Flow cell

Abstract

An example flow cell includes a substrate, a plurality of chambers defined on or within the substrate, and a plurality of recesses defined within the substrate and within the periphery of each of the plurality of chambers. The recesses are separated by interstitial regions. A primer is attached within each of the plurality of recesses, and a capture site is disposed within each of the plurality of chambers.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 798,348, filed January 29, 2019, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] There are various methods and applications in which it is desirable to create a library of fragmented and tagged DNA molecules from double-stranded DNA (dsDNA) target molecules. Often, the purpose is to generate smaller DNA molecules (e.g., DNA fragments) from larger dsDNA molecules for use as templates in DNA sequencing reactions. The templates may be short read lengths obtained. During data analysis, overlapping short sequence reads can be aligned to reconstruct a longer nucleic acid sequence. In some instances, a pre-sequencing step (such as barcoding specific nucleic acid molecules) can be used to simplify data analysis. Summary of the Invention

[0003] A first aspect disclosed herein is a flow cell comprising a substrate, a plurality of chambers defined on or within the substrate, a plurality of recesses defined within the substrate and within the periphery of each of the plurality of chambers, the recesses being separated by interstitial regions (interstitial regions), a primer attached within each of the plurality of recesses, and a capture site located within each of the plurality of chambers.

[0004] In one example of the first aspect, the capture site is a well defined in the substrate, the well having an opening dimension larger than the opening dimension of each of the plurality of recesses. In one embodiment, the flow cell further comprises a chemical capture agent in the well. In another example, the flow cell further comprises capture beads in the well, the capture beads being coated with the chemical capture agent.

[0005] In one example of the first embodiment, the capture sites are protrusions having chemical capture agents on their surfaces.

[0006] In one example of the first embodiment, the capture site comprises a chemical capture agent disposed on a portion of the interstitial region.

[0007] In one example of the first embodiment, the flow cell further includes a hydrophobic material disposed on the substrate, the hydrophobic material defining each of the plurality of chambers.

[0008] In one example of the first embodiment, each of the plurality of chambers has a height sufficient to block lateral diffusion of released library fragments between adjacent chambers.

[0009] In one example of the first embodiment, the flow cell further comprises a polymer layer in each of the recesses, and the primers are attached to the polymer layer.

[0010] In one example of the first aspect, each of the plurality of chambers has an opening dimension in the range of about 1 μm to about 1000 μm, and each of the plurality of recesses has an opening dimension in the range of about 1 nm to about 2 μm, and the chamber opening dimension is larger than the recess opening dimension.

[0011] In an example of the first embodiment, the plurality of chambers are arranged in a first pattern across the substrate, and each subset of the plurality of recesses are arranged in a second pattern within each of the chambers.

[0012] In an example of the first embodiment, a plurality of chambers is patterned in a substrate.

[0013] It should be understood that any of the features of the flow cells disclosed herein may be combined together in any desired manner and / or configuration.

[0014] A second aspect disclosed herein is a method comprising introducing into a flow cell a plurality of complexes comprising a plurality of chambers, a plurality of recesses within each of the plurality of chambers, a primer attached to the interior of each of the plurality of recesses, and a capture site within each of the plurality of chambers, wherein each of the complexes comprises a carrier and a sequencible nucleic acid fragment bound to or contained within the carrier, one of the complexes binds to a respective one of the capture sites in at least some of the plurality of chambers, and the carrier of the complex releases the sequencible nucleic acid fragment therefrom, whereby walls of each of the chambers restrict transport and seeding of the sequencible nucleic acid fragment to a respective recess within each of the respective chambers.

[0015] It is understood that any features of this method can be combined together in any desired manner. Furthermore, it is understood that any combination of features of this method and / or flow cell can be combined together and / or with any of the embodiments disclosed herein.

[0016] A third aspect disclosed herein is a method comprising: introducing a plurality of complexes into a flow cell comprising a plurality of chambers, each chamber having a depth that is at least about 50% of the average diameter of the complexes, and a primer attached within each of the plurality of chambers, wherein each of the plurality of complexes comprises a carrier and a sequenceable nucleic acid fragment from the same template nucleic acid attached to or encapsulated within the carrier, thereby capturing at least some of the complexes in at least some of the plurality of chambers; washing away non-captured complexes from the flow cell; and causing the carriers of captured complexes to release the sequenceable nucleic acid fragments into the respective chambers in which each complex was captured, without introducing an external immobilization agent into the flow cell, thereby limiting transport and seeding of the sequenceable nucleic acid fragments by the depth.

[0017] In one example of the third embodiment, the depth is greater than or equal to about 10 μm.

[0018] In one example of the third embodiment, each chamber has a lower surface, and one of: the primer is attached to a polymer layer across the lower surface; or the primer is attached to a plurality of spatially separated polymer islands each located on the lower surface.

[0019] In one example of the third embodiment, each chamber has a bottom surface and a plurality of recesses defined therein, and a primer is respectively attached to the polymer layer within each of the recesses.

[0020] In one example of the third embodiment, the carrier is a solid support and causing the release comprises introducing a cleavage agent into the flow cell or heating the flow cell above the annealing temperature of the first sequence of the sequenceable nucleic acid fragment.

[0021] In one example of the third embodiment, the carrier is a hydrogel support and causing the release comprises heating the flow cell, introducing a cleaving agent into the flow cell, or a combination thereof.

[0022] It is understood that any features of the methods can be combined together in any desired manner. Furthermore, it is understood that any combination of method and / or flow cell features can be used together and / or in combination with any of the embodiments disclosed herein.

[0023] A fourth aspect disclosed herein is a method comprising: introducing a plurality of complexes into a flow cell comprising a plurality of chambers, each chamber having a depth of about 5 μm or less, a capture site within each of the plurality of chambers, and a primer attached to the interior of each of the plurality of chambers, wherein each of the plurality of complexes comprises a carrier bead and a sequenceable nucleic acid fragment from the same template nucleic acid attached to the carrier bead, whereby at least some of the complexes are immobilized at their respective capture sites; washing non-immobilized complexes from the flow cell; introducing an external immobilization agent into the plurality of chambers; and causing the carrier beads of the captured complexes to release the sequenceable nucleic acid fragments into the respective chambers in which each complex is captured, whereby transport and seeding of the sequenceable nucleic acid fragments are limited by the external immobilization agent.

[0024] In one example of the fourth embodiment, the external immobilization agent is a gas diffusion barrier; a liquid diffusion barrier selected from the group consisting of mineral oil and silicone oil; a viscous medium diffusion barrier selected from the group consisting of glycerol and sucrose; or a combination thereof.

[0025] In one example of the fourth embodiment, the capture site is a capture site primer, and each of the complexes comprises a complementary primer capable of hybridizing to the capture site primer.

[0026] In one example of the fourth embodiment, the capture site comprises a first member of a binding pair and each of the complexes comprises a second member of the binding pair.

[0027] In one example of the fourth embodiment, each chamber has a lower surface and one of the following: the primer is attached to a polymer layer across the lower surface; or the primer is attached to a plurality of spatially separated polymer islands each located on the lower surface.

[0028] In one example of the fourth embodiment, each chamber has a bottom surface and a plurality of recesses defined therein, and a primer is respectively attached to the polymer layer within each of the recesses.

[0029] In one example of the fourth embodiment, causing the release comprises introducing a cleaving agent to the flow cell.

[0030] It is understood that any features of this method can be combined together in any desired manner. Furthermore, it is understood that any combination of features of this method and / or flow cell can be used together and / or combined with any of the embodiments disclosed herein.

[0031] Furthermore, it should be understood that any feature of any method and / or any flow cell can be combined together in any desired manner and / or can be combined with any embodiment disclosed herein to achieve at least the benefits as described herein.

[0032] Features of embodiments of the present disclosure will become apparent by reference to the following detailed description and drawings, in which like reference numerals correspond to similar, though perhaps not identical, inventive components. For the sake of brevity, symbols or features having previously described functions may not be described with reference to other figures in which they appear. [Brief explanation of the drawings]

[0033] [Figure 1] FIG. 1 is a perspective view of a portion of an example of a flow cell. [Figure 2] 2A and 2B are cross-sectional views taken along lines 2A-2A and 2B-2B, respectively, of FIG. 1, showing different examples of flow cell chamber architectures. [Figure 3] 3A-3C are cross-sectional views showing different examples of capture sites that can be used in a flow cell. [Figure 4]4A-4C are schematic diagrams of different examples of the conjugates disclosed herein. [Figure 5] FIG. 5 is a schematic flow diagram including steps (i) to (iii) showing an example of a method for forming a hydrogel matrix within a chamber of a flow cell. [Figure 6] Figures 6A-6C show, respectively, Figure 6A) a micrograph of a complex in the microchamber, Figure 6B) a micrograph of clusters generated from a library seeded from the complex in the microchamber of Figure 6A, and Figure 6C) a fluorescence micrograph of real-time analysis of the microchamber of Figure 6B during the first sequencing run. Figure 6D) A diagram showing islands obtained from reads from the microchamber shown in Figure 6C. [Figure 7] Figures 7A and 7B show micrographs of five different sections and portions of two different lanes of the flow cell a) after complex introduction (Figure 7A), and b) during real-time analysis of the first sequencing run (Figure 7B), where the portions within sections 1-5 of lane 1 are labeled (i)-(v), respectively, and the portions within sections 1-5 of lane 2 are labeled (vi)-(x), respectively. [Figure 8] Figure 8 shows several micrographs illustrating 1) flow cell sample encapsulation by hydrogel formation, 2) flow cell sample dissolution, 3) flow cell DNA extraction, and 4) flow cell library preparation. [Figure 9] FIG. 9 is a black and white version of the originally colored fluorescence microscope images of the different microchambers on the flow cell after a sequencing run was performed. [Figure 10] Figures 10A and 10B show micrographs of five different sections and two different lanes of the flow cell a) after encapsulation of the encapsulation matrix precursor (Figure 10A) and b) after hydrogel formation (Figure 10B), where the portions within sections 1-5 of lane 1 are labeled (i)-(v), respectively, and the portions within sections 1-5 of lane 2 are labeled (vi)-(x), respectively. [Figure 11]11A and 11B show bright field images of flow cell surfaces prepared with fluids having different bead concentrations and the same air flow rate. [Figure 12] 12A-12E show bright field images of flow cell surfaces prepared with fluids having different bead concentrations and the same air flow rate. [Figure 13] FIG. 13 is a graph of water domain diameter (e.g., droplet, puddle island, liquid layer) (in μm) versus bead density (beads / mm), where the data is extrapolated from FIGS. 12A to 12E. [Figure 14] 14A and 14B depict bright field images of flow cell surfaces prepared with fluids having the same bead concentration and different air flow rates. [Figure 15] 15A-15D show bright field images of flow cell surfaces prepared with fluids having the same bead concentration and different salt concentrations, but with the same air flow rate. [Figure 16] FIG. 16 is a graph of water domain diameter (e.g., droplet, paddle island, liquid layer) (in μm) versus salt concentration (mM Na+), where the data is extrapolated from FIGS. 15A through 15D. DETAILED DESCRIPTION OF THE INVENTION

[0034] The flow cells disclosed herein have a specific architecture that allows for spatial separation of individual libraries on the flow cell. Individual libraries contain deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) fragments of the same size (e.g., <1000 bp) of a larger nucleic acid sample, with the fragments having adapters attached to their respective ends. In some examples disclosed herein, the library is contained on or in a carrier that is introduced into the flow cell. In some other examples disclosed herein, the library is formed in situ on the flow cell after the sample is introduced into the flow cell. In some examples, the flow cell architecture includes individual capture sites that can capture individual carriers or samples. These capture sites are located in individual chambers, thus allowing for spatial separation of carriers (and thus libraries within or on carriers) or samples across the flow cell within individual chambers. In other examples, the flow cell architecture includes chambers without capture sites. In these other examples, the chambers themselves can physically confine one or more carriers or samples.

[0035] The spatial separation and confinement of carriers can help achieve spatial separation and confinement of libraries contained within or on the carriers. The spatial separation and confinement of samples can help achieve spatial separation and confinement of libraries generated on the flow cell from samples. In any of these examples, libraries released from individual carriers or formed on the flow cell from individual samples can be contained within specific chambers. In this way, the chamber structure reduces random binding of library fragments across the flow cell surface. Furthermore, the transport and seeding of library fragments, as well as subsequent cluster generation, can also be confined within each chamber. In this way, confinement can result in substantially uniform seeding of library fragments and therefore even substantially homogenized cluster density. During sequencing, individual clusters generate "spatial clouds" of fluorescent signal as nucleotides are incorporated into the cluster's respective template strand. Confining clusters to chambers can at least reduce spatial cloud crosstalk and / or overlap and can also improve spatial cloud identification. Furthermore, since the reads obtained from any individual chamber can be generated from the same sample, they can be used to reconstruct the sample by bioinformatically stitching short reads together.

[0036] The flow cell architecture disclosed herein can also improve overall utilization of surface area. definition

[0037] Terms used herein will be understood to have their ordinary meaning in the relevant art unless otherwise specified. Some terms used herein and their meanings are described below.

[0038] As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless the context clearly dictates otherwise, and the term "comprising," as used herein, is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended, and does not exclude additional, unrecited elements or method steps.

[0039] References throughout the specification to "one example," "another example," "an embodiment," etc., mean that a particular element (e.g., a feature, structure, composition, configuration, and / or characteristic) described in connection with an example is included in at least one example described herein and may or may not be present in other examples. Also, it should be understood that elements described with respect to any example can be combined in any suitable manner in various examples, unless the context clearly dictates otherwise.

[0040] The terms "substantially" and "about," as used throughout this disclosure, including the claims, are used to describe and account for small variations due to processing variations, etc. For example, these terms can refer to ±2% or less from the specified value, e.g., ±1% or less from the specified value, e.g., ±0.5% or less from the specified value, e.g., ±0.2% or less from the specified value, e.g., ±0.1% or less from the specified value, e.g., ±0.05% or less from the specified value, or ±5% or less from the specified value.

[0041] Adapter: A linear oligonucleotide sequence that can be fused to a nucleic acid molecule, for example, by ligation or tagmentation. In some embodiments, the adapter is substantially non-complementary to the 3' or 5' end of any target sequence introduced into the flow cell. Suitable adapter lengths can range from about 10 nucleotides to about 100 nucleotides, or from about 12 nucleotides to about 60 nucleotides, or from about 15 nucleotides to about 50 nucleotides. Adapters can include any combination of nucleotides and / or nucleic acids. In some examples, adapters include one or more cleavable groups at one or more positions. In some examples, adapters can include sequences complementary to at least a portion of a primer, e.g., a primer including a universal nucleotide sequence (e.g., a P5 or P7 sequence). In some examples, adapters can include an index or barcode sequence to assist in downstream error correction, identification, or sequencing. The index can be unique to the sample or source (e.g., fragment) of the nucleic acid molecule. In some examples, adapters can include a sequencing primer sequence or a sequencing binding site. A combination of different adapters can be incorporated into a nucleic acid molecule, such as a DNA fragment.

[0042] Capture site: A portion of a flow cell surface modified with physical and / or chemical properties that allows for the localization of either a complex or a sample. In one example, a capture site can include a chemical capture agent.

[0043] Support: A hydrogel support that can have a sequencing library contained therein, or a solid support that can have sequenceable nucleic acid fragments bound to its surface.

[0044] Chemical capture agent: A material, molecule, or moiety that can adhere to, retain, or bind to a target molecule (i.e., a complex or sample). One example of a chemical capture agent includes a capture nucleic acid (e.g., a capture oligonucleotide) that is complementary to at least a portion of the target nucleic acid of the target molecule or that is bound to the target molecule. Another example of a chemical capture agent is a linker. In the case of a natural DNA or RNA sample, the linker may include a nucleic acid-binding moiety on one end, such as an intercalator, that binds via charge or hydrophobic interactions. In the case of a cellular sample, the linker may include a cell membrane-binding moiety (e.g., an antigen for a surface protein) or a membrane-spanning moiety (e.g., a phospholipid at one end). Yet another example of a chemical capture agent includes a member of a receptor-ligand binding pair (e.g., avidin, streptavidin, biotin, lectin, carbohydrate, nucleic acid-binding protein, epitope, antibody, etc.) that can bind to the target molecule (or a linking moiety bound to the target molecule). Another example of a chemical capture agent is a chemical reagent that can form electrostatic interactions, hydrogen bonds, or homovalent bonds with a target molecule (e.g., thiol-disulfide exchange, click chemistry, Diels-Alder, etc.).

[0045] Complex: A support, such as a hydrogel support or a solid support, and a sequenceable nucleic acid fragment bound to or contained within the support. The support may also contain one member of a binding pair, the other member of which is part of the capture site.

[0046] External fixation agent: A gas, liquid, or viscous medium that is immiscible with the complex or sample introduced into the flow cell chamber. A gaseous external fixation agent can be used to generate droplets around the complex or sample. An example of a gaseous external fixation agent is air directed at an appropriate flow rate through the flow cell. For example, air can be used to aspirate a fluid containing the complex or sample from the flow cell, forming droplets of the liquid containing the complex or sample. The droplets formed act as a diffusion barrier. A liquid or viscous medium is used to prevent the diffusion of a sequencing library released from the complex or formed in the chamber, for example, on the flow cell surface. The external fixation agent can form a diffusion barrier because the sequencing library or any other polynucleotides have little to no solvation in the external fixation agent. Examples of liquid external fixation agents include hydrophobic oils such as mineral oil, silicon oil, perfluorinated oil, fluorocarbon oil (e.g., FLUORINERT™ FC40 from 3M), or combinations thereof. Examples of external fixatives that form viscous media include buffers containing polymers (e.g., polyethylene glycol, polyvinylpyrrolidone, etc.), dextran, sucrose, glycerin, etc. In some instances, the viscous media is a temperature-responsive gel. A temperature-responsive gel is non-viscous at the non-seeding temperature and becomes viscous at the seeding temperature. Examples of temperature-responsive gels include poly(N-isopropylacrylamide) and polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO) / Laponite nanoparticle composites.

[0047] Fragment: A portion or fragment of genetic material (e.g., DNA, RNA, etc.).

[0048] Hydrogel or Hydrogel Matrix: A colloidal material containing organic polymers (natural or synthetic) crosslinked via covalent, ionic, or hydrogen bonds to form a three-dimensional open lattice structure that traps water molecules to form a gel. In one example, a hydrogel contains about 60% to about 90% fluid, such as water, and about 10% to about 30% polymer. Hydrogels can be porous, i.e., open / void. Porosity is the volume fraction (dimensionless) of a hydrogel, i.e., measures the void space of the material and is the fraction of void volume relative to the total volume, expressed as a percentage between 0 and 100% (or a fraction between 0 and 1). In one example, the porosity of a hydrogel can range from about 50% (0.5) to about 99% (0.99). The porosity may be sufficient to allow diffusion of reagents (e.g., enzymes, chemicals, and smaller sized oligonucleotides (less than 50 base pairs, e.g., primers) but prevent diffusion of larger sized nucleic acid molecules (e.g., samples, fragments, etc.).

[0049] Hydrogel support: A hydrogel having at least a substantially spherical shape (eg, hydrogel beads) within which a sequencing library can be contained.

[0050] Nucleic Acid Molecule: A polymeric form of nucleotides of any length which can contain ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. The term can refer to single- or double-stranded polynucleotides.

[0051] A "target" or "template" nucleic acid molecule can refer to the sequence to be analyzed.

[0052] Nucleotides in nucleic acid molecules can include naturally occurring nucleic acids and their functional analogs. Examples of functional analogs can hybridize to nucleic acids in a sequence-specific manner or serve as templates for replicating specific nucleotide sequences. Naturally occurring nucleotides generally have a backbone containing phosphodiester bonds. Analog structures can have alternative backbone linkages, including any of a variety known in the art. Naturally occurring nucleotides generally have a deoxyribose sugar (e.g., found in DNA) or a ribose sugar (e.g., found in RNA). Analog structures can have alternative sugar moieties, including any of a variety known in the art. Nucleotides can contain natural or unnatural bases. Natural DNA can contain one or more of adenine, thymine, cytosine, and / or guanine, while natural RNA can contain one or more of adenine, uracil, cytosine, and / or guanine. Any unnatural base can be used, such as locked nucleic acids (LNAs) and bridged nucleic acids (BNAs).

[0053] Primer: A nucleic acid molecule that can hybridize with a target sequence of interest. In one example, a primer serves as a substrate onto which nucleotides can be polymerized by a polymerase. For example, an amplification primer serves as a starting point for template amplification and cluster generation. In yet another example, a primer can serve as a starting point for DNA or RNA synthesis. For example, a sequencing primer can hybridize to a synthesized nucleic acid template strand to initiate synthesis of a new strand complementary to the synthesized nucleic acid template strand. A primer can contain any combination of nucleotides or their analogs. In some examples, a primer is a single-stranded oligonucleotide or polynucleotide.

[0054] Sample: A source of genetic material, such as a cell, microbiome, or nucleic acid. In some examples, the cell is a single cell, including a prokaryotic or eukaryotic cell. In some examples, the cell is a mammalian cell, a human cell, or a bacterial cell. In some examples, the nucleic acid is a long-chain DNA molecule, including viral nucleic acid, bacterial nucleic acid, or mammalian nucleic acid. In some examples, the sample is bound (as fragments) via a transposon insert bound to the surface of a solid support (e.g., a bead).

[0055] Sequenable nucleic acid fragment: A portion (fragment) of genetic material with adapters at the 3' and 5' ends. In a sequenceable nucleic acid fragment, each adapter contains a known universal sequence (e.g., complementary to at least a portion of a primer on a flow cell) and a sequencing primer sequence. Both adapters can also contain index (barcode or tag) sequences. In one example, the P5 side can contain a bead index, and the P7 side can contain a sample index. The sequenceable nucleic acid fragment can be attached, for example, via insertion to a transposon attached to the surface of a solid support (e.g., a bead), or can be directly immobilized via a binding pair or other cleavable linker. The sequenceable nucleic acid fragment can also be contained within a hydrogel support.

[0056] Seeding: Immobilization of adapted fragments (e.g., sequenceable nucleic acid fragments) in an exemplary chamber of a flow cell disclosed herein.

[0057] Sequencing library: A collection of nucleic acid fragments or fragment amplicons of one or more target nucleic acid molecules. In some examples, the fragments are linked to one or more adapters at their 3' and 5' ends. In some examples, the sequencing library is prepared from one or more target nucleic acid molecules and is part of a complex. In other examples, the sequencing library is prepared on a flow cell surface using a sample.

[0058] Solid Support: A small, rigid or semi-rigid body having a shape, characterized as, for example, a sphere, ellipsoid, microsphere, or other recognized particulate shape, whether having regular or irregular dimensions. The solid support can have a sequencing library attached to it. Exemplary materials useful for the solid support include, but are not limited to, glass; plastics, such as acrylic, polystyrene or copolymers of styrene with other materials, polypropylene, polyethylene, polybutylene, polyurethane, or polytetrafluoroethylene (Teflon® from Chemours); polysaccharides or cross-linked polysaccharides, such as agarose or Sepharose; nylon; nitrocellulose; resins; silica or silica-based materials, including silicon and modified silicon; carbon fiber; metals; inorganic glass; fiber optic bundles, or various other polymers. Exemplary solid supports include controlled pore glass beads, paramagnetic or other magnetic beads, triazol, sepharose beads, nanocrystals, and others known in the art, for example, as described in the Microsphere Detection Guide from Bangs Laboratories, Fishers Ind.

[0059] Tagmentation: The modification of nucleic acid molecules (e.g., DNA or RNA samples) by transposomes to fragment the nucleic acid molecules and then ligate adapters to the 5' and 3' ends of the fragments in a single step. The tagmentation reaction can be used to prepare sequencing libraries, particularly complexes containing solid supports. The tagmentation reaction combines random sample fragmentation and adapter ligation into a single step, thereby increasing the efficiency of the sequencing library preparation process.

[0060] Transposome: A complex formed between an integrating enzyme (e.g., an integrase or transposase) and a nucleic acid containing an integration recognition site (e.g., a transposase recognition site).

[0061] Universal nucleotide sequence: A region of sequence common to two or more nucleic acid molecules, where the molecules also have regions that differ from each other. Universal sequences present in different members of a collection of molecules can enable capture of several different nucleic acids using a population of universal capture nucleic acids (i.e., adapters with sequences complementary to at least a portion of the primers). Similarly, universal sequences present in different members of a collection of molecules can enable amplification or replication of several different nucleic acids using a population of universal sequencing primer sequences. Flow Cell Architecture

[0062] A portion of an example flow cell 10 is shown in Figure 1. Flow cell 10 includes a substrate 12, a plurality of chambers 14 defined on or within substrate 12, a plurality of recesses 16 defined within substrate 12 and within the periphery of each of the plurality of chambers 14, a primer 20 attached within each of the recesses 16, and a capture site 22 located within each of the plurality of chambers 14.

[0063] The substrate 12 is generally rigid and insoluble in aqueous liquids. The substrate 12 may be a single layer or multi-layer structure. Examples of suitable substrates 12 include epoxy siloxane, polyhedral oligomeric silsequioxane (POSS) or derivatives thereof, glass, modified glass, plastic, nylon, ceramics / ceramic oxides, silica (silicon oxide (SiO2)), fused silica, silica-based materials, aluminum silicate, silicon, modified silicon (e.g., boron-doped p+ silicon), silicon nitride (Si3N4), tantalum pentoxide (TaO5) or other tantalum oxides (TaO X), hafnium oxide (HaO2), inorganic glass, etc. Some examples of plastics suitable for the substrate 12 include acrylic, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, polytetrafluoroethylene (e.g., TEFLON® from Chemours Co.), cyclic olefin / cycloolefin polymer (COP) (e.g., ZEONOR® from Zeon), polyimide, etc. The substrate 12 may also be glass or silicon or POSS with a coating layer of tantalum oxide or another ceramic oxide on its surface. The substrate 12 may also be glass or silicon with a coating layer of POSS on its surface. Another example of a suitable substrate 12 is a silicon-on-insulator substrate.

[0064] Substrate 12 may be in the form of a wafer, panel, rectangular sheet, die, or any other suitable configuration. In one embodiment, substrate 12 may be a circular wafer or panel having a diameter ranging from about 2 mm to about 300 mm. As a more specific example, substrate 12 is a wafer having a diameter ranging from about 200 mm to about 300 mm. In another example, substrate 12 may be a rectangular sheet or panel having a maximum dimension of up to about 10 feet (about 3 meters). As a specific example, substrate 12 is a die having a width ranging from about 0.1 mm to about 10 mm. While exemplary dimensions are provided, it should be understood that substrate 12 having any suitable dimensions may be used.

[0065] Multiple chambers 14 may be defined on or within substrate 12 .

[0066] An example of a chamber 14 defined on a substrate 12 is shown in Figure 2A. In the embodiments disclosed herein, the chamber 14 includes: i) a substrate surface S 12 defines the bottom surface of the chamber 14, and ii) a separator 18 is disposed on the substrate 12 and defines the walls W of the chamber 14. 18When a silicon-on-insulator substrate is used with separator 18, the walls W of chamber 14 are considered to be "defined" on substrate 12. 18 may be defined in part by the outermost silicone layer of the substrate and the separator 18.

[0067] The separator 18 may be a hydrophobic material such as a fluorinated polymer, a perfluorinated polymer, a silicone polymer, or a mixture thereof. The polymer backbone of the hydrophobic material may be carbon or silicon, or a combination thereof. In some examples, the fluorinated polymer is an amorphous fluoropolymer (some commercially available have the following terminal functional groups: A type: -COOH, M type: -CONH-Si(OR)). n The separator 18 may be made of a hydrophobic polymer, such as polytetrafluoroethylene (e.g., Teflon® from Chemours), parylene (e.g., grades A, F, and HT), fluorinated hydrocarbons, fluoroacrylic copolymers (e.g., Fluoropel™ from Cytonix), (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane (FOTS), fluorosilanes, or plasma-deposited fluorocarbons, or the CYTOP® series from AGC Chemicals, containing one of these mixtures. As another example, the hydrophobic polymer or hydrophobic polymer layer may include a hydrophobic hydrocarbon such as 1-heptadecene. In some examples, the silicone polymer is polydimethylsiloxane or another siloxane. It may be particularly desirable to utilize a hydrophobic material for the separator 18 when the material in the recess 16 is hydrophilic. Other polymers can be used as the separator 18, as long as the resulting structure is capable of inducing pearling of the liquid moving across the structure. Alternatively, if the material in the recess 16 is / is hydrophobic, it may be desirable to use a hydrophilic material for the separator 18. The hydrophobic or hydrophilic properties of the separator 18 may help guide the reagents towards the recess 16.

[0068] In one example, the separator material 18 may be deposited on the substrate 12 and then patterned using photolithography. In an example where the substrate 12 is a silicon-on-insulator substrate, photolithography may be used to pattern the separator material 18 and the outermost silicon layer. As one example, a mask (e.g., photoresist) may be used to define the spaces / locations where the separator material 18 will be deposited. The separator material 18 may then be deposited and the mask may be removed (e.g., via lift-off, dissolution, or another suitable technique). As another example, the separator material 18 may be deposited and then a mask may be deposited on the separator material 18. The mask may be patterned using photolithography, and any exposed portions of the separator material 18 may be removed via plasma etching or dry etching with oxygen gas. The mask may then be removed to reveal the remaining separator material 18. In yet another example, the separator material 18 may be laminated to the substrate 12 or transferred to the substrate 12 from a mold or sacrificial layer. In yet another example, the separator 18 can be printed using microcontact printing (using a stamp), aerosol printing, or inkjet printing.

[0069] An example of a chamber 14 defined in the substrate 12 is shown in Figure 2B. In the embodiments disclosed herein, the chamber 14 includes: i) a substrate surface S 12 defines a gap region around the chamber 14; and ii) another substrate surface S' 12 defines the bottom surface of the chamber 14, and iii) the substrate 12 defines the walls W of the chamber 14. 12 is considered to be "defined" by substrate 12 when it also defines the

[0070] In this example, the chambers 14 may be patterned into the substrate 12. Patterning may include etching the chambers 14 into the substrate 12 and / or using imprint lithography.

[0071] Whether formed on or within the substrate 12, the chambers 14 may be distributed throughout the substrate 12 in any suitable pattern or layout. Many different layouts of the chambers 14 are envisioned, including regular, repetitive, and irregular patterns. In one example, the chambers 14 are arranged in a hexagonal grid for close packing and increased density. Other layouts may include, for example, parallelogram layouts (i.e., rectangular, square, etc.), triangular layouts, circular layouts, etc. In some examples, the layout or pattern may be an x-y format of chambers 14 in rows and columns (as shown in FIG. 1).

[0072] The chamber 14 can have any suitable shape, such as a circle (as shown in FIG. 1), an oval, a polygon (eg, a triangle, a square, a pentagon, etc.), or the like.

[0073] The size of each chamber 14 may be characterized by its opening area, diameter, and / or length and width. As shown in FIG. 1, the flow cell 10 has multiple recesses 16 located within each of the chambers 14. Thus, the size of the chamber 14 is greater than the size of each recess 16. In other words, the dimension(s) of the chamber 14 are greater than the dimensions of each recess 16. In this example, "dimension" refers to the area occupied by each chamber opening or recess, and / or the diameter of the chamber 14 or recess 16, and / or the length and width of each chamber 14 or recess 16. In the example shown in FIG. 1, the opening area and diameter of each chamber 14 are greater than the opening area and diameter of each recess 16. The opening area and diameter or length and width of each chamber 14 depend on the number of recesses 16 disposed within the chamber 14 and the size of the capture sites 22 disposed within the chamber 14.

[0074] The area occupied by each chamber opening can be selected to allow complexes (examples of which are shown in Figures 4A-4C) to enter chamber 14 and attach to capture sites 22 within chamber 14. For example, the area of ​​each chamber opening can be at least about 1 μm 2 , at least about 10 μm 2 , at least about 100 μm 2 The footprint of each chamber opening may be greater than or between the numbers specified above.

[0075] In some examples, the diameter or length and width of each chamber 14 can be at least about 1 μm, at least about 10 μm, at least about 20 μm, at least about 30 μm, at least about 40 μm, at least about 50 μm, at least about 100 μm, or more. An example of a chamber diameter is in the range of about 1 μm to about 1000 μm. Another example of a chamber diameter is in the range of about 10 μm to about 50 μm. It should be understood that when chamber 14 has a length and a width, the length and width may be the same or different.

[0076] The chambers 14 may also have a depth that depends on the technique used to form the chambers 14. For example, the depth of each chamber 14 can be a monolayer thick if microcontact, aerosol, or inkjet printing is used to form the chamber walls W18. In other examples, the depth of each chamber 14 can be about 1 μm, about 10 μm, about 50 μm, or more. In another example, the depth is at least about 50% of the average diameter of the complexes introduced into the chamber 14. In one example, the depth can range from about 10 μm to about 30 μm. This depth is sufficient to block lateral diffusion of released library fragments between adjacent chambers 14, thus maintaining the released library fragments within the chamber 14 without an external immobilization agent. In other examples, the depth is 5 μm or less. It should be understood that the depth of each chamber 14 may be greater than, less than, or somewhere in between the values ​​specified above.

[0077] The adjacent chamber 14 is connected to the surface S of the additional material 18. 18 (shown in FIG. 2A) or by the surface S of the substrate 12 12 (shown in FIG. 2B ). The average chamber pitch represents the distance from the center of one chamber 14 to the center of an adjacent chamber 14 (center-to-center spacing) or from the edge of one chamber 14 to the edge of an adjacent chamber 14 (edge-to-edge spacing). The layout or pattern of the chambers 14 may be regular, such that the coefficient of variation around the average pitch is small, or the layout or pattern may be irregular, in which case the coefficient of variation may be relatively large. In either case, the average pitch may be, for example, at least about 1 μm, at least about 5 μm, at least about 10 μm, at least about 100 μm, or more. In one example, the average pitch is twice the diameter of the chambers 14. The average pitch of a particular pattern of chambers 14 may be between one of the lower values ​​and one of the upper values ​​selected from the range above. While example average chamber pitch values ​​are provided, it should be understood that other average chamber pitch values ​​may be used.

[0078] A plurality of recesses 16 may be defined in the substrate 12. In the embodiments disclosed herein, the recesses 16 are formed by: i) a recess in the substrate surface S 12 or S' 12 defines gap regions 24 separating the recesses 16; and ii) another substrate surface S''. 12 ii) defines the bottom of the recess 16, and iii) the substrate 12 also defines the walls of the recess 16.

[0079] The recesses 16 may be patterned in the substrate 12. Patterning may include etching the recesses 16 into the substrate 12 and / or using imprint lithography.

[0080] Each subset of recesses 16 may be distributed across each of the chambers 14 in any suitable pattern or layout. The pattern of recesses 16 in each chamber 14 may be the same, or different patterns of recesses 16 may be used in different chambers 14. Many different patterns / layouts of recesses 16 are contemplated, including regular, repetitive, and irregular patterns. In one example, the recesses 16 are arranged in a hexagonal grid to increase density. Other layouts may include, for example, parallelogram layouts (i.e., rectangular, square, etc.), triangular layouts, circular layouts, etc. In the example illustrated in FIG. 1 , the recesses 16 in each chamber 14 are arranged in a circular pattern around the capture site 22. As shown in FIG. 1 , the multiple chambers 14 may be arranged in a first pattern (e.g., 2x2) across the substrate 12, and the recesses of each subset are arranged in a second pattern (e.g., circular) within each of the chambers 14.

[0081] Each recess 16 can have any suitable shape (and corresponding three-dimensional shape), such as a circle (as shown in FIG. 1), an oval, a polygon (eg, a triangle, a square, a pentagon, etc.), etc.

[0082] The size of each recess 16 may be characterized by its opening area, diameter, and / or length and width. As shown in Figure 1, flow cell 10 has multiple recesses 16 located within each of chambers 14. As such, the size of each recess 16 is smaller than the size of the chamber 14 in which it is located.

[0083] The area occupied by each recess opening can be selected so that the complex cannot enter recess 16. For example, the area of ​​each recess opening can be at least about 1×10 -4 μm 2 , at least 1 x 10 -3 μm 2 , at least about 1 x 10 -2 μm 2 , at least about 0.1 μm 2 , at least about 0.5 μm2 , at least about 1 μm 2 , or at least about 4 μm 2 The area occupied by each recess opening may be less than or between the above values.

[0084] In some examples, the diameter or length and width of each recess 16 can be at least about 1 nm, at least about 50 nm, at least about 100 nm, at least about 500 nm, or even larger, as long as the dimensions are smaller than the diameter or length and width of the chamber. An example recess diameter is in the range of about 1 nm to about 500 nm. Another example recess diameter is in the range of about 300 nm to about 2 μm.

[0085] The recesses 16 may also have a depth. By way of example, the depth of each recess 16 may be at least about 10 nm, at least about 50 nm, at least about 1 μm, or up to about 2 μm. In some examples, the depth is about 0.4 μm. It should be understood that the depth of each recess 16 may be greater than, less than, or between the values ​​specified above.

[0086] In one embodiment, the aspect ratio (diameter:depth) of the recesses 16 may range from about 1:1 to about 1:2, or from about 1:1.25 to about 1:1.75.

[0087] Adjacent recesses 16 may be separated by interstitial regions 24 within a given chamber 14. The average recess pitch represents the distance from the center of one recess 16 to the center of an adjacent recess 16 (center-to-center spacing) or from the edge of one recess 16 to the edge of an adjacent recess 16 (edge-to-edge spacing). The layout or pattern of recesses 16 can be regular, resulting in a small coefficient of variation around the average pitch, or the layout or pattern can be irregular, resulting in a relatively large coefficient of variation. In either case, the average pitch can be, for example, at least about 10 nm, at least about 0.1 μm, at least about 0.5 μm, or more, depending on the dimensions of the chamber 14. Alternatively, or additionally, the average pitch can be, for example, up to about 0.5 μm, or up to about 0.1 μm or less. The average pitch of a particular pattern of recesses 16 can be between one of the lower values ​​selected from the above range and one of the upper values.

[0088] A primer 20 is attached to each recess 16. The primer 20 is attached to at least the bottom of each recess 16 (i.e., the surface S''). 12 ) present in layer 26. Primers 20 can form within each recess 16 a lawn of capture oligonucleotides that can bind to the adaptors of sequenceable nucleic acid fragments.

[0089] In one embodiment, primers 20 can be immobilized to layer 26 by a single-point covalent bond at or near the 5' end of primer 20. This bond allows i) the adapter-specific portion of primer 20 to anneal to its cognate sequenceable nucleic acid fragment and ii) there is no 3' hydroxyl group for primer extension. Any suitable covalent bond can be used for this purpose. Examples of terminal primers that can be used include alkyne-terminated primers, tetrazine-terminated primers, azide-terminated primers, amino-terminated primers, epoxy- or glycidyl-terminated primers, thiophosphate-terminated primers, thiol-terminated primers, aldehyde-terminated primers, hydrazine-terminated primers, phosphoramidite-terminated primers, and triazolinedione-terminated primers. In another example, primers 20 can be immobilized to layer 26 via non-covalent interactions. In one embodiment, each primer 20 can include a linking molecule (e.g., biotin) that can be non-covalently bound to layer 26. In some examples, two different primers 20 are used. Specific examples of suitable primers 20 include the P5 and P7 primers used on the surface of commercially available flow cells sold by Illumina Inc. for sequencing on HISEQ™, HISEQX™, MISEQ™, MISEQDX™, MINISEQ™, NEXTSEQ™, NEXTSEQDX™, NOVASEQ™, GENOME ANALYZER™, ISEQ™, and other instrument platforms.

[0090] In one embodiment, layer 26 is a material that can non-covalently bind to a linking molecule attached to primer 20. By way of example, the linking molecule is biotin and layer 26 is avidin, streptavidin, etc. In this example, layer 26 can be applied by microcontact printing or aerosol printing, deposition and polishing, or another suitable selective deposition technique.

[0091] In another example, layer 26 is a polymer that can be covalently bonded to primer 20. The bottom surface of recess 16 (e.g., S'') 12 ) may be activated and then a polymer may be applied to form layer 26 .

[0092] In some examples, activation can include applying a silane or silane derivative (e.g., norbornene silane). In other examples, activation can include plasma ashing to generate surface-activating agents (e.g., —OH groups) that can attach to the polymer used to form layer 26.

[0093] Examples of polymers that can be used to form layer 26 include acrylamide copolymers, such as poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide, PAZAM). PAZAM and some other forms of acrylamide copolymers have the following structure (I): [ka] is represented by where: R A is selected from the group consisting of azide, optionally substituted amino, optionally substituted alkenyl, optionally substituted hydrazine, optionally substituted hydrazine, carboxyl, hydroxyl, optionally substituted tetrazole, optionally substituted tetrazine, oxide nitrile, nitrone, and thiol; R B is H or optionally substituted alkyl, R C , R D , and R E are each independently selected from the group consisting of H and optionally substituted alkyl; -(CH2) p - is optionally substituted; p is an integer ranging from 1 to 50; n is an integer ranging from 1 to 50,000; m is an integer ranging from 1 to 100,000. Those skilled in the art will recognize that the sequence of repeating "n" and "m" features in structure (I) is representative and that the monomer subunits may be present in any order in the polymer structure (e.g., random, block, patterned, or combinations thereof).

[0094] In some instances, the PAZAM is a linear polymer. In other instances, the PAZAM is a lightly cross-linked polymer.

[0095] In another example, the polymer that can be used to form layer 26 can be a variation of structure (I). In one example, the acrylamide unit is N,N-dimethylacrylamide. [ka] In this example, the acrylamide unit of structure (I) may be substituted with: [ka] where R D , R E , and R F are each H or C1-C6 alkyl, and R G and R H are each C1-C6 alkyl (instead of H as in acrylamide). In this example, q may be an integer ranging from 1 to 100,000. In another example, N,N-dimethylacrylamide may be used in addition to the acrylamide units. In this example, structure (I) includes, in addition to the recurring "n" and "m" features, [ka] where R D , R E , and R F are each H or C1-C6 alkyl, and R G and R Hare each C1 to C6 alkyl. In this embodiment, q may be an integer ranging from 1 to 100,000.

[0096] Other polymers or molecules may adhere to the surface S'' 12 It should be understood that any suitable polymer may be used to form layer 26, so long as it is functionalized to interact with the subsequently applied primer 20. Other examples of suitable polymers for layer 26 include polymers with colloidal structures, such as agarose; or polymer mesh structures, such as gelatin; or cross-linked polymeric structures, such as polyacrylamide polymers and copolymers, silane-free acrylamides (SFAs), or azido-type SFAs. Examples of suitable polyacrylamide polymers can be synthesized from acrylamide and acrylic acid or vinyl-containing acrylic acid, or from monomers that form a [2+2] photocycloaddition reaction. Still other examples of suitable polymers for layer 26 include mixed copolymers of acrylamide and acrylate.

[0097] The method used to functionalize recess 16 with the polymer forming layer 26 may depend on whether chamber 14 is defined on or in substrate 12 .

[0098] For example, if the chamber 14 is defined on the substrate 12 by the separator 18, the substrate surface S 12 and S'' 12 The surface S may be treated to functionalize the recess 16, and then the separator 18 may be applied to the surface S to define the chamber 14. 12 It should be understood that the silane or silane derivative can be applied to the substrate surface S using vapor deposition, spin coating, or other deposition methods. 12 and S'' 12 In another embodiment, the substrate surface S 12 and S'' 12The polymer (forming layer 26) may then be applied to the activated substrate surface S using spin coating, immersion or dip coating, or flow of material under positive or negative pressure, or another suitable technique. 12 and S'' 12 In one example, the polymer may be present in a mixture (e.g., water or ethanol and water). Depending on the polymer, the applied mixture may be subjected to a curing process to form a cured surface S. 12 and S'' 12 A (covalently bonded) layer 26 can be formed across the surface S. In one embodiment, curing can occur for a time ranging from about 1 millisecond to about several days at a temperature ranging from room temperature (e.g., about 25°C) to about 95°C. 12 Polishing is performed to remove layer 26 from surface S'' 12 In these embodiments, the separator 18 can then be applied to the surface S as described herein. 12 It may be formed on (for example, by photolithography, printing, film transfer, lamination, etc.).

[0099] For another example, it should be understood that if chambers 14 are defined in substrate 12, selective deposition techniques may be used to functionalize recesses 16. In this example, the silane or silane derivative, followed by the polymer mixture, may be deposited by microcontact printing, aerosol printing, or inkjet printing.

[0100] A grafting process may be performed to graft the primer 20 onto the layer 26 within the recess 16. In one example, grafting may include flow-through deposition (e.g., using a temporarily bonded lid), dunk coating, spray coating, puddle spencing, or another suitable method of attaching the primer 20 to the layer 26 within the recess 16. Each of these example techniques may use a primer solution or mixture, which may include a primer, water, a buffer, and a catalyst. In either grafting method, the primer 20 reacts with reactive groups on the polymer layer 26 within the recess 16 and adheres to the interstitial regions 24, other substrate surfaces S. 12 Or S' 12 , or has no affinity for the separator 18. Thus, the primer 20 selectively grafts onto the polymer layer 26 within the recess 16.

[0101] The embodiments of flow cell 10 disclosed herein include a capture site 22 located within each of the plurality of chambers 14. An example of a capture site 22 is shown in Figures 1, 2A, and 2B, and another example of a capture site 22 is shown in Figures 3A-3C.

[0102] Capture sites 22 are physically and / or chemically capable of immobilizing complexes or samples within a particular chamber 16. In the example shown in FIG. 3A, physical immobilization is possible. Chemical immobilization involves a chemical capture agent 28, as defined herein. If capture sites 22 are capable of chemical immobilization, the chemical capture agent 28 used may depend, in part, on the complex or sample introduced into flow cell 10.

[0103] In some of the examples disclosed herein, capture site 22 can capture complexes that are introduced to flow cell 10. In other examples disclosed herein, capture site 22 can capture samples that then undergo further processing on the flow cell surface to generate a library.

[0104] 1, 2A, and 2B, capture site 22 is formed in the center of chamber 14. It should be understood that capture site 22 can be located at any desired location within chamber 14, which may depend on the placement of recess 16. The locations of capture sites 22 across substrate 12 may be uniform (e.g., each capture site 22 may be in substantially the same location (e.g., center, far left, etc.) within each chamber 14) or non-uniform (e.g., capture sites 22 may be in different locations within different chambers 24).

[0105] The capture site 22 may have any suitable shape, geometry, and dimensions, which may depend at least in part on the structure of the capture site 22 (e.g., patch, well, protrusion, etc.), the dimensions of the chamber 14 in which the capture site 22 is formed, and the type of complex or sample to be captured by the capture site 22.

[0106] In the example shown in FIGS. 1, 2A, and 2B, capture site 22 is a chemical capture agent 28 applied to a portion of interstitial region 24. Any example of chemical capture agent 28 disclosed herein may be used. In one embodiment, chemical capture agent 28 may be deposited at a desired location using microcontact printing, aerosol printing, or the like. In another example, a mask (e.g., photoresist) may be used to define the spaces / locations where chemical capture agent 28 is to be deposited. Chemical capture agent 28 may then be deposited, and the mask may be removed (e.g., via lift-off, dissolution, or another suitable technique). In this embodiment, chemical capture agent 28 may form a monolayer or thin layer of chemical capture agent 28, which may be referred to as a patch.

[0107] Other examples of capture sites 22 are shown in Figures 3A, 3B, and 3C. It should be understood that any of the capture sites 22 may be used in any example of flow cell 10, including those having chambers 14 (and including additional material 18) defined on substrate 12 or those having chambers 14 defined within substrate 12.

[0108] 3A and 3B, capture sites 22 include wells 30 defined in substrate 12. The wells may be "defined" in substrate 12 in the same manner as recesses 16. Wells 30 may be formed using etching, photolithography, and / or imprinting, depending on the substrate 12 used. In one example, wells 30 may be formed simultaneously with recesses 16.

[0109] The wells 30 can have any suitable shape and geometry, including any of those described herein for the recesses 16 .

[0110] In the example shown in FIG. 3A , the well 30 has an opening dimension larger than the opening dimension of each of the plurality of recesses 16. In this example, "opening dimension" refers to the area occupied by each well opening and recess opening, and / or the diameter of each well opening and recess opening, and / or the length and width of each well opening and recess opening. The opening dimension of the well 30 may depend on the size of the complex or sample to be introduced. In the example shown in FIG. 3A , the recess 16 is smaller than the well 30, in part, so that it cannot physically accommodate the complex or sample. In FIG. 3A , the depth of the recess 16 is smaller than the depth of the well 30, but it should be understood that the diameter or length and width may also be smaller. In other examples, the well 30 may be similar in size to or the same as the recess 16. In one example, the chemical capture agent 28 includes a primer 20, and therefore, any of the recesses 16 can function as a well 30 for capturing the complex or sample.

[0111] In some instances, wells 30 do not have additional chemical capture agents 28 added thereto. In these examples, the opening dimensions allow complexes or samples to self-assemble into wells 30 rather than into recesses 16 by size exclusion.

[0112] In other examples, wells 30 have additional chemical capture agents 28 added (as shown in phantom in FIG. 3A ). Any example of chemical capture agent 28 disclosed herein may be used. In one example, chemical capture agent 28 may be deposited in wells 30 using microcontact printing. In another example, chemical capture agent 28 may be deposited in wells 30 using a mask (e.g., photoresist). In these examples, the opening dimensions allow complexes or samples to self-assemble in wells 30, rather than in recesses 16, by size exclusion and by binding affinity between chemical capture agent 28 and complexes or samples introduced into flow cell 10.

[0113] The capture site 22 in FIG. 3B includes a well 30 and a capture bead 32 having a chemical capture agent 28 on its surface. The capture bead 32 can be sized to fit within the well 30 but not within the recess 16. In some examples, the capture bead 32 may be flush with or extend slightly above the adjacent interstitial region 24, so that complexes or samples that eventually attach to it are not trapped within the well 30. In one example, the capture bead 32 is selected from the group consisting of silicon dioxide, superparamagnetic material, polystyrene, and acrylate. Any of the examples of chemical capture agents 28 disclosed herein may be used on the surface of the capture bead 32 or may be coated onto the capture bead 32 before being introduced into the well 30.

[0114] The depth of well 30 (FIG. 3A or 3B) can vary depending on whether chemical capture agent 28 is introduced therein and whether capture beads 32 are introduced therein. The depth can be selected to accommodate at least these materials (i.e., the material is contained within well 30). In one example, the depth of well 30 ranges from about 1 nm to about 5 μm. In other examples, the depth of well 30 ranges from about 1 nm to about 100 nm, or from about 1 μm to about 5 μm. Other depths are possible.

[0115] In FIG. 3C, capture sites 22 are located on the substrate 12 or on the surface S of the substrate 12. 12 The protrusions 34 are three-dimensional structures that extend outward (upward) from adjacent surfaces. When the protrusions 34 are formed in the substrate 12, the substrate 12 is patterned (e.g., by via etching, photolithography, imprinting, etc.) to extend above the adjacent surrounding interstitial regions 24. When the protrusions 34 are formed on the substrate 12, additional material 18 is introduced into the adjacent surrounding substrate surface S. 12 The insulating film is patterned (eg, via etching, photolithography, imprinting, etc.) to extend above the insulating film.

[0116] Any suitable three-dimensional shape may be used for the protrusions 34, preferably having at least a substantially flat upper surface. Exemplary convex shapes include spheres, cylinders, cubes, polygonal prisms (e.g., rectangular prisms, hexagonal prisms, etc.), and the like.

[0117] 3C, chemical scavenger 28 is applied to the top surface of protrusion 34. Any of the examples of chemical scavenger 28 disclosed herein can be used, and any deposition technique can be used to apply chemical scavenger 28 to the top surface of protrusion 34.

[0118] In some instances, it may be desirable to have one capture site 22 per chamber 14. In other instances, it may be desirable to have multiple, separate capture sites 22 per chamber 14. The number of capture sites 22 in an individual chamber 14 can help control the number of complexes captured in a given chamber 14.

[0119] 2A and 2B, the flow cell 10 may also include a lid 36 bonded to the separator 18 or substrate 12. The lid 36 may be arranged to define a single flow path (in fluid communication with multiple chambers 14) or multiple fluid separation flow paths (each in fluid communication with a subset of the multiple chambers 14).

[0120] The lid 36 may be any material that is transparent to the excitation light directed toward the recess 16. By way of example, the lid may be glass (e.g., borosilicate, fused silica, etc.), plastic, etc. A commercially available example of a suitable borosilicate glass is D263® available from Schott North America, Inc. Commercially available examples of suitable plastic materials, i.e., cycloolefin polymers, are ZEONOR® products available from Zeon Chemicals LP.

[0121] The lid 36 may be bonded using any suitable technique, such as laser bonding, diffusion bonding, anodic bonding, eutectic bonding, plasma activated bonding, glass frit bonding, or other methods known in the art. In one embodiment, a spacer layer may be used to bond the lid 36 to portions of the separator 18 or substrate 12. The spacer layer may be any material that seals at least a portion of the separator 18 or substrate 12 and the lid 36 to one another.

[0122] Although not shown, it should be understood that one or more additional layers may be incorporated between the substrate 12 and the lid 36, or between the substrate 12 and the recess 16. These additional layers may be selected to function as a planar waveguide for excitation of the recess 16 with an evanescent field.

[0123] It should be understood that other flow cell architectures are also contemplated herein. As an example, a flow cell 10 may include a chamber 14 and a capture site 22, but may not include a recess 16. In these examples, the primer 20 may be attached to the bottom surface of the chamber 14 rather than to a separate recess 16. The bottom surface of the chamber 14 may be functionalized with a layer 26 and a primer 20. In some examples, the layer 26 may be applied to the entire bottom surface (except where the capture site 22 is formed). In these examples, at least a substantially uniform primer 20 may be formed across the bottom surface of the chamber 14. In other examples, the layer 26 may be applied as islands (e.g., circular, triangular, rectangular, etc. in shape) that are spatially separated from one another within the chamber 14. Library fragments released from specific complexes captured on the flow cell 10, or library fragments formed in situ on the flow cell 10, may be randomly distributed within the chamber 14 (as opposed to being confined within a recess 16 within the chamber 14). Examples of this flow cell architecture are shown in Figures 5(i)-5(iii).

[0124] As another example, flow cell 10 can include capture sites 22 without chambers 14 or recesses 16. In these examples, capture sites 22 can be arranged in a desired geometric shape across the substrate, and primers 20 can be attached to the surface of substrate 12 around capture sites 22. Library fragments released from specific complexes captured on flow cell 10, or library fragments formed in situ on flow cell 10, can be randomly distributed on substrate 12, and confinement of the released library fragments can be achieved by controlled reaction-diffusion.

[0125] As yet another example, flow cell 10 can include recesses 16 and capture sites 22, but not chambers 14. In these examples, capture sites 22 can be arranged in a desired geometric shape across substrate 12, and primers 20 can be attached to the interior of each recess 16, as described herein. Library fragments released from a particular complex captured on flow cell 10, or formed in situ on flow cell 10, can be randomly distributed in recesses 16 near the complex, and restriction (confinement) of the released library fragments can be achieved by controlling reaction-diffusion.

[0126] In yet another example, the walls of chamber 14 have a height sufficient to capture one or more of the complexes or samples introduced into flow cell 10, and thus do not include capture sites 22. A height sufficient to capture one or more of the complexes or samples corresponds to a chamber depth that is at least about 50% of the average diameter of the complexes or samples introduced into flow cell 10. In one example, the height or depth of the walls of chamber 14 is 10 μm or greater. In this example, the number / amount of complexes or samples in a given chamber 14 is random and determined by a Poisson distribution.

[0127] It should be understood that in any of the embodiments disclosed herein, primer 20 may not be located on the sidewalls of recess 16 and / or chamber 14, in part because layer 26 may not be located on the sidewalls. This helps prevent library fragments from seeding onto the sidewalls.

[0128] The flow cell architecture disclosed herein can be used in a variety of applications, including sequencing technologies, such as linked long-read sequencing applications, high-throughput protein biomarker studies, microbiome studies, or single-cell omics. For example, the flow cell architecture and methods disclosed herein can be used to analyze the binding of DNA-labeled antibodies. In this example, the antibody label is attached to a unique DNA sequence with a P5 / P7 adapter, which is introduced into the flow cell architecture. The antibody can be cleaved, and the released P5 / P7 primers are seeded onto the flow cell. The seeded primers allow for identification of which antibody has bound. Conjugates for use in flow cell architecture

[0129] The flow cell architecture may be particularly suitable for use with the exemplary complexes disclosed herein. As described herein, a complex includes a support (e.g., a hydrogel support or a solid support) and a sequenceable nucleic acid fragment attached to or contained within the support. Examples of suitable complexes are shown in Figures 4A-4C. While several exemplary methods for creating complexes are described, it should be understood that other methods may be used as long as the sequenceable nucleic acid fragment is attached to or contained within the support.

[0130] FIG. 4A shows a complex 40A comprising a solid support 42 and a sequenceable nucleic acid fragment 44 bound to the solid support 42.

[0131] In one example, to form this complex 40A, an adapter sequence (52, 52') is attached to the solid support 42 via one member 46 of a binding pair. In one embodiment, the adapter sequence includes a first sequence (e.g., a P5' sequence) complementary to at least a portion of one of the primers 20 on the flow cells 10A-10I, a first sequencing primer sequence (e.g., a read 1 sequencing primer sequence). As previously described, the adapter sequence may be attached to one member 46 of the binding pair (e.g., biotin), thereby binding to the surface of the solid support 42 (which includes the other member of the binding pair (e.g., avidin, streptavidin, etc.)). The adapter sequence may also include an index sequence.

[0132] The Y-adapter may be mixed with a transposase enzyme (e.g., two Tn5 molecules) to form a transposome. The Y-adapter may include two mosaic end sequences that hybridize to each other. One of the mosaic end sequences may be linked to a second sequence (e.g., a P5' sequence) complementary to at least a portion of one of the primers 36 on flow cells 10A-10I, a second sequencing primer sequence (e.g., a read2 sequencing primer sequence), and optionally an index / barcode sequence. The second sequencing primer sequence and the second sequence together constitute the adapter sequence 48, 48'.

[0133] A tagging (labeling) process can then be performed. A liquid (e.g., a labeling buffer) containing a sample (e.g., DNA) can be added to the solid support 42, which has transposomes and adapter sequences attached to it. When the sample contacts the transposomes, the DNA is tagged (fragmented and tagged with adapter sequences 52, 52' on the solid support 42) and attached to a Y adapter (e.g., by ligation of free mosaic end sequences). Continuous tagging of the sample results in multiple bridge molecules between transposomes. Further extension and ligation are performed to complete the sequenceable fragment, ensuring that fragments 50, 50' are attached to sequences 48 and 48'. The transposase enzyme can then be removed by sodium dodecyl sulfate (SDS) treatment, heating, or proteinase K digestion.

[0134] The resulting complex 40A is shown in Figure 4A. The crosslinked molecules are sequenceable nucleic acid fragments 44, each of which contains a fragment 50, 50' and an adapter sequence 48, 52, or 48' and 50' attached to either end. The adapter sequence 52, 52' was originally attached to the solid support 42 and contains a first sequencing primer sequence, a first sequence complementary to a flow cell primer, and one member of the bound complex 46. The adapter sequence 48, 48' is derived from a Y-adapter and contains a second sequence complementary to another flow cell primer and a second sequencing primer sequence. Because each sequenceable nucleic acid fragment 44 contains the appropriate adapter for amplification (e.g., bridge amplification) and sequencing, PCR amplification is not performed. In this way, these fragments 44 are sequenceable. Furthermore, because the library fragments 44 are from the same sample, the fragments 44 may be suitable for linked long read applications.

[0135] Figure 4B shows another complex 40B, which includes a solid support 42 and sequenceable nucleic acid fragments 44' bound to the solid support 42. In one embodiment, a PCR-free nucleotide library is created in a tube, and then the library is hybridized to the solid support 42 in the tube. In the example shown in Figure 4B, a primer with one member of a binding pair is added to the library fragments in the tube, and then the sequenceable nucleic acid fragments 44' are bound to the solid support 42. In another embodiment, the solid support 42 may have primers bound to it via a binding pair (e.g., avidin on the support 42 and biotin bound to the primers). These primers hybridize to the library fragments (thus, the primer and the member of the binding pair are on one end of the fragment, but not the other). In another example, extension can be performed using a strand-displacing enzyme. This results in a completely double-stranded library (e.g., without forks or Y-adapters, as shown in Figure 4B). The sequenceable nucleic acid fragments 44' can be released onto a flow cell via denaturation. Because the library fragments 44' are created before being attached to the solid support 42, the fragments 44' may not be from the same sample and therefore may not be suitable for linked long read applications.

[0136] FIG. 4C shows an example of a complex 40C that includes a hydrogel support 70 and a sequenceable nucleic acid fragment 44'' contained within the hydrogel support 70.

[0137] To form this complex 40C, fluids containing hydrogel monomers and / or polymers and / or crosslinkers are mixed in the presence of a sample (e.g., genetic material). The fluids can be loaded into mineral oil or another suitable hydrophobic fluid and emulsified to form droplets. A radical initiator may be added to polymerize and / or crosslink the hydrogel monomers and / or polymers to form the hydrogel support 70. Examples of suitable monomers, polymers, crosslinkers, and initiators are described with reference to Figures 5(i)-(iii).

[0138] The sample becomes encapsulated within the hydrogel support because its size is sufficient to prevent it from passing through the pores of the hydrogel beads. In some examples, the sample is DNA or RNA and is at least about 100 nucleotides in length (e.g., 1000 nucleotides or more, 10,000 nucleotides or more, 500,000 nucleotides or more, etc.). In some examples, the pore size of the hydrogel support 70 refers to the average diameter or average effective diameter of the cross-section of the pores based on measurements of multiple pores. The effective diameter of a non-circular cross-section is equal to the diameter of a circular cross-section having the same cross-sectional area as the non-circular cross-section. In one example, the pore size ranges from about 10 nm to about 100 nm.

[0139] Library preparation can then occur within the hydrogel support 70. Multiple reagent exchanges may occur through the pores of the hydrogel support 70. The sample and any library fragments generated therefrom are maintained within the hydrogel matrix. Library preparation can include adding adapters that fragment the sample and result in sequenceable fragments 44''.

[0140] In one example, library preparation can be performed via tagging performed within the hydrogel support 70. The resulting complex 40C is shown in FIG. 4C. The adapter sequences include appropriate adapters for bridge amplification and sequencing, and thus the resulting fragments 44'' are sequenceable. In another example, library preparation can be performed using polymerase extension, resulting in a double-stranded library. The library in this example must be denatured before forming and seeding the hydrogel support 70. Methods involving complexes

[0141] Some examples of the methods disclosed herein utilize the example flow cell 10 disclosed herein and any one of complexes 40A, 40B, or 40C. As described above, each of complexes 40A, 40B, or 40C contains sequenceable fragments obtained from the same sample of genetic material. When one or several complexes are isolated within each chamber, spatial co-localization of libraries from the same sample is achieved.

[0142] In a first example method, the flow cell 10 includes multiple chambers 14 but does not include a capture site 22. Rather, the chambers 14 themselves function as capture sites for complexes 40A, 40B, or 40C introduced into the flow cell. Each chamber 14 can function as a capture site, for example, if its depth is at least about 50% of the average diameter of the introduced complexes 40A, 40B, or 40C. In one example, the depth is at least about 10 μm (approximately 10 μm or greater). In this example, the flow cell 10 may have primers 20 attached to the bottom surface of the chambers 14 or may include a recess 16 with the primers 20 contained therein. In these examples, the number of complexes 40A, 40B, or 40C trapped in any given chamber 14 may be random or determined by a Poisson distribution.

[0143] In this first example method, complexes 40A, 40B, or 40C are introduced into the flow cell 10, for example, through one or more input ports. The complexes 40A, 40B, or 40C may be introduced into a fluid, such as a Tris-HCl buffer or a 0.5x saline sodium citrate (SSC) buffer. At least some of the complexes 40A, 40B, or 40C from the fluid settle into at least some of the chambers 14. It should be understood that some complexes 40A, 40B, or 40C do not settle, and these complexes 40A, 40B, or 40C are removed from the flow cell before further processing is performed. It should also be understood that some chambers 14 may receive one or more of the complexes 40A, 40B, or 40C, while others of the chambers 14 may not receive the complexes 40A, 40B, or 40C. The distribution of complexes 40A, 40B, or 40C in this example is random, in part due to the lack of capture sites 22.

[0144] The method of this first example then includes washing the non-captured complexes 40A, 40B, or 40C from the flow cell. Washing can include introducing a fluid into the flow cell 10. The flow can push any unsettled complexes 40A, 40B, or 40C through the outlet port of the flow cell. The deep chamber 14 can prevent the settled complexes 40A, 40B, or 40C from becoming part of the outlet flow.

[0145] The exemplary method then includes causing the carrier (e.g., solid support 42 or hydrogel support 70) of the captured complex 40A, 40B, or 40C to release the sequenceable nucleic acid fragment 44, 44', or 44'' into the respective chamber 14 in which the respective complex 40A, 40B, or 40C is captured; in this example, the transport and seeding of the sequenceable nucleic acid fragment 44, 44', or 44'' is limited by the depth of the respective chamber 14, and therefore, no external immobilization agent is introduced into the flow cell.

[0146] Depending on the complex 40A, 40B, or 40C used, the carrier (i.e., support 42 or 70) that releases the sequenceable nucleic acid fragments 44, 44', or 44" can be varied. In one example, the carrier is a solid support 42, and the cause involves introducing a cleaving agent into the flow cell. The cleaving agent can initiate chemical, enzymatic, or photochemical release of the sequenceable nucleic acid fragments 44, 44' from the solid support 42. In these examples, another stimulus, such as heat or light, can trigger the cleaving agent to release the library fragments 44 or 44' from the solid support 42. As an example, free biotin can be introduced as a cleaving agent and heated to approximately 92°C to induce biotin-oligo release from the solid support 42.

[0147] In other examples, complex 40C is used, and thus the carrier is a hydrogel support 70. In these other examples, causing library release can include heating the flow cell 10, introducing a cleaving agent to the flow cell 10, or a combination thereof. Heating to release the library fragments 44" from the hydrogel support 70 can include heating to a temperature of about 90°C. The entire flow cell 10 can be heated, and when complex 40C is heated, the hydrogel support 70 can decompose and release the fragments 44". In some examples, the cleaving agent can include one or more moieties capable of depolymerizing the hydrogel support 70 and releasing the sequenceable fragments 44" therefrom. Exemplary cleaving agents include dithiothreitol (DTT), tris-(2-carboxyethyl)phosphine (TCEP), or tris-(3-hydroxypropyl)phosphine (THP). In other examples, the cleaving agent is light. In these examples, the crosslinker used to form the hydrogel support 70 can include a photocleavable moiety, and exposure of the complex 40C in the chamber 14 to light of an appropriate wavelength can cleave this moiety and degrade the hydrogel support 70.

[0148] As previously mentioned, transport and seeding of sequenceable nucleic acid fragments 44, 44', or 44'' is limited by the depth of the respective chamber 14, and therefore fragments 44, 44', or 44'' of any particular complex 40A, 40B, or 40C are confined to the chamber 14 in which the particular complex 40A, 40B, or 40C is confined.

[0149] Using the flow cell architecture disclosed herein, primers 20 on the surface of flow cell 10 can be seeded with released sequenceable nucleic acid fragments 44, 44', or 44''. In one example, seeding is achieved through hybridization between a first or second sequence of fragment 44, 44', or 44'', the complement of primer 20, and chamber 14. Seeding can be performed at a hybridization temperature appropriate for fragment 44, 44', or 44'' and primer 20.

[0150] The location of the sequenceable nucleic acid fragments 44, 44', or 44" seed within each chamber 14 depends in part on how the primers 20 are attached within the chamber 14. In some examples of the flow cell 10, each chamber 14 has a bottom surface and the primers 20 are attached to the polymer layer 26 across the bottom surface, or the primers 20 are each attached to a plurality of spatially separated polymer islands disposed on the bottom surface. In these examples, the sequenceable nucleic acid fragments 44, 44', or 44" are seeded across the bottom surface of the chamber 14 or across each of the islands. In other examples, each chamber 14 has a bottom surface and a plurality of recesses 16 defined therein, and the primers 20 are each attached to the polymer layer 26 within each of the recesses 16. In these examples, the sequenceable nucleic acid fragments 44, 44', or 44" are seeded across the polymer layer 26 within each of the recesses 16.

[0151] In another exemplary method (second exemplary method), flow cell 10 includes a plurality of chambers 14, a capture site 22 within each of the plurality of chambers 14, and a primer 20 attached to the interior of each of the plurality of chambers 14. In this example, flow cell 10 may or may not include recess 16.

[0152] In this second exemplary method, the depth of each chamber 14 is 5 μm or less. At such shallow depths, capture sites 22 may be included to immobilize a single complex 40A, 40B, or 40C within a single chamber 14. It should be understood that while each chamber 14 has a capture site 22, some of the chambers 14 may not receive a complex 40A, 40B, or 40C during a given run of the method.

[0153] In this second exemplary method, complexes 40A, 40B, or 40C are introduced into the flow cell 10, for example, through one or more input ports. Complexes 40A, 40B, or 40C may be introduced in a fluid, such as a buffer solution, as disclosed herein. In this example, each capture site 22 and complex 40A, 40B, or 40C are members of a binding pair; therefore, one complex 40A, 40B, or 40C binds to one capture site 22 in each of the chambers 14. More specifically, a capture site 22 may include a first member of a binding pair, and each of complexes 40A, 40B, or 40C may include a second member of the binding pair. As one specific example, the capture site 22 is a capture site primer (e.g., a capture oligonucleotide), and each of complexes 40A, 40B, or 40C includes a complementary primer that can hybridize to the capture site primer. As another example, capture site 22 can include avidin, and biotin can be attached to the surface of complex 40A, 40B, or 40C.

[0154] This second exemplary method then involves washing non-immobilized complexes 40A, 40B, or 40C from the flow cell 10. Washing can include introducing any suitable buffer into the flow cell 10. The flow can push any complexes 40A, 40B, or 40C that are not attached to capture sites 22 out through the exit port of the flow cell 10.

[0155] This second exemplary method then includes introducing an external fixation agent into the flow cell 10, and specifically into the plurality of chambers 14. In one example, the external fixation agent is air or a liquid or viscous medium that is immiscible with the fluid complexes 40A, 40B, or 40C introduced into the flow cell chambers 14.

[0156] Using air to aspirate wash fluid from the flow cell 10 can create droplets that surround the complexes 40A, 40B, or 40C and form a diffusion barrier. A liquid or viscous external immobilization agent at least partially surrounds the deposited complexes 40A, 40B, or 40C within the chamber 14. By at least partially surrounding the complexes 40A, 40B, or 40C, the external immobilization agent inhibits diffusion of the sequenceable nucleic acid fragments 44, 44', or 44'' outside the chamber 14 as the fragments 44, 44', or 44'' are released. If the external immobilization agent is a temperature-responsive material, increasing the temperature to the seeding temperature can cause the agent to become more viscous, forming a form that can prevent library diffusion.

[0157] It should be understood that while any of the external immobilization agents disclosed herein may be used, in one example the external immobilization agent is a liquid diffusion barrier selected from the group consisting of mineral oil and silicone oil, viscous medium diffusion barriers selected from the group consisting of glycerol and sucrose, and combinations thereof.

[0158] The exemplary method then includes causing the carrier (e.g., solid support 42 or hydrogel support 70) of the captured complex 40A, 40B, or 40C to release the sequenceable nucleic acid fragment 44, 44', or 44'' into the respective chamber 14 in which the immobilized 40A, 40B, or 40C, respectively, was captured, where in this example, the transport and seeding of the sequenceable nucleic acid fragment 44, 44', or 44'' is restricted by the external immobilization agent.

[0159] Triggering the support (i.e., support 42 or 70) to release the sequenceable nucleic acid fragments 44, 44', or 44" can vary depending on the complex 40A, 40B, or 40C used. In one embodiment, the support is solid support 42, and the triggering includes introducing a cleavage agent into flow cell 10 (as described in the first embodiment) and using another stimulus to trigger the cleavage agent to release library fragments 44 or 44' from solid support 42. In other examples, complex 40C is used, and thus the support is hydrogel support 70. In these other examples, triggering library release can include heating the flow cell, introducing a cleavage agent into the flow cell, or a combination thereof (as described in the method of the first example).

[0160] As previously mentioned, transport and seeding of sequenceable nucleic acid fragments 44, 44', 44" in this second exemplary method is restricted by the external immobilization agent. Thus, fragments 44, 44', or 44" of any particular complex 40A, 40B, or 40C are restricted to chamber 14 in which the particular complex 40A, 40B, or 40C is confined because the external immobilization agent at least partially surrounds the complex 40A, 40B, or 40C.

[0161] Using the flow cell architecture disclosed herein, primers 20 on the surface of flow cell 10 can be seeded with released sequenceable nucleic acid fragments 44, 44', or 44'', with seeding being achieved via hybridization between a first or second sequence of fragment 44, 44', or 44'' and one of the primers 20 complementary to chamber 14. Seeding can be performed at a hybridization temperature appropriate for fragment 44, 44', or 44'' and primer 20.

[0162] The location of the sequenceable nucleic acid fragment 44, 44', or 44" seed within each chamber 14 depends in part on how the primer 20 is attached within the chamber 14. In some examples of flow cells, each chamber 14 has a bottom surface, and the primer 20 is attached to the polymer layer 26 across the bottom surface, or the primer 20 is attached to each of a plurality of spatially separated polymer islands disposed on the bottom surface. In these examples, the sequenceable nucleic acid fragment 44, 44', or 44" seed spans (across) the bottom surface of the chamber 14 or spans each of the islands. In other examples, each chamber 14 has a bottom surface and a plurality of recesses defined therein, and a primer is attached to the polymer layer 26 within each recess 16, respectively. In these examples, the sequenceable nucleic acid fragment 44, 44', or 44" seed spans the polymer layer 26 within each of the recesses 16.

[0163] In yet another example of the present method (referred to as the third example method), any of the examples of flow cell 10 shown in Figures 1, 2A, 2B, and 3A-3C can be used. In this third exemplary method, capture sites 22 can be included to immobilize a single complex 40A, 40B, or 40C within a single chamber 14, and the depth of each chamber 14 can be sufficient to restrict the transport and seeding of sequenceable nucleic acid fragments 44, 44', or 44'' to their respective recesses 16 within the respective chamber 14.

[0164] In this third exemplary method, complexes 40A, 40B, or 40C are introduced into flow cell 10, for example, through one or more input ports. Complexes 40A, 40B, or 40C may be introduced in a fluid, such as a buffer solution, as disclosed herein. In this example, each capture site 22 and complex 40A, 40B, or 40C is a member of a binding pair; therefore, one complex 40A, 40B, or 40C binds to one capture site 22 in at least some of chambers 14. While each chamber 14 has a capture site 22, it should be understood that some of the chambers 14 may not receive complexes 40A, 40B, or 40C during any given run of the method.

[0165] This third exemplary method then involves washing non-captured complexes 40A, 40B, or 40C from the flow cell 10. Washing can include introducing a buffer into the flow cell. The flow can push any complexes 40A, 40B, or 40C that are not immobilized at capture sites 22 out through the exit port of the flow cell 10.

[0166] The exemplary method then includes causing the carrier (e.g., solid support 42 or hydrogel support 70) of the captured complex 40A, 40B, or 40C to release the sequenceable nucleic acid fragment 44, 44', or 44'' into the respective chamber 14 in which the respective complex 40A, 40B, or 40C is captured; in this example, the transport and seeding of the sequenceable nucleic acid fragment 44, 44', or 44'' is limited by the depth of the respective chamber 14, and therefore, no external immobilization agent is introduced into the flow cell 10.

[0167] Depending on the complex 40A, 40B, or 40C used, causing the release of sequenceable nucleic acid fragments 44, 44', or 44" onto the support (i.e., support 42 or 70) can vary. In one example, the support is solid support 42, and the cause includes introducing a cleaving agent into flow cell 10 and exposing flow cell 10 to an external stimulus (as described in the first exemplary method). In other examples, complex 40C is used, and thus the support is hydrogel support 70. In these other examples, causing library release can include heating the flow cell, introducing a cleaving agent into the flow cell, or a combination thereof (as described in the first exemplary method).

[0168] As previously mentioned, transport and seeding of sequenceable nucleic acid fragments 44, 44', or 44" is limited by the depth of the respective chamber 14; therefore, fragments 44, 44', or 44" of any particular complex 40A, 40B, or 40C are restricted to the chamber 14 in which the particular complex 40A, 40B, or 40C is confined. In this particular example, because flow cell 10 contains primers 20 within recesses 16, seeding of fragments 44, 44', or 44" occurs within recesses 16 and not over interstitial region 24.

[0169] In another exemplary method (fourth exemplary method), the flow cell 10 may be of any of the architectures disclosed herein and includes at least a capture site 22 and a primer 20 .

[0170] As with other methods disclosed herein, complexes 40A or 40B (including solid support 42) are introduced into flow cell 10, for example, via one or more inlets. Complexes 40A or 40B may be introduced in a fluid, such as a buffer solution, as disclosed herein. In this example, each capture site 22 and complex 40A or 40B are members of a binding pair, such that one complex 40A or 40B binds to one capture site 22. More specifically, capture site 22 may include a first member of a binding pair, and each complex 40A or 40B may include a second member of the binding pair. As one specific example, capture site 22 is a capture site primer (e.g., a capture oligonucleotide), and each complex 40A or 40B includes a complementary primer that can hybridize to the capture site primer. As another specific example, capture site 22 may include avidin, and biotin may be attached to the surface of complex 40A or 40B.

[0171] This fourth exemplary method then involves introducing an external immobilization agent into the flow cell 10.

[0172] In one example, the external immobilization agent is air. Introducing air into the flow cell 10 aspirates most of the fluid (in which complexes 40A or 40B have been introduced) and also pushes non-immobilized complexes 40A or 40B out of the flow cell 10. The immobilized complexes 40A or 40B act as fluidic pinning centers, thus retaining a portion of the liquid from the fluid on their respective surfaces. The retained fluid forms droplets surrounding each complex 40A or 40B. In some examples, the droplets are liquid films with a tent-like shape (e.g., a center whose height is set by the seeding particles and whose meniscus profile is set by the strong surface tension of the liquid). This droplet creates a diffusion barrier for the library fragments 44, 44' released from complexes 40A or 40B.

[0173] In some instances, droplets formed around complexes 40A or 40B may be adjacent, thus forming a shallow liquid layer across the flow cell surface. In other instances, droplets formed around complexes 40A or 40B may be spatially isolated from one another. In yet other instances, some of the droplets may be adjacent, forming islands of liquid that are spatially isolated from other islands of adjacent droplets. Increasing the density of complexes 40A or 40B on the flow cell surface facilitates the formation of a shallow liquid layer.

[0174] The droplet configuration can be controlled by selecting a solid support 42 with a desired diameter, applying the complexes 40A or 40B at a particular surface density, adjusting the air flow rate and / or velocity through the flow cell 10, and selecting a fluid to have desired physical properties (e.g., viscosity, surface tension, etc.). For example, more complexes 40A or 40B immobilized within the flow cell 10 (e.g., a higher surface density) results in a shallower liquid layer with a more uniform thickness. In contrast, when fewer complexes 40A or 40B are immobilized within the flow cell 10, the droplets tend to be isolated from each other or form islands of liquid (e.g., small, separated puddles surrounding each group of complexes 10A or 10B).

[0175] Air may be introduced into the flow cell 10 at a predetermined flow rate. In one embodiment, the flow rate ranges from about 100 μL / min to about 2000 μL / min. As previously described, the air flow can be adjusted to vary the droplet configuration. For example, the air flow rate can affect the size and / or diameter of the liquid surrounding the composite 40A or 40B. As an example, a higher air flow rate may result in a continuous shallow liquid layer, while a lower air flow rate may result in liquid droplets that are isolated from each other or form several islands of liquid.

[0176] The physical properties of the fluid can also alter the droplet configuration. For example, a higher surface tension fluid has a greater affinity to adhere to the surface of the composite 40A or 40B, which results in greater wetting and a larger meniscus. In some instances, the liquid used to introduce the composite 40A or 40B has a surface tension of approximately 20 mN m -1 ~Approx. 90mN m -1 For example, a 6 M NaCl solution has a surface tension of approximately 83 mN m -1 The surface tension of a 55% aqueous sucrose solution is approximately 76 mN m -1 and water has a surface tension of 72.86 mN m -1 and water with surfactant added has a surface tension of 20 mN m -1 to approximately 72 mN m -1 ethanol has a surface tension in the range of 22.39 mN m -1 However, the surface tension may vary depending on the fluid used to introduce the complex 40A or 40B.

[0177] The peak height of either droplet (in the form of isolated droplets, paddle islands, or liquid layers) is less than the height of complexes 40A or 40B. In one embodiment, the peak height of either droplet (in the form of isolated droplets, paddle islands, or liquid layers) is about 3 μm or less. The shallow droplet height allows the released library fragments to spread along the XY plane of flow cell 10, rather than across the entire flow path where they become captured by surface primers 20. This improves the localization of the library fragments near, and released from, complexes 40A, 40B.

[0178] In another example, the external immobilization agent is a liquid or viscous medium that is immiscible with complexes 40A or 40B introduced into flow cell 10. In this example, non-immobilized complexes 10A or 10B can be washed out of flow cell 10 before introducing the liquid or viscous medium. Washing can include introducing any suitable buffer into flow cell 10. The flow can push any complexes 40A or 40B that are not attached to capture sites 22 out through the exit port of flow cell 10.

[0179] The liquid or viscous external immobilization agent can be introduced in an amount that forms a shallow layer (e.g., about 3 μm or less) that at least partially surrounds the deposited complexes 40A or 40B within the flow cell 10. By at least partially surrounding the complexes 40A or 40B, the external immobilization agent inhibits diffusion of the sequenceable nucleic acid fragments 44, 44′, or 44″ out of the shallow layer. If the external immobilization agent is a temperature-responsive material, increasing the temperature to the seeding temperature may cause the agent to become more viscous, resulting in a form that can prevent library diffusion.

[0180] This exemplary method then involves causing the carrier (e.g., solid support 42) of immobilized complex 40A or 40B to release sequenceable nucleic acid fragments 44, 44', or 44" from the solid support 42. As an example, a cleavage agent can be introduced into flow cell 10 (as described in the first exemplary method) and another stimulus can be applied to trigger the cleavage agent to release library fragments 44 or 44' from solid support 42, in this example, the transport and seeding of sequenceable nucleic acid fragments 44, 44', or 44" being restricted by droplets formed around complex 40A or 40B.

[0181] Using the flow cell architecture disclosed herein, primers 20 on the surface of flow cell 10 can be seeded with released sequenceable nucleic acid fragments 44, 44', or 44'' as described herein.

[0182] In any of the example methods involving complex 40A, 40B, or 40C (e.g., the first, second, third, or fourth methods described herein), the seed sequencing library can be amplified using cluster generation.

[0183] In one example of cluster generation, a sequenceable nucleic acid fragment 44, 44', or 44'' is copied from a hybridized primer 20 by 3' extension using a high-fidelity (hi-fi) DNA polymerase; the original sequenceable nucleic acid fragment 44, 44', or 44'' is denatured; the copy remains immobilized within chamber 14. Isothermal bridge amplification or some other form of amplification can be used to amplify the immobilized copy. For example, the copied template loops over to hybridize to an adjacent complementary primer 20, and the polymerase copies the copied template to form a double-stranded bridge, which is denatured to form two double-stranded strands. These two strands loop over adjacent complementary primers 20, hybridize, and are again extended to form two new double-stranded loops. This process is repeated on each template copy by cycles of isothermal denaturation and amplification, creating a dense clonal cluster. Each cluster of double-stranded bridges is denatured. In one embodiment, the reverse strand is removed by specific base cleavage, leaving the forward template polynucleotide strand. It should be understood that clustering results in the formation of several template sequenceable nucleic acid fragments, for example, within each chamber 14, and in some instances, within each recess 16 within each chamber 14. An example of this clustering is bridge amplification, which is one example of amplification that can be performed. It should be understood that other amplification techniques, such as the ExAmp workflow (Illumina Inc.), may also be used.

[0184] After cluster generation, sequencing can be performed. Any example of a flow cell 10 disclosed herein may be used with a variety of sequencing approaches or techniques, including techniques often referred to as sequencing-by-synthesis (SBS), cyclic array sequencing, sequencing-by-ligation, pyrosequencing, etc.

[0185] As one example, sequencing-by-synthesis (SBS) reactions can be performed on a system such as HISEQ™, HISEQX™, MISEQ™, MISEQDX™, MINISEQ™, NOVASEQ™, NEXTSEQDX™, ISEQ™, NEXTSEQ™, or other sequencing systems from Illumina (San Diego, CA).

[0186] A sequencing primer can be introduced that hybridizes to a complementary sequence on the template polynucleotide strand. This sequencing primer prepares the template polynucleotide strand for sequencing. In SBS, the extension of the sequencing primer along the template sequenceable nucleic acid fragment (template polynucleotide strand) is monitored to determine the sequence of nucleotides in the template. The 3' end of the template and any flow cell-bound primers 20 (not bound to the copy) can be blocked to prevent interference with the sequencing reaction, particularly to prevent undesired priming. The underlying chemical process can be polymerization (e.g., catalyzed by a polymerase enzyme) or ligation (e.g., catalyzed by a ligase enzyme).

[0187] In certain polymerase-based SBS processes, fluorescently labeled nucleotides are added to a sequencing primer (thereby extending the sequencing primer) in a template-dependent manner, so that detection of the order and type of nucleotides added to the sequencing primer can be used to determine the sequence of the template. More specifically, one of the nucleotides is incorporated into a nascent strand complementary to the template polynucleotide strand by a respective polymerase, extending the sequencing primer. For example, to initiate the first SBS cycle, one or more labeled nucleotides, a DNA polymerase, or the like can be delivered into / through a flow cell 10, where sequencing primer extension incorporates the labeled nucleotide. This incorporation can be detected through an imaging event. During the imaging event, an illumination system (not shown) may provide excitation light to the flow cell 10.

[0188] In some instances, the fluorescently labeled nucleotide can further comprise a reversible termination property, which terminates further primer extension once the nucleotide is added to the template.For example, a nucleotide analogue with a reversible terminator moiety can be added to the template so that subsequent extension cannot occur until a deblocking agent is delivered to remove the residue (moiety).Therefore, in instances using reversible termination, a deblocking agent can be delivered to the flow cell, etc. (after detection has occurred).

[0189] Washing can be performed between the various fluid delivery steps. The SBS cycle is then repeated n times to extend the template by n nucleotides, thereby allowing detection of a sequence of length n.

[0190] Although SBS is described in the detailed description, it should be understood that the flow cell 10 described herein may be utilized with other sequencing protocols, for genotyping, or in other chemical and / or biological applications. Methods involving complex formation on a flow cell structure

[0191] Another example of the methods disclosed herein does not utilize the composites 40A, 40B, or 40C shown in Figures 4A-4C. Rather, a hydrogel matrix is ​​formed in situ within chamber 14 of the flow cell. This example is described with reference to Figures 5(i)-5(iii).

[0192] In this example, the flow cell 10' may be part of a sequencing kit that includes the flow cell 10' and various reagents for forming a hydrogel matrix 74 within the chambers 14 of the flow cell 10'. One example of a sequencing kit includes a flow cell 10' that includes a plurality of chambers 14 (e.g., formed in or on a substrate 12, as shown in Figures 1, 2A, and 2B) and a primer 20 attached to the interior of each of the plurality of chambers 14. In the example shown in Figures 5(i)-5(iii), each chamber has a bottom surface, and the primers 20 are attached to a polymer layer 26 across the bottom surface. Although not shown, the polymer layer 26 (on the bottom chamber surface) may alternatively be in the form of a plurality of spatially separated polymer islands, and a respective primer 20 may be attached to each island. In yet another example of the flow cell 10', a plurality of recesses 16 (as defined herein for the flow cell 10) may be defined in the bottom surface of the chamber, and a primer 20 may be attached to the polymer layer 26 in each of the recesses 16.

[0193] As shown in FIG. 5( i ), the flow cell 10 ′ also includes an example of a capture site 22 ′, where the capture site 22 ′ is configured to capture a sample 72 .

[0194] The capture site 22' may be any example of a chemical capture agent disclosed herein that can attach to a sample 72 introduced into the flow cell 10'. In the case of a natural DNA or RNA sample 72, the capture site 22' may include a linker with a nucleic acid-binding moiety at one end, such as an intercalator that binds via charge or hydrophobic interactions, or one member of a binding pair (if the sample 72 includes the other member), or an oligonucleotide that can hybridize to the sample 72. In the case of a cellular sample 72, the linker may include a cell membrane-binding moiety (e.g., an antigen for a surface protein) or a membrane-spanning moiety (e.g., a phospholipid at one end).

[0195] Although not shown in Figures 5(i) to 5(iii), the flow cell 10' can include a recess 16 within each of the chambers 14, and a primer 20 can be mounted within each of the recesses 16.

[0196] The sequencing kit also includes an encapsulation (hydrogel) matrix precursor composition comprising a fluid, a monomer or polymer containing radical-generating and chain-extending functional groups, a radical source, and a crosslinker. The encapsulation (hydrogel) matrix precursor composition does not include sample 72. In one example, the encapsulation (hydrogel) matrix precursor composition includes about 2% (w / v) to about 20% (w / v) of the monomer or polymer, about 1 wt% to about 10 wt% of the crosslinker, and about 0.1% (w / v) to about 10% (w / v) of the radical source. When included in the composition, the radical initiator may be present in an amount of about 0.1% (w / v) to about 10% (w / v).

[0197] The fluid of the encapsulation (hydrogel) matrix precursor composition can be water (eg, ion-exchanged water).

[0198] When a monomer is used in the encapsulation (hydrogel) matrix precursor composition, the monomer is selected from the group consisting of acrylamide, N,N'-bis(acryloyl)cystamine, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, ethylene glycol diallyl ether, ethylene glycol diacrylate, trimethylolpropane trimethacrylate, ethoxylated trimethylol diacrylate, ethoxylated pentaerythritol tetraacrylate, collagen monomer, and combinations thereof. When a polymer is used in the encapsulation (hydrogel) matrix precursor composition, the polymer is selected from the group consisting of polyethylene glycol thiol, polyethylene glycol acrylate, polyethylene glycol diacrylate, polyethylene glycol (e.g., having a weight average molecular weight ranging from about 100 to about 200,000), polypropylene oxide, polyacrylic acid, poly(hydroxyethyl methacrylate), poly(methyl methacrylate), poly(N-isopropylacrylamide), poly(lactic acid), poly(lactic-co-glycolic acid), polycaprolactone, poly(vinyl sulfonic acid), poly(L-aspartic acid), poly(L-asglutamic acid), polylysine, agar, agarose, alginic acid, heparin, alginate sulfate, dextran sulfate, hyaluronic acid, pectin, carrageenan, gelatin, chitosan, cellulose, collagen polymers, and combinations thereof. Both monomers and polymers may also be used in combination within the encapsulation (hydrogel) matrix precursor composition.

[0199] The radical source is a molecule that generates radicals upon decomposition. In one example, the radical source is selected from the group consisting of potassium persulfate, ammonium persulfate, 4,4'-azobis(4-cyanovaleric acid), 1,1'-azobis(cyclohexanecarbonitrile), azobisisobutyronitrile, 2,2'-azobis(2-methylpropionitrile), 2,2'-azobis(2-methylpropionitrile), peroxide, riboflavin, 3-(dimethylamino)propionitrile, and combinations thereof.

[0200] The crosslinker forms bonds, such as disulfide bonds, within the polymers of the hydrogel matrix. The crosslinker can be reversible, in that it can be crosslinked and uncrosslinked depending on the chemicals to which it is exposed. For example, a reversible crosslinker is a bisacrylamide crosslinker containing disulfide bonds, which can be decomposed with a reducing agent such as DTT, TCEP, or THP (phosphine). In one embodiment, the crosslinker is selected from the group consisting of acrylamide, N,N'-bis(acryloyl)cystamine, bisacrylamide, 1,4-diaroylpiperazine, N-N'-diallyl-L-thaldiamide, and N-N'-(1,2-dihydroxyethylene)-bis-acrylamide.

[0201] The sequencing kit also includes a radical initiator, either as part of the encapsulation matrix precursor composition or as a separate component. In one embodiment, the radical initiator may be a photoinitiator. Examples of photoinitiators include azobisisobutyronitrile, benzoyl peroxide, and eosin-5-isothiocyanate. This type of radical initiator may be included in the encapsulation (hydrogel) matrix precursor composition because it does not initiate crosslinking until exposed to light of an appropriate wavelength. In another example, the radical initiator may initiate crosslinking upon exposure to a radical source in the encapsulation (hydrogel) matrix precursor composition. In these examples, the radical initiator is kept separate from the encapsulation (hydrogel) matrix precursor composition until it is desired to form a hydrogel matrix on the flow cell 10'. An example of this type of radical initiator is tetramethylethylenediamine (TEMED).

[0202] In one embodiment, the sequencing kit may further include a sample fluid comprising water and sample 72 (eg, genetic material).

[0203] In one embodiment, the sequencing kit can further include a library preparation solution comprising adapter sequences and transposomes.

[0204] In an example method of using the sequencing kit, a sample fluid (including a sample of genetic material 72) is introduced into the flow cell 10', for example, via an input port (FIG. 5(i)). Through the capture sites 22' in each chamber 14, at least some of the genetic material (sample 72) enters at least some of the multiple chambers 14. The sample 72 is immobilized at the capture sites 22'.

[0205] The sample fluid liquid, including any unbound sample 72, can then be removed. Removal can include introducing a wash buffer (e.g., TRIS HCl) into the flow cell 10'. The flow can push any unbound sample 72 out through an outlet port of the flow cell 10'.

[0206] An example of this method involves introducing an encapsulation matrix precursor composition 76 into flow cell 10' (Figure 5(ii)). At least some of the encapsulation matrix precursor composition 76 enters at least some of the chambers 14 containing sample 72.

[0207] The method then includes encapsulating the sample 72 (i.e., genetic material) within the hydrogel matrix 74 within at least some of the chambers 14 by initiating crosslinking or crosslinking and polymerization of the encapsulation matrix precursor composition 76 contained within at least some of the chambers 14 (Figure 5(iii)).

[0208] Prior to encapsulation, an external fixation agent can be introduced into the flow cell 10'. This agent can remove the encapsulation matrix precursor composition 76 from the flow cell 10', except for the composition 76 that has entered the chamber. This creates a barrier during hydrogel matrix formation. Any of the examples of external fixation agents disclosed herein can be used.

[0209] If encapsulation matrix precursor composition 76 includes a photoinitiator (e.g., a UV-irradiated radical initiator), encapsulation involves exposing flow cell 10' to UV radiation. This exposure initiates radical generation, which in turn initiates cross-linking or cross-linking and polymerization of components within encapsulation matrix precursor composition 76 remaining within chamber 14. The cross-linking or cross-linking and polymerization forms a hydrogel matrix 74 within chamber 14. This encapsulates sample 72 within hydrogel matrix 74 within chamber 14.

[0210] If a radical initiator is used that initiates crosslinking when exposed to a radical source, the radical initiator is introduced separately from the encapsulation matrix precursor composition 76. In these examples, encapsulation involves exposing the flow cell 10' to the radical initiator. In this example, the radical initiator may be introduced along with the external immobilization agent. The radical initiator in the external immobilization agent initiates radical generation in the encapsulation matrix precursor composition 76, which in turn initiates crosslinking or crosslinking and polymerization of components in the encapsulation matrix precursor composition 76 remaining in the chamber 14. The crosslinking or crosslinking and polymerization forms a hydrogel matrix 74 within the chamber 14. This encapsulates the sample 72 within the hydrogel matrix 74 within the chamber 14.

[0211] Library preparation can be performed on the surface of the flow cell 10'. The external immobilization agent can be removed and a buffer solution can be introduced into the flow cell 10' along with the library preparation solution. Library preparation can be performed as described with reference to Figure 4C.

[0212] Seeding and clustering and sequencing can then be performed according to the examples disclosed herein.

[0213] To further illustrate the present disclosure, examples are presented herein. It should be understood that these examples are provided for illustrative purposes and should not be construed as limiting the scope of the disclosure. Non-limiting Examples Example 1

[0214] A hydrophobic layer (CYTOP® S) was deposited on the outermost silicon layer of a silicon-on-insulator substrate, and a positive photoresist was deposited on the hydrophobic layer. Using photolithography, 50 μm diameter microchambers were patterned in the photoresist. The hydrophobic layer and the outermost silicon layer were then etched following the photoresist pattern. The photoresist was then lifted off.

[0215] The microchambers in the substrate were silylated and PAZAM was deposited on them. Unattached PAZAM was washed away, and then P5 and P7 primers were implanted onto the PAZAM in the microchambers. A lid was attached to the substrate.

[0216] A complex similar to that shown in Figure 4A was prepared with an average diameter of 3 µm. The fragments on a particular bead were derived from the same long DNA molecule (derived from the PhiX genome). The library fragments were bound to a solid support via decio-biotin oligos, which have a weaker affinity for streptavidin than biotin on the bead surface. The library fragments contained P5 and P7' sequences, read 1 and read 2 sequences, along with an index sequence. This complex was loaded into a microchamber.

[0217] FIG. 6A is a micrograph showing one of the complexes in one of the microchambers.

[0218] Seeding was initiated by releasing the library fragments from the complex, and clustering was performed using bridge amplification. Figure 6B shows a fluorescence micrograph of clusters derived from the seeded library from the complex in Figure 6A.

[0219] The first sequencing run was performed, and real-time analysis (RTA) of the microchamber in Figure 6B is shown in Figure 6C. Figure 6D shows an example of an island obtained from the readouts inside the microchamber in Figure 6C. The results in Figure 6D show that all reads originated from the same long DNA fragment. Example 2

[0220] A flow cell was fabricated using a two-lane glass substrate. Hydrophobic material was deposited on the glass substrate. A positive photoresist was applied to the hydrophobic material. The positive photoresist was exposed and developed to define a circular pattern outlining the different microchambers in each of the two lanes. The hydrophobic material beneath the undeveloped resist was etched away, exposing the surface of each microchamber. With the developed photoresist in place, the microchambers were silylated and PAZAM was deposited on top of them. Unattached PAZAM was removed with the photoresist using a lift-off technique. P5 and P7 primers were then grafted onto the PAZAM in the microchambers. A lid was attached to the substrate using a UV-curable adhesive.

[0221] The microchambers in this example were prepared in two different lanes along the length of the flow cell, with different diameters and pitches. Table 1 shows the diameter and pitch of each lane.

[0222] [Table 1]

[0223] The complex used in this example contained a solid support and sequenceable nucleic acid fragments bound to the solid support via an avidin-biotin linker. The library fragments of the complex were similar to those shown in Figure 4B. A PCR-free library was prepared in a tube according to the TruSeq™ platform (Illumina, Inc.) protocol. The library was bound to beads via hybridization to the P7 primer, which was bound to the beads via biotin.

[0224] The complexes were introduced into the flow cell by flowing hybridization buffer containing the complexes (200 μL in hybridization buffer) through the flow cell channel. Figure 7A is a micrograph showing a magnified portion of each section of the flow cell lane after complex introduction. Lane and section identifications correspond to Table 1. As shown, one or more complexes were isolated in at least some of the microwells.

[0225] Seeding was initiated by releasing the library fragments from the complex. Library release was initiated by heating the flow cell above the melting temperature of the P7 primer. The fragments were hybridized and the first strand extension was performed. The solid support and non-hybridized fragments were removed with 0.2 M sodium solution. Clustering was then performed using bridge amplification. The first base sequence was performed, and real-time analysis (RTA) of the microchambers is shown in Figure 7B. These results indicate that there was no crosstalk between the microchambers that received the complex and those that did not. Example 3

[0226] The flow cell was formed in a manner similar to that described in Example 2.

[0227] E. coli stained with Sytox Green and dispersed in TRIS HCl wash buffer (pH 8.1) was mixed with the encapsulation matrix precursor and introduced into the flow cell. The flow cell was then washed. The encapsulation matrix precursor composition contained acrylamide monomer, potassium persulfate (KPS), and bis(acrylamido)amide. Mineral oil containing a radical initiator (TEMED) was then introduced. The oil forced the encapsulation matrix into at least some of the microchambers and initiated crosslinking. Figure 8(1) shows a micrograph of several microchambers after hydrogel formation. As shown in the figure, hydrogel had formed in most of the microchambers.

[0228] The hydrogel matrix allowed reagents to freely exchange from the matrix but retained the bacteria within. To demonstrate this, washing was performed, followed by cell lysis by introducing lysozyme into the flow cell. The flow cell was heated to approximately 37°C for approximately 30 minutes to activate the lysozyme. Figure 8(2) shows a micrograph of the microscope chamber (Figure 8(1)) after cell lysis.

[0229] DNA extraction was performed in the presence of proteinase K (ProK), which digests contaminating proteins. Figure 8 (3) is a photomicrograph of the microscope chamber (Figure 8 (2)) after DNA extraction.

[0230] Library preparation was performed using a transposon (Illumina NEXTERA™ DNA Library Prep Kit) with a mixture of P7-ME (mosaic end) / ME' and P5-ME / ME' adapters. After labeling, proteins were removed with SDS, and an extension reaction was performed with PCR enzyme to generate a double-stranded library.

[0231] Figure 8(4) is a micrograph of the microscope chamber (Figure 8(3)) after library preparation. These results demonstrate that in situ sample encapsulation and hydrogel formation occur on the flow cell surface. Example 4

[0232] A glass substrate was used, and circular nanorecesses were etched into it. CYTOP® S was used as the hydrophobic polymer (separator 18) and deposited across the glass substrate, including within the nanorecesses. A photoresist (Shipley S-1805) was applied to the hydrophobic polymer, which was then developed to define the circular pattern of the hydrophobic polymer. The hydrophobic polymer was then removed from the nanorecesses not covered by the photoresist using plasma etching. The exposed nanorecesses were then silylated and covered with a gel material. The gel material was PAZAM. A lift-off process was then used to remove the photoresist and any gel material above the photoresist, thereby exposing the underlying hydrophobic polymer. The hydrophobic polymer extended from approximately 1 μm to approximately 2 μm in the Z direction above the interstitial region. The Z direction refers to the Z axis of a Cartesian coordinate system in three-dimensional space. In this example, the hydrophobic polymer defined a circular chamber with a diameter of 50 μm. Primer grafting was performed to attach P5 and P7 primers to PAZAM in the nanorecesses.

[0233] Complexes similar to those shown in Figure 4A were prepared. Fragments on a particular bead were derived from DNA molecules of the same length. Library fragments were bound to a solid support via decio-biotin oligos, which have a weaker affinity for biotin than streptavidin on the bead surface. These complexes were then loaded into microchambers. Binding of the complexes to the microchamber surface was achieved using anchors (e.g., a complementary primer bearing biotin hybridized to a P5 primer bound to the gel material, or an alkyne-PEG-biotin linker covalently bound to a free azide on the gel material using click chemistry). Free biotin in a saline-sodium citrate buffer containing sodium dodecyl sulfate was introduced, and the flow cell was heated to approximately 80 °C to release the library from each complex. Air was drawn through the flow cell to push the free biotin solution out. Due to the hydrophobic / hydrophilic surface structure, droplets formed within the microchamber as the liquid was pushed out by the air. The droplets prevented the library fragments from diffusing to adjacent microchambers.

[0234] The released library fragments were then hybridized to surface primers in the microchambers, and an extension step was performed to generate complementary copies. Cluster generation was achieved by bridge amplification, followed by sequencing on the flow cell.

[0235] Figure 9 shows a portion of the flow cell after data analysis of a sequencing run. The original colors represented islands or short reads grouped based on their proximity on the reference genome. Because each color was isolated to a specific microchamber, it was concluded that the short reads in a given microchamber were from the same genomic DNA fragment and therefore the same complex. These results demonstrate that the microchambers can confine complexes and released library fragments within their respective chambers. Example 5

[0236] This example demonstrates the formation of a hydrogel flow cell.

[0237] A two-lane glass substrate was used. Microchambers of different sizes were etched into a glass slide. The diameter and pitch of the microchambers in each lane were the same as those listed in Table 1. A hydrophobic material was deposited on the glass substrate. A positive photoresist was applied to the hydrophobic material. The positive photoresist was exposed and developed to define the hydrophobic material covering the interstitial spaces between the microchambers. The hydrophobic material beneath the undeveloped resist (and therefore within the microchambers) was etched away, exposing the surface of each microchamber. With the developed photoresist in place, the microchambers were silylated and PAZAM was deposited on top of them. The unattached PAZAM was removed with the photoresist using a lift-off technique. P5 and P7 primers were then grafted onto the PAZAM in the microchambers. A lid was attached to the substrate using a UV-curable adhesive.

[0238] The encapsulation matrix precursor was introduced into the flow cell. The flow cell was then washed. The encapsulation matrix precursor composition included N,N'-bis(acryloyl)cystamine, acrylamide, and 2,2'-azobis(2-methylpropionamidine) dihydrochloride. Evanchian oil was then introduced into the flow cell. The oil forced the encapsulation matrix into at least some of the microchambers. Figure 10A shows micrographs of different sized microchambers (see Table 1 for diameters and pitch) in different sections (1-5) of two lanes after encapsulation. The lighter areas in each image 10A(i)-10A(x) indicate the microchambers, and the darker areas indicate the stroma. Figures 10A(i)-10A(x) show that the stroma is relatively transparent to the encapsulation matrix, regardless of the size of the microchamber. The flow cell was then exposed to UV light to initiate hydrogel formation. The flow cell was then washed with TRISHCl wash buffer (pH 8.1). Figure 10B shows micrographs of differently sized microchambers in different sections (1-5) of two lanes after hydrogel formation. The lighter areas in each image 10B(i)-10B(x) indicate the microchambers, and the darker areas indicate the stroma. Figures 10B(i)-10B(x) show that hydrogels formed in at least some of the microchambers, regardless of their size. Example 6

[0239] In this example, two single-lane glass substrate flow cells were used, with PAZAM and P5 and P7 primers present on the substrate surface.

[0240] Complexes similar to those shown in Figure 4A were prepared. Fragments on a particular bead were derived from the same long DNA molecule. Library fragments were attached to a solid support via desthiobiotin oligos. The complexes were loaded into flow cell lanes. Binding of the complexes to the lane surface was achieved using anchors (e.g., complementary primers bearing biotin hybridized to P5 primers bound to the gel material, or alkyne-PEG-biotin linkers covalently linked to free azides on the gel material using click chemistry).

[0241] Free biotin in a saline-sodium citrate buffer containing sodium dodecyl sulfate was introduced into each flow cell. In the comparative flow cell, the buffer filled the flow channel. In the example flow cell, air was introduced into the flow channel at a flow rate of 100 μL / min to aspirate the buffer, forming a shallow liquid layer around the complexes. The flow cell was heated to a temperature ranging from approximately 72 °C to approximately 77 °C to release the library from each complex.

[0242] After library release and seeding, images of the cloud profile were acquired. Although not reproduced here, these images showed that when library release and seeding occurred in bulk liquid (comparative example), a portion of the library fragments detached from the released solid support and migrated upward within the channel. These library fragments were either lost in the bulk volume or seeded on the opposite side of the channel from which they were released, thus reducing the number of library fragments per cluster cloud and the overall number seeded (total number = 1229). In contrast, with a shallow liquid layer, all library fragments that detached from the solid surface were confined by the shallow liquid layer, resulting in more 2D localized seeding and a higher number of seeded library fragments (total number = 2017).

[0243] The flow cell of this example also showed significantly reduced cross-sectional surface contamination of library fragments from one surface of the flow cell to the opposite surface of the flow cell.

[0244] To test the effect of complex surface density on droplet / paddle island / shallow layer formation, 3 μm diameter streptavidin-coated magnetic beads were introduced into a single-lane glass substrate flow cell coated with biotin. Fluids with two different bead concentrations (saline sodium citrate buffer with sodium dodecyl sulfate) were introduced into the flow cell. One fluid had a higher bead concentration of approximately 30,000 beads / μL, and the other had a lower bead concentration of approximately 6000 beads / μL.

[0245] Air was introduced into each flow channel at a flow rate of 100 μL / min, aspirating the buffer solution and forming droplets around the magnetic beads. Bright-field images were taken of each flow cell and are shown in Figures 11A and 11B. Figure 11A shows the flow cell surface after aspiration of a fluid with a higher bead concentration, and Figure 11B shows the flow cell surface after aspiration of a fluid with a lower bead concentration.

[0246] Figure 11A shows a denser population of immobilized beads and several paddle islands formed from adjacent droplets. Figure 11B shows a less dense population of immobilized beads and fewer paddle islands and several isolated droplets. Comparing Figures 11A and 11B, it is clear that the larger number of beads (or complexes) on the flow cell surface allows the liquid droplets to merge and form a more uniform liquid layer across the flow cell surface.

[0247] Similar experiments were performed with five fluids containing different bead concentrations. Each fluid was prepared from the same initial fluid: saline-sodium citrate buffer with sodium dodecyl sulfate at a bead concentration of 10 mg / mL (approximately 700,000 beads / µL). The initial fluid was diluted differently to produce five fluids: Fluid 1 = 250x diluted, Fluid 2 = 500x diluted, Fluid 3 = 2,000x diluted, Fluid 4 = 8,000x diluted, and Fluid 5 = 32,000x diluted. Air was introduced into each channel at a flow rate of 100 µL / min, aspirating the buffer solution and forming droplets around the magnetic beads. Light field images were taken for each flow cell and are shown in Figure 12A (fluid 1 = 250x dilution), Figure 12B (fluid 2 = 500x dilution), Figure 12C (fluid 3 = 2,000x dilution), Figure 12D (fluid 4 = 8,000x dilution), and Figure 12E (fluid 5 = 32,000x dilution). As shown in the figures, increasing bead surface density correlated with increasing bead concentration in the fluid. As shown in Figure 13, the average diameter of the fluid (liquid) domains around individual beads gradually increased as the bead surface density decreased.

[0248] To test the effect of air flow rate on droplet / puddle island / shallow liquid layer formation, 3 μm diameter streptavidin-coated magnetic beads were introduced into a single-lane glass substrate flow cell coated with biotin. Fluids with different bead concentrations (saline sodium citrate buffer with sodium dodecyl sulfate) were introduced into the flow cell. One fluid had a lower bead concentration of approximately 7200 beads / μL, while the other had a higher bead concentration of approximately 14,100 beads / μL.

[0249] Two different air flow rates were used: 2,000 μL / min and 200 μL / min. Bright-field images were taken of each flow cell and are shown in Figures 14A and 14B. Figure 14A shows the flow cell surface after aspirating fluid at a higher air flow rate, and Figure 14B shows the flow cell surface after aspirating fluid at a lower air flow rate. At higher flow rates (2,000 μL / min), the liquid layer was more continuous, as shown in Figure 14A, whereas at lower flow rates (200 μL / min), more separated paddle islands formed.

[0250] Another experiment was performed using four fluids with the same bead concentration but different salt concentrations. Each fluid was a saline sodium citrate buffer with sodium dodecyl sulfate at a bead concentration of 10 mg / mL (approximately 700,000 beads / µL). The sodium concentration in each solution was different; for example, fluid 6 = 750 mM Na + , fluid 7=375mM Na + , fluid 8=127.5mM Na + , and fluid 9 = 93.8 mM Na + Air was introduced into each channel at a flow rate of 100 μL / min, aspirating the buffer solution and forming droplets around the magnetic beads. Bright-field images were taken for each flow cell, as shown in Figure 15A (fluid 6 = 750 mM Na + ), Figure 15B (Fluid 7 = 375 mM Na + ), Figure 15C (Fluid 8 = 127.5 mM Na + ), and Figure 15D (Fluid 9 = 93.8 mM Na + ) As shown, increasing salt concentration did not appear to affect the droplets formed. The average diameter of the fluid domains around individual beads remained substantially consistent across different salt concentrations (e.g., within 10 μM), as shown in FIG. Notes

[0251] Furthermore, ranges provided herein should be understood to include the stated range and any value or subrange within the stated range, as if expressly set forth. For example, a range expressed as about 2 mm to about 300 mm should be interpreted to include not only the explicitly recited range of about 2 mm to about 300 mm, but also individual values ​​such as about 15 mm, 22.5 mm, 245 mm, etc., and subranges such as about 20 mm to about 225 mm.

[0252] It is understood that all combinations of the foregoing concepts, and additional concepts detailed below (where such concepts are not mutually inconsistent), are contemplated as 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 part of the inventive subject matter disclosed herein. It is also understood that terminology used expressly herein, and which may also appear in any disclosures incorporated by reference, shall have a meaning that is most consistent with the particular concepts disclosed herein.

[0253] While several embodiments have been described in detail, it is understood that the disclosed embodiments may be modified, and therefore the above description should be considered non-limiting.

Claims

1. A substrate; a plurality of chambers defined on or within the substrate; a plurality of recesses defined in the substrate and within a perimeter of each of the plurality of chambers, the recesses being separated by interstitial regions, each recess having a bottom surface; an amplification primer attached to the bottom surface within each of the plurality of recesses; a capture site disposed within each of the plurality of chambers; the capture site is part of a flow cell that is physically and / or chemically modified for localization of a complex or sample; and (i) a well defined in the substrate, the well having an opening dimension greater than an opening dimension of each of the plurality of recesses; (ii) protrusions having chemical scavenger agents on their surfaces; or (iii) a chemical sequestrant disposed in a portion of the interstitial region; The chemical capture agent is a material, molecule or moiety for adhering to, retaining or binding to a complex or sample, flow cell.

2. The flow cell of claim 1 , further comprising a chemical capture agent within the well when the capture site is a well defined within the substrate.

3. 10. The flow cell of claim 1, further comprising a capture bead within the well when the capture site is a well defined within the substrate, the capture bead being coated with a chemical capture agent.

4. The flow cell of claim 1 , further comprising a hydrophobic material disposed on the substrate, the hydrophobic material defining each of the plurality of chambers.

5. 5. The flow cell of claim 1, wherein each of the plurality of chambers has a height sufficient to block lateral diffusion of released library fragments between adjacent chambers.

6. 6. The flow cell of claim 1, further comprising a polymer layer in each said well, said amplification primers being attached to said polymer layer.

7. each of the plurality of chambers has an opening size in the range of 1 μm to 1000 μm; each of the plurality of recesses has an opening dimension in the range of 1 nm to 2 μm; 7. The flow cell of claim 1, wherein the opening dimension of the chamber is larger than the opening dimension of the recess.

8. the plurality of chambers are arranged in a first pattern across the substrate; 8. The flow cell of claim 1, wherein each subset of the plurality of recesses is arranged in a second pattern within each of the chambers.

9. 9. The flow cell of claim 1, wherein the plurality of chambers are patterned in the substrate.

10. a plurality of chambers; a plurality of recesses within each of the plurality of chambers, the recesses being separated by interstitial regions, each recess having a bottom surface; an amplification primer attached to the bottom surface within each of the plurality of recesses; a capture site in each of the plurality of chambers; introducing a plurality of conjugates into a flow cell comprising: The capture site is (i) a well having an opening dimension larger than the opening dimension of each of the plurality of recesses; (ii) protrusions having chemical scavenger agents on their surfaces; or (iii) a chemical sequestrant disposed in a portion of the interstitial region; Each of the complexes comprises: A carrier; a sequenceable nucleic acid fragment attached to or contained within the carrier, whereby one of the complexes is attached to each one of the capture sites in at least some of the plurality of chambers; introducing a plurality of complexes comprising: allowing each carrier of the complex to release the sequenceable nucleic acid fragment therefrom, whereby the walls of each chamber confine transport and seeding of the sequenceable nucleic acid fragment to each recess within each chamber; A method comprising:

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