, the three-dimensional matrix may be produced from silk fibroin or polymer fibers (such as cellulose/cellulose products) and their constructs (e.g., paper) upon which tufts may be formed.

A large hydrogel network (such as that shown in fig. 15) can be constructed by polymerizing functional hydrogels within a flow-through cell to form a continuous polymer network with uniformly distributed functional groups that support cluster formation. In one embodiment, PAZAM is reacted with DBCO functionalized PEG. The density of the network can be controlled by adjusting the concentration of PAZAM and the relative concentration of PAZAM: DBCO-PEG to optimize the density of functional groups and the diffusion properties of the hydrogel. The method uses a soft polymer network as a three-dimensional scaffold with nucleic acid anchoring points defining the positions at which clusters are located within a hydrogel matrix. Fig. 19A-14D depict exemplary embodiments of methods of forming a hydrogel within a flow-through cell by polymerizing PAZAM + di DBCO-PEG.

A three-dimensional matrix of solid or porous particles (such as that shown in fig. 16) provides a robust network in which sequencing reactions occur on the surface of the particles or inside the particles and diffusion of reagents occurs in interstitial regions between the particles. The clusters are located on the surface of the particle or throughout the particle. In this embodiment, the particles are designed to provide optimal surface area, modulus, and optical clarity. Examples include porous hydrogel beads (e.g., acrylamide gel), solid polymer particles (e.g., polystyrene), polymer core shells, and inorganic materials (e.g., silica particles); all of these have grafting primers on their surface and/or throughout their three-dimensional structure.

Referring to fig. 20, in one exemplary embodiment, hydrogel beads with oligonucleotides are fabricated using simple droplet generation in combination with copolymerization of acrylamide and acrylamide-based modified oligonucleotides (commercially available from Integrated DNA Technologies, inc.). Fig. 20 depicts the copolymerization of acrylamide and acrylamide-based modified oligonucleotides into large polyacrylamide beads. Since the hydrogel beads are slightly larger in size (-120 μm) than the height between the top and bottom surfaces of a typical flow cell (e.g., 100 μmm), the hydrogel beads can be tightly packed and trapped within the flow cell channels without any form of chemical attachment thereto. Fig. 21A is a bright field micrograph depicting hydrogel beads on a slide; and FIG. 21B is a bright field microscopic image depicting the stacking in HiSeq^TMHydrogel beads within the flow cell. To demonstrate that reagents can readily diffuse into and out of porous hydrogels made by the disclosed methodsGel beads, complementary strands labeled with a dye are hybridized to oligonucleotide-labeled acrylamide beads to detect fluorescence throughout the hydrogel matrix. Control standard acrylamide beads that were not grafted with oligonucleotides did not show a fluorescent signal when incubated with the complementary dye-labeled strand, confirming that the signal detected for the oligonucleotide-modified acrylamide beads was driven by hybridization and that oligonucleotides can remain within the beads in the absence of hydrogel-bound complementary strand. FIG. 22A is a fluorescence micrograph of standard acrylamide beads after incubation with a dye-labeled complementary strand; and figure 17B is a fluorescence micrograph of the oligonucleotide-modified acrylamide beads after incubation with the dye-labeled complementary strand.

This embodiment can be easily converted to a simple method for performing long reads, where the hydrogel beads with grafted primers encapsulate long DNA library fragments (-100 kb) and act as reaction vessels for enzymatic processes (such as tagging, ligation, and clustering) to generate unique, spatially isolated linked read clusters. The internally tagged and ligated DNA fragments bind to the P5 primer and P7 primer distributed within the hydrogel beads to allow bridge amplification assisted clusters to form throughout the three-dimensional hydrogel structure and unique spatial barcodes to be formed. FIG. 23A depicts a hydrogel bead in which long DNA fragments have been encapsulated to be captured within a flowcell; FIG. 23B depicts an enzymatic process for library preparation performed within the trapped hydrogel beads of FIG. 23A; and figure 23C depicts an amplified library that produces clusters of linked reads distributed three-dimensionally within each hydrogel bead.

Certain embodiments utilizing a three-dimensional matrix of solid or porous particles include particles having complex physical and chemical structures outside the flow cell that are directed into the flow cell and immobilized in the flow cell by cross-linking. Clonal amplification of a nucleic acid library can initially occur on beads outside of the flow cell, which 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 the methods previously described to form a scaffold within the flow-through cell. Figure 24A depicts template capture and extension occurring on a hydrogel bead carrying oligonucleotides; and figure 24B depicts clonal amplification of library inserts on hydrogel beads, which were used to generate clusters. Figure 25A depicts clustered beads delivered into a flow cell in a hydrogel precursor solution; and fig. 25B depicts the immobilization of clustered beads within a cross-linked hydrogel matrix for maintaining the spatial position of the beads in three dimensions during sequencing and subsequent imaging.

By using external hydrogel bead preparation, spatially co-localized regions of orthogonal linearization chemistry can be prepared in solution by using specific bead designs. Such beads can then be delivered into a flow cell and used for simultaneous three-dimensional forward/reverse strand sequencing. This is achieved using hydrogel particles with spatially separated oligonucleotide primers that have unique linearization chemistry. Particle dimers with different surface chemistries have been demonstrated using continuous flow reactions where precursor particles are synthesized and functionalized prior to dimerization. Dimeric particles may be used for spatially linked three-dimensional forward and reverse reads. Figure 26 depicts dimer particles with different orthogonal linearization chemistries; and figure 27 depicts a prior art system for synthesizing similar dimer particles.

Some embodiments of the disclosed systems and methods provide a functional three-dimensional scaffold by utilizing columnar columns (such as those depicted in fig. 17) that extend in a vertical direction from the bottom of the flow-through cell to the top of the flow-through cell. These pillar-like pillars may be fabricated using top-down microfabrication techniques, such as photolithography; depositing a thin film; and selective etching. The surface of the pillars may be functionalized using the previously described methods, including PAZAM, for providing a rigid network of chemically active pillars, while allowing liquid flow and chemical diffusion to occur throughout the interstitial regions between the pillars. Certain embodiments utilize pillars fabricated to have alternating material compositions in the Z-direction. Such pillars can be selectively functionalized on the surface of one of the two materials, allowing control of the spatial distribution of the clusters in the Z direction to limit polyclonality and facilitate optical imaging. In this embodiment, the manifold of Z-slices used for optical interrogation is organized in space in a systematic manner. Fig. 28A and 28B depict spatial control of clusters in three dimensions using a three-dimensional matrix with columnar pillars of alternating material composition in the Z-direction.

Some embodiments utilize microporous crystalline materials to create a scaffold on a flow cell (such as depicted in fig. 18). Microporous crystalline materials have a well-defined structure comprising pores that are ordered and aligned in one direction. Thus, these materials essentially provide multiple fluid channels, with each well representing a single fluid channel. The surface of many porous materials can be functionalized; thus, microporous materials with aligned pores can be used as a matrix for three-dimensional sequencing, where chemical reactions occur on the walls of the pores. Referring to fig. 18, the directions of both fluid flow and optical imaging occur in the Z-direction, as viewed down the long axis of the bore. Microporous silicon is one example of a material that can be fabricated with pre-oriented pores of controlled size. The lateral dimensions and thickness of the microporous silicon membrane are easily controlled by the choice of precursor wafer, and the flow cell can be prepared by mounting the microporous silicon membrane onto a separate fluidic unit.

Some embodiments utilize a polymer scaffold for three-dimensional sequencing on a flow cell. Fig. 29A-29D depict a simplified exemplary method for producing a polymer scaffold in which unpolymerized monomer solution is embedded with salt particles having a predetermined size distribution. The salt particles displace the monomers, creating a three-dimensional network within the solution. The monomer solution is polymerized to form a three-dimensional polymer scaffold surrounding the salt particles, and the salt particles are dissolved, thereby creating a random three-dimensional array of pores that define the scaffold. Such stents may be activated and coated with a hydrogel (such as PAZAM). Although such stents need not be of an equally spaced multi-layered structure (such as those previously described), a suitable imaging strategy will image the entire stent, and image processing will then be used to identify the different clusters. The salt particles used in this exemplary embodiment may be doped with passivated metal particles. After dissolution of the salt particles, these particles will remain in a fixed position in the scaffold. During imaging, these particles can be used to provide a point with which a cluster can be aligned, thereby essentially serving as a reference point.

FIG. 30 is a flow chart depicting an exemplary method for making a permeable three-dimensional substrate on a flow cell. The method 2500 includes embedding an oligonucleotide within a permeable three-dimensional matrix at block 2502; and introducing the permeable three-dimensional oligonucleotide-containing matrix into a flow cell at block 2504, wherein the flow cell comprises at least one channel for receiving the permeable three-dimensional oligonucleotide-containing matrix.

FIG. 31 is a flow chart depicting a first exemplary method for three-dimensional sequencing of a nucleic acid library. Method 2600 includes loading a polymer precursor solution into the flow cell at block 2602, wherein the polymer precursor solution comprises monomers and oligonucleotides; at block 2604, polymerizing the polymer precursor solution to create a permeable three-dimensional matrix within the flow cell; at block 2606, diffusing a sequencing library into the permeable three-dimensional polymer matrix, wherein the sequencing library comprises nucleic acid fragments; diffusing an enzyme and a reagent into the permeable three-dimensional polymer matrix at block 2608; at block 2610, hybridizing the nucleic acid fragments to the oligonucleotides in the permeable three-dimensional polymer matrix; at block 2612, clonally amplifying the hybridized nucleic acid fragments to generate clusters for sequencing within the permeable three-dimensional polymer matrix; at block 2614, sequencing the clusters within the permeable three-dimensional polymer matrix; and at block 2614, optically imaging the sequenced clusters within the three-dimensional matrix in a plurality of discrete two-dimensional slices to characterize the sequencing library, wherein the plurality of discrete two-dimensional slices represent an entire three-dimensional internal volume of the flow cell.

FIG. 32 is a flow diagram depicting a second exemplary method for three-dimensional sequencing of a nucleic acid library. The method 2700 includes loading a polymer precursor solution into the flow cell at block 2702, wherein the polymer precursor solution comprises a monomer, a crosslinker, a photoinitiator, and an oligonucleotide; at block 2604, polymerizing the polymer precursor solution using ultraviolet light to create a permeable three-dimensional matrix within the flow cell; at block 2606, diffusing a sequencing library into the permeable three-dimensional polymer matrix, wherein the sequencing library comprises nucleic acid fragments; diffusing an enzyme and a reagent into the permeable three-dimensional polymer matrix at block 2608; at block 2610, hybridizing the nucleic acid fragments to the oligonucleotides in the permeable three-dimensional polymer matrix; at block 2612, clonally amplifying the hybridized nucleic acid fragments to generate clusters for sequencing within the permeable three-dimensional polymer matrix; at block 2614, sequencing the clusters within the permeable three-dimensional polymer matrix; and at block 2616, imaging the sequenced clusters within the three-dimensional matrix in a plurality of discrete two-dimensional slices using a confocal microscope or a light sheet illumination microscope to characterize the sequencing library, wherein the plurality of discrete two-dimensional slices represent an entire three-dimensional internal volume of the flow-through cell.

Functionalization of hydrogel structures on flow cells

Various embodiments of the disclosed systems, devices, and methods may be used to create a reversible hydrogel structure within a fluidic channel on a flow cell that may be used to introduce temporary functional surfaces within the flow cell in addition to pre-existing sequencing surfaces. These temporary functional surfaces can be used in a variety of applications including, for example: (i) enriching target DNA; (ii) clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) screening; and (iii) highly multiplexed screening applications using DNA-conjugated antigens. Using the methods disclosed herein (including those discussed above), hydrogel microcolumns decorated with streptavidin moieties are fabricated on flow-through cells. As discussed in more detail below, biotinylated capture oligonucleotides bind to streptavidin and immobilize the target library molecules to a hydrogel structure for library enrichment on a flow-through cell. Similarly, proteins and oligonucleotides can be attached to hydrogel columns using biotin-streptavidin linkages to enable a variety of other screening processes, such as CRISPR screening. The disclosed embodiments provide a much larger surface area for the binding reaction due to the porous nature of the hydrogel and allow the entire volume of the flow cell to be screened for binding events rather than just the surface, resulting in higher binding capacity and reaction rates.

An exemplary embodiment is shown in fig. 33, which depicts the formation of hydrogel micropillars on a channel within a flow cell using the methods described previously for fabricating hydrogel microstructures on flow cells. In fig. 33, the flow cell 400 is shown inserted into a cassette 460. An array 450 of individual hydrogel micropillars 452 has been formed within the channel 422 and is visible in the bright field micrograph at the bottom of fig. 33. In an exemplary embodiment, the hydrogel micropillars 452 are formed by copolymerization of acrylamide monomer with N, N' -bis (acryloyl) cystamine crosslinker. Control of the spatial patterning is achieved by initiating the polymerization reaction using the photoinitiator lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (LAP) by directing ultraviolet light through a photomask that has been positioned over channels 422 on flow cell 400. Fig. 34A-34C depict the fabrication of a hydrogel micropillar 452 on a flow cell, where a hydrogel precursor solution containing acrylamide and crosslinker monomers and a photoinitiator is introduced into the flow cell 400 (fig. 34A) and then exposed to ultraviolet light through a photomask 418 (fig. 34B) that has been pre-patterned with desired features (e.g., openings with a particular geometry) to form a hydrogel column (fig. 34C). In fig. 34A to 34C, the flow cell 400 includes a narrow channel 422 in which hydrogel micro-pillars 452 are formed and a wide channel 424. Hydrogel micropillars 452 are attached to both the upper surface 412 and the lower surface 414 of the narrow channel 422.

The flow cell 400 is provided with two types of oligonucleotides (e.g., P5 and P7), referred to as surface primers or sequencing primers, bound to the upper and lower surfaces of the flow cell. The sequences of these surface primers are complementary to the library adaptors, and fragments of the DNA library are captured by these oligonucleotides. As used herein, P5 and P7 refer to the universal P5 or P7 sequences or P5 or P7 primers used for capture purposes and/or amplification purposes. The P5 sequence includes the sequence defined by SEQ ID NO:1(AATGATACGGCGACCACCGA) and the P7 sequence includes the sequence defined by SEQ ID NO:2 (CAAGCAGAAGACGGCATACGA).

Example 5: PAZAM conjugated biotin

In the exemplary embodiment shown in fig. 35A-36E, a functionalized reversible hydrogel structure is formed within the channel on the flow-through cell by utilizing poly (N- (5-azidoacetamidylpentyl) acrylamide-co-acrylamide) (PAZAM) into which an azide moiety has been incorporated, and an azide-alkyne click reaction. The azide-alkyne click reaction involves a copper-catalyzed reaction of the azide with the alkyne to form a 5-membered heteroatom ring: cu (i) catalyzed azido-alkyne cycloaddition (CuAAC). The azide-alkyne click reaction can be photoinitiated using cu (II) and a photoinitiator system (such as a type II photoinitiator system, e.g., camphorquinone), which can use 470nm blue light as an excitation source.

In this exemplary embodiment, a UV mediated copolymerization of acrylamide, a crosslinking agent, and PAZAM, into which an azide moiety has been incorporated, is first used, in MiSeq^TMA hydrogel microcolumn is fabricated within a flow cell (or any other suitable flow cell). The biotin-polyethylene glycol (PEG) -alkyne complex is then clicked onto the azide moiety of PAZAM and used to bind streptavidin. The multiple binding sites of streptavidin then allow the immobilized streptavidin to immobilize an oligonucleotide capture probe that in turn hybridizes to the sequencing library molecule of interest introduced into the flow cell. Any unhybridized library fragments were washed out of the flow cell. The bound sequencing library fragments are then eluted from the hydrogel mini-column located in a narrow channel (non-sequencing region) of the flow cell and seeded in a wide channel (sequencing region) of the flow cell in preparation for amplification, clustering, sequencing-by-synthesis or sequencing by another method.

Fig. 35A-35E depict an exemplary embodiment of a method for fabricating a functionalized hydrogel structure on a flow cell. In FIG. 35A, a hydrogel precursor solution containing 10% Polyacrylamide (PA), a cross-linking agent, and 0.25% PAZAM into which an azide moiety has been incorporated is loadedOnto the flow cell 400. In fig. 35A, flow cell 400 is a MiSeq with both narrow channels 422 and wide channels 424^TMA flow-through cell. In fig. 35B, a photomask 418 comprising a plurality of 200 μm openings formed therein is placed over the flow cell 400, and the flow cell is then exposed to ultraviolet light for 10 seconds to copolymerize acrylamide and PAZAM and form an array 450 of azide-functionalized hydrogel micropillars 452 in the narrow channel 422. In fig. 35C, 5 μ M biotin-PEG-alkyne was clicked onto the azide moiety during Blackpool incubation. In FIG. 35D, streptavidin labeled with fluorescein (1:500) binds to biotin in the hydrogel micropillars 452. In fig. 35E, streptavidin is bound to biotinylated capture oligonucleotides to enable immobilization of target sequencing library molecules that have been tagged with a sequence complementary to the sequence of the capture oligonucleotides during library preparation.

Fig. 36A-36F are a series of bright field and fluorescence micrographs (488nm excitation) depicting fluorescein-streptavidin staining of biotinylated hydrogel micropillars. FIG. 36A is a 4X bright field micrograph of the PA/PAZAM control (no biotin). FIG. 36B is a 4X bright field micrograph of PA/PAZAM plus Blackpool. FIG. 36C is a 4X fluorescence micrograph of the PA/PAZAM control (no biotin) after five minutes of reaction time. FIG. 36D is a 4X fluorescence micrograph of PA/PAZAM plus Blackpool after five minutes reaction time. FIG. 36E is a 4X fluorescence micrograph of the PA/PAZAM control (no biotin) after ten minute reaction time at 40 ℃. FIG. 36F is a 4X fluorescence micrograph of PA/PAZAM plus Blackpool after ten minute reaction time at 40 ℃. Fig. 36D and 7F show binding of fluorescein-labeled streptavidin to a biotin-conjugated hydrogel microcolumn. In control experiments (fig. 36C and fig. 36E), the polyacrylamide/PAZAM microcolumns without biotin did not bind to fluorescein-labeled streptavidin. These figures clearly show that in this particular example, the biotin-conjugated (functionalized) hydrogel microcolumn effectively binds the target of interest, i.e., fluorescein-labeled streptavidin.

Example 6: streptavidin-acrylamide copolymer

In the exemplary embodiment shown in fig. 37A-37D, a functionalized reversible hydrogel structure is formed within a channel on a flow-through cell by photopolymerization of an acrylamide monomer, a crosslinker, and a streptavidin-labeled acrylamide monomer. The streptavidin functional group of the hydrogel binds to biotinylated capture oligonucleotides, which in turn hybridize to the sequence library molecules of interest introduced into the flow cell. Unhybridized library fragments were washed out of the flow cell. The bound sequencing library fragments are then eluted from the hydrogel mini-column located in a narrow channel (non-sequencing region) of the flow cell and seeded in a wide channel (sequencing region) of the flow cell in preparation for amplification, clustering, sequencing-by-synthesis or sequencing by another method.

In fig. 37A, a hydrogel precursor solution containing 10% Polyacrylamide (PA) and 0.25% streptavidin-labeled acrylamide monomers is loaded onto a flow cell 400. In fig. 37A, flow cell 400 is a MiSeq with both narrow channels 422 and wide channels 424^TMA flow-through cell. In fig. 37B, a photomask 418 comprising a plurality of 200 μm openings formed therein is placed over the flow cell 400, and the flow cell is then exposed to ultraviolet light for 10 seconds to copolymerize the acrylamide streptavidin labeled acrylamide monomer and form an array 450 of streptavidin functionalized hydrogel micropillars 452 in the narrow channels 422. In fig. 37C, biotinylated capture oligonucleotides are bound to streptavidin moieties in the hydrogel structure, and target library molecules are hybridized to the biotinylated capture oligonucleotides and immobilized on hydrogel microposts. In fig. 37D, the immobilized target molecules are eluted from the capture oligonucleotides and seeded on the wide channel 424 of the flow cell 400 for amplification, clustering, sequencing-by-synthesis, or sequencing by another method.

Referring to fig. 38A to 38C, streptavidin on the surface of the hydrogel column 452 can be detected by incubation with biotinylated primers P5 and P7. Fig. 38A depicts biotinylated P5 primer and P7 primer (908 and 906, respectively) bound to streptavidin-functionalized hydrogel microposts 452. FIG. 38B depicts biotinylated P5 primer and P7 primer (908 and 906, respectively) incubated with TET-labeled complementary P5 'and P7' oligonucleotides (909 and 907, respectively). FIG. 38C depicts TET-labeled complementary P5 'and P7' oligonucleotides (909 and 907, respectively) hybridized to biotinylated P5 and P7 primers (908 and 906, respectively). Control hydrogel minicolumns made without streptavidin showed no staining with TET-labeled primers, whereas streptavidin-containing columns showed staining with TET-labeled primers.

FIG. 39A is a bright field micrograph showing hydrogel micropillars 452 incubated with TET-P5 'and TET-P7' in the absence of biotin-P5 oligonucleotide and biotin-P7 oligonucleotide. FIG. 39B is a fluorescence micrograph (488nm excitation) showing hydrogel micropillars 452 incubated with TET-P5 'and TET-P7' in the absence of biotin-P5 oligonucleotide and biotin-P7 oligonucleotide, where uniform staining of the P5 primer and P7 primer on the flow cell surface was observed. FIG. 39C is a bright field micrograph showing hydrogel micropillars 452 incubated with TET-P5 'and TET-P7' after incubation with biotin-P5 oligonucleotide and biotin-P7 oligonucleotide. FIG. 39D is a fluorescence micrograph (488nm excitation) showing hydrogel microposts 452 incubated with TET-P5 'and TET-P7' after incubation with biotin-P5 and biotin-P7 oligonucleotides, where TET staining was observed to localize to the edges of the hydrogel microposts, indicating that the TET labeled oligonucleotides had hybridized to the streptavidin-conjugated biotinylated P5 and P7 primers.

When TET-P5 'oligo/TET-P7' oligo was incubated with streptavidin-containing microcolumns previously incubated with biotin-P5 oligo and biotin-P7 oligo, depletion of TET-labeled primers was observed in the interstitial spaces between the microcolumns on the flow-through cell as fluorescence increased at the surface of the microcolumns and penetrated the hydrogel. Fig. 40A is a fluorescence micrograph depicting the level of TET-P5 'oligonucleotide/TET-P7' oligonucleotide in the interstitial space between hydrogel micropillars at an incubation time of one minute, and fig. 40B is a graph depicting the level of TET-P5 'oligonucleotide/TET-P7' oligonucleotide in the interstitial space between hydrogel micropillars at an incubation time of one minute, with distances shown on the X-axis and levels shown on the Y-axis. Figure 40C is a fluorescence micrograph depicting the level of TET-P5 'oligonucleotide/TET-P7' oligonucleotide in the interstitial space between hydrogel micropillars at an incubation time of five minutes, and figure 40D is a graph depicting the level of TET-P5 'oligonucleotide/TET-P7' oligonucleotide in the interstitial space between hydrogel micropillars at an incubation time of five minutes, with distances shown on the X-axis and levels shown on the Y-axis. Figure 40E is a fluorescence micrograph depicting the level of TET-P5 'oligonucleotide/TET-P7' oligonucleotide in the interstitial space between hydrogel micropillars at an incubation time of ten minutes, and figure 40F is a graph depicting the level of TET-P5 'oligonucleotide/TET-P7' oligonucleotide in the interstitial space between hydrogel micropillars at an incubation time of ten minutes, with distances shown on the X-axis and levels shown on the Y-axis. The fluorescence intensity distribution represented by these micrographs demonstrates that binding of TET-labeled oligonucleotides to the hydrogel surface depletes the oligonucleotides in the interstitial regions between the micropillars.

Referring to fig. 41A to 41C, to demonstrate the ability to capture and release target library molecules using the above exemplary embodiment, the PhiX library was incubated with biotinylated P5 primer and P7 primer (1: 10). Figure 41A depicts the hybridization of the P7 'and P5' regions (902 and 904, respectively) of the sequencing library molecules to biotinylated P5 and P7 oligonucleotides (906 and 908, respectively). Fig. 41B depicts capture of

sequencing library molecules

902 and 904 with streptavidin-functionalized hydrogel column 452 attached to the surface of flow cell 400. Figure 41C depicts the inoculation of bound

sequencing library molecules

902 and 904 in the following manner: incubation at 85 ℃ denatures hybridized

biotinylated primers

906 and 908, and then heating to 20 ℃ allows

library molecules

902 and 904 to hybridize to surface

primers

510 and 912, respectively. The hybridized PhiX library (1pM) was then incubated in either an untreated flow cell (control) or a streptavidin column patterned flow cell. After incubation with library molecules hybridized to biotin-primers, the flow-through cell was washed with HT-1, followed by inoculation (5 min at 80 ℃, 5 min at 60 ℃,2 min at 40 ℃ and 2 min at 20 ℃), first extension (AMS-1, 5 min at 50 ℃) and 24 bridge amplification cycles. Although the control flow cell did not show clusters (see fig. 42A-42B), the streptavidin-column flow cell showed a high cluster density (see fig. 42C-42F), demonstrating successful capture of the library hybridized with biotin-P5/biotin-P7.

Fig. 42A is a bright field micrograph of an untreated flow cell (control) and fig. 42B is a fluorescence micrograph (488nm) of a sytox (thermofisher scientific) stained untreated flow cell (control), with clusters not shown. Fig. 42C is a bright field micrograph of a flow cell with streptavidin micropillars, and fig. 42D is a fluorescence micrograph (488nm) of a SYTOX-stained flow cell with streptavidin micropillars. Fig. 42E is a bright field micrograph of the hydrogel micropillars of fig. 42C. FIG. 42F is a fluorescence micrograph (488nm) of the SYTOX stained microcolumn of FIG. 42D. Fluorescence micrographs of SYTOX stained flow cells showed that while the untreated flow cell did not show clusters, the flow cell with the streptavidin-hydrogel column exhibited a high cluster density and a column "footprint" in which the hydrogel micropillars had been patterned. Furthermore, when only one channel (narrow channel) of a flow cell is patterned with a streptavidin column, the clusters show a density gradient within the same flow cell, wherein the cluster density is higher close to the column. Fig. 43A depicts a flow cell 400 in a cassette 460 in which streptavidin micropillars have been formed in narrow channels 422, but not in wide channels 422. When the streptavidin column was only in MiSeq^TMWhen patterned in narrow channels of a flow-through cell, the cluster density forms a gradient from high to low from close to the micropillars (narrow channels) to far from the micropillars (wide channels). Fig. 43B is a micrograph of a wide channel 424 stained with a SYTOX dye after 24 cycles of bridge amplification, and fig. 43C is a micrograph of a narrow channel 422 stained with a SYTOX dye after 24 cycles of bridge amplification. The following description provides some additional examples related to the methods provided herein. They are not necessarily part of the non-limiting working examples provided above.

Fig. 44 is a flow diagram depicting a first method for preparing a functionalized three-dimensional polymer structure on a flow-through cell. The method 1500 includes loading a polymer precursor solution into a flow cell at block 1902, wherein the polymer precursor solution comprises a monomer, a crosslinking agent, a photoinitiator, and a functionalized polymer, such as PAZAM containing an azide moiety, and wherein the flow cell includes at least one channel for receiving the polymer precursor solution, and wherein the at least one channel has an upper interior surface and a lower interior surface; at block 1904, a photomask is placed over the at least one channel, wherein the photomask comprises a series of openings formed therein; and irradiating the polymer precursor solution through a photomask with a light source at block 1906, wherein the light source emits light at a wavelength that activates the photoinitiator, and wherein activation of the photoinitiator polymerizes the polymer precursor solution below the opening in the photomask and forms a three-dimensional polymer structure extending from an upper interior surface to a lower interior surface of the at least one channel.

Fig. 45 is a flow diagram depicting a second method for preparing a functionalized three-dimensional polymer structure on a flow-through cell. The method 1600 includes loading a polymer precursor solution into a flow cell at block 1602, wherein the polymer precursor solution comprises a monomer, a cross-linking agent, a photoinitiator, and a PAZAM containing an azide moiety, and wherein the flow cell includes at least one channel for receiving the polymer precursor solution, and wherein the at least one channel has an upper interior surface and a lower interior surface; placing a photomask over the at least one channel at block 1604, wherein the photomask comprises a series of apertures formed therein; irradiating the polymer precursor solution through a photomask with a light source at block 1606, wherein the light source emits light at a wavelength that activates the photoinitiator, and wherein activation of the photoinitiator polymerizes the polymer precursor solution under the openings in the photomask and forms a three-dimensional polymer structure extending from an upper interior surface to a lower interior surface of the at least one channel; reacting the biotin-PEG-alkyne complex with the azide moiety in the PAZAM in the three-dimensional polymer structure using an azide-alkyne click reaction at block 1608; binding streptavidin to biotin in the biotin-PEG-alkyne complex at block 1610; and binding biotinylated capture oligonucleotides to streptavidin at block 1612, wherein the biotinylated capture oligonucleotides are specific for the target molecules of interest in the sequencing library.

Fig. 46 is a flow diagram depicting a third method for preparing a functionalized three-dimensional polymer structure on a flow cell. The method 1700 includes loading a polymer precursor solution into a flow cell at block 1702, wherein the polymer precursor solution comprises a monomer, a crosslinker, a photoinitiator, and a streptavidin-labeled acrylamide monomer, and wherein the flow cell includes at least one channel for receiving the polymer precursor solution, and wherein the at least one channel has an upper interior surface and a lower interior surface; placing a photomask over the at least one channel at block 1704, wherein the photomask includes a series of apertures formed therein; irradiating the polymer precursor solution through a photomask with a light source at block 1706, wherein the light source emits light at a wavelength that activates the photoinitiator, and wherein activation of the photoinitiator polymerizes the polymer precursor solution below the openings in the photomask and forms a three-dimensional polymer structure extending from an upper interior surface to a lower interior surface of the at least one channel; binding biotinylated capture oligonucleotides to streptavidin in the three-dimensional polymer structure at block 1708, wherein the biotinylated capture oligonucleotides are specific for and bind to target molecules of interest in a sequencing library; and eluting the bound target molecules at block 1710 and seeding the eluted target molecules on a surface of the flow cell on which the oligonucleotides are bound.

The above description is provided to enable any person skilled in the art to practice the various configurations described herein. While the subject technology has been described in detail with reference to various figures and configurations, it should be understood that these figures and configurations are for illustrative purposes only and should not be taken as limiting the scope of the subject technology.

All documents and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, treatises, and web pages, regardless of the format in which they are presented, are expressly incorporated by reference in their entirety. If one or more of the incorporated references and similar materials differ or contradict this application, including but not limited to defined terms, term usage, described techniques, and the like, this application controls.

As used herein, the singular forms "a", "an" and "the" refer to both the singular and the plural, unless the context clearly dictates otherwise. As used herein, the term "comprising" 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, particularly suitable methods and materials are described herein. The recitation of numerical ranges by endpoints includes all numbers subsumed within that range unless the context indicates otherwise. Furthermore, references to "one implementation" are not intended to be interpreted as excluding the existence of additional implementations that also incorporate the recited features. Furthermore, unless explicitly stated to the contrary, implementations "comprising" or "having" one or more elements having a particular property may include additional elements, whether or not they have that property.

The terms "substantially" and "about" are used throughout this specification to describe and account for small fluctuations, such as small fluctuations due to variations in processing. For example, they may 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%.

There may be many other ways to implement the subject technology. The various functions and elements described herein may be divided differently than 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 the generic principles defined herein may be applied to other implementations. Accordingly, many changes and modifications may be made to the subject technology by one of ordinary skill in the art without departing from the scope of the subject technology. For example, a different number of a given module or unit may be employed, one or more different 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.

Underlined and/or italicized headings and subheadings are used for convenience only, do not limit the subject technology, and are not cited in connection with an explanation of the description of the subject technology. All structural and functional equivalents to the various embodied elements 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 are 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.

It should be understood 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.

Claims

1. A method for making a three-dimensional polymeric structure on a flow cell, the method comprising:

the polymer precursor solution is loaded onto the flow cell,

wherein the polymer precursor solution comprises a monomer, a crosslinking agent, and a photoinitiator,

wherein the flow cell comprises at least one channel for receiving the polymer precursor solution, and

wherein the at least one channel has an upper inner surface and a lower inner surface; and

irradiating the polymer precursor solution through a patterned photomask using light of a wavelength sufficient to activate the photoinitiator, wherein activation of the photoinitiator polymerizes at least some of the polymer precursor solution below openings in the patterned photomask and forms a three-dimensional polymer structure extending from the upper interior surface to the lower interior surface of the at least one channel.

2. The method of claim 1, further comprising a light source, wherein the light source is an ultraviolet light source.

3. The method of any one of claims 1 to 2, further comprising cutting at least some of the three-dimensional polymeric structures from the flow cell using heat, a cutting chemical, or a combination of heat and a cutting chemical.

4. The method of any one of claims 1 to 3, wherein the monomer is a compound of formula I:

wherein each R²Independently is hydrogen or (C)_1-6) An alkyl group.

5. The method of any one of claims 1 to 4, wherein the crosslinking agent is a compound of formula II:

wherein:

each n is independently an integer from 1 to 6; and is

Each R¹Independently is hydrogen or (C)_1-6) An alkyl group.

6. The method of any one of claims 1 to 5, wherein the photoinitiator is a diazosulfonate initiator; monoacylphosphine oxide (MAPO) salts; bisacylphosphine oxide (BAPO) salts; or a combination thereof.

7. The method of any one of claims 1 to 6, wherein the monomer is acrylamide, the crosslinker is N, N' -bis (acryloyl) cystamine (BACy), and the photoinitiator is lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (LAP).

8. The method of any one of claims 1 to 7, wherein the polymer precursor solution comprises polyethylene glycol (PEG) -thiol, PEG-acrylate, acrylamide, N' -bis (acryloyl) cystamine (BACy), PEG, polypropylene oxide (PPO), polyacrylic 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), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronic acid, pectin, carrageenan, and mixtures thereof, Gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinylsulfone, diethylene glycol diallyl ether, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylolpropane trimethacrylate, ethoxylated trimethylol triacrylate, ethoxylated pentaerythritol tetraacrylate, or combinations thereof.

9. The method of any one of claims 1 to 8, wherein the polymer precursor solution comprises polyethylene glycol (PEG) -thiol/PEG-acrylate; acrylamide/N, N' -bis (acryloyl) cystamine (BACy); PEG/polypropylene oxide (PPO), or combinations thereof.

10. The method of any of claims 1-9, wherein the photomask comprises polyethylene terephthalate, carbon ink, a chemically etched metal film, or a combination thereof.

11. The method of any one of claims 1 to 10, wherein the three-dimensional polymeric structure is cylindrical.

12. The method of any one of claims 1 to 11, wherein the three-dimensional polymeric structure is inverted C-shaped.

13. The method of any one of claims 1 to 12, further comprising reacting a bifunctional linker having a first end and a second end with the functionalized polymer, wherein the first end of the bifunctional linker is chemically or enzymatically attached to the functionalized polymer, and wherein the second end of the bifunctional linker selectively binds a predetermined type of molecule.

14. The method of claim 13, wherein the functionalized polymer is poly (N- (5-azidoacetamidylpentyl) acrylamide-co-acrylamide (PAZAM) containing an azide moiety, and wherein the bifunctional linker is a biotin-PEG-alkyne complex, and the method further comprises reacting the biotin-PEG-alkyne complex with the azide moiety in the PAZAM using an azide-alkyne click reaction.

15. The method of claim 14, further comprising binding streptavidin to biotin in the biotin-PEG-alkyne complex.

16. The method of claim 15, further comprising binding a biotinylated capture oligonucleotide to the streptavidin, wherein the biotinylated capture oligonucleotide is specific for a target of interest in a sequencing library.

17. A method of three-dimensional sequencing using a three-dimensional sequencing matrix on a flow cell, the method comprising:

loading a polymer precursor solution into a flow cell, wherein the polymer precursor solution comprises monomers and oligonucleotides;

polymerizing the polymer precursor solution to produce a permeable three-dimensional matrix within the flow cell;

diffusing a sequencing library into the permeable three-dimensional polymer matrix, wherein the sequencing library comprises nucleic acid fragments;

diffusing an enzyme and a reagent into the permeable three-dimensional polymer matrix;

hybridizing the nucleic acid fragments to the oligonucleotides in the permeable three-dimensional polymer matrix to produce hybridized nucleic acid fragments;

clonally amplifying the hybridized nucleic acid fragments to produce 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 a three-dimensional matrix in a plurality of discrete two-dimensional slices to characterize the sequencing library, wherein the plurality of discrete two-dimensional slices represent the entire three-dimensional internal volume of the flow cell.

18. The method of claim 17, further comprising fragmenting the released nucleic acid and ligating an adaptor to the ends of the nucleic acid fragments.

19. The method of claim 18, further comprising seeding the nucleic acid fragments on the upper and lower surfaces of the at least one sequencing channel by:

introducing a diffusion barrier layer into the at least one channel,

heating the flow cell to a temperature at which the polymeric structure is cleaved and the nucleic acid fragments are released therefrom,

hybridizing said nucleic acid fragments to said oligonucleotides on said upper surface and said lower surface of said at least one channel, and

washing the cut polymeric structure out of the flow cell.

20. A flow-through cell, the flow-through cell comprising:

a channel, wherein the channel comprises an upper interior surface having a primer coated thereon and a lower interior surface having a primer coated thereon; and

a reversible, permeable three-dimensional polymeric structure formed from a polymer precursor solution in the channel, wherein the three-dimensional polymeric structure extends from the upper interior surface of the channel to the lower interior surface of the channel.