Sequencing kits

Abstract

The embodiment of sequencing kit comprises flow cell, encapsulation matrix precursor composition and free radical initiator.Flow cell comprises multiple chambers and primers attached in each of these multiple chambers.Encapsulation matrix precursor composition is made up of fluid, monomer or polymer, free radical source and cross-linking agent comprising free radical generation and chain extension functional group.Free radical initiator is the part of encapsulation matrix precursor composition or a separate component.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 798,354, filed January 29, 2019; the contents of which are incorporated herein by reference in their entirety. Background Art

[0003] There are multiple methods and applications for the library of fragmented and labeled DNA molecules that need to be generated from double-stranded DNA (dsDNA) target molecules. Generally, the purpose is to produce smaller DNA molecules (e.g., DNA fragments) from larger dsDNA molecules used as templates in DNA sequencing reactions. This template can make it possible to obtain short reading lengths. In the data analysis process, overlapping short sequence reading fragments can be compared to rebuild longer nucleic acid sequences. In some cases, a pre-sequencing step (such as barcoding of specific nucleic acid molecules) can be used to simplify data analysis. Summary of the invention

[0004] The first aspect disclosed herein is a sequencing kit comprising: a flow cell comprising: a plurality of chambers; and a primer attached within each of the plurality of chambers; an encapsulation matrix precursor composition consisting of: a fluid; a monomer or polymer comprising free radical generating and chain extending functional groups; a free radical source; and a cross-linking agent; and a free radical initiator as part of the encapsulation matrix precursor composition or as a separate component.

[0005] In an embodiment of the first aspect, one of the following is present: the monomer is selected from 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 monomers and combinations thereof; or the polymer is selected from polyethylene glycol-thiol, polyethylene glycol-acrylate, polyethylene glycol diacrylate, polyethylene glycol, 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-glutamic acid), polylysine and combinations thereof; or any combination of the monomer and polymer used together.

[0006] In an embodiment of the first aspect, the polymer includes a first polymer and a second polymer; the first polymer is selected from the group consisting of polyethylene glycol-thiol, polyethylene glycol-acrylate, polyethylene glycol diacrylate, polyethylene glycol, polypropylene oxide, polyacrylic acid, poly(hydroxyethyl methacrylate), poly(methyl methacrylate), poly(N-isopropylacrylamide), poly(lactic acid), poly(lactic-co-glycolic acid), polycaprolactone, poly(ethylene sulfonic acid), poly(L-aspartic acid), poly(L-glutamic acid), polylysine, and combinations thereof; the second polymer is selected from the group consisting of agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronic acid, pectin, carrageenan, gelatin, chitosan, cellulose, collagen polymers, and combinations thereof.

[0007] In an embodiment of the first aspect, the free radical initiator is tetramethylethylenediamine.

[0008] In an embodiment of the first aspect, the free radical initiator is a photoinitiator and is included in the encapsulation matrix precursor composition.

[0009] In one embodiment of the first aspect, the free radical source is selected from 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.

[0010] In an embodiment of the first aspect, the cross-linking agent is selected from acrylamide, N,N'-bis(acryloyl)cystamine, bisacrylamide, 1,4-dipropionylpiperazine, N-N'-diallyl L-tartaric acid diamide and N-N'-(1,2-dihydroxyethylene)-bisacrylamide.

[0011] In an embodiment of the first aspect, each chamber has a bottom surface, and the primer is attached to the polymer layer over the entire bottom surface.

[0012] In an embodiment of the first aspect, each chamber has a bottom surface, and wherein the primers are respectively attached to a plurality of spaced-apart polymer islands located on the bottom surface.

[0013] In an embodiment of the first aspect, each chamber has a bottom surface and a plurality of recesses defined therein, and the primers are respectively attached to the polymer layer within each recess.

[0014] In an embodiment of the first aspect, the sequencing kit further comprises a library preparation solution comprising an adapter sequence and a transposome.

[0015] In an embodiment of the first aspect, the sequencing kit further comprises a sample fluid comprising genetic material.

[0016] It will be appreciated that any features of the sequencing kit may be combined together in any desired manner.

[0017] A second aspect disclosed herein is a method comprising introducing a fluid containing genetic material into a flow cell, the flow cell comprising: a plurality of chambers; and primers attached within each of the plurality of chambers; whereby at least some of the genetic material enters at least some of the plurality of chambers; removing liquid of the fluid from the flow cell; introducing an encapsulation matrix precursor composition into the flow cell, the encapsulation matrix precursor composition comprising: a monomer or polymer containing free radical generating and chain extending functional groups; a free radical source; and a cross-linking agent; whereby at least some of the encapsulation matrix precursor composition enters at least some of the chambers containing the genetic material; and encapsulating the genetic material in a hydrogel matrix in at least some of the chambers by initiating cross-linking or cross-linking and polymerization of the encapsulation matrix precursor composition contained in the at least some of the chambers.

[0018] In an embodiment of the second aspect, the encapsulation matrix precursor composition further comprises an ultraviolet free radical initiator, and wherein producing the hydrogel matrix comprises exposing the flow cell to ultraviolet radiation.

[0019] In an embodiment of the second aspect, the crosslinking or the crosslinking and polymerization of the encapsulating matrix precursor composition contained in at least some of the chambers comprises introducing a free radical initiator into the flow cell.

[0020] It should be understood that any features of the method can be combined in any desired manner. In addition, it should be understood that any combination of features of the method and / or sequencing kit can be used together, and / or combined with any embodiment disclosed herein.

[0021] A third aspect disclosed herein is a sequencing kit comprising a flow cell comprising a plurality of chambers and primers attached within each of the plurality of chambers; and an encapsulation matrix precursor composition consisting of a fluid and a polymer selected from the group consisting of agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronic acid, pectin, carrageenan, gelatin, chitosan, cellulose, collagen polymers, and combinations thereof.

[0022] In an embodiment of the third aspect, the polymer is alginate and the fluid is a calcium-containing solution.

[0023] Embodiments of the third aspect further include a library preparation solution comprising an adaptor sequence and a transposome.

[0024] Embodiments of the third aspect further include a sample fluid comprising genetic material.

[0025] It should be understood that any features of the sequencing kit can be combined in any desired manner. In addition, it should be understood that any combination of features of the sequencing kit and / or the method and / or another sequencing kit can be used together and / or combined with any embodiment disclosed herein.

[0026] A fourth aspect disclosed herein is a method comprising introducing a fluid containing genetic material into a flow cell, the flow cell comprising a plurality of chambers and a primer attached within each of the plurality of chambers, whereby at least some of the genetic material enters at least some of the plurality of chambers; introducing an encapsulation matrix precursor composition into the flow cell, the encapsulation matrix precursor composition comprising a fluid and a polymer selected from the group consisting of agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronic acid, pectin, carrageenan, gelatin, chitosan, cellulose, collagen polymers, and combinations thereof, whereby at least some of the encapsulation matrix precursor composition enters at least some of the chambers containing the genetic material; flushing the flow cell with a liquid external fixative at a temperature ranging from about 40° C. to about 80° C.; and exposing the flow cell to a gelling temperature of the polymer in at least some of the chambers, thereby encapsulating the genetic material in a hydrogel matrix in at least some of the chambers.

[0027] Embodiments of the fourth aspect further comprise heating the flow cell to a temperature in the range of about 40°C to about 80°C during the introduction of the fluid and the encapsulation matrix precursor composition and during the flushing process.

[0028] In an embodiment of the fourth aspect, exposing the flow cell to a gelling temperature of the polymer comprises cooling the flow cell to the gelling temperature, and maintaining the flow cell at the gelling temperature for a predetermined time.

[0029] In an embodiment of the fourth aspect, exposing the flow cell to a gelling temperature of the polymer comprises heating the flow cell to the gelling temperature, and maintaining the flow cell at the gelling temperature for a predetermined time.

[0030] It should be understood that any features of the method can be combined in any desired manner. In addition, it should be understood that any combination of features of any or both of the method and / or another method and / or the sequencing kit can be used together, and / or combined with any embodiment disclosed herein.

[0031] Still further, it should be understood that any features of any of the described sequencing kits and / or any of the described methods may be combined together in any desired manner and / or may be combined with any embodiments disclosed herein to achieve at least the benefits described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Features of the embodiments of the present disclosure will become clear by reference to the following detailed description and accompanying drawings, in which like reference numerals correspond to similar but possibly different components. For the sake of brevity, reference numerals or features having previously described functions may or may not be described in conjunction with other drawings in which they appear.

[0033] Figure 1 is a perspective view of a portion of an embodiment of a flow cell;

[0034] Figure 2A and 2B Along Figure 1 2A-2A and 2B-2B of FIG. 1 , which illustrate different embodiments of the chamber architecture of the flow cell;

[0035] FIG. 3A to FIG. 3C are cross-sectional views showing different embodiments of capture sites that can be used in a flow cell;

[0036] FIG. 4A to FIG. 4C are schematic diagrams of various embodiments of the composites disclosed herein;

[0037] Figure 5 is a schematic flow chart including (i) to (iii) showing an embodiment of a method in which a hydrogel matrix is ​​formed within a chamber of a flow cell;

[0038] FIG. 6A to FIG. 6C Depicted separately Fig. 6A ) Micrograph of the complex in the microchamber; Figure 6B ) from Fig. 6A A micrograph of clusters generated by a seeded library of complexes in a microchamber of FIG. 1 ; and Figure 6C ) during the first base sequencing run Figure 6B Fluorescence micrographs of real-time analysis of the microchamber;

[0039] Fig.6D Shown from Figure 6C Islands obtained from read fragments of the indicated microchambers;

[0040] Fig. 7A and Figure 7B Depicted is a) after the complex is introduced ( Fig. 7A ) and b) during real-time analysis of the first base sequencing run ( Figure 7B ) Micrographs of portions of 5 different sections and 2 different lanes of the flow cell, wherein the portions in sections 1-5 of lane 1 are labeled (i)-(v), respectively, and the portions in sections 1-5 of lane 2 are labeled (vi)-(x), respectively;

[0041] Figure 8Several micrographs are shown, which show 1) flow cell sample encapsulation by hydrogel formation, 2) flow cell sample lysis, 3) flow cell DNA extraction, and 4) flow cell library preparation;

[0042] Fig. 9 is a black and white version of the original color fluorescence microscopy images of the different microchambers on the flow cell after a sequencing run; and

[0043] Fig. 10A and 10B Depicted are a) after encapsulation of the encapsulation matrix precursor ( Fig. 10A ) and b) after hydrogel formation ( Fig. 10B ) Micrographs of portions of 5 different sections and 2 different lanes of a flow cell, wherein portions of sections 1-5 of lane 1 are labeled (i)-(v), respectively, and portions of sections 1-5 of lane 2 are labeled (vi)-(x), respectively. DETAILED DESCRIPTION

[0044] The flow cell disclosed herein has a specific architecture that allows spatial isolation of a single library on the flow cell. A single library includes a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) fragment of similar size (e.g., less than 1000bp) of a larger nucleic acid sample, and the fragment has an attached adapter at the corresponding end. In some embodiments disclosed herein, the library is included on or in a carrier introduced into the flow cell. In some other embodiments disclosed herein, after the sample is introduced into the flow cell, a library is formed in situ on the flow cell. In some embodiments, the flow cell architecture includes a single capture site that can capture a single carrier or sample. These capture sites are located in a single chamber, and therefore the carrier (and therefore the library in or on the carrier) or sample on the flow cell can be spatially isolated in a single chamber. In other embodiments, the flow cell architecture includes a chamber without a capture site. In these other embodiments, the chamber itself can physically restrict one or more carriers or samples.

[0045] The spatial isolation and restriction of the carrier can help to achieve the spatial isolation and restriction of the library contained in or on the carrier. The spatial isolation and restriction of the sample help to achieve the spatial isolation and restriction of the library generated from the sample on the flow cell. In any of these embodiments, the library released from a single carrier or formed from a single sample on the flow cell can be contained in a specific chamber. Therefore, the chamber architecture reduces the random combination of library fragments on the entire flow cell surface. In addition, the transportation and inoculation of library fragments and subsequent cluster generation may also be limited in each chamber. Therefore, restriction can lead to substantially uniform library fragment inoculation, and thus obtain substantially uniform cluster density. In the sequencing process, as nucleotides are incorporated into the corresponding template chain of the cluster, a single cluster produces a "spatial cloud" of fluorescent signals. Clusters are restricted in chambers at least can reduce spatial cloud crosstalk and / or overlap, and can also improve the recognition of spatial clouds. Further, since the reading fragments obtained from any single chamber may be produced by the same sample, they can be used to reconstruct samples by stitching together short reading fragments in a bioinformatic manner.

[0046] The flow cell architecture disclosed herein can also improve the overall utilization of the surface area.

[0047] definition

[0048] Unless otherwise specified, the terms used in this article should be understood to take the usual meaning in the relevant field. Several terms used in this article and their meanings are as follows.

[0049] As used herein, the singular forms "a", "an", and "the" refer to the singular as well as the plural, unless the context clearly dictates otherwise. The term "comprising" as used herein is synonymous with "including", "containing", or "having the characteristics of..." and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0050] References throughout the specification to "one embodiment," "another embodiment," "an embodiment," etc. mean that particular elements (e.g., features, structures, compositions, configurations, and / or characteristics) described in conjunction with the embodiment are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the described elements for any embodiment may be combined in various embodiments in any suitable manner unless the context clearly dictates otherwise.

[0051] The terms "substantially" and "approximately" as used throughout this disclosure, including the claims, are used to describe and account for small fluctuations, such as due to variations in processing. For example, these terms may refer to less than or equal to 5% of a specified value, such as less than or equal to 2% of a specified value, such as less than or equal to 1% of a specified value, such as less than or equal to 0.5% of a specified value, such as less than or equal to 0.2% of a specified value, such as less than or equal to 0.1% of a specified value, such as less than or equal to 0.05% of a specified value.

[0052] Adaptor: A linear oligonucleotide sequence that can be fused to a nucleic acid molecule, for example, by connection or tagmentation. In some embodiments, the adaptor is substantially non-complementary to the 3' end or 5' end of any target sequence introduced into the flow cell. Suitable adaptor lengths may 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. The adaptor may include any combination of nucleotides and / or nucleic acids. In some embodiments, the adaptor includes one or more cleavable groups at one or more positions. In some embodiments, the adaptor may include a sequence complementary to at least a portion of a primer, such as a primer including a universal nucleotide sequence (e.g., a P5 or P7 sequence). In some embodiments, the adaptor may include an index or barcode sequence that facilitates downstream error correction, identification, or sequencing. The index may be unique to a sample or source of a nucleic acid molecule (e.g., a fragment). In some embodiments, the adaptor may include a sequencing primer sequence or a sequencing binding site. A combination of different adaptors may be incorporated into a nucleic acid molecule, such as a DNA fragment.

[0053] Capture site: A portion of a flow cell surface that is physically modified and / or chemically altered to allow localization of a complex or sample. In one embodiment, a capture site may include a chemical capture agent.

[0054] Support: A hydrogel support that can have a sequencing library contained therein or a solid support that can have sequencing-ready nucleic acid fragments attached to its surface.

[0055] Chemical capture agent: a material, molecule or part capable of attaching, retaining or binding to a target molecule (i.e., a complex or sample). An exemplary chemical capture agent includes a capture nucleic acid (e.g., a capture oligonucleotide) that is complementary to at least a portion of a target nucleic acid or connected to a target molecule. Another exemplary chemical capture agent is a connector. For a raw DNA or RNA sample, a connector may include a nucleic acid binding portion at one end, such as an intercalator that binds by charge or hydrophobic interactions. For a cell sample, a connector may include a cell membrane binding portion (e.g., an antigen for a surface protein) or a membrane penetrating portion (e.g., a phospholipid at one end). Yet another exemplary chemical capture agent includes a member of a receptor-ligand binding pair that is capable of binding to a target molecule (or a connecting portion connected to a target molecule) (e.g., avidin, streptavidin, biotin, lectin, carbohydrate, nucleic acid binding protein, epitope, antibody, etc.). Yet another example of a chemical capture agent is a chemical reagent that is capable of forming an electrostatic interaction, a hydrogen bond or a covalent bond with a target molecule (e.g., thiol-disulfide exchange, click chemistry, Diels-Alder, etc.).

[0056] Complex: A support (such as a hydrogel support or a solid support) and a sequencing-ready nucleic acid fragment attached to or contained within the support. The support may also include one member of a binding pair, the other member of which is part of a capture site.

[0057] External fixative: A gas, liquid or viscous medium that is immiscible with the complex or sample that has been introduced into the flow cell chamber. A gaseous external fixative can be used to create droplets around the complex or sample. An example of a gaseous external fixative is air directed through the flow cell at a suitable flow rate. For example, air can be used to draw a fluid containing the complex or sample from the flow cell, which forms droplets of the liquid containing the complex or sample. The droplets formed act as a diffusion barrier. The liquid or viscous medium is used to prevent the diffusion of the sequencing library released from the complex or formed in the chamber, such as on the surface of the flow cell. The external fixative can form a diffusion barrier because the sequencing library or any other polynucleotide is barely solvated in the external fixative. Examples of external fixatives in liquid form include hydrophobic oils, such as mineral oil, silicone oil, perfluorinated oil, fluorinated carbon oil (e.g., FLUORINERT from 3M). TM FC40) or a combination thereof. Examples of external fixatives in the form of viscous media include buffers containing polymers (e.g., polyethylene glycol, polyvinyl pyrrolidone, etc.), dextran, sucrose, glycerol, etc. In certain embodiments, the viscous medium is a temperature responsive gel. The temperature responsive gel is non-viscous at non-inoculation temperatures and becomes a viscous medium at inoculation temperatures. Examples of temperature responsive gels include poly (N-isopropylacrylamide) and polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO) / hectorite nanoparticle composites.

[0058] Fragment: A part or segment of genetic material (such as DNA, RNA, etc.).

[0059] Hydrogel or hydrogel matrix: A colloidal material comprising an organic polymer (natural or synthetic) that is cross-linked by covalent, ionic or hydrogen bonds to form a three-dimensional open grid structure that encapsulates water molecules to form a gel. In one embodiment, the hydrogel comprises about 60% to about 90% of a fluid, such as water, and about 10% to about 30% of a polymer. The hydrogel may be porous, i.e., comprise open / void space. Porosity is the fractional volume (dimensionless) of the hydrogel, i.e., measures the void space in the material and is the fraction of the void volume relative to the total volume, as a percentage of 0-100% (or a fraction of 0-1). In one embodiment, the porosity of the hydrogel may be in the range of about 50% (0.5) to about 99% (0.99). 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 prohibits diffusion of larger-sized nucleic acid molecules (e.g., samples, fragments, etc.).

[0060] Hydrogel support: A hydrogel having an at least substantially spherical shape (eg, a hydrogel bead) within which a sequencing library may be contained.

[0061] Nucleic acid molecule: A polymeric form of nucleotides of any length and may include ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. The term may refer to a single-stranded or double-stranded polynucleotide.

[0062] A "target" or "template" nucleic acid molecule may refer to the sequence to be analyzed.

[0063] Nucleotides in nucleic acid molecules can include naturally occurring nucleic acids and functional analogs thereof. Examples of functional analogs can hybridize with nucleic acids in a sequence-specific manner, or can be used as templates for replication of specific nucleotide sequences. Naturally occurring nucleotides typically have a backbone comprising a phosphodiester bond. Analog structures can have alternative backbone connections, including any of a plurality of species known in the art. Naturally occurring nucleotides typically have deoxyribose (e.g., found in DNA) or ribose (e.g., found in RNA). Analog structures can have alternative sugar moieties, including any of a plurality of species known in the art. Nucleotides can include natural or non-natural bases. Natural DNS can include one or more of adenine, thymine, cytosine and / or guanine, and natural RNA can include one or more of adenine, uracil, cytosine and / or guanine. Any non-natural bases can be used, such as locked nucleic acids (LNA) and bridged nucleic acids (BNA).

[0064] Primer: A nucleic acid molecule capable of hybridizing with a target sequence of interest. In one embodiment, a primer is used as a substrate on which nucleotides can be polymerized by a polymerase. For example, an amplification primer is used as a starting point for template amplification and cluster generation. In yet another embodiment, a primer can be used as a starting point for DNA or RNA synthesis. For example, a sequencing primer can hybridize with a synthetic nucleic acid template strand to initiate the synthesis of a new strand complementary to the synthetic nucleic acid template strand. A primer can include any combination of nucleotides or their analogs. In some embodiments, a primer is a single-stranded oligonucleotide or polynucleotide.

[0065] Sample: Any source of genetic material, such as a cell, a microbiome, or a nucleic acid. In some embodiments, the cell is a single cell including a prokaryotic or eukaryotic cell. In some embodiments, the cell is a mammalian cell, a human cell, or a bacterial cell. In some embodiments, the nucleic acid is a long DNA molecule, including a viral nucleic acid, a bacterial nucleic acid, or a mammalian nucleic acid. In some embodiments, the sample is bound (as a fragment) by insertion into a transposon bound to the surface of a solid support (e.g., a bead).

[0066] Sequencing-ready nucleic acid fragment: A portion (fragment) of genetic material with adapters at the 3' and 5' ends. In the sequencing-ready nucleic acid fragment, each adapter includes a known universal sequence (e.g., which is complementary to at least a portion of the primer on the flow cell) and a sequencing primer sequence. Both adapters may also include an index (barcode or tag) sequence. In one embodiment, the P5 side may include a bead index, and the P7 side may include a sample index. The sequencing-ready nucleic acid fragment can be bound by insertion of a transposon, where the inserted DNA molecule is fixed to the surface of a solid support (e.g., a bead); or directly fixed by a binding pair or other cleavable linker; or by hybridization binding, where complementary adapter sequences are present on the surface of a solid support.

[0067] Seeding: Immobilization of adapted fragments (eg, sequencing-ready nucleic acid fragments) in a chamber of a flow cell embodiment disclosed herein.

[0068] Sequencing library: A collection of nucleic acid fragments of one or more target nucleic acid molecules, or amplicons of fragments. In some embodiments, the fragments are connected to one or more adapters at their 3' and 5' ends. In some embodiments, a sequencing library is prepared from one or more target nucleic acid molecules and is part of a complex. In other embodiments, a sequencing library is prepared on a flow cell surface using a sample.

[0069] Solid support: A small object made of a rigid or semi-rigid material, whose shape has the characteristics of a sphere, ellipsoid, microsphere, or other recognized particle shape, whether it has regular or irregular dimensions. The solid support can have a sequencing library attached to it. Example materials that can be used for solid supports include, but are not limited to, glass; plastics such as acrylic, polystyrene or a copolymer of styrene and another material, polypropylene, polyethylene, polybutylene, polyurethane, or polytetrafluoroethylene (from Chemours Co. ); polysaccharides or cross-linked polysaccharides, such as agarose or Sepharose; nylon; nitrocellulose; resins; silica or silica-based materials, including silicon and modified silicon; carbon fibers, metals; inorganic glass; fiber optic bundles or various other polymers. Exemplary solid supports include controlled pore glass beads, paramagnetic or other magnetic beads, thoria sols, Sepharose beads, nanocrystals, and other substances known in the art, such as described in the Microsphere Detection Guide from Bangs Laboratories, Fishers Ind.

[0070] Tagmentation: Modification of nucleic acid molecules (e.g., DNA or RNA samples) by transposomes to fragment the nucleic acid molecules and attach adapters to the 5' and 3' ends of the fragments in a single step. Tagmentation reactions can be used to prepare sequencing libraries, particularly complexes that include solid supports. Tagmentation reactions combine random sample fragmentation and adapter ligation into a single step, which improves the efficiency of the sequencing library preparation process.

[0071] Transposome: A complex formed between an integrating enzyme (eg, an integrase or a transposase) and a nucleic acid comprising an integration recognition site (eg, a transposase recognition site).

[0072] Universal nucleotide sequence: A sequence region shared by 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 allow for the capture of several different nucleic acids using a population of universal capture nucleic acids (i.e., adapters having a sequence that is complementary to at least a portion of a primer). Similarly, universal sequences present in different members of a collection of molecules can allow for the amplification or replication of several different nucleic acids using a population of universal sequence binding sites (sequencing primer sequences).

[0073] Flow cell architecture

[0074] Figure 1A portion of an example flow cell 10 is shown. The flow cell 10 includes a substrate 12, a plurality of chambers 14 defined on or in the substrate 12, a plurality of recesses 16 defined in the substrate 12 and within the perimeter of each of the plurality of chambers 14, a primer 20 attached within each recess 16, and a capture site 22 located within each of the plurality of chambers 14.

[0075] The substrate 12 is generally rigid and insoluble in aqueous liquids. The substrate 12 may be a single layer or a multilayer structure. Examples of suitable substrates 12 include epoxysiloxane, polyhedral oligomeric silsesquioxane (POSS) or its derivatives, glass, modified glass, plastic, nylon, ceramic / ceramic oxide, silica (silicon dioxide), fused silica, silicon dioxide-based materials, aluminum silicate, silicon, modified silicon (e.g., boron-doped p+ silicon), silicon nitride (Si 3 N 4 ), tantalum pentoxide (TaO 5 ) or other tantalum oxides (TaO x ), hafnium oxide (HfO 2 ), inorganic glass, etc. Some examples of plastics suitable for substrate 12 include acrylic, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, polytetrafluoroethylene (such as from Chemours Co. ), cycloolefins / cycloolefin polymers (COP) (e.g. from Zeon ), polyimide, etc. The substrate 12 may also be glass or silicon or POSS, with a coating of tantalum oxide or another ceramic oxide on its surface. The substrate 12 may also be glass or silicon, with a POSS coating on its surface. Another example of a suitable substrate 12 is a silicon-on-insulator substrate.

[0076] The form of substrate 12 can be a wafer, panel, rectangular sheet, mold or any other suitable configuration. In one embodiment, substrate 12 can be a circular wafer or panel with a diameter range of about 2 mm to about 300 mm. As a more specific example, substrate 12 is a wafer with a diameter range of about 200 mm to about 300 mm. In another embodiment, substrate 12 can be a rectangular sheet or panel with a maximum size of up to about 10 feet (~3 meters). As a specific example, substrate 12 is a mold with a width range of about 0.1 mm to about 10 mm. Although example sizes have been provided, it should be understood that substrate 12 with any suitable size can be used.

[0077] A plurality of chambers 14 may be defined on or in the substrate 12 .

[0078] Figure 2A An embodiment of a chamber 14 defined on a substrate 12 is shown. In the embodiments disclosed herein, when i) the substrate surface S 12ii) an insulating material 18 is located on the substrate 12 and defines the walls of the chamber 14. 18 When the silicon-on-insulator substrate is used with the isolation material 18, the walls of the chamber 14 are considered to be "defined" on the substrate 12. 18 It may be defined in part by the outermost silicon layer of the substrate and the isolation material 18 .

[0079] The isolation material 18 can be a hydrophobic material, such as a fluorinated polymer, a perfluorinated polymer, a silicon polymer, or a mixture thereof. The polymer backbone of the hydrophobic material can be carbon or silicon, or a combination thereof. In some embodiments, the fluorinated polymer is an amorphous fluoropolymer (commercially available examples of which include fluoropolymers from AGC Chemicals). series, which have one of the following terminal functional groups: A type: -COOH, M type: -CONH-Si(OR) n Or S type: -CF 3 ), polytetrafluoroethylene (e.g., from Chemours Co. ), parylen (such as A-grade, F-grade, HT-grade), fluorinated hydrocarbons, fluorinated acrylic copolymers (such as FLUOROPEL from Cytonix TM ), (tridecafluoro-1,1,2,2-tetrafluorooctyl)trifluorosilane (FOTS), fluorosilane or plasma deposited fluorocarbon, or a mixture thereof. As another example, the hydrophobic polymer or hydrophobic polymer layer may include a hydrophobic hydrocarbon, such as 1-heptadecyne. In some embodiments, the silicon polymer is polydimethylsiloxane or another siloxane. When the material in the recess 16 is hydrophilic, it may be particularly desirable to use a hydrophobic material for the isolation material 18. Other polymers may be used as the isolation material 18 as long as the resulting structure is capable of inducing pearling of a liquid moving over the structure. Alternatively, if the material in the recess 16 is hydrophobic, it may be desirable to use a hydrophilic material for the isolation material 18. The hydrophobic or hydrophilic properties of the isolation material 18 may help direct the reagent to the recess 16.

[0080] In one embodiment, the isolation material 18 may be deposited on the substrate 12 and then patterned using photolithography. In an embodiment where the substrate 12 is a silicon-on-insulator substrate, the isolation material 18 and the outermost silicon layer may be patterned using photolithography. As an embodiment, a mask (e.g., a photoresist) may be used to define the space / position where the isolation material 18 is deposited. The separated material 18 may then be deposited, and the mask may be removed (e.g., by lift-off, dissolution, or another suitable technique). As another embodiment, the isolation material 18 may be deposited, and then a mask may be deposited on the isolation material 18. The mask may be patterned using photolithography, and any exposed portion of the isolation material 18 may be removed by plasma etching or dry etching of oxygen. The mask may then be removed to reveal the remaining isolation material 18. In yet another embodiment, the isolation material 18 may be laminated to the substrate 12, or transferred to the substrate 12 from a mold or sacrificial layer. In yet another embodiment, the isolation material 18 may be printed using microcontact printing (using a stamp), aerosol printing, or inkjet printing.

[0081] Figure 2B An embodiment of a chamber 14 defined in a substrate 12 is shown. In the embodiments disclosed herein, when i) the substrate surface S 12 defining a gap region around the chamber 14, ii) another substrate surface S' 12 iii) the base 12 further defines the walls of the chamber 14. 12 , chamber 14 is considered to be “defined” in substrate 12 .

[0082] In this embodiment, chamber 14 may be patterned into the substrate. Patterning may include etching chamber 14 into substrate 12 and / or using imprint lithography.

[0083] Whether formed on or in substrate 12, chambers 14 may be distributed throughout substrate 12 in any suitable pattern or layout. Many different layouts of chambers 14 are contemplated, including regular, repeating, and irregular patterns. In one embodiment, 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 embodiments, the layout or pattern may be an xy format of chambers 14 in rows and columns (e.g., Figure 1 shown).

[0084] Chamber 14 may have any suitable shape, such as a circular shape (eg, Figure 1 as shown), ellipse, polygon (e.g., triangle, quadrilateral, pentagon, etc.), and the like.

[0085] The size of each chamber 14 can be characterized by its opening area, diameter and / or length and width. Figure 1 As shown, the flow cell 10 has a plurality of recesses 16 located within each chamber 14. Therefore, the size of the chamber 14 is greater than the size of each recess 16. In other words, the dimensions of the chamber 14 are greater than the dimensions of each recess 16. In this embodiment, "dimensions" refers to the area occupied by each chamber opening or recess opening, and / or the diameter of the chamber 14 or recess 16, and / or the length and width of each chamber 14 or recess 16. Figure 1 In the illustrated embodiment, the open area and diameter of each chamber 14 is greater than the open area and diameter of each recess 16. The open area, diameter or length and width of each chamber 14 depends on the number of recesses 16 to be located in the chamber 14 and the size of the capture sites 22 to be located in the chamber 14.

[0086] The area occupied by each chamber opening can be selected so that the composite (an example of which is shown in FIG. 4A to FIG. 4C ) can enter chamber 14 and attach to capture site 22 in chamber 14. In one embodiment, 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 area occupied by each chamber opening may be greater than or between the above specified values.

[0087] In some cases, the diameter or length and width of each chamber 14 may 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. When chamber 14 has a length and a width, it should be understood that the length and width can be the same or different.

[0088] Chamber 14 may also have a depth depending on the technique used to form chamber 14. For example, when microcontact, aerosol, or inkjet printing is used to form chamber walls. 18 When the depth of each chamber 14 is about 1 μm, about 10 μm, about 50 μm or more, the depth of each chamber 14 can be a monolayer thickness. For other embodiments, the depth of each chamber 14 can be about 1 μm, about 10 μm, about 50 μm or more. In another embodiment, the depth is at least about 50% of the average diameter of the complex to be introduced into the chamber 14. In one embodiment, the depth can be in the range of about 10 μm to about 30 μm. The depth is sufficient to block the lateral diffusion of the released library fragments between adjacent chambers 14, thereby keeping the released library fragments in the chamber 14 without any external fixative. In another embodiment, the depth is about 5 μm or less. It should be understood that the depth of each chamber 14 can be greater than, less than, or between the above-mentioned specified values.

[0089] Adjacent chambers 14 can be separated by the surface S of the additional material 18. 18 ( Figure 2A ) or through the surface S of the substrate 12 12 ( Figure 2B The average chamber spacing represents the spacing from the center of one chamber 14 to the center of an adjacent chamber 14 (center-to-center spacing) or the spacing 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 so that the coefficient of variation of the average spacing 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 spacing 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 greater. In one embodiment, the average spacing is twice the diameter of the chamber 14. The average spacing of a particular pattern of chambers 14 may be between one of the lower limits and one of the upper limits selected from the above range. Although exemplary average chamber spacing values ​​have been provided, it should be understood that other average chamber spacing values ​​may also be used.

[0090] A plurality of recesses 16 may be defined in the substrate 12. In the embodiments disclosed herein, when i) the substrate surface S 12 or S' 12 defining gap regions 24 separating the recesses 16, ii) another substrate surface S" 12 The recess 16 is considered to be “defined in” the substrate 12 when iii) the substrate 12 also defines the bottom surface of the recess 16 , and iii) the substrate 12 also defines the walls of the recess 16 .

[0091] Recesses 16 may be patterned into substrate 12. Patterning may include etching recesses 16 into substrate 12 and / or using imprint lithography.

[0092] The respective subsets of recesses 16 may be distributed in each chamber 14 in any suitable pattern or layout. The pattern of recesses 16 may be the same in each chamber 14, or different patterns of recesses 16 may be used in different chambers 14. Many different patterns / layouts of recesses 16 are contemplated, including regular, repeating, and irregular patterns. In one embodiment, the recesses 16 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, and the like. Figure 1 In the illustrated embodiment, the depressions 16 in each chamber 14 are arranged in a circular pattern around the capture site 22. Figure 1 As shown in , a plurality of chambers 14 may be arranged in a first pattern (eg, 2x2) across substrate 12 , and a corresponding subset of recesses may be arranged in a second pattern (eg, circular) within each chamber 14 .

[0093] Each recess 16 may have any suitable shape (and corresponding three-dimensional geometry), such as a circle (e.g. Figure 1 ), an ellipse, a polygon (e.g., a triangle, a quadrilateral, a pentagon, etc.), and the like.

[0094] The size of each recess 16 can be characterized by its opening area, diameter and / or length and width. Figure 1 As shown, the flow cell 10 has a plurality of depressions 16 located within each chamber 14. Thus, the size of each depression 16 is smaller than the size of the chamber 14 in which it is located.

[0095] The area occupied by each recess opening can be selected so that the composite cannot enter the recess 16. In one embodiment, the area of ​​each recess opening can be at least about 1×10 -4 μm 2 , at least about 1×10 -3 μm 2 , at least about 1×10 -2 μm 2 , at least about 0.1 μm 2 , at least about 0.5 μm 2 , at least about 1 μm 2 or at least about 4 μm 2 The area occupied by each recessed opening may be less than or between the above specified values.

[0096] In some cases, the diameter or length and width of each depression 16 can be at least about 1 nanometer, at least about 50 nanometers, at least about 100 nanometers, at least about 500 nanometers, or more, as long as the dimensions are smaller than the diameter or length and width of the chamber. An example range of depression diameters is about 1 nanometer to about 500 nanometers. Another example range of depression diameters is about 300 nanometers to about 2 μm.

[0097] The recesses 16 may also have a depth. As an example, the depth of each recess 16 may be at least about 10 nanometers, at least about 50 nanometers, at least about 1 μm, up to about 2 μm. In some embodiments, 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 above specified values.

[0098] In one embodiment, the aspect ratio (diameter:depth) of the depressions 16 may be in the range of about 1:1 to about 1:2, or in the range of about 1:1.25 to about 1:1.75.

[0099] Adjacent recesses 16 may be separated by gap regions 24 within a given chamber 14. The average recess spacing represents the spacing from the center of one recess 16 to the center of an adjacent recess 16 (center-to-center spacing) or the spacing from the edge of one recess 16 to the edge of an adjacent recess 16 (edge-to-edge spacing). The layout or pattern of the recesses 16 may be regular, so that the coefficient of variation of the average spacing is small, or the layout or pattern may be irregular, in which case the coefficient of variation may be relatively large. In either case, depending on the dimensions of the chamber 14, the average spacing may be, for example, at least about 10 nanometers, at least about 0.1 μm, at least about 0.5 μm, or greater. Alternatively or additionally, the average spacing may be, for example, at most about 0.5 μm, at most about 0.1 μm, or less. The average spacing of a particular recess 16 pattern may be between one of the lower and upper limits selected from the above range.

[0100] Primer 20 is also attached within each recess 16. Primer 20 can be any forward amplification primer or reverse amplification primer, including primers that can be attached to at least the bottom of each recess 16 (i.e., surface S" 12 Primers 20 may form a lawn of capture oligonucleotides within each recess 16, which may bind to adapters of sequencing-ready nucleic acid fragments.

[0101] In one embodiment, primer 20 can be fixed to layer 26 by a single point covalent attachment at or near the 5' end of primer 20. This attachment allows i) the adaptor-specific portion of primer 20 to be free for annealing to a homologous sequencing-ready nucleic acid fragment and ii) the 3' hydroxyl group to be free for primer extension. Any suitable covalent attachment can be used for this purpose. Examples of end-capped primers that can be used include alkyne-terminated primers, tetrazine-terminated primers, azide-terminated primers, amino-terminated primers, epoxy- or glycidyl-terminated primers, phosphorothioate-terminated primers, thiol-terminated primers, aldehyde-terminated primers, hydrazine-terminated primers, phosphoramidite-terminated primers, and triazolinedione-terminated primers. In another embodiment, primer 20 can be fixed to layer 26 by non-covalent interactions. In one embodiment, each primer 20 can include a linker molecule (e.g., biotin) that can non-covalently bind to layer 26. In some embodiments, two different primers 20 are used. Specific examples of suitable primers 20 include primers for use in HISEQ. TM HISEQX TM MISEQ TM MISEQDX TM MINISEQ TM 、NEXTSEQ TM 、NEXTSEQDX TM 、NOVASEQ TM 、GENOME ANALYZERTM 、ISEQ TM The P5 and P7 primers are used on the surface of commercial flow cells sold by Illumina Inc. for sequencing on PCR and other instrument platforms.

[0102] In one embodiment, layer 26 is a substance capable of non-covalently binding to a linker molecule attached to primer 20. As one example, the linker molecule is biotin, and layer 26 is avidin, streptavidin, etc. In this embodiment, layer 26 can be applied by microcontact printing or aerosol printing, deposition and polishing, or another suitable selective deposition technique.

[0103] In another embodiment, layer 26 is a polymer capable of being covalently attached to primer 20. The bottom surface of recess 16 (e.g., S" 12 ) can be activated and then a polymer can be applied to form layer 26.

[0104] In some embodiments, activation may include applying a silane or silane derivative (eg, norbornene silane). In other embodiments, activation may include plasma ashing to produce a surface active agent (eg, -OH groups) that can adhere to the polymer used to form layer 26.

[0105] One example of a polymer that can be used to form layer 26 includes an acrylamide copolymer, such as poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide, PAZAM. PAZAM and some other forms of acrylamide copolymers are represented by the following structure (I):

[0106]

[0107] in:

[0108] R A is selected from the group consisting of azido, optionally substituted amino, optionally substituted alkenyl, optionally substituted hydrazone, optionally substituted hydrazine, carboxyl, hydroxyl, optionally substituted tetrazole, optionally substituted tetrazine, nitrile oxide, nitrone and thiol;

[0109] R B is hydrogen or optionally substituted alkyl;

[0110] R C , R D and R E are each independently selected from H and optionally substituted alkyl;

[0111] Each-(CH 2 ) p - may be optionally substituted;

[0112] p is an integer ranging from 1 to 50;

[0113] n is an integer in the range of 1 to 50,000; and

[0114] m is an integer in the range of 1 to 100,000.

[0115] One of ordinary skill in the art will recognize that the arrangement of the repeating "n" and "m" features in structure (I) is representative and that the monomer subunits may be present in the polymer structure in any order (e.g., random, block, patterned, or a combination thereof).

[0116] In some embodiments, PAZAM is a linear polymer. In other embodiments, PAZAM is a lightly cross-linked polymer.

[0117] In other embodiments, the polymer that can be used to form layer 26 can be a variation of structure (I). In one embodiment, the acrylamide unit can be N,N-dimethylacrylamide. In this embodiment, the acrylamide unit in structure (I) can be replaced by Substitute, where R D , R E and R F Each is H or C1-C6 alkyl, and R G and R H Each is a C1-C6 alkyl group (rather than H as in the case of acrylamide). In this embodiment, q can be an integer in the range of 1 to 100,000. In another embodiment, in addition to the acrylamide units. N,N-dimethylacrylamide can be used. In this embodiment, in addition to the recurring "n" and "m" features, structure (I) can include Where R D , R E and R F Each is H or C1-C6 alkyl, and R G and R H Each is a C1-C6 alkyl group. In this embodiment, q may be an integer in the range of 1 to 100,000.

[0118] It should be understood that other polymers or molecules may be used to form layer 26, provided they are functionalized to bond with surface S″. 12and subsequently applied primer 20. Other examples of polymers suitable for layer 26 include those with colloidal structures, such as agarose; or polymer mesh structures, such as gelatin; or cross-linked polymer structures, such as polyacrylamide polymers and copolymers, silane-free acrylamide (SFA) or azid forms of SFA. 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 polymers suitable for layer 26 include mixed copolymers of acrylamide and acrylate.

[0119] The method used to functionalize recesses 16 with the polymer that forms layer 26 may depend on whether chambers 14 are defined on or in substrate 12 .

[0120] For example, when chamber 14 is defined on substrate 12 by insulating material 18, it should be understood that substrate surface S 12 and S” 12 Treatment may be performed to functionalize the recesses 16, and then the isolation material 18 may be attached to the surface S 12 To define chamber 14. In one embodiment, silane or silane derivatives can be deposited on 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” 12 The polymer (which will form layer 26) can then be applied to the activated substrate surface S using spin coating, or dipping or immersion coating, or flow of material under positive or negative pressure, or another suitable technique. 12 and S” 12 In one embodiment, the polymer may be present in a mixture (e.g., with water or with ethanol and water). Depending on the polymer, the applied mixture may be exposed to a curing process to form a solid phase on the surface S 12 and S” 12 In one embodiment, curing can be performed at a temperature ranging from room temperature (e.g., about 25° C.) to about 95° C. for a period of about 1 millisecond to about several days. Polishing can then be performed to remove the surface S 12 Removing layer 26, while making surface S" 12 In these embodiments, the isolation material 18 may then be formed on the surface S as described herein. 12 on (e.g., photolithography, printing, film transfer or lamination, etc.).

[0121] For another embodiment, when chambers 14 are defined in substrate 12, it will be appreciated that selective deposition techniques may be used to functionalize recesses 16. In this embodiment, the silane or silane derivative and then polymer mixture may be deposited by microcontact printing, aerosol printing, or inkjet printing.

[0122] A grafting process may be performed to graft the primer 20 onto the layer 26 in the recess 16. In one embodiment, the grafting may include flow through deposition (e.g., using a temporarily bonded cap), dunk coating, spray coating, puddle dispensing, or another suitable method by which the primer 20 is attached to the layer 26 in the recess 16. Each of these exemplary techniques may utilize a primer solution or mixture that may include a primer, water, a buffer, and a catalyst. Using any of the grafting methods, the primer 20 reacts with the reactive groups of the polymer layer 26 in the recess 16 and is bonded to the interstitial region 24, other substrate surfaces S 12 or S' 12 Or the isolation material 18 has no affinity. Therefore, the primer 20 is selectively grafted to the polymer layer 26 in the recess 16.

[0123] Examples of flow cells 10 disclosed herein include capture sites 22 located within each of the plurality of chambers 14 . Figure 1 , Figure 2A and Figure 2B One embodiment of a capture site 22 is shown, and FIG. 3A to FIG. 3C Other embodiments of capture sites 22 are shown.

[0124] Capture sites 22 can physically and / or chemically immobilize a complex or sample within a particular chamber 14. Physical immobilization utilizes Figure 3A The illustrated embodiment is possible. Chemical immobilization involves a chemical capture agent 28 as defined herein. When the capture sites 22 are capable of chemical immobilization, the chemical capture agent 28 used may depend in part on the complex or sample to be introduced into the flow cell 10 .

[0125] In some embodiments disclosed herein, capture sites 22 are capable of capturing complexes introduced into flow cell 10. In other embodiments disclosed herein, capture sites 22 are capable of capturing samples that are then further processed on the flow cell surface to generate a library.

[0126] exist Figure 1 , Figure 2A and Figure 2B, the capture site 22 is formed at the center of the chamber 14. It should be understood that the capture site 22 can be located at any desired location within the chamber 14, which may depend on the arrangement of the recesses 16. The location of the capture site 22 across the substrate 12 can be uniform (e.g., each capture site 22 is substantially at the same location within each chamber 14 (e.g., center, leftmost, etc.)) or can be non-uniform (e.g., capture sites 22 are located at different locations within different chambers 14).

[0127] The capture site 22 may have any suitable shape, geometry, and size, which may depend at least in part on the configuration of the capture site 22 (e.g., a patch, a hole, a 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.

[0128] exist Figure 1 , Figure 2A and Figure 2B In the illustrated embodiment, the capture site 22 is a chemical capture agent 28 applied to a portion of the gap region 24. Any example of the chemical capture agent 28 disclosed herein can be used. In one embodiment, the chemical capture agent 28 can be deposited at the desired location using microcontact printing, aerosol printing, etc. In another embodiment, a mask (e.g., a photoresist) can be used to define the space / position where the chemical capture agent 28 will be deposited. The chemical capture agent 28 can then be deposited and the mask removed (e.g., by stripping, dissolving, or another suitable technique). In this embodiment, the chemical capture agent 28 can form a single layer or thin layer of the chemical capture agent 28, which can be referred to as a patch.

[0129] Figure 3A , Figure 3B and Figure 3C Other embodiments of capture sites 22 are shown. It should be understood that any capture site 22 may be used in any embodiment of flow cell 10, including those having chambers 14 defined on substrate 12 (and including additional material 18) or those having chambers 14 defined in substrate 12.

[0130] exist Figure 3A and Figure 3B In the embodiment of the present invention, the capture site 22 includes a hole 30 defined in the substrate 12. The hole can be "defined" in the substrate 12 in the same manner as the depression 16. Depending on the substrate 12 used, the hole 30 can be formed using etching, photolithography and / or embossing. In one embodiment, the hole 30 can be formed simultaneously with the depression 16.

[0131] Aperture 30 may have any suitable shape and geometry, including any of those described herein for recess 16 .

[0132] exist Figure 3AIn the illustrated embodiment, the opening dimension of the well 30 is greater than the opening dimension of each of the plurality of recesses 16. In this embodiment, the "opening dimension" refers to the area occupied by each well opening and each recess opening, and / or the diameter of each well opening and each recess opening, and / or the length and width of each well opening and each recess opening. The opening dimension of the well 30 may depend on the size of the compound or sample to be introduced therein. Figure 3A In the illustrated embodiment, the recess 16 is smaller than the well 30, in part so that the recess 16 cannot physically contain the compound or sample. Figure 3A In one embodiment, the depth of the depression 16 is less than the depth of the well 30, although it should be understood that the diameter or length and width may also be smaller. In other embodiments, the size of the well 30 may be similar or the same as the size of the depression 16. In one embodiment, the chemical capture agent 28 includes the primer 20, and thus any depression 16 may be used as a well 30 to capture the complex or sample.

[0133] In some embodiments, the wells 30 do not have additional chemical capture agents 28 added thereto. In these embodiments, the opening dimensions enable the complex or sample to self-assemble into the wells 30 rather than into the recesses 16 by size exclusion.

[0134] In other embodiments, the well 30 has additional chemical capture agents 28 added thereto (e.g. Figure 3A ). Any example of the chemical capture agent 28 disclosed herein can be used. In one embodiment, the chemical capture agent 28 can be deposited in the well 30 using microcontact printing. In another embodiment, a mask (e.g., a photoresist) can be used to deposit the chemical capture agent 28 in the well 30. In these embodiments, the opening dimensions enable the complex or sample to self-assemble into the well 30, rather than into the recess 16, by size exclusion and by binding affinity between the chemical capture agent 28 and the complex or sample introduced into the flow cell 10.

[0135] Figure 3B The capture site 22 in the apparatus comprises a hole 30 and a capture bead 32 having a chemical capture agent 28 on its surface. The capture bead 32 may be sized to fit into the hole 30 rather than the recess 16. In some embodiments, the capture bead 32 may be coplanar with the adjacent gap region 24 or extend slightly above the adjacent gap region 24 so that the complex or sample ultimately attached thereto is not confined within the hole 30. In one embodiment, the capture bead 32 is selected from silica, superparamagnetic materials, polystyrene, and acrylates. Any example of a chemical capture agent 28 disclosed herein may be used on the surface of the capture bead 32 and may be coated on the capture bead 32 before the capture bead 32 is introduced into the hole 30.

[0136] The depth of hole 30 (in Figure 3AOr 3B) can vary depending on whether the chemical capture agent 28 is introduced therein and whether the capture bead 32 is introduced therein. The depth can be selected to at least accommodate these materials (i.e., the material is contained within the hole 30). In one embodiment, the depth of the hole 30 ranges from about 1 nanometer to about 5 microns. In other embodiments, the depth of the hole 30 ranges from about 1 nanometer to about 100 nanometers, or from about 1 micron to about 5 microns. Other depths are also possible.

[0137] exist Figure 3C In the embodiment, the capture site 22 includes a portion of the substrate 12 or a portion of the surface S of the substrate 12. 12 The protrusion 34 is a three-dimensional structure extending outward (upward) from the adjacent surface. When the protrusion 34 is formed in the substrate 12, the substrate 12 is patterned (e.g., by etching, photolithography, embossing, etc.) so that it extends above the adjacent surrounding gap region 24. When the protrusion 34 is formed on the substrate 12, the additional material 18 is patterned (e.g., by etching, photolithography, embossing, etc.) so that it extends to the adjacent surrounding substrate surface S 12 Above.

[0138] Although any suitable three-dimensional geometry can be used for protrusion 34, a geometry having at least a substantially flat top surface may be desirable. Exemplary protrusion geometries include spheres, cylinders, cubes, polygonal prisms (e.g., rectangular prisms, hexagonal prisms, etc.), and the like.

[0139] like Figure 3C As shown, the chemical capture agent 28 is applied to the top surface of the protrusion 34. Any example of the chemical capture agent 28 disclosed herein may be used, and the chemical capture agent 28 may be applied to the top surface of the protrusion 34 using any deposition technique.

[0140] In some cases, it may be desirable to have one capture site 22 per chamber 14. In other cases, it may be desirable to have multiple, isolated capture sites 22 per chamber 14. The number of capture sites 22 in a single chamber 14 can help control the number of complexes captured within a given chamber 14.

[0141] See again Figure 2A and Figure 2B , the flow cell 10 may further include a cover 36 bonded to the isolation material 18 or the substrate 12. The cover 36 may be positioned so that it defines a single flow channel (in fluid communication with the plurality of chambers 14) or a plurality of fluidically isolated flow channels (each of which is in fluid communication with a subset of the plurality of chambers 14).

[0142] The cover 36 may be any material that is transparent to the excitation light directed toward the recess 16. For example, the cover may be glass (e.g., borosilicate, fused silica, etc.), plastic, etc. A commercially available example of a suitable borosilicate glass is D Available from Schott North America, Inc. Commercially available examples of suitable plastic materials (ie, cycloolefin polymers) are available from Zeon Chemicals LP. product.

[0143] The lid 36 may be bonded using any suitable technique, such as laser welding, diffusion bonding, anodic bonding, eutectic bonding, plasma activated bonding, glass paste 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 isolation material 18 or substrate 12. The spacer layer may be any material that seals at least some of the isolation material 18 or substrate 12 with the lid 36.

[0144] Although not shown, it will be appreciated that one or more additional layers may be added between substrate 12 and cover 36 or between substrate 12 and recess 16. These additional layers may be selected to act as a planar waveguide for exciting recess 16 with an evanescent field.

[0145] It should be understood that other flow cell architectures are also contemplated herein. As an example, flow cell 10 may include chamber 14 and capture site 22, but not recess 16. In these embodiments, primer 20 may be attached to the bottom surface of chamber 14, rather than in discrete recess 16. The bottom surface of chamber 14 may be functionalized with layer 26 and primer 20. In certain embodiments, layer 26 may be applied to the entire bottom surface (except where capture site 22 is formed). In these embodiments, a primer 20 lawn may be formed that is at least substantially uniform on the entire bottom surface of chamber 14. In other embodiments, layer 26 may be applied as an island (e.g., a shape such as a circle, triangle, rectangle) that is spatially isolated from one another in chamber 14. The library fragments released from the specific complex captured on flow cell 10 or the library fragments formed in situ on flow cell 10 may be randomly distributed in chamber 14 (contrary to being confined in the recess 16 of chamber 14). Figure 5 (i) to Figure 5 (iii) shows an example of such a flow cell architecture.

[0146] As another example, the flow cell 10 may include capture sites 22 without chambers 14 or recesses 16. In these examples, the capture sites 22 may be positioned in a desired geometry throughout the substrate, and the primers 20 may be attached to the surface of the substrate 12 around the capture sites 22. Library fragments released from specific complexes captured on the flow cell 10 or formed in situ on the flow cell 10 may be randomly distributed on the substrate 12, and confinement of the released library fragments may be achieved by controlling reaction-diffusion.

[0147] As yet another example, the flow cell 10 may include recesses 16 and capture sites 22, but not chambers 14. In these examples, the capture sites 22 may be positioned in a desired geometry throughout the substrate 12, and the primers 20 may be attached as described herein within each recess 16. Library fragments released from a specific complex captured on the flow cell 10 or formed in situ on the flow cell 10 may be randomly distributed in the recesses 16 near the complex, and confinement of the released library fragments may be achieved by controlling reaction-diffusion.

[0148] In still other embodiments, capture sites 22 are not included because the walls of chamber 14 have a height sufficient to capture one or more of the complexes or samples introduced into flow cell 10. The height sufficient to capture one or more complexes or samples corresponds to a chamber depth of at least about 50% of the average diameter of the complexes or samples introduced into flow cell 10. In one embodiment, the height of the wall or the depth of chamber 14 is 10 microns or more. In this embodiment, the number / amount of complexes or samples in a given chamber 14 is random and will be determined by a Poisson distribution.

[0149] In any embodiments disclosed herein, it should be understood that primers 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 being seeded on the sidewalls.

[0150] The flow cell architecture disclosed herein can be used for a variety of applications, including sequencing technology, such as linked long read sequencing applications, high-throughput protein biomarker research, microbiome research, or single-cell omics. For example, the flow cell architecture and method disclosed herein can be used to analyze the binding of antibodies labeled with DNA. In this embodiment, the antibody tag is attached to a unique DNA sequence with a P5 / P7 adapter, which is introduced into the flow cell architecture. The antibody can be cut, and the released P5 / P7 primer is inoculated onto the flow cell. The inoculated primer enables identification of which antibody is attached.

[0151] Composites for flow cell architecture

[0152] Flow cell architectures may be particularly suitable for use with the complex embodiments disclosed herein. As described herein, the complex includes a support (e.g., a hydrogel support or a solid support) and a sequencing-ready nucleic acid fragment attached to or contained within the support. Examples of suitable complexes are shown in FIG. 4A to FIG. 4C Although some exemplary methods for preparing the complexes are described, it should be understood that other methods may be used as long as the sequencing-ready nucleic acid fragments are attached to or contained within a vector.

[0153] Figure 4A A complex 40A including a solid support 42 and a sequencing-ready nucleic acid fragment 44 attached to the solid support 42 is shown.

[0154] In one embodiment, to form the complex 40A, an adapter sequence (52, 52') is bound to the solid support 42 via one member of the binding pair 46. In one embodiment, the adapter sequence includes a first sequencing primer sequence (e.g., a read fragment 1 sequencing primer sequence), a first sequence complementary to at least a portion of one of the primers 20 on the flow cell 10A-10I (e.g., a P5' sequence). As described, the adapter sequence is bound to one member of the binding pair 46 (e.g., biotin) so that it can bind 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 can also include an index sequence.

[0155] The Y-adapter can be mixed with a transposase (e.g., two Tn5 molecules) to form a transposome. The Y-adapter may include two splicing end sequences that hybridize to each other. One of the splicing end sequences may be attached to a second sequencing primer sequence (e.g., a read fragment 2 sequencing primer sequence), a second sequence complementary to at least a portion of one of the primers 36 on the flow cell 10A-10I (e.g., a P5' sequence), and an optional index / barcode sequence. The second sequencing primer sequence and the second sequence together constitute an adapter sequence 48, 48'.

[0156] The labeling process can then be performed. A fluid (e.g., labeling buffer) comprising a sample (e.g., DNA) can be added to a transposome and a solid support 42 having an adapter sequence bound thereto. When the sample contacts the transposome, the DNA is labeled (fragmented and labeled with adapter sequences 52, 52' on the solid support 42) and is bound to the Y-adapter (e.g., by connection of free spliced ​​end sequences). The free spliced ​​end sequences of the Y-adapter are connected to the adapter sequences on the solid support 42. The sequential labeling of the sample results in multiple bridging molecules between the transposomes. To complete the sequencing preparation fragments, further extension and connection are performed to ensure that the fragments 50, 50' are attached to the sequences 48 and 48'. The transposase can then be removed by sodium dodecyl sulfate (SDS) treatment or heating or proteinase K digestion.

[0157] The resulting complex 40A is shown in Figure 4A In. The bridging molecules are sequencing-ready nucleic acid fragments 42, each of which includes a fragment 50, 50' and an adapter sequence 48 and 52 or 48' and 50' attached to either end. The adapter sequences 52, 52' are those sequences that are initially bound to the solid support 42, and include a first sequencing primer sequence, a first sequence complementary to a flow cell primer, and a member 46 of the binding complex. The adapter sequences 48, 48' are from the Y-adapter and include a second sequence complementary to another flow cell primer and a second sequencing primer sequence. Because each sequencing-ready nucleic acid fragment 44 includes a suitable adapter for amplification (e.g., bridge amplification) and sequencing, PCR amplification is not performed. Therefore, these fragments 44 are ready for sequencing. In addition, because the library fragments 44 are from the same sample, the fragments 44 may be suitable for linked long-read applications.

[0158] Figure 4B Another complex 40B is shown, which includes a solid support 42 and a sequencing-ready nucleic acid fragment 44' attached to the solid support 42. In one embodiment, a PCR-free nucleotide library is established in a tube and then hybridized to the solid support 42 in the tube. Figure 4B In the embodiment shown, a primer having one member of a binding pair is added to the library fragments in the tube, and then the sequencing-ready nucleic acid fragments 44' are bound to the solid support 42. In another embodiment, the solid support 42 can have primers attached to it via a binding pair (e.g., avidin on the support 42 and biotin attached to the primer). These primers hybridize to the library fragments (and thus the primer and binding pair member are located at one end of the fragments but not the other). In another embodiment, extension can be performed using a strand displacement enzyme. This will produce a completely double-stranded library (e.g., no forked or Y-shaped adapters, such as Figure 4B). Sequencing-ready nucleic acid fragments 44' may be released on the flow cell by denaturation. Because library fragments 44' are generated before being attached to solid support 42, fragments 44' may not be from the same sample and therefore may not be suitable for linked long read applications.

[0159] Figure 4C An embodiment of a complex 40C is shown, which includes a hydrogel support 70 and a sequencing-ready nucleic acid fragment 44" contained within the hydrogel support 70.

[0160] In some embodiments of forming the composite 40, a fluid containing hydrogel monomers and / or polymers and a crosslinking agent is mixed in the presence of a sample (e.g., genetic material). The fluid can be loaded into mineral oil or another suitable hydrophobic fluid and emulsified to produce droplets. A free radical initiator can be added to polymerize and / or crosslink the hydrogel monomers and / or polymers and form a hydrogel support 70. Examples of suitable monomers, polymers, crosslinking agents, and initiators are shown in FIG. Figure 5 (i) to Figure 5 (iii) Provide a description.

[0161] In other embodiments of forming the composite 40C, a fluid containing a hydrogel polymer is mixed with a sample (e.g., genetic material) and loaded onto a flow cell. The flow cell may be exposed to heating or cooling so that the hydrogel polymer forms a gel (e.g., hydrogel support 70). Examples of suitable polymers and temperatures are further described below.

[0162] The sample becomes encapsulated in the hydrogel support 70 because its size is sufficient to prevent it from passing through the pores of the hydrogel beads. In some embodiments, the sample is DNA or RNA and is at least about 100 nucleotides in length (e.g., 1,000 or more, 10,000 or more, 500,000 or more, etc.). In some embodiments, based on the measurement of multiple pores, the pore size of the hydrogel support 70 refers to the average diameter or average effective diameter of the pore cross section. 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 embodiment, the pore size ranges from about 10 nanometers to about 100 nanometers.

[0163] Library preparation can then be performed within the hydrogel support 70. Multiple reagent exchanges can be performed through the pores of the hydrogel support 70. The sample and any library fragments generated thereby are maintained within the hydrogel matrix. Library preparation can involve fragmenting the sample and adding adapters, which will produce sequencing-ready fragments 44".

[0164] In one embodiment, library preparation can be performed by tagmentation performed within a hydrogel support 70. The resulting complex 40C is shown in Figure 4CThe adapter sequence includes appropriate adapters for bridge amplification and sequencing, and the resulting fragments 44" are therefore ready for sequencing. In another embodiment, library preparation can be performed using polymerase extension, which results in a double-stranded library. This example library requires denaturation prior to release from the hydrogel support 70 and seeding.

[0165] Methods involving complexes

[0166] Some embodiments of the methods disclosed herein utilize embodiments of the flow cell 10 disclosed herein and any one of the complexes 40A, 40B, or 40C. As described above, each of the complexes 40A, 40B, or 40C includes sequencing-ready fragments obtained from the same genetic material sample. When one or several complexes are isolated in corresponding chambers, spatial co-localization of libraries from the same sample is achieved.

[0167] In a first exemplary method, the flow cell 10 includes a plurality of chambers 14, but does not include capture sites 22. Instead, the chambers 14 themselves serve as capture sites for complexes 40A, 40B, or 40C introduced into the flow cell. Each chamber 14 can serve as a capture site, for example, when the depth is at least about 50% of the average diameter of the complexes 40A, 40B, or 40C to be introduced therein. In one embodiment, the depth is at least about 10 microns (about 10 microns or greater). In this embodiment, the flow cell 10 may have primers 20 attached to the bottom surface of the chambers 14, or may include recesses 16 in which the primers 20 are contained. In these embodiments, the number of complexes 40A, 40B, or 40C that become captured in any given chamber 14 may be random and determined by a Poisson distribution.

[0168] In this first exemplary method, complex 40A, 40B or 40C is introduced into flow cell 10, for example, through one or more input ports. Complex 40A, 40B or 40C can be introduced into a fluid, such as Tris-HCl buffer or 0.5x sodium citrate saline (SSC) buffer. At least some complexes 40A, 40B or 40C from the fluid will settle in at least some chambers 14. It should be understood that some complexes 40A, 40B or 40C may not precipitate, and before further processing, these complexes 40A, 40B or 40C are removed from the flow cell. It should also be understood that some chambers 14 may receive one or more complexes 40A, 40B or 40C, while other chambers 14 may not receive complexes 40A, 40B or 40C. In the present embodiment, the distribution of complexes 40A, 40B or 40C is random, in part because of the lack of capture sites 22.

[0169] The first exemplary method then includes washing the non-captured complexes 40A, 40B, or 40C from the flow cell. Washing may include introducing a fluid into the flow cell 10. The flow may push any unsettled complexes 40A, 40B, or 40C out through the outlet port of the flow cell. The deep chamber 14 may prevent any settled complexes 40A, 40B, or 40C from becoming part of the outlet flow.

[0170] The method embodiment then includes causing the carrier (e.g., solid support 42 or hydrogel support 70) of the captured complexes 40A, 40B, or 40C to release the sequencing-ready nucleic acid fragments 44, 44', or 44" into the corresponding chamber 14 where each complex 40A, 40B, or 40C was captured. In this embodiment, the transport and seeding of the sequencing-ready nucleic acid fragments 44, 44', or 44" is limited by the depth of the corresponding chamber 14, and therefore the external fixative is not introduced into the flow cell.

[0171] Causing the carrier (i.e., support 42 or 70) to release the sequencing-ready nucleic acid fragments 44, 44' or 44" can vary depending on the complex 40A, 40B or 40C used. In one embodiment, the carrier is a solid support 42, and the causing includes introducing a cleavage agent into the flow cell. The cleavage agent can initiate chemical, enzymatic, or photochemical release of the sequencing-ready nucleic acid fragments 44, 44' from the solid support 42. In these embodiments, another stimulus, such as heat or light, can trigger the cleavage agent to release the library fragments 44 or 44' from the solid support 42. As an example, free biotin can be introduced as a cleavage agent, and heating to about 92°C can be used to induce release of the biotin-oligomer from the solid support 42.

[0172] In other embodiments, complex 40C is used, and thus the carrier is a hydrogel support 70. In these other embodiments, causing the library to release may involve heating the flow cell 10, introducing a cleavage agent to the flow cell 10, or a combination thereof. Heating to release the library fragments 44" from the hydrogel support 70 may include heating to a temperature of about 90°C. The entire flow cell 10 may be heated, and when the complex 40C is heated, the hydrogel support 70 may degrade to release the fragments 44". In some embodiments, the cleavage agent may include one or more components that can depolymerize the hydrogel support 70 and release the sequencing-ready fragments 44" therefrom. For example, the cleavage agent includes dithiothreitol (DTT), tris-(2-carboxyethyl)phosphine (TCEP), or tris-(3-hydroxypropyl)phosphine (THP). In other embodiments, the cleavage agent is light. In these embodiments, the cross-linking agent used to form the hydrogel support 70 may include a light-cleavable portion, and exposure of the complex 40C in the chamber 14 to light of an appropriate wavelength may cleave the portion and degrade the hydrogel support 70.

[0173] As described, transport and seeding of sequencing-ready nucleic acid fragments 44, 44' or 44" are restricted by the depth of the corresponding chamber 14. Thus, the fragments 44, 44' or 44" of any particular complex 40A, 40B or 40C are confined to the chamber 14 in which that particular complex 40A, 40B or 40C is confined.

[0174] Using the flow cell architecture disclosed herein, the primers 20 on the surface of the flow cell 10 can be seeded with the released sequencing-ready nucleic acid fragments 44, 44' or 44". In one embodiment, the seeding is accomplished by hybridization between the first or second sequence of the fragment 44, 44' or 44" and the complementary sequence in the primer 20 of the chamber 14. The seeding can be performed at a suitable hybridization temperature for the fragment 44, 44' or 44" and the primer 20.

[0175] The location at which the sequencing-ready nucleic acid fragments 44, 44', or 44" are seeded in the corresponding chamber 14 depends in part on how the primers 20 are attached within the chamber 14. In some embodiments of the flow cell 10, each chamber 14 has a bottom surface, and the primers 20 are attached to the polymer layer 26 on the entire bottom surface, or the primers 20 are respectively attached to multiple spatially isolated polymer islands located on the bottom surface. In these embodiments, the sequencing-ready nucleic acid fragments 44, 44', or 44" are respectively seeded on the entire bottom surface of the chamber 14 or on each island. In other embodiments, each chamber 14 has a bottom surface and a plurality of recesses 16 defined therein, and the primers 20 are respectively attached to the polymer layer 26 within each recess 16. In these embodiments, the sequencing-ready nucleic acid fragments 44, 44', or 44" are seeded on the polymer layer 26 within each recess 16.

[0176] In another exemplary method (referred to as the second exemplary method), the 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 within each of the plurality of chambers 14. In this embodiment, the flow cell 10 may or may not include a recess 16.

[0177] In this second exemplary method, the depth of each chamber 14 is about 5 microns or less. With such a shallow depth, capture sites 22 can be included to immobilize a single complex 40A, 40B, or 40C in a single chamber 14. Although each chamber 14 has a capture site 22, it should be understood that during any given run of the method, some chambers 14 may not receive a complex 40A, 40B, or 40C.

[0178] In this second exemplary method, complex 40A, 40B or 40C is introduced into flow cell 10, for example, through one or more input ports. Complex 40A, 40B or 40C can be introduced into fluid, such as buffer disclosed herein. In this embodiment, corresponding capture site 22 and complex 40A, 40B or 40C are members of binding pairs, so that a complex 40A, 40B or 40C is combined with a capture site 22 in each chamber 14. More specifically, capture site 22 can include a first member of the binding pair, and each complex 40A, 40B or 40C can include a second member of the binding pair. As a specific embodiment, capture site 22 is a capture site primer (e.g., capture oligonucleotide), and each complex 40A, 40B or 40C includes a complementary primer that can hybridize with the capture site primer. As another specific embodiment, capture site 22 can include avidin, and biotin can be attached to the surface of complex 40A, 40B or 40C.

[0179] The second exemplary method then includes washing the 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 the capture sites 22 out through the outlet port of the flow cell 10.

[0180] The second exemplary method then includes introducing an external fixative into the flow cell 10, and specifically, into the plurality of chambers 14. In one embodiment, the external fixative is air or a liquid medium or a viscous medium that is immiscible with the composite 40A, 40B, or 40C of the fluids that have been introduced into the flow cell chambers 14.

[0181] Using air to draw the wash fluid out of the flow cell 10 can create droplets around the complex 40A, 40B, or 40C and form a diffusion barrier. A liquid or viscous external fixative at least partially surrounds the complex 40A, 40B, or 40C attached within the chamber 14. By at least partially surrounding the complex 40A, 40B, or 40C, the external fixative inhibits diffusion of the sequencing-ready nucleic acid fragments 44, 44', or 44" outside the chamber 14 when the fragments 44, 44', or 44" are released. When the external fixative reagent is a temperature-responsive material, raising the temperature to the seeding temperature can make the reagent more viscous and in a form that prevents diffusion of the library.

[0182] It should be understood that any external fixative disclosed herein may be used, but in one embodiment, the external fixative is a liquid diffusion barrier selected from mineral oil and silicone oil, a viscous medium diffusion barrier selected from glycerol and sucrose, and combinations thereof.

[0183] This embodiment of the method then includes causing the carrier (e.g., solid support 42 or hydrogel support 70) of the captured complexes 40A, 40B or 40C to release the sequencing-ready nucleic acid fragments 44, 44' or 44" into the corresponding chamber 14, wherein each fixed 40A, 40B or 40C is captured. In this embodiment, the transport and seeding of the sequencing-ready nucleic acid fragments 44, 44' or 44" is restricted by the external fixative.

[0184] Depending on the complex 40A, 40B, or 40C used, causing the support (i.e., support 42 or 70) to release the sequencing-ready nucleic acid fragments 44, 44', or 44" can vary. In one embodiment, the support is a solid support 42, and the causing includes introducing a cleavage agent to the flow cell 10 (as described in the first exemplary method), and using another stimulus to trigger the cleavage agent to release the library fragments 44 or 44' from the solid support 42. In other embodiments, complex 40C is used, and thus the support is a hydrogel support 70. In these other embodiments, causing the library release can include heating the flow cell, introducing a cleavage agent to the flow cell, or a combination thereof (as described in the first exemplary method).

[0185] As described above, in this second exemplary method, transport and seeding of sequencing-ready nucleic acid fragments 44, 44' or 44" are confined by an external fixative. Thus, the 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 because the external fixative at least partially surrounds the complex 40A, 40B or 40C.

[0186] Using the flow cell architecture disclosed herein, primers 20 on the surface of the flow cell 10 can be seeded with the released sequencing-ready nucleic acid fragments 44, 44' or 44". The seeding is accomplished by hybridization between the first or second sequence of the fragment 44, 44' or 44" and the complementary sequence of the primer 20 in the chamber 14. The seeding can be performed at a suitable hybridization temperature for the fragment 44, 44' or 44" and the primer 20.

[0187] The location at which the sequencing-ready nucleic acid fragments 44, 44' or 44" are seeded in the corresponding chamber 14 depends in part on how the primers 20 are attached within the chamber 14. In some embodiments of the flow cell, each chamber 14 has a bottom surface, and the primers 20 are attached to the polymer layer 26 of the entire bottom surface, or the primers 20 are respectively attached to multiple spatially isolated polymer islands located on the bottom surface. In these embodiments, the sequencing-ready nucleic acid fragments 44, 44' or 44" are respectively seeded on the bottom surface of the chamber 14 or on each island. In other embodiments, each chamber 14 has a bottom surface and a plurality of recesses defined therein, and wherein the primers are respectively attached to the polymer layer 26 within each recess 16. In these embodiments, the sequencing-ready nucleic acid fragments 44, 44' or 44" are seeded on the polymer layer 26 within each recess 16.

[0188] In yet another embodiment of the method (referred to as the third exemplary method), the Figure 1 , Figure 2A , Figure 2B and FIG. 3A to FIG. 3C Any embodiment of the flow cell 10 shown. In this third exemplary method, a capture site 22 can be included to immobilize a single complex 40A, 40B, or 40C in a single chamber 14, and the depth of each chamber 14 can be sufficient to restrict the transport and seeding of the sequencing-ready nucleic acid fragment 44, 44', or 44" to the corresponding recess 16 within each corresponding chamber 14.

[0189] In this third exemplary method, complex 40A, 40B or 40C is introduced into flow cell 10, for example, through one or more input ports. Complex 40A, 40B or 40C can be introduced into a fluid, such as a buffer disclosed herein. In this embodiment, the corresponding capture site 22 and complex 40A, 40B or 40C are members of a binding pair, such that one complex 40A, 40B or 40C binds to one capture site 22 in at least some chambers 14. Although each chamber 14 has a capture site 22, it should be understood that some chambers 14 may not receive complex 40A, 40B or 40C during any given run of the method.

[0190] The third exemplary method then includes washing the 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 the capture sites 22 through the outlet port of the flow cell 10.

[0191] This embodiment of the method then includes causing the carrier (e.g., solid support 42 or hydrogel support 70) of the captured complexes 40A, 40B, or 40C to release the sequencing-ready nucleic acid fragments 44, 44', or 44" into the corresponding chamber 14 in which each complex 40A, 40B, or 40C is captured. In this embodiment, the transport and seeding of the sequencing-ready nucleic acid fragments 44, 44', or 44" is limited by the depth of the corresponding chamber 14, and therefore external fixatives are not introduced into the flow cell 10.

[0192] Depending on the complex 40A, 40B, or 40C used, causing the carrier (i.e., support 42 or 70) to release the sequencing-ready nucleic acid fragments 44, 44', or 44" can vary. In one embodiment, the carrier is a solid support 42, and the causing includes introducing a cleavage agent to the flow cell 10 (as described in the first exemplary method) and exposing the flow cell 10 to an external stimulus. In other embodiments, complex 40C is used, and thus the carrier is a hydrogel support 70. In these other embodiments, causing the library to release may involve heating the flow cell, introducing a cleavage agent to the flow cell, or a combination thereof (as described in the first exemplary method).

[0193] As described, transport and seeding of sequencing-ready nucleic acid fragments 44, 44', or 44" are restricted by the depth of the corresponding chamber 14. Therefore, the fragments 44, 44', or 44" of any particular complex 40A, 40B, or 40C will be confined to the chamber 14 in which the particular complex 40A, 40B, or 40C is confined. In this particular embodiment, because the flow cell 10 includes primers 20 in recesses 16, seeding of the fragments 44, 44', or 44" occurs within the recesses 16 rather than on the interstitial region 24.

[0194] In any of the embodiments of the methods involving complexes 40A, 40B, or 40C (eg, the first, second, or third methods described herein), the seeded sequencing library can be amplified using cluster generation.

[0195] In one embodiment of cluster generation, a sequencing-ready nucleic acid fragment 44, 44' or 44" is copied by 3' extension from a hybridized primer 20 using a high-fidelity DNA polymerase. The original sequencing-ready nucleic acid fragment 44, 44' or 44" is denatured, thereby immobilizing the copy within the chamber 14. Isothermal bridge amplification or some other form of amplification can be used to amplify the immobilized copy. For example, the copied template is looped to hybridize with an adjacent complementary primer 20, and the polymerase replicates the copied template to form a double-stranded bridge, which denatures to form two single-stranded chains. The two chains form a loop and hybridize with the adjacent complementary primer 20, and are extended again to form two new double-stranded loops. The process is repeated on each template copy through cycles of isothermal denaturation and amplification to produce dense clonal clusters. 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 chain. It should be understood that clustering results in the formation of several template sequencing-ready nucleic acid fragments, such as in each chamber 14, and in some cases, within each recess 16 in each chamber 14. This cluster embodiment is a bridge amplification, and is one example of an amplification that may be performed. It will be appreciated that other amplification techniques may be used, such as the Exclusion Amplification (ExAmp) protocol (Illumina Inc.).

[0196] After cluster generation, sequencing can be performed.Any of the embodiments of the flow cell 10 disclosed herein can be used in a variety of sequencing methods or techniques, including techniques generally referred to as sequencing by synthesis (SBS), cycle array sequencing, sequencing by ligation, pyrosequencing, and the like.

[0197] For example, a sequencing-by-synthesis (SBS) reaction can be performed in a HISEQ TM HISEQX TM MISEQ TM MISEQDX TM MINISEQ TM 、NOVASEQ TM 、NEXTSEQDX TM 、ISEQ TM 、NEXTSEQ TM or other sequencer systems from Illumina (San Diego, CA).

[0198] A sequencing primer that hybridizes with a complementary sequence on a template polynucleotide chain can be introduced. The sequencing primer prepares the template polynucleotide chain for sequencing. In SBS, the sequencing primer is monitored along the extension of the template sequencing-prepared nucleic acid fragment (template polynucleotide chain) to determine the sequence of nucleotides in the template. The 3' end of the template and any primer 20 (not attached to the copy) bound by the flow cell can be closed to prevent interference with the sequencing reaction, and in particular to prevent undesirable initiation. The basic chemical process can be polymerization (e.g., polymerase catalysis) or connection (e.g., ligase catalysis).

[0199] In a specific polymerase-based SBS process, fluorescently labeled nucleotides are added to the sequencing primer (thereby extending the sequencing primer) in a template-dependent manner so that the order and type of nucleotides added to the sequencing primer can be used to determine the sequence of the template. More specifically, a nucleotide is incorporated into the extended sequencing primer and the nascent chain complementary to the template polynucleotide chain by a corresponding polymerase. For example, to start the first SBS cycle, one or more labeled nucleotides, DNA polymerases, etc. can be transported to / through the flow cell 10, etc., where the sequencing primer extension causes the labeled nucleotides to be incorporated. This incorporation can be detected by an imaging event. During the imaging event, an illumination system (not shown) can provide excitation light to the flow cell 10.

[0200] In some embodiments, the fluorescently labeled nucleotides may further include a reversible termination property that terminates further primer extension when the nucleotides are added to the template. For example, a nucleotide analog having a reversible terminator portion may be added to the template such that subsequent extension cannot occur until a deblocking agent is delivered to remove the portion. Thus, for embodiments using reversible termination, a deblocking agent may be delivered to a flow cell, etc. (after detection occurs).

[0201] Washing may be performed between each fluid transfer step.The SBS cycle may then be repeated n times to extend the template by n nucleotides, thereby detecting a sequence of length n.

[0202] Although SBS has been described in detail, it will be appreciated that the flow cell 10 described herein may be used in other sequencing protocols, for genotyping or other chemical and / or biological applications.

[0203] Methods involving complex formation on a flow cell architecture

[0204] Other embodiments of the methods disclosed herein do not use FIG. 4A to FIG. 4C In contrast, the hydrogel matrix is ​​formed in situ within chamber 14 of the flow cell. Figure 5 (i) to Figure 5 (iii) Describe an embodiment.

[0205] In this embodiment, the flow cell 10' can be part of a sequencing kit that includes the flow cell 10' and various reagents for forming the hydrogel matrix 74 within the chambers 14 of the flow cell 10'. An embodiment of the sequencing kit includes a flow cell 10' that includes a plurality of chambers 14 (e.g., as described in reference Figure 1 , Figure 2A and Figure 2B The primer 20 formed in or on the substrate 12 and attached to each of the plurality of chambers 14. Figure 5 (i) to Figure 5 (iii) In the illustrated embodiment, each chamber has a bottom surface, and the primer 20 is attached to the polymer layer 26 of the entire bottom surface. Although not shown, the polymer layer 26 (on the chamber bottom surface) may alternatively be in the form of a plurality of spaced-apart polymer islands, and the primer 20 is attached to each island, respectively. In yet another embodiment 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 the primer 20 is attached to the polymer layer 26 in each recess 16.

[0206] like Figure 5 As shown in (i), the flow cell 10 ′ further includes an embodiment of a capture site 22 ′, wherein the capture site 22 ′ is configured to capture a sample 72 .

[0207] The capture site 22' can be any example of a chemical capture agent disclosed herein, which can be attached to a sample 72 introduced into the flow cell 10'. For a natural DNA or RNA sample 72, the capture site 22' can include a linker having a nucleic acid binding portion at one end, such as an intercalator that binds by charge or hydrophobic interactions, or one member of a binding pair (where the sample 72 includes the other member), or an oligonucleotide that can hybridize with the sample 72, etc. For a cell sample 72, the linker can include a cell membrane binding portion (e.g., an antigen to a surface protein) or a membrane penetrating portion (e.g., a phospholipid at one end).

[0208] Although not in Figure 5 (i) to Figure 5 As shown in (iii), the flow cell 10 ′ may include a recess 16 within each chamber 14 , and a primer 20 may be attached within each recess 16 .

[0209] This embodiment of the sequencing kit also includes an encapsulated (hydrogel) matrix precursor composition consisting of a fluid, a monomer or polymer (including free radical generation and chain extension functional groups), a free radical source and a cross-linking agent. The encapsulated (hydrogel) matrix precursor composition does not include sample 72. In one embodiment, the encapsulated (hydrogel) matrix precursor composition includes about 2% (w / v) to about 20% (w / v) of a monomer or polymer, about 1% by weight to about 10% by weight of a cross-linking agent and about 0.1% (w / v) to about 10% (w / v) of a free radical source. When included in the composition, the free radical initiator can be present in an amount of about 0.1% (w / v) to about 10% (w / v).

[0210] The fluid encapsulating the (hydrogel) matrix precursor composition may be water (eg, deionized water).

[0211] When the monomer is used in the encapsulating (hydrogel) matrix precursor composition, the monomer is selected from 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 monomers and combinations thereof. In some embodiments, when a polymer is used in the encapsulation (hydrogel) matrix precursor composition, the polymer is selected from polyethylene glycol-thiol, polyethylene glycol-acrylate, polyethylene glycol diacrylate, polyethylene glycol (e.g., with 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-glutamic acid), polylysine, and combinations thereof. In other embodiments, when a polymer is used for the encapsulation (hydrogel) matrix precursor composition, the polymer includes a first polymer and a second polymer; wherein the first polymer is selected from polyethylene glycol-thiol, polyethylene glycol-acrylate, polyethylene glycol diacrylate, polyethylene glycol, polyoxypropylene, polyacrylic acid, poly (hydroxyethyl methacrylate), poly (methyl methacrylate), poly (N-isopropylacrylamide), poly (lactic acid), poly (lactic acid-co-glycolic acid), polycaprolactone, poly (ethylene sulfonic acid), poly (L-aspartic acid), poly (L-glutamic acid), polylysine and combinations thereof; and the second polymer is selected from agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronic acid, pectin, carrageenan, gelatin, chitosan, cellulose, collagen polymers and combinations thereof. Any of the monomers and polymers may also be used in combination in the encapsulation (hydrogel) matrix precursor composition.

[0212] The free radical source is a molecule that generates free radicals when decomposed. In one embodiment, the free radical source is selected from 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.

[0213] The cross-linking agent forms a bond, such as a disulfide bond, in the polymer of the hydrogel matrix. The cross-linking agent can be reversible because it can be cross-linked or uncross-linked depending on the chemical substance to which it is exposed. For example, a reversible cross-linking agent is a bisacrylamide cross-linking agent containing a disulfide bond, which can be decomposed with a reducing agent such as DTT, TCEP or THP (phosphine). In one embodiment, the cross-linking agent is selected from acrylamide, N, N'-bis (acryloyl) cystamine, bisacrylamide, 1,4-diacyl piperazine, N-N'-diallyl L-tartrate diamide and N-N'-(1,2-dihydroxyethylene)-bisacrylamide.

[0214] This embodiment of the sequencing kit also includes a free radical initiator as part of the encapsulated matrix precursor composition or as a separate component. In one embodiment, the free radical initiator can be a photoinitiator. Examples of photoinitiators include azobisisobutyronitrile, benzoyl peroxide, eosin-5-isothiocyanate. This type of free radical initiator can be included in the encapsulated (hydrogel) matrix precursor composition because it will not cause crosslinking until it is exposed to light of an appropriate wavelength. In another embodiment, when exposed to a free radical source in the encapsulated (hydrogel) matrix precursor composition, the free radical initiator can cause crosslinking. In these embodiments, the free radical initiator is kept separate from the encapsulated (hydrogel) matrix precursor composition until it is desired to form a hydrogel matrix on the flow cell 10'. The example of this type of free radical initiator is tetramethylethylenediamine (TEMED).

[0215] In one embodiment, the sequencing kit may further include a sample fluid comprising water and a sample 72 (eg, genetic material).

[0216] In one embodiment, the sequencing kit may further include a library preparation solution comprising an adaptor sequence and a transposome.

[0217] In an embodiment of the method of using this embodiment of the sequencing kit, a sample fluid (including a sample 72 of genetic material) is introduced into the flow cell 10' ( Figure 5 (i)). At least some of the genetic material (sample 72) enters at least some of the plurality of chambers 14 through the capture sites 22' in the corresponding chambers 14. The sample 72 is fixed to the capture sites 22'.

[0218] The liquid of the sample fluid, 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'. This flow can push any unbound sample 72 out through the outlet port of the flow cell 10'.

[0219] This embodiment of the method includes introducing an encapsulating matrix precursor composition 76 into a flow cell 10' ( Figure 5 (ii)). At least some of the encapsulating matrix precursor composition 76 enters at least some of the chambers 14 containing the sample 72.

[0220] The method then includes encapsulating the sample 72 (i.e., genetic material) in the hydrogel matrix 74 in at least some of the chambers 14 by initiating crosslinking or crosslinking and polymerization of the encapsulation matrix precursor composition 76 contained in at least some of the chambers 14. Figure 5 (iii)).

[0221] Prior to encapsulation, an external fixative may be introduced into the flow cell 10'. This agent may remove the encapsulation matrix precursor composition 76 from the flow cell 10', except for the composition 76 that has entered the chamber. This forms a barrier during the hydrogel matrix formation process. Any example of an external fixative disclosed herein may be used.

[0222] When the encapsulation matrix precursor composition 76 includes a photoinitiator (e.g., an ultraviolet free radical initiator), encapsulation involves exposing the flow cell 10' to ultraviolet radiation. The exposure initiates free radical generation, which in turn initiates cross-linking or cross-linking and polymerization of the components in the encapsulation matrix precursor composition 76 retained in the chamber 14. The cross-linking or cross-linking 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.

[0223] When a free radical initiator that initiates crosslinking when exposed to a free radical source is used, the free radical initiator is introduced separately from the encapsulation matrix precursor composition 76. In these embodiments, encapsulation includes exposing the flow cell 10' to the free radical initiator. In this embodiment, the free radical initiator can be introduced together with an external fixative. The free radical initiator in the external fixative initiates the generation of free radicals in the encapsulation matrix precursor composition 76, which then initiates the crosslinking or crosslinking and polymerization of the components in the encapsulation matrix precursor composition 76 retained in the chamber 14. Crosslinking forms or crosslinking and polymerization forms a hydrogel matrix 74 in the chamber 14. This encapsulates the sample 72 in the hydrogel matrix 74 in the chamber 14.

[0224] Library preparation can be performed on the surface of the flow cell 10'. The external fixative can be removed, and a buffer can be introduced into the flow cell 10' along with the library preparation solution. Library preparation can be performed as described in Figure 4C Proceed as described.

[0225] Then, seeding, clustering and sequencing can be performed according to the embodiments disclosed herein.

[0226] In another embodiment, the hydrogel matrix is ​​formed in situ in the chamber 14 of the flow cell 10' in the absence of a cross-linking agent or a free radical initiator. In this embodiment, the polymer in the encapsulating (hydrogel) matrix precursor composition is capable of forming a gel when exposed to a gelling temperature.

[0227] In this embodiment, the flow cell 10' may be part of a sequencing kit including the flow cell 10' and an encapsulating (hydrogel) matrix precursor composition.

[0228] The flow cell 10' includes a plurality of chambers 14 (e.g., as shown in FIG. Figure 1 , Figure 2A and Figure 2B The primers 20 are formed in or on the substrate 12 and attached within each of the plurality of chambers 14. It should be understood that any embodiment of the flow cell disclosed herein may be used.

[0229] Sequencing kit also includes encapsulation (hydrogel) matrix precursor composition made up of fluid and polymer, and described polymer is selected from agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronic acid, pectin, carrageenan, gelatin, chitosan, cellulose, collagen polymer and combination thereof.When being exposed to specific gelling temperature, each of these polymers can form gel.Encapsulation (hydrogel) matrix precursor composition does not include sample.In one embodiment, encapsulation (hydrogel) matrix precursor composition comprises polymer of about 0.1% (w / v) to about 10% (w / v).In another embodiment, encapsulation (hydrogel) matrix precursor composition comprises polymer of about 2% (w / v) to about 8% (w / v).The fluid of encapsulation (hydrogel) matrix precursor composition can be water (for example, deionized water) or buffer solution (for example, phosphate buffered saline (PBS)).When polymer is alginate, fluid is calcium-containing solution, because calcium ions are cross-linked with alginate in gel formation process. In one embodiment, the molar concentration of calcium ions may be in the range of about 1 mM to about 10 mM.

[0230] In one embodiment, the sequencing kit may further include a sample fluid comprising water and a sample 72 (eg, genetic material).

[0231] In one embodiment, the sequencing kit may further include a library preparation solution comprising an adaptor sequence and a transposome.

[0232] In one embodiment of the method for this embodiment using a sequencing kit, sample fluid (comprising the sample 72 of genetic material) and encapsulation (hydrogel) matrix precursor composition can be mixed before introducing into the flow cell. In one embodiment, sample fluid is by rotating the required genetic material, being suspended in the buffer solution, and solution is heated to a desired temperature (e.g., 42 ℃) to prepare. Individually, polymer solution (e.g., 2.5% agarose in the PBS) is prepared under the temperature (e.g., 80 ℃) raised. The sample fluid then concentrated is combined with encapsulation (hydrogel) matrix precursor composition with a desired ratio. In one embodiment, the mixture of sample fluid and encapsulation (hydrogel) matrix precursor composition contains approximately 2% polymer.

[0233] The combination of sample fluid and encapsulating (hydrogel) matrix precursor composition can be introduced into the flow cell 10 ′, for example, through an input port. The polymer and sample enter at least some of the chambers 14 .

[0234] This embodiment of the method then includes flushing the flow cell 10' with a liquid external fixative at a temperature in the range of about 40°C to about 80°C. In other embodiments, the flow cell 10' may be flushed with a liquid external fixative at a temperature in the range of about 40°C to about 70°C or about 40°C to about 50°C. Any example of a liquid external fixative may be used. In one embodiment, the liquid external fixative is mineral oil and the temperature is about 42°C. It is desirable to flush the flow cell 10' to remove any non-specifically attached polymer. Heating the liquid external fixative may help wash the polymer away from the interstitial region without removing a sample of the polymer from the chamber 14.

[0235] In an embodiment of the method, the temperature of the flow cell is raised or lowered to the gelation temperature of the polymer in at least some of the chambers 14. At this temperature, the polymer gels and forms a hydrogel matrix that encapsulates the genetic material. In one embodiment, exposing the flow cell 10' to the gelation temperature of the polymer includes cooling the flow cell 10' to the gelation temperature and maintaining the flow cell at the gelation temperature for a predetermined time. For example, the gelation temperature may be about 25°C or lower, and the predetermined time may be up to about 30 minutes. When agarose is the polymer, the gelation temperature may be in the range of about 4°C to about 20°C, and the predetermined time is about 10 minutes. In another embodiment, exposing the flow cell 10' to the gelation temperature of the polymer includes heating the flow cell 10' to the gelation temperature and maintaining the flow cell at the gelation temperature for a predetermined time (e.g., up to about 10 minutes).

[0236] Depending on the sample used, cell lysis and DNA extraction can then be performed.

[0237] Library preparation can also be performed on the surface of the flow cell 10'. The external fixative can be removed, and a buffer can be introduced into the flow cell 10' along with the library preparation solution. Figure 4C Proceed as described.

[0238] Then, seeding, clustering and sequencing can be performed according to the embodiments disclosed herein.

[0239] In order to further illustrate the present disclosure, examples are given herein. It should be understood that these examples are provided for illustrative purposes and should not be construed as limiting the scope of the present disclosure.

[0240] Non-limiting working examples

[0241] Example 1

[0242] A hydrophobic layer ( S), and deposit a positive photoresist on the hydrophobic layer. Using photolithography, a microchamber with a diameter of 50 microns is then patterned in the photoresist. The hydrophobic layer and the outermost silicon layer are then etched according to the photoresist pattern. The photoresist is then stripped.

[0243] The microchambers in the substrate were silanized and PAZAM was deposited on them. Unattached PAZAM was washed away and then P5 and P7 primers were grafted onto PAZAM in the microchambers. The lid was attached to the substrate.

[0244] Prepared similar to Figure 4A The complex shown has an average diameter of 3 microns. The fragments on a particular bead come from the same long DNA molecule (from the PhiX genome). The library fragments are attached to the solid support via desthiobiotin oligomers, which have a weaker affinity for streptavidin on the bead surface than biotin. The library fragments include P5 and P7' sequences as well as an index sequence, and read fragment 1 and read fragment 2 sequences. The complex is loaded into a microchamber.

[0245] Fig. 6A is a photomicrograph of one of the composites illustrating the interior of one of the microchambers.

[0246] Seeding was initiated by releasing library fragments from the complex and clustering was performed using bridge amplification. Figure 6B It is derived from Fig. 6A Fluorescence micrographs of clusters of the seeded library of complexes.

[0247] Perform first base sequencing, and Figure 6B The real-time analysis (RTA) of the microchamber is shown in Figure 6C middle. Fig.6D Shows from Figure 6C Example of an island obtained by reading fragments inside a microchamber. Fig.6D The results in show that all read fragments originate from the same segment of the long DNA fragment.

[0248] Example 2

[0249] A flow cell was prepared using a glass substrate with two lanes. 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 a circular pattern outlining a different microchamber in each of the two lanes. Any hydrophobic material beneath the non-developed resist was etched away. This exposed the surface of each microchamber. With the developed photoresist in place, the microchambers were silanized and PAZAM was deposited on top of them. The non-attached PAZAM was removed along with the photoresist using a lift-off technique. P5 and P7 primers were then grafted to the PAZAM in the microchambers. The lid was bonded to the substrate using a UV curable adhesive.

[0250] The microchambers in this example have different diameters and spacings and are prepared in two different lanes along the length of the flow cell. Table 1 shows the diameter and spacing of each lane.

[0251] Table 1

[0252]

[0253] The complex used in this example includes a solid support and a sequencing-ready nucleic acid fragment attached to the solid support via an avidin-biotin linker. The library fragment of the complex is similar to Figure 4B According to TruSeq TM The ELISA platform (Illumina, Inc.) protocol prepares PCR-free libraries in test tubes. The libraries are bound to the beads by hybridization with the P7 primers, which are attached to the beads via biotin.

[0254] The complexes were introduced into the flow cell by flowing hybridization buffer (200 μL in hybridization buffer) containing the complexes through the flow cell channels. Fig. 7A is a photomicrograph showing a magnified portion of various sections of a flow cell lane after introduction of the complexes. The lane and section designations correspond to Table 1. As shown, one or more complexes are sequestered in at least some of the microwells.

[0255] Seeding was initiated by releasing library fragments from the complex. Library release was initiated by heating the flow cell above the melting temperature of the P7 primer. Fragments hybridized and first strand extension was performed. The solid support and non-hybridized fragments were removed with 0.2 M sodium hydroxide solution. Clustering was then performed using bridge amplification. First base sequencing was performed and real-time analysis (RTA) of the microchamber was shown in Figure 7B These results suggest that there is no crosstalk between the microcompartments that received the complexes and those that did not.

[0256] Example 3

[0257] A flow cell was formed in a similar manner as described in Example 2.

[0258] Escherichia coli dyed with Sytox green and dispersed in TRIS HCl washing buffer (pH 8.1) are mixed with encapsulation matrix precursor and introduced into the flow cell. The flow cell is then washed. The encapsulation matrix precursor composition includes acrylamide monomer, potassium persulfate (KPS) and bisacrylamide. Mineral oil containing a free radical initiator (TEMED) is then introduced. The oil forces the encapsulation matrix into at least some of the microchambers and initiates crosslinking. Figure 8 Number 1 in FIG. 1 is a micrograph of some microchambers after hydrogel formation. As shown, hydrogels were formed in most of the microchambers.

[0259] The hydrogel matrix allows reagents to be freely exchanged in and out of the matrix, but retains the bacteria within. To demonstrate this, washing was performed and then cell lysis was performed by introducing lysozyme into the flow cell. The flow cell was heated to about 37°C for about 30 minutes to activate the lysozyme. Figure 8 Number 2 in FIG. 1 is a micrograph of the microchamber after cell lysis (of number 1 ).

[0260] DNA extraction was performed in the presence of proteinase K (ProK), which digests contaminating proteins. Figure 8 Number 3 in the figure is a micrograph of the microchamber after DNA extraction (number 2).

[0261] Transposomes (NEXTERA from Illumina, Inc. TM The library was prepared by using a DNA library preparation kit) with P7-ME (splice end) / ME' and P5-ME / ME' adapter mixture. After tagmentation, SDS was used to remove proteins, and PCR enzymes were used for extension reaction to generate double-stranded libraries.

[0262] Figure 8 Figure 4 in Figure 4 is a micrograph of the microchamber after library preparation (Figure 3). These results demonstrate that in situ sample encapsulation and hydrogel formation can be performed on the flow cell surface.

[0263] Example 4

[0264] A glass substrate is used and circular nano-depressions are etched into it. S is a hydrophobic polymer (isolation material 18), which is deposited on the entire glass substrate, including in the nano-depression. A photoresist (Shipley S-1805) is applied to the hydrophobic polymer and developed to define a circular pattern of the hydrophobic polymer. The hydrophobic polymer is removed from the nano-depression that is not covered by the photoresist by plasma etching. The exposed nano-depression is then silanized and coated with a gel material. The gel material is PAZAM. The photoresist and any gel material on the photoresist are then removed using a stripping process. This shows the hydrophobic polymer of the lower layer. The hydrophobic polymer extends from about 1 micron to about 2 microns along the Z direction on the gap region. The Z direction refers to the Z axis of the Cartesian coordinate system of the three-dimensional space. In the present embodiment, the hydrophobic polymer defines a circular chamber with a diameter of 50 microns. Primer grafting was performed to attach P5 and P7 primers to the PAZAM in the nano-depression.

[0265] Prepared similar to Figure 4A The complex shown. The fragments on a specific bead come from the same long DNA molecule. The library fragments are attached to a solid support by desthiobiotin oligomers, which have a weaker affinity for streptavidin on the bead surface than biotin. The complex is loaded into a microchamber. The attachment of the complex to the surface of the microchamber is accomplished by anchoring (e.g., a complementary primer with biotin hybridized to a P5 primer attached to a gel material, or an alkyne-PEG-biotin linker covalently linked to a free azide on the gel material using click chemistry). Free biotin in a sodium citrate saline buffer containing sodium dodecyl sulfate is introduced, and the flow cell is heated to about 80°C to release the library from the corresponding complex. Air is drawn through the flow cell to push out the free biotin solution. Due to the hydrophobic / hydrophilic surface structure, droplets are formed in the microchamber when the liquid is pushed out by the air. Droplets prevent library fragments from diffusing to adjacent microchambers.

[0266] The released library fragments are then hybridized to the surface primers in the microchamber and an extension step is performed to generate complementary copies. Cluster generation is performed by bridge amplification. Sequencing is then performed on the flow cell.

[0267] Fig. 9 A portion of the flow cell after data analysis of a sequencing run is shown. The original colors represent islands, or short read fragments grouped together according to their proximity on the reference genome. Since the corresponding colors are isolated to specific microchambers, it is concluded that the short read fragments in a given microchamber come from the same segment of genomic DNA, and therefore from the same complex. These results indicate that the microchambers are able to confine the complex and the released library fragments to the corresponding chambers.

[0268] Example 5

[0269] This example demonstrates hydrogel formation on a flow cell.

[0270] A glass substrate with two lanes is used. Microchambers of different sizes are etched into a glass slide. The diameter and spacing of the microchambers in each lane are the same as shown in Table 1. A hydrophobic material is deposited on a glass substrate. A positive photoresist is applied to the hydrophobic material. The positive photoresist is exposed and developed to define the covering hydrophobic material on the gap between the microchambers. Any hydrophobic material under the non-developing resist (and therefore in the microchamber) is etched away. This exposes the surface of each microchamber. With the developed photoresist in place, the microchamber is silanized and PAZAM is deposited thereon. The non-attached PAZAM is removed together with the photoresist using a stripping technique. Then, P5 and P7 primers are grafted to PAZAM in the microchamber. The cover is bonded to the substrate using a UV-cured adhesive.

[0271] The encapsulation matrix precursor is introduced into the flow cell. The flow cell is then washed. The encapsulation matrix precursor composition includes N, N'-bis (acryloyl) cystamine, acrylamide and 2,2'-azobis (2-methyl propionamidine) dihydrochloride. Evagreen oil is then introduced into the flow cell. The oil forces the encapsulation matrix into at least some of the microchambers. Fig. 10A are micrographs of chambers of different sizes (diameter and spacing see Table 1) in different sections (1-5) of two lanes after encapsulation. The lighter areas of each image 10A(i)-10A(x) depict the chambers, while the darker areas depict the gaps. Fig. 10A (i) to Fig. 10A (x) depicts the gaps versus no encapsulating matrix regardless of the size of the microchamber. The flow cell was then exposed to UV radiation to initiate hydrogel formation. The flow cell was then washed with TRIS HCl wash buffer (pH 8.1). Fig. 10B are micrographs of chambers of different sizes in different sections (1-5) of two lanes after hydrogel formation. The brighter areas of each image 10B(i)-10B(x) depict the chambers, while the darker areas depict the gaps. Fig. 10B (i) to Fig. 10B (x) shows that hydrogels are formed in at least some of the microchambers, regardless of the size of the microchambers.

[0272] In addition, it should be understood that the ranges provided herein include the range and any value or sub-range within the range, as if they were explicitly listed. For example, a range expressed as about 2 mm to about 300 mm should be interpreted as including not only the explicitly listed limits of about 2 mm to about 300 mm, but also individual values, such as about 15 mm, 22.5 mm, 245 mm, etc., and sub-ranges, such as about 20 mm to about 225 mm, etc.

[0273] It should be understood that all combinations of the foregoing concepts and additional concepts discussed in more detail below (provided that these concepts are not mutually inconsistent) are contemplated as part of the inventive subject matter disclosed herein. In particular, all combinations of the claimed subject matter appearing at the end of this disclosure are considered to be part of the inventive subject matter disclosed herein. It should also be understood that the terms explicitly used herein (which may also appear in any disclosure incorporated by reference) should be given the meaning most consistent with the specific concepts disclosed herein.

[0274] While several embodiments have been described in detail, it should be understood that the disclosed embodiments may be modified. Therefore, the foregoing description should be considered non-limiting.

Claims

1. A sequencing kit, comprising: A flow cell comprising: Multiple chambers; a layer located within each of the plurality of chambers; and a primer attached to the layer within each of the plurality of chambers; an encapsulating matrix precursor composition, at least some of which is in at least some of said chambers and consists of: fluid; Monomers or polymers including free radical generating and chain extending functional groups; Free radical sources; and cross-linking agent; and A free radical initiator as part of the encapsulating matrix precursor composition or as a separate component.

2. The sequencing kit according to claim 1, wherein the kit comprises one of the following: The monomer is selected from 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 a combination thereof; or The polymer is selected from polyethylene glycol-thiol, polyethylene glycol-acrylate, polyethylene glycol diacrylate, polyethylene glycol, polypropylene oxide, polyacrylic acid, poly(hydroxyethyl methacrylate), poly(methyl methacrylate), poly(N-isopropylacrylamide), poly(lactic acid), poly(lactic acid-co-glycolic acid), polycaprolactone, poly(ethylene sulfonic acid), poly(L-aspartic acid), poly(L-glutamic acid), polylysine and a combination thereof; or Any combination of the monomers and the polymers are used together.

3. The sequencing kit according to claim 1, wherein: The polymer includes a first polymer and a second polymer; The first polymer is selected from the group consisting of polyethylene glycol-thiol, polyethylene glycol-acrylate, polyethylene glycol diacrylate, polyethylene glycol, polypropylene oxide, polyacrylic acid, poly(hydroxyethyl methacrylate), poly(methyl methacrylate), poly(N-isopropylacrylamide), poly(lactic acid), poly(lactic-co-glycolic acid), polycaprolactone, poly(ethylene sulfonic acid), poly(L-aspartic acid), poly(L-glutamic acid), polylysine, and combinations thereof; and The second polymer is selected from the group consisting of agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronic acid, pectin, carrageenan, gelatin, chitosan, cellulose, collagen polymers, and combinations thereof.

4. The sequencing kit according to any one of claims 1, 2 or 3, wherein the free radical initiator is tetramethylethylenediamine.

5. The sequencing kit according to any one of claims 1, 2 or 3, wherein the free radical initiator is a photoinitiator and is included in the encapsulation matrix precursor composition.

6. A sequencing kit according to any one of claims 1, 2 or 3, wherein the free radical source is selected from potassium persulfate, ammonium persulfate, 4,4'-azobis(4-cyanovaleric acid), 1,1'-azobis(cyclohexanecarbonitrile), azobisisobutyronitrile, 2,2'-azobis(2-methylpropionitrile), peroxide, riboflavin, 3-(dimethylamino)propionitrile and combinations thereof.

7. The sequencing kit according to any one of claims 1, 2 or 3, wherein the cross-linking agent is selected from acrylamide, N,N'-bis(acryloyl)cystamine, bisacrylamide, 1,4-dipropionylpiperazine, N-N'-diallyl L-tartrate diamide and N-N'-(1,2-dihydroxyethylene)-bisacrylamide.

8. The sequencing kit according to any one of claims 1, 2 or 3, wherein each chamber has a bottom surface, and wherein the layer is a polymer layer attached to the entire bottom surface.

9. The sequencing kit of any one of claims 1, 2 or 3, wherein each chamber has a bottom surface, and wherein the layer is a plurality of spatially isolated polymer islands located on the bottom surface.

10. The sequencing kit according to any one of claims 1, 2 or 3, wherein each chamber has a bottom surface and a plurality of recesses defined therein, and wherein the layer is a polymer layer within each of the recesses.

11. The sequencing kit according to any one of claims 1, 2 or 3, further comprising a library preparation solution comprising an adapter sequence and a transposome.

12. A sequencing kit as defined in any one of claims 1, 2 or 3, further comprising a sample fluid comprising genetic material.

13. A method comprising: A fluid containing genetic material is introduced into a flow cell comprising: Multiple chambers; a layer located within each of the plurality of chambers; and a primer attached to the layer within each of the plurality of chambers; whereby at least some of said genetic material enters at least some of said plurality of chambers; removing liquid of the fluid from the flow cell; introducing into the flow cell an encapsulating matrix precursor composition comprising: Monomers or polymers including free radical generating and chain extending functional groups; Free radical sources; and Cross-linking agent; whereby at least some of said encapsulation matrix precursor composition enters at least some of said chambers containing said genetic material and said attachment primers; and The genetic material is encapsulated in the hydrogel matrix in the at least some of the compartments by initiating cross-linking or cross-linking and polymerization of the encapsulation matrix precursor composition contained in the at least some of the compartments.

14. The method of claim 13, wherein the encapsulation matrix precursor composition further comprises an ultraviolet free radical initiator, and wherein generating the hydrogel matrix is ​​accomplished by exposing the flow cell to ultraviolet radiation.

15. The method of claim 13, wherein the cross-linking or the cross-linking and polymerization of the encapsulating matrix precursor composition contained in the at least some of the chambers is accomplished by introducing a free radical initiator into the flow cell.

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